The Expanding Horizons in Thrombosis and Hemostasis

Vivian Xiaoyan Du The Expanding Horizons in Thrombosis and Hemostasis Thesis University Utrecht ISBN: 978-90-8891-881-0 Cover design: Andrea Almering Layout: Vivian X. Du Printing: Uitgeverij BOXPress Copy right © X.Du, Utrecht 2014 The Expanding Horizons in Thrombosis and Hemostasis

De Uitbreiding van de Horizon van Trombose en Hemostase (met een samenvatting in het Nederlands)

血栓与凝血新进展 （含中文概要）

Proefschrift

ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof. dr. G. J. van der Zwaan, ingevolge het besluit van het collage voor promoties in het openbaar te verdedigen op maandag 16 juni 2014 des middags te 2.30 uur

door

Xiaoyan Du

geboren op 23 september 1980 te Dongping county, China Promotor: Prof. dr. Ph. G. de Groot Co-promotor: Dr. B. de Laat

The studies described in this thesis were supported by a grant from Sanquin Blood Supply awarded to Dr. Bas de Laat . Financial support by the Netherlands Heart Foundation for the publication of this thesis is gratefully acknowledged. Financial support by Synapse B.V. and Phenom-World for publication of this thesis is gratefully acknowledged. Additional financial support by Stago BNL, CASLO ApS, Bayer B.V. and ChipSoft B.V. is gratefully acknowledged. “As far as we can discern, the sole purpose of human existence is to kindle a light in the darkness of mere being.” Carl Jung

To Iwan My father Pa en Ma Fernhout Dear Uncle and Aunt Du Beoordelingscommissie: prof. dr. J. A. G. van Strijp prof. dr. R. H. W. M. Derksen prof. dr. C. E. Hack prof. dr. H. ten Cate prof. dr. J. W. N. Akkerman

Paranimfen Arjan Barendrecht Silvie Sebastian Contents

Chapter 1 General introduction 9

Chapter 2 New insights into the role of erythrocytes in 29 thrombus formation Semin Thromb Hemost. 2014; 40: 72-80 chapter 3 Identification of intercellular adhesion molecule 4 on 49 erythrocytes as mediator of -erythrocyte interaction in thrombus formaiton Submitted

Chapter 4 Functional characterization of the ICAM-4-αIIbβ3 73 interaction on platelet function Prepared for submition

Chapter 5 Platelet apoptosis by cold-induced glycoprotein 91 Ibα clustering J Thromb Haemost. 2010; 8: 2554-2562

Chapter 6 Accelerated uptake of VWF/platelet complexes in 111 contribute to VWD-type 2B associated thrombocytopenia Blood. 2013;122: 2893-2902

Chapter 7 From antibody to clinical phenotype, the black box of 135 the antiphospholipid syndrome: pathogenic mechanisms of antiphospholipid syndrome Thromb Res. 2013; 132: 319-326

Chapter 8 Indications for a protective function of 163 beta2-glycoprotein I in thrombotic thrombocytopenic purpura Br J Haematol. 2012; 159: 94-103

Chapter 9 General discussion 181 Summary/Samenvatting/概要 195 Acknoledgement/Dankwoord 203 List of publications and curriculum vitae 209 Abbreviations 213

1

General introduction Chapter 1 Chapter

9 General introduction General The hemostatic system Blood is constantly circulating through our organs and tissues inside the vascular system. Upon vascular damage, the blood vessel integrity and the blood flow is disturbed and must be restored within limited time to prevent exsanguination. The system responsible for the arrest of bleeding and the restoration of vascular integrity is known as the hemostatic system. The hemostatic system seals a leak in the vessel by platelet plug formation (platelet adhesion and aggregation) and fibrin formation (coagulation) at the site of the vessel damage. Platelet plug formation and fibrin formation are essential to the arrest of bleeding and to the subsequent vascular repair. However, exaggerated response at site of atherosclerotic plaque rupture or at site of chronic vascular inflammation in the veins can lead to arterial or venous thrombosis.1-4 Hemostasis is therefore tightly regulated and an imbalance will lead to either bleeding or thrombosis.

Chapter 1 Chapter The platelet plug formation (also known as primary hemostasis) starts when adhere to subendothelial proteins (such as collagen) exposed to the flowing blood 10 upon vascular damage. The adhered platelets become activated and undergo a shape change accompanied by the secretion of platelet granule contents, such as

adenosine diphosphate (ADP) and serotonine, and synthesis of thromboxane (Tx)A2. These substances activate adjacent platelets and amplify platelet activation reaction. The activation of platelets lead to a conformational change in integerin alpha(IIb)

beta(3) (αIIbβ3) which increases its affinity for its soluble ligands fibrinogen and von Willebrand factor (VWF). Fibrinogen or VWF will then act as bridging proteins General introduction General to interact with activated integrin αIIbβ3 thereby forms a plug. At low shear rate,

fibrinogen is the predominant adhesive surface for platelets via integrinα IIbβ3, whereas at high shear rate VWF plays a more important role via platelet receptor glycoprotein Ib.5;6 The exposure of tissue factor (TF) at the site of vascular injury will initiate the coagulation cascade, which leads to thrombin formation and subsequently fibrin formation. The formation of fibrin around or inside a platelet plug will stabilize the plug and make the plug resistant to the shear force applied by the blood flow. New horizons in understanding thrombosis and hemostasis Erythrocytes in thrombosis and hemostasis

Erythrocytes, also known as red blood cells (RBCs), are the most abundant cells in our blood stream. They occupy approximately 40% of the total blood volume. Erythrocytes are produced in bone marrow from erythroid progenitor cells. Mature human erythrocytes lack cell nucleus and most organelles. In resting state, erythrocytes possess a biconcave shape. However, under the influence of high blood shear or in the capillary systems, they can be dramatically flexible. The principal function of erythrocytes is to transfer gas (oxygen and carbon dioxide) throughout the body. Erythrocytes take up oxygen in the lungs and release it at tissues. They take up the carbon dioxide produced by the peripheral tissues and release it in the lungs. Erythrocytes are generally considered as innocent bystanders in thrombosis and hemostasis. In a classic thrombus formation model (which mainly concerns endothelial cells, platelets and soluble coagulation factors), erythrocytes are passively entrapped inside a developing thrombus as they flow through the vasculature. Although erythrocytes seem to be smooth in shape and innate in function, they possess numerous adhesion molecules on the cell membrane7-9 and some signal transduction pathways inside the cytoplasma.10-15 Evidence has accumulated in the last years indicating that erythrocytes may influence the hemostatic balance in an active and well regulated manner. A systematic and up-to-date review about the involvement of erythrocytes in thrombosis and hemostasis as well as the underlying mechanisms is addressed in chapter 2 of this thesis. Historical background regarding the involvement of erythrocytes in thrombosis and hemostasis

The first publication suggesting that erythrocytes might play an active rolein 1 Chapter hemostasis dates back to 1910. In this year, Dr. Duke reported that thrombocytopenic 11 patients showed improvement in bleeding time after , even though their platelet counts were not increased after the transfusion.16 A few years later, Hellem et al. readdressed this issue in controlled studies aimed at assessing the possible impact of hematocrit on hemostasis by studying the bleeding time. They observed a decrease in bleeding time upon transfusion of washed erythrocytes.17 Later on, more clinical observations have shown that various bleeding disorders can

be treated by an elevation of erythrocyte counts despite of the unchanged or even introduction General decreased platelet count.18-20 In contrast to patients with low hematocrit who have an increased risk for bleeding, patients with increased hematocrit as in the condition called polycythemia vera are known to suffer from thrombosis. Increased hematocrit has been identified to be the only factor predictive of thrombotic events in these patients. 21-23 Thrombotic complications can have various underlying pathologies. In the last 100 years, erythrocytes have been implicated to be a contributor to the pro-thrombotic state of both erythroid diseases (hereditary erythrocyte abnormalities, such as sickle cell disease and thalassemia) and nonerythroid diseases (such as acute coronary syndromes and venous thrombosis) (Table 1). In 1991 Santos and Valles et al. made the first laboratory finding that indicated an interaction between erythrocyte and platelet. Their results demonstrated that intact erythrocytes can enhance platelet reactivity.24 Since then, this research group published several interesting papers regarding the cross-talking between erythrocyte and platelets. The presence of erythrocytes were shown to induce a twofold increase in platelet thromboxane B2

(TXB2) synthesis upon collagen stimulation, indicating that erythrocytes actively modulate platelet eicosanoid formation and thereby activation.25;26 This effect of erythrocyte on platelet activation was shown to be positively related with aspirin resistance, and thus is clinically relevant.26-31 In 1999, Andrews and Low summarized the clinical and laboratory evidence supporting the involvement of erythrocyte in thrombus formation in a detailed review. 32 In 2002, Goel and Diamond make the first observation that erythrocyte can adhere to immobilized platelets at depressed shear rates.33 In 2003, the study from Hermand et al. showed for the first time that erythrocyte intercellular adhesion molecule-4 (ICAM-4) is a ligand for platelet 34;35 activated integrin αIIbβ3. This finding has laid the biomolecule basis for a receptor/ ligand mediated interaction between erythrocyte and platelets. While the research line of erythrocyte-platelet interaction has been lying in silence in the last 10 years, new breakthroughs have emerged supporting a direct involvement of erythrocyte in coagulation.36-38 In 2012, research from Whelihan et al. has demonstrated that erythrocyte can provide procoagulant surface for prothrombin activation through the meizothrombin pathway.38 A detailed literature review regarding the contribution of erythrocytes in coagulation can be found in the chapter 2 of this thesis. In 2012, the group of Motto et al. demonstrated that erythrocytes were the first and predominant

Chapter 1 Chapter cell type present at the site of injury in a ferric chloride based thrombosis model in mice39 This finding further strengthened the idea for the active involvement of 12 erythrocytes in thrombosis. Figure 1 in this chapter summarized the major findings about the role of erythrocytes in thrombosis and hemostasis in a chronological order. General introduction General Although the erythrocyte-platelet interaction has been observed more than 10 years ago, the underlying mechanisms and the physiological relevance of this phenomenon are still unclear. In our study, which is described in chapter 3, we confirmed a direct erythrocyte adhesion to platelets under conditions of low shear flow. We found that this interaction between erythrocyte and platelets is at least partially mediated via

ICAM-4 on erythrocytes and activated αIIbβ3 on platelets. In addition, we have studied influence of this direct erythrocyte-platelet interaction on thrombus formation. In order to further characterize the influence of ICAM-4- IIbα β3 interaction on platelet function, we constructed an ICAM-4 peptide libary and looked into the effect of each peptide in this libary on platelet function. The findings of this study are described in chapter 4. In short, we found that 64P90K on domain I and 112P138L on domain II are the crucial regions on ICAM-4 to mediate erythrocyte-platelet interaction. Two peptides located on both regions of ICAM-4 are able to modulate

αIIbβ3 conformational change from an inactive form to an active form (high affinity for fibrinogen binding). Interactions between ICAM-4 and α β are like to induce IIb 3 1 Chapter signalling transduction in platelets and further activate platelets. 13 Platelet storage in Platelets play a very important role in hemostasis. A normal human platelet count in blood ranges from 150 to 450 x 109 /L. Various diseases are known to cause decreased platelet counts (platelet count < 50 x 109/L) which are known under the general term thrombocytopenia. These diseases include blood cancer (e.g. leukemia or myeloma), von Willebrand disease (VWD) type 2B, autoimmune diseases, infections, and so on. When platelet count is < 10 x 109/L, patients should receive prophylactic platelet introduction General transfusion to reduce the risk of bleeding.59;60 In addition, platelet transfusions are routinely applied as prophylaxis for treatment of bleeding in thrombocytopenic patients undergoing invasive surgery or chemotherapy. The use of prophylactic significantly reduced the mortality rate caused by bleeding and in the meanwhile profoundly increased the demand for platelet concentrates (PCs). Nowadays, platelet concentrates can be prepared by blood banks via 3 independent methods, namely the platelet-rich plasma (PRP) method, the buffy coat method, and the aphaeresis method.61 The PRP method is mainly used in the United States. is collected in disposable plastic bags, centrifuged at slow speed to sediment red and white blood cells and to concentrate most platelets in the supernatant plasma (PRP). The PRP is centrifuged at a higher speed to sediment the platelets. Most plasma is removed and platelets are resuspended in a small volume of plasma or synthetic medium and stored at room temperature with mild rotation.61 The buffy coat method is mainly used in Europe and Canada. Whole blood is centrifuged at a high speed and separated into red blood cells (RBCs), plasma and a buffy coat containing fraction. Buffy coats from 4-8 ABO-matched donors are subsequently pooled and centrifugated to isolate the platelet fraction. Both the PRP and the buffy coat methods include a leukocyte reduction step to decrease the risk of recipient Chapter 1 Chapter

14

Figure 1. Major findings about the role of erythrocytes in thrombosis and hemostasis.

alloimmunization. Aphaeresis method is used when patients are at risk for transfusion-

General introduction General related infectious diseases. Blood from the donor is processed with a cell separator. Platelets are collected into citrate in plastic bags, while the other blood cells and most of the plasma are returned to the donor. Aphaeresis machines apply leukocyte reduction during . Unlike other blood products, such as packed and plasma, which are stored in cold conditions, the storage of platelets is currently carried out at room temperature (22-24 °C) with slight agitation. The shelf life of platelet concentrates is 7 days in maximum. The current storage conditions for platelet concentrates are far from ideal. Firstly, platelets stored under room temperature undergo changes known as the platelet storage lesion (PSL), which is defined as the sum of all the deterioration processes due to the storage. PSL can be monitored by changes in platelet morphology, activation, metabolism, senescence and apoptosis. Secondly, room temperature storage favors bacterial growth and thus limits the product quality. It is estimated that one out of 5000 units of platelet concentrates is contaminated with bacteria and more than 10% of the transfusions with the contaminated platelet resulted in sepsis.62 Facing these profound disadvantages of storage platelets in room temperature, one may wonder why platelet products are not stored at low temperatures (0-4°C). Low temperature or cold storage suppresses metabolic activity and reduces the risk of bacterial growth.63;64 The main scientific reason for excluding cold storage as the condition for preserving platelet concentrates lies in the fact that transfused platelets stored at low temperature are rapidly clearly from the circulation.65 Hoffmeister and colleagues have demonstrated that this rapid clearance of cold platelets is mediated by a mechanism that involves the VWF receptor –glycoprotein (GP) Ibα, which is a member of the GPIb-V-IX complex.66 They showed that upon chilling of platelets, GPIbα changed its localization (also known as GPIb cluster) and became recognizable by the integrin αMβ2/MAC-1 on macrophages, promoting platelet by macrophages. Chilling promoted deglycosylation of GPIbα, resulting in exposure of β-N-Acetyl-D-Glucosamine (β-GN) residues which are recognized by the αM- 67 lectin domain of αMβ2. Besides the increased phagocytosis due to the interaction between platelet GPIbα and the integrin αMβ2/MAC-1 on the , chilling has also been shown to influence the programmed platelet death — apoptosis. Platelets stored at 0-4 °C undergo increased apoptotic process, such as the activation of pro- apoptotic Bad and Bax, the depolarization of the inner mitochondrial membrane Chapter 1 Chapter potential and the cytochrome c release.68;69 15 Since the demand for prophylactic platelet transfusion has been increasing, finding the proper approaches to improve the quality of platelet concentrates is of great importance for the health of the patients and of great economical gain for the whole society. Under this circumstances, we set up our study to further investigate the clearance of platelets stored in cold conditions. We aimed to further understand the mechanisms of cold induced platelet apoptosis and to imply our findings in improving

the quality of cold stored platelets. The findings of this study are described in detail introduction General in chapter 5 of this thesis. Von Willebrand factor and von Willebrand Disease

Von Willebrand factor is a multimeric protein that contributes to the recruitment of platelets to the injured vessel wall. It is produced in endothelial cells and . In Megakaryocytes, VWF and its propeptide are stored in granules, while in endothelial cells they are stored in endothelial-specific Weibel-Palade bodies.70 Endothelial cells are the main source of VWF that is found in subendothelial tissues and in plasma. VWF circulates in an inactive state in our blood stream. However, binding to exposed subendothelial matrix (such as collagen and fibronectin), together with high shear stress converts VWF into its platelet-binding conformation (active VWF). Active VWF binds to its receptor GPIbα on platelets and thus serves as a bridge between exposed matrix and platelets. This capture of platelets by matrix- bound active VWF is particularly important in hemostasis in arterial vessels where the shear stress is too high for platelets to bind to the matrix proteins directly.71 The contribution of VWF to coagulation also concerns the stabilization of factor VIII (FVIII). FVIII is an essential element of the coagulation cascade, where it acts as a cofactor for activated factor IX (FIX).72 Complex formation between FVIII and VWF stabilizes the heterodimeric structure of FVIII and prevents the premature clearance of FVIII.73 VWF stored in endothelial cells or platelets (in the alpha granules) is different from that circulates in plasma. While the plasma VWF is inactive, the freshly-secreted VWF is able to interact with platelet GPIbα promptly. Another name for this freshly- secreted VWF is ultra large (UL) –VWF because its molecule weight is significantly larger than that in plasma. The UL-VWF multimers expose high-affinity binding sites for GPIbα,74 whereas these sites are unavailable in plasma VWF multimers unless high shear force is applied or interaction with the matrix protein has occurred. After secretion, UL-VWF is cleaved into smaller derivatives by a metalloproteased called ADMTS-13 into smaller VWF multimers in various sizes which form the normal plasma VWF. 75;76 Deficiency of VWF leads to von Willebrand disease (VWD) which is characterized by defects in blood clotting and formation of platelet plugs at sites of vascular injury. VWD is inherited in an autosomal dominant manner and can be divided into two Chapter 1 Chapter general categories based on quantitative or qualitative defects within the VWF 16 protein. 77 Quantitative VWD is the result of a total disruption of gene function by deletion of a segment of the gene or a nonsense mutation. VWD type 1 is the most common form of VWD, taking up 70-80% of the cases. Patients with this type of VWD generally have mild deficiency in VWF antigen levels (1-40% of normal).78;79 VWD type 3 is a relatively rare disease (approximately 1 in 1 million). Patients of this type suffer from a pronounced bleeding disorder with very little or no detectable plasma or platelet VWF as well as a secondary deficiency of FVIII.80;81

General introduction General VWD type 2 is a qualitative VWF deficiency in which a mutation in the VWF gene results in single amino acid substitutions that interfere with the protein structure and function.82 VWF type 2 has 4 subclasses: type 2A, 2B, 2M and 2N. While type 2A, 2M and 2N leads to impaired VWF-platelet interactions, the 2B subtype is characterized by enhanced interactions between VWF and platelets, which is caused by gain-of-function mutations within the VWF A1 domain.82;83 VWF -type 2B patients lack high molecular weight (HMW)-VWF multimers and have thrombocytopenia. The thrombocytopenia in these patients is associated with spontaneous platelet aggregates. However, molecular mechanism underlying the thrombocytopenia remains unknown. In the attempt to fill this knowledge gap and having a successful murine model for VWF-type 2B at hand, we studied the clearance of VWF/platelet in the type 2B mice. Our study provided for the first time solid evidence showing that the increased removal of VWF/platelet complex by macrophages is one of the mechanisms underlying thrombocytopenia in VWD-type 2B (chapter 6 of this thesis). Antiphospholipid syndrome

The antiphospholipid syndrome (APS) is a non-inflammatory autoimmune disease with a wide variety of clinical features. It refers to patients with arterial Chapter 1 Chapter

17 General introduction General

or venous thrombosis, pregnancy loss, and thrombocytopenia, in which so-called antiphospholipid antibodies (aPLs) are repeatedly detected in their plasma. The diagnosis of APS requires at least one of the clinical manifestations (thrombosis and/or recurrent pregnancy loss) in combination with the persistent presence of aPLs in the plasma of the patient (the serological criteria) (Table 2).84 The presence of aPLs can be determined by either the prolongation of phospholipid–dependent coagulation test (lupus anticoagulant, LAC) or solid phase immune assays such as anti-cardiolipin enzyme-linked immunosorbent assay (ELISA). The term antiphospholipid syndrome arose from an observation made by Harris and Hughes.91 They noticed that the presence of a prolonged clotting assay or the presence of anticardiolipin antibodies in patients with thrombosis and/or recurrent pregnancy loss often coincides with systemic lupus erythromatosus patients that tested false positive for syphilis in a Venereal Disease Research Laboratory (VDRL)- flocculation test. The main antigen in this test was a phospholipid called cardiolipin extracted from beef heart. The antibodies responsible for this false positive reaction in the syphilis assay were thus called anticardiolipin antibodies (aCLs). Later, it is discovered that the so-called antiphospholipid (or anticardiolipin) antibodies are not directed against phospholipids but to proteins that bound to phospholipids,92;93 such 94-96 97;98 as the plasma protein beta(2)-glycoprotein I (β2GPI), prothrombin, protein 99 99-101 102-107 108-111 Chapter 1 Chapter C, protein S, annexin V, Factor XII, and high/low molecular weight 112-114 18 kininogen in complex with phosphatidylethanolamine (PE). Antibodies directed

against β2GPI are now generally considered to be the most clinically relevant aPL

in antiphospholipid syndrome, especially those anti- β2GPI that elicit LAC activity. 115-117 Although the classification criteria for APS seems to be clear to be used as inclusion criteria for patient related studies, the pathological mechanism that connect the serological criteria and the clinical manifestations are far from clear. In the last decade, General introduction General A B

Figure 2 Structure of β2GPI with its 5 domains and its two confirmations. (A) β2GPI in a closed confirmation. The

5 domains are labeled as I to V. Beta(2)-GPI in this confirmation does not bind to phospholipids.(B) β2GPI in an open (fish-hook) confirmation. Beta(2)GPI in this confirmation possesses a positively charged patch and can bind to phospholipids. researchers took great efforts to unravel the mechanism by which aPLs can induce thrombosis or pregnancy loss. A vast number of studies have emerged proposing a potpourri of pathogenic mechanisms. aPLs were shown to induce thrombosis by targeting various systems via various receptors and pathways, such as the cellular blood compartment (platelets and ), the vascular wall (endothelial cells), the plasma compartment (annexin V, prothrombin, β2GPI), and even the metabolic pathways. Due to the diversity in the mechanisms and the differences in the methodology, the focus of the mechanistical studies in the APS field seems to be largely diffused. It is hard to imagine that aPLs can exert such a diversity of effects. The relationship between aPLs and the clinical manifestations thus by far remains to be a mysterious “black box”. In order to gain some insights in this black box, we performed a meta-analysis on 124 mechanistical studies. The methodology and the results of this study are described in the chapter 7 of this thesis. Beta(2)-GP I beyond the antiphospholipid syndrome Chapter 1 Chapter Beta(2)-GPI or apolipoprotein H is a highly abundant protein in plasma (3-6 µM). It consists of 326 amino acids arranged in five short consensus repeat domains (Figure 19 2).118;119 The first 4 domains contain 60 amino acids each, while the 5th domain is aberrant with an ‘extra’ six-residue insertion and a C-terminal extension of 19 amino acids, which is C-terminally cross-linked by an additional C-terminal disulfide bond. The extra amino acids are responsible for the formation of a large positive charged patch within the fifth domain ofβ 2GPI that forms the binding site for anionic phospholipids.120 In the middle of this positive loop there is a flexible hydrophobic loop with a classic Trp-Lys motive,119 often observed in proteins at the site of introduction General insertion into cellular membranes. The relatively recent research has shown that 121 β2GPI can exist in two conformations — closed and open conformations. β2GPI in plasma is predominantly in a closed conformation, which cannot be recognized by aPLs (Figure 2A). Less than 1% of the plasma β2GPI is in an open fish-hook shaped structure (Figure 2B).121 This fish-hook confirmation can be recognized by aPLs.119;122

The clinical relevant epitope for the aPLs is located within the first domain ofβ 2GPI around amino acids Arg39-Arg43.95;117 Beta(2)-GPI is best known as the antigen against which aPLs are directed.123 However, research evidence indicates that its biological function may be beyond its role in antiphospholipid syndrome. The biological function of β2GPI was first reported in 1985 by Schousboe, who have demonstrated that this protein inhibits the activation 124 of the contact system of the blood coagulation. Preincubation of β2GPI with an ellagic acid-phospholipid suspension (Cephotest) reduced the Cephotest induced coagulation. The addition of β2GPI to either normal or β2GPI-deficient plasma gave similar inhibitory effects on Cephotest triggered coagulation.124 In 2011, Agar and colleagues published an important finding describing β2GPI as a component 125 of innate immunity. In this study, β2GPI was shown to be able to neutralize and clear lipopolysaccharide (LPS, a major constituent of the outer membrane of Gram- negative bacteria) from the circulation. LPS plays an important role in activating the host’s immune response by binding to monocytes and other cells. The in vitro study

of Agar et al. showed that β2GPI inhibited LPS-induced expression of tissue factor

and IL-6 on monocytes and endothelial cells. The binding of β2GPI to LPS caused

a conformational change in β2GPI that led to binding of the β2GPI-LPS complex to and ultimately clearance of this complex.125 Furthermore, study from 126 Hulstein et al. demonstrated that β2GPI can interact with active VWF. Their results

showed that β2GPI inhibited VWF-induced platelet aggregation. In the presence

of β2GPI, platelet adhesion to VWF-coated surface was decreased by 50%. β2GPI binds to the A1 domain of VWF preferably when this domain is in its GP1b-binding

conformation (such as the A1 domains in UL-VWF). The binding of β2GPI to VWF prevents VWF from binding to GP1bα which explains the impaired platelet-VWF

interactions in the presence of β2GPI. Thrombotic thrombocytopenic purpura (TTP) is a disease characterized with increased UL-VWF in the circulation. In order to further explore the physiological role of β GPI – VWF interaction, we set up a study Chapter 1 Chapter 2

to investigate whether β2GPI could regulate the UL-VWF function in patients with 20 TTP. The results of this sutdy are described in detail in chapter 8. Outline of this thesis Chapter 1: general introduction Part I: erythrocyte-platelet interaction in thrombus formation Chapter 2: a detailed and up to date review about the involvement of erythrocyte in

General introduction General thrombosis and hemostasis Chapter 3: ICAM-4 mediated erythrocyte-platelet interaction

Chapter 4: ICAM-4-αIIbβ3 interaction on platelet function Part II: platelet clearance in cold storage and in VWF-type 2B Chapter 5: platelet apoptosis by cold-induced glycoprotein Ibα clustering Chapter 6: clearance of VWF/platelet complexes in VWD-type 2B

Part III: antiphospholipid syndrome and β2GPI Chapter 7: pathogenic mechanisms of the antiphospholipid syndrome

Chapter 8: indications for a protective function of β2GPI in TTP Chapter 9: general discussion

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Blood 1991;78:154-162. 26. Valles J, Santos MT, Aznar J et al. Platelet-erythrocyte interactions enhance alpha(IIb)beta(3) integrin receptor activation and P-selectin expression during platelet recruitment: down-regulation by aspirin ex vivo. Blood 2002;99:3978-3984. 27. Santos MT, Valles J, Aznar J et al. Prothrombotic effects of erythrocytes on platelet reactivity. Reduction by aspirin. Circulation 1997;95:63-68. 28. Santos MT, Valles J, Aznar J et al. Aspirin therapy for inhibition of platelet reactivity in the Chapter 1 Chapter presence of erythrocytes in patients with vascular disease. J.Lab Clin.Med. 2006;147:220-227. 22 29. Santos MT, Valles J, Lago A et al. Residual platelet thromboxane A2 and prothrombotic effects of erythrocytes are important determinants of aspirin resistance in patients with vascular disease. J.Thromb.Haemost. 2008;6:615-621. 30. Valles J, Santos MT, Aznar J et al. Modulatory effect of erythrocytes on the platelet reactivity to collagen in IDDM patients. Diabetes 1997;46:1047-1053. 31. Valles J, Santos MT, Aznar J et al. Erythrocyte promotion of platelet reactivity decreases the effectiveness of aspirin as an antithrombotic therapeutic modality: the effect of low-dose aspirin is

General introduction General less than optimal in patients with vascular disease due to prothrombotic effects of erythrocytes on platelet reactivity. Circulation 1998;97:350-355. 32. 32. Andrews DA, Low PS. Role of red blood cells in thrombosis. Curr.Opin.Hematol. 1999;6:76-82. 33. Goel MS, Diamond SL. Adhesion of normal erythrocytes at depressed venous shear rates to activated , activated platelets, and fibrin polymerized from plasma. Blood 2002;100:3797-3803. 34. Hermand P, Gane P, Huet M et al. Red cell ICAM-4 is a novel ligand for platelet-activated alpha IIbbeta 3 integrin. J.Biol.Chem. 2003;278:4892-4898. 35. Hermand P, Gane P, Callebaut I et al. Integrin receptor specificity for human red cell ICAM-4 ligand. Critical residues for alphaIIbeta3 binding. Eur.J.Biochem. 2004;271:3729-3740. 36. Ninivaggi M, Apitz-Castro R, Dargaud Y et al. Whole-blood thrombin generation monitored with a calibrated automated thrombogram-based assay. Clin.Chem. 2012;58:1252-1259. 37. Peyrou V, Lormeau JC, Herault JP et al. Contribution of erythrocytes to thrombin generation in whole blood. Thromb.Haemost. 1999;81:400-406. 38. Whelihan MF, Zachary V, Orfeo T, Mann KG. Prothrombin activation in blood coagulation: the erythrocyte contribution to thrombin generation. Blood 2012;120:3837-3845. 39. Barr JD, Chauhan AK, Schaeffer GV, Hansen JK, Motto DG. Red blood cells mediate the onset of thrombosis in the ferric chloride murine model. Blood 2013;121:3733-3741. 40. Gallagher PG, Chang SH, Rettig MP et al. Altered erythrocyte endothelial adherence and membrane phospholipid asymmetry in hereditary hydrocytosis. Blood 2003;101:4625-4627. 41. Barbui T, Finazzi MC, Finazzi G. Front-line therapy in polycythemia vera and essential thrombocythemia. Blood Rev. 2012;26:205-211. 42. Fujita H, Sakuma R, Tomiyama J et al. Relationship between clotting activity and phosphatidylserine expression on erythrocyte membranes in polycythemia vera patients with the JAK2 V617F mutation. Arch.Physiol Biochem. 2011;117:231-235. 43. Marchioli R, Finazzi G, Specchia G et al. Cardiovascular events and intensity of treatment in polycythemia vera. N.Engl.J.Med. 2013;368:22-33. 44. Odievre MH, Bony V, Benkerrou M et al. Modulation of erythroid adhesion receptor expression by hydroxyurea in children with sickle cell disease. Haematologica 2008;93:502-510. 45. Setty BN, Rao AK, Stuart MJ. Thrombophilia in sickle cell disease: the red cell connection. Blood 2001;98:3228-3233. 46. Setty BN, Kulkarni S, Stuart MJ. Role of erythrocyte phosphatidylserine in sickle red cell- endothelial adhesion. Blood 2002;99:1564-1571. Chapter 1 Chapter 47. Eldor A, Rachmilewitz EA. The hypercoagulable state in thalassemia. Blood 2002;99:36-43. 48. Goldschmidt N, Spectre G, Brill A et al. Increased platelet adhesion under flow conditions is 23 induced by both thalassemic platelets and red blood cells. Thromb.Haemost. 2008;100:864-870. 49. Mannucci PM. Red cells playing as activated platelets in thalassemia intermedia. J.Thromb. Haemost. 2010;8:2149-2151. 50. Ruf A, Pick M, Deutsch V et al. In-vivo platelet activation correlates with red cell anionic phospholipid exposure in patients with beta-thalassaemia major. Br.J.Haematol. 1997;98:51-56. 51. Tripodi A, Cappellini MD, Chantarangkul V et al. Hypercoagulability in splenectomized General introduction General thalassemic patients detected by whole-blood thromboelastometry, but not by thrombin generation in platelet-poor plasma. Haematologica 2009;94:1520-1527. 52. Arbel Y, Banai S, Benhorin J et al. Erythrocyte aggregation as a cause of slow flow in patients of acute coronary syndromes. Int.J.Cardiol. 2012;154:322-327. 53. Yunoki K, Naruko T, Komatsu R et al. Enhanced expression of haemoglobin scavenger receptor in accumulated macrophages of culprit lesions in acute coronary syndromes. Eur.Heart J. 2009;30:1844-1852. 54. Yunoki K, Naruko T, Sugioka K et al. Erythrocyte-rich thrombus aspirated from patients with ST-elevation myocardial infarction: association with oxidative stress and its impact on myocardial reperfusion. Eur.Heart J. 2012;33:1480-1490. 55. Gao C, Xie R, Yu C et al. Procoagulant activity of erythrocytes and platelets through phosphatidylserine exposure and microparticles release in patients with nephrotic syndrome. Thromb.Haemost. 2012;107:681-689. 56. Bonomini M, Sirolli V, Merciaro G et al. Red blood cells may contribute to hypercoagulability in uraemia via enhanced surface exposure of phosphatidylserine. Nephrol.Dial.Transplant. 2005;20:361-366. 57. Braekkan SK, Mathiesen EB, Njolstad I, Wilsgaard T, Hansen JB. Hematocrit and risk of venous thromboembolism in a general population. The Tromso study. Haematologica 2010;95:270-275. 58. Yu FT, Armstrong JK, Tripette J, Meiselman HJ, Cloutier G. A local increase in red blood cell aggregation can trigger deep vein thrombosis: evidence based on quantitative cellular ultrasound imaging. J.Thromb.Haemost. 2011;9:481-488. 59. Schiffer CA, Anderson KC, Bennett CL et al. Platelet transfusion for patients with cancer: clinical practice guidelines of the American Society of Clinical Oncology. J.Clin.Oncol. 2001;19:1519- 1538. 60. Slichter SJ. Evidence-based platelet transfusion guidelines. Hematology.Am.Soc.Hematol.Educ. Program. 2007172-178. 61. Tynngard N. Preparation, storage and quality control of platelet concentrates. Transfus.Apher.Sci. 2009;41:97-104. 62. Eder AF, Kennedy JM, Dy BA et al. Bacterial screening of platelets and the residual risk of septic transfusion reactions: the American Red Cross experience (2004-2006). Transfusion 2007;47:1134-1142. 63. Currie LM, Harper JR, Allan H, Connor J. Inhibition of cytokine accumulation and bacterial growth during storage of platelet concentrates at 4 degrees C with retention of in vitro functional activity. Transfusion 1997;37:18-24. Chapter 1 Chapter 64. Slichter SJ. In vitro measurements of platelet concentrates stored at 4 and 22 degree C: correlation 24 with posttransfusion platelet viability and function. Vox Sang. 1981;40 Suppl 1:72-86. 65. Murphy S, Gardner FH. Effect of storage temperature on maintenance of platelet viability-- deleterious effect of refrigerated storage. N.Engl.J.Med. 1969;280:1094-1098. 66. Hoffmeister KM, Felbinger TW, Falet H et al. The clearance mechanism of chilled blood platelets. Cell 2003;112:87-97. 67. Josefsson EC, Gebhard HH, Stossel TP, Hartwig JH, Hoffmeister KM. The alphaMbeta2 integrin alphaM lectin domain mediates the phagocytosis of chilled platelets. J.Biol. General introduction General Chem. 2005;280:18025-18032. 68. Babic AM, Josefsson EC, Bergmeier W et al. In vitro function and phagocytosis of galactosylated platelet concentrates after long-term refrigeration. Transfusion 2007;47:442-451. 69. Wandall HH, Hoffmeister KM, Sorensen AL et al. Galactosylation does not prevent the rapid clearance of long-term, 4 degrees C-stored platelets. Blood 2008;111:3249-3256. 70. Wagner DD, Saffaripour S, Bonfanti R et al. Induction of specific storage organelles by von Willebrand factor propolypeptide. Cell 1991;64:403-413. 71. Sadler JE. Biochemistry and genetics of von Willebrand factor. Annu.Rev.Biochem. 1998;67:395- 424. 72. Noe DA. A mathematical model of coagulation factor VIII kinetics. Haemostasis 1996;26:289- 303. 73. Federici AB. The factor VIII/von Willebrand factor complex: basic and clinical issues. Haematologica 2003;88:EREP02. 74. Arya M, Anvari B, Romo GM et al. Ultralarge multimers of von Willebrand factor form spontaneous high-strength bonds with the platelet glycoprotein Ib-IX complex: studies using optical tweezers. Blood 2002;99:3971-3977. 75. Dent JA, Galbusera M, Ruggeri ZM. Heterogeneity of plasma von Willebrand factor multimers resulting from proteolysis of the constituent subunit. J.Clin.Invest 1991;88:774-782. 76. Zheng X, Chung D, Takayama TK et al. Structure of von Willebrand factor-cleaving protease (ADAMTS13), a metalloprotease involved in thrombotic thrombocytopenic purpura. J.Biol. Chem. 2001;276:41059-41063. 77. Sadler JE. A revised classification of von Willebrand disease. For the Subcommittee onvon Willebrand Factor of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis. Thromb.Haemost. 1994;71:520-525. 78. Lillicrap D. Von Willebrand disease - phenotype versus genotype: deficiency versus disease. Thromb.Res. 2007;120 Suppl 1:S11-S16. 79. Sadler JE. Von Willebrand disease type 1: a diagnosis in search of a disease. Blood 2003;101:2089- 2093. 80. Eikenboom JC, Ploos van Amstel HK, Reitsma PH, Briet E. Mutations in severe, type III von Willebrand’s disease in the Dutch population: candidate missense and nonsense mutations associated with reduced levels of von Willebrand factor messenger RNA. Thromb.Haemost. 1992;68:448-454. 81. Ngo KY, Glotz VT, Koziol JA et al. Homozygous and heterozygous deletions of the von Willebrand Chapter 1 Chapter factor gene in patients and carriers of severe von Willebrand disease. Proc.Natl.Acad.Sci.U.S.A 1988;85:2753-2757. 25 82. Sadler JE. New concepts in von Willebrand disease. Annu.Rev.Med. 2005;56:173-191. 83. Sadler JE, Budde U, Eikenboom JC et al. Update on the pathophysiology and classification of von Willebrand disease: a report of the Subcommittee on von Willebrand Factor. J.Thromb.Haemost. 2006;4:2103-2114. 84. Miyakis S, Lockshin MD, Atsumi T et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J.Thromb.Haemost. 2006;4:295-306. introduction General 85. Wisloff F, Jacobsen EM, Liestol S. Laboratory diagnosis of the antiphospholipid syndrome. Thromb.Res. 2002;108:263-271. 86. Brandt JT, Triplett DA, Alving B, Scharrer I. Criteria for the diagnosis of lupus anticoagulants: an update. On behalf of the Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibody of the Scientific and Standardisation Committee of the ISTH. Thromb.Haemost. 1995;74:1185-1190. 87. Tincani A, Allegri F, Sanmarco M et al. Anticardiolipin antibody assay: a methodological analysis for a better consensus in routine determinations--a cooperative project of the European Antiphospholipid Forum. Thromb.Haemost. 2001;86:575-583. 88. Harris EN, Pierangeli SS. Revisiting the anticardiolipin test and its standardization. Lupus 2002;11:269-275. 89. Wong RC, Favaloro EJ, Adelstein S et al. Consensus guidelines on anti-beta 2 glycoprotein I testing and reporting. Pathology 2008;40:58-63. 90. Reber G, Tincani A, Sanmarco M, de MP, Boffa MC. Proposals for the measurement of anti-beta2- glycoprotein I antibodies. Standardization group of the European Forum on Antiphospholipid Antibodies. J.Thromb.Haemost. 2004;2:1860-1862. 91. Harris EN, Gharavi AE, Boey ML et al. Anticardiolipin antibodies: detection by radioimmunoassay and association with thrombosis in systemic lupus erythematosus. Lancet 1983;2:1211-1214. 92. Bevers EM. Anticardiolipin antibody cofactor. Lancet 1991;337:550. 93. Bevers EM, Galli M. Cofactors involved in the antiphospholipid syndrome. Lupus 1992;1:51-53. 94. Galli M, Comfurius P, Maassen C et al. Anticardiolipin antibodies (ACA) directed not to cardiolipin but to a plasma protein cofactor. Lancet 1990;335:1544-1547. 95. McNeil HP, Simpson RJ, Chesterman CN, Krilis SA. Anti-phospholipid antibodies are directed against a complex antigen that includes a lipid-binding inhibitor of coagulation: beta 2-glycoprotein I (apolipoprotein H). Proc.Natl.Acad.Sci.U.S.A 1990;87:4120-4124. 96. Matsuura E, Igarashi Y, Fujimoto M, Ichikawa K, Koike T. Anticardiolipin cofactor(s) and differential diagnosis of autoimmune disease. Lancet 1990;336:177-178. 97. Bevers EM, Galli M, Barbui T, Comfurius P, Zwaal RF. Lupus anticoagulant IgG’s (LA) are not directed to phospholipids only, but to a complex of lipid-bound human prothrombin. Thromb. Haemost. 1991;66:629-632. 98. Galli M, Beretta G, Daldossi M, Bevers EM, Barbui T. Different anticoagulant and immunological properties of anti-prothrombin antibodies in patients with antiphospholipid antibodies. Thromb. Haemost. 1997;77:486-491.

Chapter 1 Chapter 99. Oosting JD, Derksen RH, Bobbink IW et al. Antiphospholipid antibodies directed against a combination of phospholipids with prothrombin, protein C, or protein S: an explanation for their 26 pathogenic mechanism? Blood 1993;81:2618-2625. 100. Sorice M, Arcieri P, Griggi T et al. Inhibition of protein S by autoantibodies in patients with acquired protein S deficiency. Thromb.Haemost. 1996;75:555-559. 101. D’Angelo A, Mazzola G, Bergmann F et al. Autoimmune protein S deficiency: a disorder predisposing to thrombosis. Haematologica 1995;80:114-121. 102. de Laat B, Derksen RH, Mackie IJ et al. Annexin A5 polymorphism (-1C-->T) and the presence of anti-annexin A5 antibodies in the antiphospholipid syndrome. Ann.Rheum.Dis. 2006;65:1468- General introduction General 1472. 103. de Laat B, Wu XX, van Lummel M et al. Correlation between antiphospholipid antibodies that recognize domain I of beta2-glycoprotein I and a reduction in the anticoagulant activity of annexin A5. Blood 2007;109:1490-1494. 104. Kuragaki C, Kidoguchi K, Nakamura N, Wada Y. Anti-annexin V antibodies in plasma and serum samples from patients with lupus anticoagulant. Am.J.Hematol. 1995;50:68. 105. Matsuda J, Saitoh N, Gohchi K, Gotoh M, Tsukamoto M. Anti-annexin V antibody in systemic lupus erythematosus patients with lupus anticoagulant and/or anticardiolipin antibody. Am.J.Hematol. 1994;47:56-58. 106. Matsuda J, Gotoh M, Saitoh N et al. Anti-annexin antibody in the sera of patients with habitual fetal loss or preeclampsia. Thromb.Res. 1994;75:105-106. 107. Nakamura N, Ban T, Yamaji K, Yoneda Y, Wada Y. Localization of the apoptosis-inducing activity of lupus anticoagulant in an annexin V-binding antibody subset. J.Clin.Invest 1998;101:1951- 1959. 108. Gallimore MJ, Jones DW, Winter M. Factor XII determinations in the presence and absence of phospholipid antibodies. Thromb.Haemost. 1998;79:87-90. 109. Jones DW, Gallimore MJ, Winter M. Pseudo factor XII deficiency and phospholipid antibodies. Thromb.Haemost. 1996;75:696-697. 110. Jones DW, Gallimore MJ, Harris SL, Winter M. Antibodies to factor XII associated with lupus anticoagulant. Thromb.Haemost. 1999;81:387-390. 111. Jones DW, Gallimore MJ, Mackie IJ, Harris SL, Winter M. Reduced factor XII levels in patients with the antiphospholipid syndrome are associated with antibodies to factor XII. Br.J.Haematol. 2000;110:721-726. 112. Sugi T, McIntyre JA. Autoantibodies to phosphatidylethanolamine (PE) recognize a kininogen-PE complex. Blood 1995;86:3083-3089. 113. Sugi T, Mclntyre JA. Phosphatidylethanolamine induces specific conformational changes in the kininogens recognizable by antiphosphatidylethanolamine antibodies. Thromb.Haemost. 1996;76:354-360. 114. Sugi T, McIntyre JA. Autoantibodies to kininogen-phosphatidylethanolamine complexes augment thrombin-induced platelet aggregation. Thromb.Res. 1996;84:97-109. 115. de Laat HB, Derksen RH, Urbanus RT, Roest M, DE Groot PG. beta2-glycoprotein I-dependent lupus anticoagulant highly correlates with thrombosis in the antiphospholipid syndrome. Blood 2004;104:3598-3602.

116. Galli M, Luciani D, Bertolini G, Barbui T. Lupus anticoagulants are stronger risk factors for 1 Chapter thrombosis than anticardiolipin antibodies in the antiphospholipid syndrome: a systematic review of the literature. Blood 2003;101:1827-1832. 27 117. de Laat B, Derksen RH, Urbanus RT, DE Groot PG. IgG antibodies that recognize epitope Gly40- Arg43 in domain I of beta 2-glycoprotein I cause LAC, and their presence correlates strongly with thrombosis. Blood 2005;105:1540-1545. 118. Lozier J, Takahashi N, Putnam FW. Complete amino acid sequence of human plasma beta 2-glycoprotein I. Proc.Natl.Acad.Sci.U.S.A 1984;81:3640-3644.

119. Bouma B, DE Groot PG, van den Elsen JM et al. Adhesion mechanism of human beta(2)- introduction General glycoprotein I to phospholipids based on its crystal structure. EMBO J. 1999;18:5166-5174. 120. Hunt JE, Simpson RJ, Krilis SA. Identification of a region of beta 2-glycoprotein I critical for lipid binding and anti-cardiolipin antibody cofactor activity. Proc.Natl.Acad.Sci.U.S.A 1993;90:2141- 2145. 121. Agar C, van Os GM, Morgelin M et al. Beta2-glycoprotein I can exist in 2 conformations: implications for our understanding of the antiphospholipid syndrome. Blood 2010;116:1336-1343. 122. Schwarzenbacher R, Zeth K, Diederichs K et al. Crystal structure of human beta2-glycoprotein I: implications for phospholipid binding and the antiphospholipid syndrome. EMBO J. 1999;18:6228- 6239. 123. McNeil HP, Simpson RJ, Chesterman CN, Krilis SA. Anti-phospholipid antibodies are directed against a complex antigen that includes a lipid-binding inhibitor of coagulation: beta 2-glycoprotein I (apolipoprotein H). Proc.Natl.Acad.Sci.U.S.A 1990;87:4120-4124. 124. Schousboe I. beta 2-Glycoprotein I: a plasma inhibitor of the contact activation of the intrinsic blood coagulation pathway. Blood 1985;66:1086-1091. 125. Agar C, DE Groot PG, Morgelin M et al. beta(2)-glycoprotein I: a novel component of innate immunity. Blood 2011;117:6939-6947. 126. Hulstein JJ, Lenting PJ, de LB et al. beta2-Glycoprotein I inhibits von Willebrand factor dependent platelet adhesion and aggregation. Blood 2007;110:1483-14 Chapter 1 Chapter

28 General introduction General 2

New insights into the role of erythrocytes in thrombus formation

Vivian X.Du,1 Dana Huskens,2 Coen Maas,1 Raed Al Dieri,2 Philip.G. de Groot2 and Bas de Laat1,2,3,4 Chapter 2 Chapter

29

1Department of Clinical Chemistry and Hematology, University Medical Center Utrecht, Utrecht, The Netherlands 2Sanquin Research, Amsterdam, The Netherlands 3Departement of Biochemistry, CARIM, University of Maastricht, Maastricht, The Netherlands 4Synapse BV, Maastricht, The Netherlands Erythrocytes in thrombus formation in thrombus Erythrocytes

Semin Thromb Hemost. 2014; 40: 72-80 Abstract The role of erythrocytes in thrombus formation has previously been regarded as passive by their influence on rheology. Erythrocytes are known, due to their abundance and size, to push platelets to the vascular wall (laminar shearing). This results in an increased platelet delivery at the vascular wall enabling platelets to seal off a vascular damage preventing excessive blood loss. Recently, there is new evidence for erythrocytes to influence thrombus formation in multiple ways besides their effect on rheology. Several groups have shown that besides platelets, erythrocytes are the main suppliers of phosphatidylserine-exposing membranes needed for coagulation

resulting in fibrin formation. In addition, our group has found that the ICAM-4-αIIb β3 interaction mediates erythrocyte-platelet interaction in flowing blood. By inhibiting this interaction we found decreased thrombin formation and decreased incorporation of erythrocytes into a thrombus. This review will provide more in-detail information of existing and new hypotheses regarding the role of erythrocytes in thrombus Chapter 2 Chapter formation. 30 Erythrocytes in thrombus formation in thrombus Erythrocytes Introduction Hemostasis is a powerful process of the human body to prevent blood loss, by sealing off sites of vascular injury. Several blood components have been shown to play pivotal roles in this process. Platelets are considered to be the key players in thrombus formation by first rolling over collagen-bound von Willebrand factor followed by formation of a stable interaction with collagen.1 The first adhered platelets bind additional platelets until the whole injury is sealed off by a platelet aggregate. Subsequently, the coagulation system stabilizes the formed platelet plug by creating a tight fibrin network, and then wound contraction takes place due to morphological changes in platelets. Erythrocytes (red blood cells, RBCs) are the most abundant cells in the blood. They are generally regarded as innocent bystanders in the process of thrombus formation, passively entrapped in a developing thrombus. However, both clinical and laboratory evidence supporting an active role of erythrocytes in thrombosis and hemostasis has been accumulating. Patients with an elevated Chapter 2 Chapter hematocrit (e.g. polycythemia vera), or with heritable erythrocyte abnormalities (e.g. hereditary stomatocytosis, β-thalassemia major, sickle cell anemia) are prone to 31 developing thrombosis.2-8 Non-erythroid diseases (e.g. diabetes, nephrotic syndrome or acquired dysfibrinogenemia) that indirectly modify the properties of erythrocytes can also promote thrombosis.9-12 There is now overwhelming evidence that red blood cells can exert their influence on hemostasis via different pathways. In this review, we will explore the possible mechanisms through which erythrocytes influence the balance of thrombosis and bleeding. Erythrocytes and rheology Multiple mechanisms have been proposed whereby erythrocytes can contribute to the processes of thrombus formation, of which erythrocyte regulated hemorheology is the best known.13 Hemorheology is the principle by which the flowing of the blood Erythrocytes in thrombus formation in thrombus Erythrocytes influences hemostasis. It is a rather complicated process and has been studied since the early thirties of the previous century. Changes of 1) hematocrit, 2) erythrocyte aggregation and 3) erythrocyte deformability can alter hemorheology. Hematocrit

The degree of disturbance of flow streamlines, and consequently the viscosity of blood, strongly depends on the concentration of the cellular elements (i.e., hematocrit). Increased hematocrit levels are caused either by an increase in the number of erythrocytes (erythrocytosis) or by dehydration.14 Erythrocytosis can be caused by diseases affecting the bone marrow (primary erythrocytosis, such as polycythemia vera) or by disease and environmental conditions that affect blood oxygen saturation (secondary erythrocytosis). In secondary erythrocytosis, the bone marrow produces more red blood cells to counterbalance the decrease inoxygen saturation.15 An increase in hematocrit results in an increase in viscosity that decreases the blood flow and can lead to the development of a thrombus.11 In addition, an elevated hematocrit promotes the transport of platelets and coagulation factors toward the vessel wall, thereby increasing collisions of platelets with the vasculature and with themselves.11;16;17 Erythrocyte aggregation

In a laminar blood flow, the highly deformable and biconcave-shaped erythrocytes push platelets to the peripheral, and they themselves migrate to the center of the flow.13 This phenomenon is also called axial migration. The axial migration of erythrocytes causes platelets to be concentrated on the edge of the vessel wall (Fig. 1A). Human erythrocytes are able to aggregate when suspended in aqueous solutions containing large plasma proteins (e.g. fibrinogen) or polymers (e.g. high molecular weight dextran).18;19 At stasis or when fluid shear rates are sufficiently low, erythrocytes tend to form regular, linear, face-to-face structures (i.e. rouleaux) or three-dimensional aggregates which are difficult to disperse (Fig. 1B).16;20 Chapter 2 Chapter Currently, the mechanisms involved in erythrocyte aggregation are not fully 32 understood, and there are two co-existing but almost mutually exclusive models.19;21 The bridging model proposes that erythrocyte aggregation occurs when bridging forces generated from the adsorption of macromolecules onto adjacent cell surfaces exceed disaggregation forces generated from electrostatic repulsion, membrane strain and mechanical shearing19;22-25 In contrast, the depletion model proposes that erythrocyte aggregation occurs because of a lower localized protein or polymer concentration near the cell surface compared to the suspending medium leading to an osmotic gradient and a depletion interaction.19;26;27 While it is not yet possible to decide the superiority between these two mechanisms, several studies favoring the depletion model have recently been published.28-31 Erythrocyte aggregation may influence various aspects of in vivo hydrodynamics.32 Erythrocytes in thrombus formation in thrombus Erythrocytes In large blood vessels under low-shear conditions, where blood can be regarded as a continuous fluid, enhanced erythrocyte aggregation increases blood viscosity and overall hydrodynamic resistance. In the microvasculature, however, enhanced erythrocyte aggregation increases axial migration of erythrocytes, resulting in an increased platelet concentration near the vessel wall, as well as a decreased local viscosity and reduced frictional resistance.33 In addition, axial accumulation of erythrocytes also promotes plasma skimming and the Fahraeus effect, which in turn may result in decreased haematocrit values, thereby reducing blood viscosity and flow resistance.34-36 This reduction in local viscosity and consequent reduction in wall shear stress leads to a lower local nitric oxide bioavailability.37;38 Nitric oxide is an important substance in maintaining the quiescent status of endothelial cells and platelets. The decrease in local nitric oxide may promote platelet activation and tilt the hemostatic balance.38 A

B Chapter 2 Chapter

Fig. 1. Schematic of blood flow at high and low shear rates. (A) Under conditions of high shear stress, the axial 33 migration of erythrocytes causes platelets to be concentrated on the peripheral of the vessel wall. Due to the high shear force, erythrocytes slightly elongate in the direction of the flow. (B) Under depressed flow shear rates, erythrocytes tend to form aggregates. The peripheral distribution of platelets disappears. Erythrocyte deformability

Deformability is the ability of an erythrocyte to alter its geometric configuration to minimize resistance to flow. A decrease in RBC deformability may increase the sensitivity for thrombus formation by rendering the erythrocytes less capable of squeezing through narrow apertures.16;39;40 More rigid erythrocytes also increase the transport of platelets towards the vascular surface.41 The cellular deformability of erythrocytes is determined by three factors: 1) cell geometry (a ratio of surface area to cellular volume); 2) cytoplasmic viscosity, principally determined by the properties formation in thrombus Erythrocytes and the concentration of hemoglobin in the cells; and 3) intrinsic viscoelastic properties of the red cell membrane (or membrane deformability). The membrane deformability in normal erythrocytes is primarily determined by the erythrocyte cytoskeleton, a network of proteins lying just beneath the cell membrane with spectrin being the most important component.42-45 The availability of metabolic energy in the form of adenosine triphosphate (ATP) is important for normal erythrocyte deformability. ATP is required by the cation pumps in the erythrocyte membrane, which serve to regulate intracellular cation and water content, thereby maintaining cell volume as well as the cell’s surface to volume ratio.44 Increased cytosolic calcium concentration is a frequently detected alteration associated with reduced erythrocyte deformability. One example of reduced erythrocyte deformability is found in sickle cell disease where erythrocytes form a sickle shape due to the aggregation or polymerization of sickle haemoglobin (HbS) upon deoxygenation.46;47 Erythrocytes and coagulation Coagulation

Coagulation is a complicated enzyme system that regulates the balance between bleeding and thrombosis. The contact (intrinsic) pathway is initiated by the exposure of a (foreign) surface. Factor XII (FXII) activation involves a complex reaction with prekallikrein and high molecular weight kininogen as cofactors, resulting in the activation of factor IX (FIXa). Factor IXa, together with activated factor VIII (FVIIIa), forms the intrinsic tenase complex, activating factor X (FXa). FXa then forms together with activated factor V (FVa) the prothrombinase complex, responsible for the conversion of the proenzyme prothrombin to the protease thrombin.48;49 The tissue factor (extrinsic) pathway, which is regarded as the major pathway of in vivo blood coagulation, starts when tissue factor (TF) is exposed to flowing blood at the site of injury. TF binds and activates factor VII (FVIIa) from the circulation. The Chapter 2 Chapter TF/FVIIa complex then cleaves its two primary substrates – FIX and FX — into two 34 serine proteases — FIXa and FXa. Together with FVIIIa, FIXa converts FX to FXa, as per the contact pathway. Subsequently, FXa forms with FVa the prothrombinase complex that converts prothrombin to thrombin .50-​52 Extra feedback loops re-enforce the coagulation cascade leading to an explosion of thrombin formation.53;54 The formation of large amounts of thrombin in a very short period is essential to cope with the flow in the blood. Many of the reactions described above require the presence of pro-coagulant surfaces. These surfaces are provided by cells expressing a negatively charged phospholipid, phosphatidylserine (PS) on their outer membrane. PS-bearing surfaces can dramatically increase the efficiency of the biochemical reactions by concentrating and co-localizing coagulation factors. Moreover, binding to a surface prevents the coagulation factor from washing away

Erythrocytes in thrombus formation in thrombus Erythrocytes by the flowing blood. At present, activated platelets bearing PS are viewed as the primary pro-coagulant surfaces.55;56 Prothrombotic membrane of erythrocytes

Although erythrocytes are major cellular components of red venous thrombi, they are often considered as passive participants in coagulation, merely providing bulk material for the obstructive clot. However, in the last few years, evidence has been accumulating that indicates erythrocytes may also play a significant role in thrombus formation by exposure of PS, which is normally localised in membrane leaflets that face the cytosol and inaccessible to blood coagulation proteins.57;58 In healthy normal erythrocytes, this asymmetrical distribution of PS across the membrane is maintained by constant flippase and floppase activity (Fig. 2A). However, under situations of apoptosis or erythrocyte damage/activation, such as high shear stress, complement attack, oxidative stress or pro-apoptotic stimulation, erythrocyte membrane loses PS asymmetry due to the activation of scramblase (Fig. 2B).11;59-61 Additionally, loss of membrane lipid asymmetry on erythrocytes is often accompanied by blebbing and subsequent shedding of microparticles (MPs) that expose PS at their surface.62 PS is an important cofactor in a number of reactions in the coagulation cascade. PS can facilitate the activation of FX to FXa and the generation of thrombin from prothrombin (Fig. 2B).63;64 However, as recently described, erythrocyte MPs do not only propagate coagulation by exposing PS but also initiate thrombin generation independently of TF in a FXII-dependent manner.65 These MPs may also be capable of promoting coagulation in a FXI-dependent manner as shown in sickle cell

A Chapter 2 Chapter

35

B Erythrocytes in thrombus formation in thrombus Erythrocytes

Fig. 2. Contribution of erythrocyte PS exposure to thrombin generation. (A) In the normal healthy erythrocyte, phosphatidylserine (PS, depicted as red) is kept in the inner leaflet of the plasma membrane by the flippase and floppase activities. (B) Under situations of apoptosis or erythrocyte damage, the erythrocyte membrane loses PS asymmetry due to the deactivation of flippase/floppase and the activation of scramblase. Exposed PS on the outer leaflet of the erythrocyte membrane can facilitate the activation of factor X (FX) to FXa and the generation of thrombin from prothrombin. disease.63;66 Besides their influence on thrombin generation, PS-exposing RBCs are also shown to be more adhesive to endothelial cells and prone to form erythrocyte aggregates in diabetes mellitus, obesity, hereditary stomatocytosis, sepsis, or chronic renal failure.67-70 Effect of hematocrit on coagulation

Until recently, it was difficult to study the effect of erythrocytes on coagulation. Coagulation assays were always performed in plasma, not in whole blood. Also thrombin generation was not possible in whole blood because variable quenching of the fluorescent signal by hemoglobin and red cell clusters can cause highly erratic signals. Nowadays, due to the development of new technical approaches in assessing blood coagulation, new light has been shed on the role of erythrocytes in coagulation.71 Peyrou et al. showed for the first time that normal erythrocytes may participate in

Chapter 2 Chapter coagulation by exposure of pro-coagulant phospholipids.72 They measured thrombin 36 generation initiated by TF in human whole blood, platelet-rich plasma (PRP), platelet- poor plasma (PPP) and in PPP supplemented with normal erythrocytes. Their results show that both the endogenous thrombin potential (ETP, the amount of thrombin which can be generated) and lag time were dependent on the platelet count or on the hematocrit. They also found that erythrocytes are able to influence the lag time to the same extent as platelets and that isolated erythrocytes showed prothrombinase activity when incubated with FXa and FVa. Later on, Horne et al. developed a TF-driven thrombin generation assay that included the cellular components of the blood.64 By using this assay, Horne et al. confirmed the finding of Peyrou that an increase in hematocrit (0-60%) correlates with an increase in ETP. Interestingly, compared to the increase in ETP observed 9

Erythrocytes in thrombus formation in thrombus Erythrocytes over the physiological range of platelet concentration (200-400 x 10 /L), the increase of ETP over physiological range of hematocrit (40-50%) was 3 times higher. This suggests that thrombin generation in non-flowing blood may be more sensitive to the erythrocyte concentration than to the platelet concentration. Using a thin-layer technology and a Rhodamin 110-based thrombin substrate, Ninivaggi et al. were able to study the influence of hematocrit on thrombin generation directly in whole blood samples.71 It is common knowledge that addition of synthetic phospholipid vesicles is essential for thrombin generation in platelet poor plasma. Addition of washed erythrocytes to PPP can restore thrombin generation. Thrombin generation in mixtures that contained washed erythrocytes (devoid of platelets and leukocytes) in PPP showed that thrombin peak values increased with hematocrit and reached a plateau value at an hematocrit of about 14%.71 These results confirmed the report of Horne et al. showing that ETP increases as the hematocrit was elevated, but only to a maximum of 14% hematocrit. Besides the apparent differences in technical approaches for measuring thrombin generation, this divergence could be attributed to the different concentrations of TF used to trigger the coagulation. An excess of TF (110 pM) was used in the study of Horne, and a sub-optimal concentration (0.5 pM) of TF was used in that of Ninivaggi. Moreover, at high TF concentration the coagulation cascade will predominantly depend on the classic extrinsic pathway. However, at low TF concentration the thrombin production will also involve factors of the intrinsic pathway. So the discrepancy between these two studies may well represent different pathways by which erythrocytes support coagulation. Erythocytes and thrombin generation

The above mentioned studies clearly showed that the erythrocyte membrane can provide a pro-coagulant surface supporting thrombin generation, but the mechanism by which prothrombin is activated on erythrocyte membranes is, however, still not entirely clear. Erythrocytes are known to express PS. About 0.5% of the erythrocyte population in healthy individuals is PS positive.73-75 A study from Whelihan et al Chapter 2 Chapter shows that the pro-coagulant effects of erythrocyte membranes were generated from this subpopulation of PS positive cells. There are roughly 550 prothrombinase binding 37 sites per PS-positive erythrocyte. Blocking the PS on the erythrocyte membrane using unlabeled bovine lactadherin could almost totally abolish the erythrocyte prothrombinase activity.73 More interestingly, with immunoassays capable of selective quantization of meizothrombin-antithrombin and alpha-thrombin-antithrombin complexes, Whelihan showed that after stimulation with TF, prothrombin activation can occur on the erythrocyte membrane through meizothrombin.73 Meizothrombin is an active but intermediate product during prothrombin activation. It can be measured in clotting blood and it will be rapidly cleaved into alpha-thrombin. Meizothrombin itself has poor fibrinogen cleavage activity. Together with surface- bound thrombomodulin, it can activate the protein C pathway and thus exert anti- coagulant activity. Since the prothrombinase activation pathway on platelets does Erythrocytes in thrombus formation in thrombus Erythrocytes not release meizothrombin, Whelihan et al for the first time identified erythrocytes as the major surface supporting meizothrombin production in blood. Based on these findings, Whelihan proposed that in normal hemostasis, erythrocytes can actively participate as both a pro- and anti- coagulant.73;76 On the one hand, erythrocytes provide PS positive surfaces for supporting prothrombin activity; on the other hand, intermediate product meizothrombin can act as an anticoagulant enzyme to prevent excessive clot formation. In a venous situation, which involves low shear flow, both effects of erythrocyte may be important to regulate the balance of coagulation.73;76 It is worth noting that the above mentioned studies have all used external TF as the trigger for coagulation, and thus reflecting the pro-coagulant activity of erythrocytes through the extrinsic pathway. With a different technique, Kawakami et al. demonstrated that normal erythrocytes can activate the intrinsic pathway by directly activating FIX.77 Interestingly, coagulation did not take place in erythrocytes resuspended in FIX-deficient PPP, while erythrocytes in FVII, FXI or FXII deficient PPP continue to support coagulation, which suggests a direct activation of FIX by erythrocyte membrane.77 Later on, the same research group identified an elastase- like enzyme on erythrocyte membranes capable of activating factor IX.78;79 Receptors involved in thrombus formation In the adherent state, erythrocytes express a multitude of adhesion receptors that in normal circulating erythrocytes are inaccessible to their protein ligands. The expression of these molecules may increase the risk of thrombosis.80 For example,

in sickle cell disease, very late antigen-4 (VLA-4, also called integrin α4β1), CD44 (hyaluronic acid receptor), basal-cell adhesion molecule or Luthrean blood group glycoprotein (B-CAM/LU), CD36 (thrombospondin receptor), CD47, and intercellular adhesion molecule 4 (ICAM-4) are the major receptors involved in the erythrocyte adhesion.80-89 In malaria, erythrocytes infected by mature forms of P. falciparum become adhesive for a number of different cells including vascular Chapter 2 Chapter endothelial cells, platelets and other infected or non-infected erythrocytes and the 38 interactions are mediated by plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1).90;91 In diabetes mellitus, a major risk factor for vascular thrombosis, erythrocytes have increased adhesion to endothelium due to a glycated erythrocyte band 3 protein. 92;93 Intracellular adhesion molecule-4

ICAM-4 is an erythroroid-specific membrane component that belongs to the family of immunoglobulin superfamily (IgSF) of proteins.94 It is also known as Landsteiner- Wiener (LW) blood group glycoprotein and is a part of the Rh macromolecule complex. The LWa and LWb phenotypes are encoded by the LWa (100Q) and LWb (100R) alleles at the locus located on chromosome 19p13.95 There is also an allele

Erythrocytes in thrombus formation in thrombus Erythrocytes that results in a null phenotype. Most Europeans are LW (a+). About 5% of Finns and fewer than 1% of other Europeans are LW (b+).96 Three phenotypes have been identified: LW (a+b-), LW (a-b+), and LW (a+b+).96 The LW (a-b-) null phenotype is very rare and the exact prevalence is unknown. ICAM-4 has been shown to

preferentially, but not exclusively, interact with the β2 part of several members of the integrin family.97,98 Interestingly, ICAM-4 has also been demonstrated to be a 99 candidate-ligand for activated αIIbβ3 on platelets. Although a minor blood group antigen, ICAM-4 may possess the ability to influence physiological and pathophysiogical conditions in erythroid and non erythroid 100 tissues. ICAM-4-αvβ3 interaction was shown to be responsible for sickle cell adhesion to endothelium and consequently leads to vascular occlusion in the microcirculation.101 Interestingly, the adhesion of sickle cells to endothelium, but not that of normal erythrocytes, can be induced by epinephrine in a protein Kinase A dependent fashion.101 Targeted gene deletion of murine ICAM-4 showed that deficient mice live and they do not show any gross abnormalities.102 Thus, their hemoglobin content, hematocrit value and other red cell indices are normal. However, the number of erythroblastic islands decreased to about half of that of normal animals. These islands are formed by a central macrophage surrounded by immature erythroid cells. Evidently, the decreased adhesion of the immature red cells to the macrophages (via ICAM-4-CD11c/CD18 interaction) results in the decreased formation of islands.

Involvement of αIIbβ3–ICAM-4 interaction in thrombus formation In our study, first presented at the annual meeting of the American Society of Hematology 2012 in Atlanta, we show for the first time an “active role” for erythrocytes in thrombus formation under flowing conditions.103 Using an in vitro perfusion system, we observed a direct erythrocyte-platelet interaction under conditions of venous flow shear rates (Fig. 3A). This adhesion of erythrocytes to platelets was proved to be at least partially depending on platelet integrin αIIbβ3 and ICAM-4 on erythrocytes (Fig.3B). Binding of erythrocytes to platelets required a higher activation state of platelets and further potentiated this activation state detected via increased P-selectin Chapter 2 Chapter expression. This indicates that both inside out-signaling and outside in signaling are 39 induced following the αIIbβ3–ICAM-4 interaction. The blockage of αIIbβ3–ICAM-4 interaction leads to reduced fibrin and thrombus formation in vitro. This finding is in line with the observation made by Santos104-106 etal. Using a two-stage in vitro system, they have shown that metabolically active erythrocytes can amplify platelet activation (demonstrated by an increased release of platelet α-granule proteins and increased synthesis of thromboxane A2 in the presence of intact erythrocytes). Santos et al. therefore proposed a biochemical messenger model to explain this phenomenon, in which the components of platelet releasate were thought to act as extracellular messenger between erythrocytes and platelets responsible for the biochemical modifications in the erythrocytes necessary to generate prothrombotic surfaces.

Based on our results, we propose a direct and receptor/ligand dependent interaction formation in thrombus Erythrocytes between platelets and erythrocytes. A peptide resembling the binding epitope of

ICAM-4 for αIIbβ3 was able to induce a higher P-selectin expression on platelets implies that the direct erythrocyte binding to platelets causes an out-side-in signaling in platelets and thus further promotes platelet activation.103

A higher platelet P-selectin expression (together with enhanced αIIbβ3 activation) in the presence of erythrocytes was also observed by the research group of Santos and Valles.104 Interestingly, both the model by Santos et al. and our receptor/ligand model support a promoting effect of the erythrocyte-platelet interaction on platelet activation. We hypothesize that at situations of platelet activation erythrocytes will bind to platelets via the αIIbβ3-ICAM-4 interaction (Fig. 3B). This interaction may on one side leads to out-side-in signaling and further activation of platelets, and on the other side may cause erythrocyte cell deformation (through shear influence and probably erythrocyte cytoskeleton rearrangement), followed by A

B Chapter 2 Chapter

40

Fig. 3. Erythrocyte binding to platelets under flow. (A) Erythrocyte binding to platelet aggregates formed on the Erythrocytes in thrombus formation in thrombus Erythrocytes surface of an endothelial monolayer. Washed erythrocytes and platelets resuspended together in physiological buffer were perfused over an endothelial monolayer for 5 minutes at shear rate of 100 s-1. Images were taken under the differential interference contrast (DIC) setting with a digital camera mounted on an inverted light microscope (Carl Zeiss Observer 1). The white triangle indicates erythrocytes, while the white arrow indicates platelet aggregates. (B) Schematic illustration of erythrocyte binding to platelets mediated via intercellular adhesion

molecule 4 (ICAM-4) and integrin αIIbβ3.

possible signaling in erythrocyte and biochemical messenger release. Conclusion Erythrocytes are more versatile than just being the suppliers of oxygen to the organs of the body. The mechanisms responsible for thrombus formation are not completely elucidated. However, within recent years, direct or indirect evidence has been accumulating which indicate a putative active contribution of erythrocytes in thrombus formation. Erythrocytes seem to be both passively and actively involved in thrombus formation. Their influence on thrombosis and hemostasis can be executed via their well-known effects on rheology and blood viscosity, but can also be carried out via their recently identified influence on thrombin formation and fibrin deposition, which in turn may be mediated by their interactions with endothelium and platelets. We have recently found evidence, further proving this hypothesis, showing that erythrocytes are able to actively bind to platelets thereby increasing the number of erythrocytes in a growing thrombus and further activating platelets. Clinical studies will show the precise clinical significance of the ICAM-4-αIIbβ3 interaction. Acknowledgements We thank the Dutch Heart Foundation (NHS 2006T5301, BdL), Sanquin Blood Supply Foundation (BdL) and the Netherlands Organization of Health Research and Development (ZonMW) (CM) for financial support. We thank Andrea Almering for her contribution in making the drawings. Chapter 2 Chapter

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Chapter 2 Chapter 1990;10:9-48 58. Yeung T, Gilbert GE, Shi J et al. Membrane 44 48. Davie EW, Fujikawa K, Kisiel W. phosphatidylserine regulates surface The coagulation cascade: initiation, charge and protein localization. Science maintenance, and regulation. Biochemistry 2008;319:210-213 1991;30:10363-10370 59. Woon LA, Holland JW, Kable EP, Roufogalis 49. Renne T, Schmaier AH, Nickel KF, BD. Ca2+ sensitivity of phospholipid Blomback M, Maas C. In vivo roles of factor scrambling in human red cell ghosts. Cell XII. Blood 2012;120:4296-4303 Calcium 1999;25:313-320. 50. Rosing J, Tans G, Govers-Riemslag 60. Dekkers DW, Comfurius P, Bevers EM, JW, Zwaal RF, Hemker HC. The role Zwaal RF. Comparison between Ca2+- of phospholipids and factor Va in the induced scrambling of various fluorescently prothrombinase complex. J.Biol.Chem. labelled lipid analogues in red blood cells. 1980;255:274-283 Biochem.J. 2002;362:741-747

Erythrocytes in thrombus formation in thrombus Erythrocytes 51. Hemker HC, Esnouf MP, Hemker PW, 61. Shi J, Shi Y, Waehrens LN et al. Lactadherin Swart AC, Macfarlane RG. Formation of detects early phosphatidylserine exposure prothrombin converting activity. Nature on immortalized leukemia cells undergoing 1967;215:248-251 programmed cell death. Cytometry A 2006;69:1193-1201 52. van DG, Tans G, Rosing J, Hemker HC. The role of phospholipid and factor VIIIa in the 62. Morel O, Jesel L, Freyssinet JM, Toti activation of bovine factor X. J.Biol.Chem. F. Cellular mechanisms underlying the 1981;256:3433-3442 formation of circulating microparticles. Arterioscler.Thromb.Vasc.Biol. 2011;31:15- 53. Gailani D, Broze GJ Jr. Factor XI activation 26 in a revised model of blood coagulation. Science 1991;253:909-912 63. van Beers EJ, Schaap MC, Berckmans RJ et al. Circulating erythrocyte-derived 54. Maas C, Meijers JC, Marquart JA, et al. microparticles are associated with Activated factor V is a cofactor for the coagulation activation in sickle cell disease. activation of factor XI by thrombin in plasma. Haematologica 2009;94:1513-1519 Proc Natl Acad Sci U S A. 2010;107:9083- 9087 64. Horne MK, III, Cullinane AM, Merryman PK, Hoddeson EK. The effect of red blood anionic phospholipid exposure in patients cells on thrombin generation. Br.J.Haematol. with beta-thalassaemia major. Br.J.Haematol. 2006;133:403-408 1997;98:51-56 65. van der Meijden PE, Van SM, Van OR 75. Wood BL, Gibson DF, Tait JF. Increased et al. Platelet- and erythrocyte-derived erythrocyte phosphatidylserine exposure microparticles trigger thrombin generation in sickle cell disease: flow-cytometric via factor XIIa. J.Thromb.Haemost. measurement and clinical associations. 2012;10:1355-1362 Blood 1996;88:1873-1880 66. Setty BN, Rao AK, Stuart MJ. Thrombophilia 76. Whelihan MF, Mann KG. The role of the in sickle cell disease: the red cell connection. red cell membrane in thrombin generation. Blood 2001;98:3228-3233 Thromb Res. 2013;131:377-382 67. Bonomini M, Sirolli V, Settefrati N et al. 77. Kawakami S, Kaibara M, Kawamoto Y, Increased erythrocyte phosphatidylserine Yamanaka K. Rheological approach to the exposure in chronic renal failure. J.Am.Soc. analysis of blood coagulation in endothelial Nephrol. 1999;10:1982-1990 cell-coated tubes: activation of the intrinsic reaction on the erythrocyte surface. 68. Bonomini M, Sirolli V, Gizzi F et al. 2 Chapter Biorheology 1995;32:521-536 Enhanced adherence of human uremic erythrocytes to vascular endothelium: role 78. Kaibara M, Iwata H, Ujiie H, Himeno 45 of phosphatidylserine exposure. Kidney Int. R, Kaibara M. Rheological analyses 2002;62:1358-1363 of coagulation of blood from different individuals with special reference to 69. Setty BN, Betal SG. Microvascular procoagulant activity of erythrocytes. Blood endothelial cells express a phosphatidylserine Coagul.Fibrinolysis 2005;16:355-363 receptor: a functionally active receptor for phosphatidylserine-positive erythrocytes. 79. Kawakami S, Kaibara M, Kawamoto Y, Blood 2008;111:905-914 Yamanaka K. Rheological approach to the analysis of blood coagulation in endothelial 70. Sola E, Vaya A, Martinez M et al. cell-coated tubes: activation of the intrinsic Erythrocyte membrane phosphatidylserine reaction on the erythrocyte surface. exposure in obesity. Obesity.(Silver.Spring) Biorheology 1995;32:521-536 2009;17:318-322

80. Wautier JL, Wautier MP. Molecular basis formation in thrombus Erythrocytes 71. Ninivaggi M, Apitz-Castro R, Dargaud Y of erythrocyte adhesion to endothelial cells et al. Whole-blood thrombin generation in diseases. Clin.Hemorheol.Microcirc. monitored with a calibrated automated 2013;53:11-21 thrombogram-based assay. Clin.Chem. 2012;58:1252-1259 81. El NW, Gauthier E, Wautier MP et al. Role of Lu/BCAM in abnormal adhesion of sickle 72. Peyrou V, Lormeau JC, Herault JP et al. red blood cells to vascular endothelium. Contribution of erythrocytes to thrombin Transfus.Clin.Biol. 2008;15:29-33 generation in whole blood. Thromb. Haemost. 1999;81:400-406 82. Hermand P, Gane P, Huet M et al. Red cell ICAM-4 is a novel ligand for platelet- 73. Whelihan MF, Zachary V, Orfeo T, Mann activated alpha IIbbeta 3 integrin. J.Biol. KG. Prothrombin activation in blood Chem. 2003;278:4892-4898 coagulation: the erythrocyte contribution to thrombin generation. Blood 2012;120:3837- 83. Hermand P, Gane P, Callebaut I et al. 3845 Integrin receptor specificity for human red cell ICAM-4 ligand. Critical residues 74. Ruf A, Pick M, Deutsch V et al. In-vivo for alphaIIbeta3 binding. Eur.J.Biochem. platelet activation correlates with red cell 2004;271:3729-3740 end products blocks hyperpermeability in diabetic rats. J.Clin.Invest 1996;97:238-243 84. Joneckis CC, Ackley RL, Orringer EP, Wayner EA, Parise LV. Integrin alpha 4 beta 94. Ihanus E, Uotila LM, Toivanen A, Varis 1 and glycoprotein IV (CD36) are expressed M, Gahmberg CG. Red-cell ICAM-4 is on circulating in sickle cell a ligand for the monocyte/macrophage anemia. Blood 1993;82:3548-3555 integrin CD11c/CD18: characterization of the binding sites on ICAM-4. Blood 85. Sugihara K, Sugihara T, Mohandas N, 2007;109:802-810 Hebbel RP. Thrombospondin mediates adherence of CD36+ sickle reticulocytes to 95. Sistonen P. Linkage of the LW blood endothelial cells. Blood 1992;80:2634-2642 group locus with the complement C3 and Lutheran blood group loci. Ann.Hum.Genet. 86. Swerlick RA, Eckman JR, Kumar A, Jeitler 1984;48:239-242 M, Wick TM. Alpha 4 beta 1-integrin expression on sickle reticulocytes: vascular 96. Sistonen P, Green CA, Lomas CG, Tippett cell adhesion molecule-1-dependent binding P. Genetic polymorphism of the LW to endothelium. Blood 1993;82:1891-1899 blood group system. Ann.Hum.Genet. 1983;47:277-284 Chapter 2 Chapter 87. van Schravendijk MR, Handunnetti SM, Barnwell JW, Howard RJ. Normal human 97. Bailly P, Tontti E, Hermand P, Cartron JP, 46 erythrocytes express CD36, an adhesion Gahmberg CG. The red cell LW blood group molecule of monocytes, platelets, and protein is an intercellular adhesion molecule endothelial cells. Blood 1992;80:2105-2114 which binds to CD11/CD18 leukocyte integrins. Eur.J.Immunol. 1995;25:3316- 88. Wautier JL, Wautier MP. Erythrocytes 3320 and platelet adhesion to endothelium are mediated by specialized molecules. Clin. 98. Ihanus E, Uotila LM, Toivanen A, Varis Hemorheol.Microcirc. 2004;30:181-184 M, Gahmberg CG. Red-cell ICAM-4 is a ligand for the monocyte/macrophage 89. Zen Q, Cottman M, Truskey G, Fraser integrin CD11c/CD18: characterization R, Telen MJ. Critical factors in basal cell of the binding sites on ICAM-4. Blood adhesion molecule/lutheran-mediated 2007;109:802-810 adhesion to laminin. J.Biol.Chem. 1999;274:728-734 99. Hermand P, Gane P, Huet M et al. Red

Erythrocytes in thrombus formation in thrombus Erythrocytes cell ICAM-4 is a novel ligand for platelet- 90. Hughes KR, Biagini GA, Craig AG. activated alpha IIbbeta 3 integrin. J.Biol. Continued cytoadherence of Plasmodium Chem. 2003;278:4892-4898 falciparum infected red blood cells after antimalarial treatment. Mol.Biochem. 100. Toivanen A, Ihanus E, Mattila M, Lutz HU, Parasitol. 2010;169:71-78 Gahmberg CG. Importance of molecular studies on major blood groups--intercellular 91. Miller LH, Good MF, Milon G. Malaria adhesion molecule-4, a blood group antigen pathogenesis. Science 1994;264:1878-1883 involved in multiple cellular interactions. 92. Grossin N, Wautier MP, Picot J, Stern DM, Biochim.Biophys.Acta 2008;1780:456-466 Wautier JL. Differential effect of plasma 101. Zennadi R, Hines PC, De Castro LM et al. or erythrocyte AGE-ligands of RAGE Epinephrine acts through erythroid signaling on expression of transcripts for receptor pathways to activate sickle cell adhesion isoforms. Diabetes Metab 2009;35:410-417 to endothelium via LW-alphavbeta3 93. Wautier JL, Zoukourian C, Chappey O interactions. Blood 2004;104:3774-3781 et al. Receptor-mediated endothelial cell 102. Lee G, Lo A, Short SA et al. Targeted dysfunction in diabetic vasculopathy. gene deletion demonstrates that the cell Soluble receptor for advanced glycation adhesion molecule ICAM-4 is critical for erythroblastic island formation. Blood 2006;108:2064-2071 103. Du VX, de Groot PG, van Wijk R, Ruggeri ZM, de Laat B. Binding of erythrocyte ICAM–4 to the platelet activated integrin αIIbβ3 leads to a direct erythrocyte-platelet adhesion under venous flow shear rate. Paper presented at:Annual Meeting of the American Society of Hematology; December 9, 2012; Atlanta, GA 104. Valles J, Santos MT, Aznar J et al. Erythrocytes metabolically enhance collagen-induced platelet responsiveness via increased thromboxane production, adenosine diphosphate release, and

recruitment. Blood 1991;78:154-162 2 Chapter 105. Valles J, Santos MT, Aznar J et al. Platelet- 47 erythrocyte interactions enhance alpha(IIb) beta(3) integrin receptor activation and P-selectin expression during platelet recruitment: down-regulation by aspirin ex vivo. Blood 2002;99:3978-3984 106. Santos MT, Valles J, Marcus AJ et al. Enhancement of platelet reactivity and modulation of eicosanoid production by intact erythrocytes. A new approach to platelet activation and recruitment. J.Clin. Invest 1991;87:571-580 Erythrocytes in thrombus formation in thrombus Erythrocytes Chapter 2 Chapter

48 Erythrocytes in thrombus formation in thrombus Erythrocytes 3

Identification of intercellular adhesion molecule 4 on erythrocytes as mediator of erythrocyte- platelet interaction in thrombus formation

Vivian X.Du,1,2 Philip.G. de Groot,1, Richard van Wijk,1 Zaverio M. Ruggeri3 and Chapter 3 Chapter Bas de Laat1,2,3,4,5 49

1Department of Clinical Chemistry and Hematology, University Medical Center Utrecht, Utrecht, The Netherlands 2Sanquin Research, Amsterdam, The Netherlands 3Roon Research Center for Arteriosclerosis and Thrombosis, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA

4Departement of Biochemistry, CARIM, University of Maastricht, Maastricht, The interaction Erythrocyte-platelet Netherlands 5Synapse BV, Maastricht, The Netherlands

Manuscript submitted for publication Abstract Erythrocytes are generally considered as passive bystanders in hemostasis predominantly based on their rheological functions. In this study we investigated additional roles of erythrocytes in hemostasis. Our in vitro studies with microfluidic perfusion devices demonstrated that erythrocytes are able to bind to adherent platelets under venous shear rates, especially when platelets were pre-activated by various stimuli. We further observed that blocking agents against the beta chain of

αIIbβ3 significantly inhibited erythrocyte binding to platelets. Erythrocyte intercellular

adhesion molecule 4 (ICAM-4) has been reported to be a ligand for activated αIIbβ3 under static conditions. Under flowing conditions we found that by blocking ICAM- 4, erythrocyte-platelet adhesion was significantly reduced. Applying flow cytometry we found a decreased fibrinogen binding to platelets and an increased P-selectin expression on platelets upon addition of an ICAM-4 peptide resembling the second extracellular domain of ICAM-4. These findings suggest that ICAM-4 competes Chapter 3 Chapter

with fibrinogen for binding to activated αIIbβ3 and that the interaction of ICAM- 50 4 peptide with activated αIIbβ3 leading to inside-out signalling increases P-selectin exposure. In addition, we observed that blocking ICAM-4 activity led to reduced thrombin generation in whole blood. Moreover, perfusion with re-calcified whole blood over collagen-coated surfaces showed a reduced fibrin deposition in the presence of a blocking ICAM-4 peptide. In conclusion, we found direct erythrocyte- platelet interactions under conditions of low shear. This interaction is at least partially

mediated via erythrocyte ICAM-4 and platelet activated integrin αIIbβ3.Interactions

between ICAM-4 and αIIbβ3 are likely to triggersignaling cascades inside platelets and promote platelet activation. Thus, ICAM-4 targeting approaches may hold promises as future therapeutic strategies in treating diseases with thrombotic complications. Erythrocyte-platelet interaction Erythrocyte-platelet Introduction Blood is composed of a cellular and plasma compartment. Despite the fact that erythrocytes are the most abundant cellular component of the human blood comprising 40% of its volume, most attention has been driven towards platelets as major contributors of thrombus formation. Platelets are thought to be the first and primary components of an initiating thrombus under conditions of flow by rolling over von Willebrand factor (VWF) via receptor glycoprotein–Ibα (GPIbα) on platelets and subsequently getting stabilized on collagen via glycoprotein VI and alpha(2)beta(1).1;2 Exposed tissue factor initiates the formation of thrombin and subsequently fibrin, stabilizing the thrombus.3 Erythrocytes have been primarily thought to be responsible for the O2 supply throughout the human body. Since the early nineties, data has accumulated for a role of erythrocytes in thrombosis. Due to their rheological properties, it has been shown that by increasing the hematocrit, excessive bleeding events can be treated.4-6 In addition, an increased hematocrit is related to an increase in thrombotic events7-9 and several erythrocyte-based diseases 3 Chapter such as sickle cell disease,10;11 thallasemia and malaria are associated with thrombotic 51 events.12-15 Lately, evidence is piling up for a more active role of erythrocytes in thrombus formation thereby possibly transforming the platelet-based mechanism of thrombus formation to a more heterogeneous mechanism involving different cell types such as erythrocytes. It was shown that erythrocytes can pre-activate or prime platelets to make them hyper-responsive to prothrombotic stimuli.16-18 The group of Motto et al. demonstrated that in a FeCl-based mouse model for thrombosis, erythrocytes were the first and predominant cell type present at the site of injury.19 Erythrocytes were shown to serve as attachment sites for platelets to adhere to the site of injury without a role for the VWF-GPIbα interaction. In addition, the group of Mann et al. interaction Erythrocyte-platelet demonstrated that platelets are not the only contributors to a prothrombotic surface. Erythrocytes also actively participate in the generation of thrombin and thereby fibrin formation by mediating the conversion of prothrombin into meizothrombin, an intermediate of thrombin.20;21 From a more molecular level, intercellular adhesion molecule 4 (ICAM-4) on the membrane of erythrocytes has been indicated to be responsible for erythroycte- platelet interaction under static conditions in vitro.22 ICAM-4 (Landsteiner-Wiener blood group glycoprotein) is an erythroroid-specific membrane component that belongs to the family of immunoglobulin superfamily of proteins.23 It is composed of two extracellular immunoglobulin(Ig)-like domains (domain 1 C2 type 1, domain 2 C2 type 2), an N-terminal I set and a membrane proximal I2 set, and a single membrane spanning domain.24;25 Although a minor blood group antigen, ICAM-4 has been shown to play an important role under physiological and phathophysiological conditions in erythroid and non erythroid tissues.26 ICAM-4 has been demonstrated to preferentially, but not exclusively, interact with the β2 part of several members 27 of the integrin family. Moreover, ICAM-4-αvβ3 interaction was responsible for sickle cell adhesion to endothelium and consequently led to vaso-occlusion in the microcirculation.28 It was also demonstrated that ICAM-4 is a candidate-ligand for 22 activated αIIbβ3 on platelets. The aim of our study was to further characterize the

ICAM-4 - αIIbβ3 interactions under flow-conditions and investigate whether and how this interaction influences thrombus formationin vitro and in vivo. Materials and methods Materials

R-phycoerythrin (RPE) labeled mouse anti-human P-selectin antibody and mouse

anti-human CD61 (integrin β3 chain) monoclonal antibody were purchased from BD biosciences (Franklin Lakes, NJ, U.S.A). Goat anti-human ICAM-4 polyclonal antibody (epitope mapping extracellular domain 2 of ICAM-4) and ICAM-4 peptide mimicking the epitope of ICAM-4 antibody were purchased from Santa Cruz Chapter 3 Chapter biotechnology inc (Delaware Avenue, CA, USA). An ICAM-4 peptide mimicking a 52 region in domain 1 of ICAM-4 was produced by CASLO ApS (Lyngby Denmark). The ICAM-4 antibody was dialyzed against PBS for 3 times before use to remove residual azide. Human VWF was purified as described.29 Human fibrinogen (plasminogen, VWF and fibronectin free) was obtained from Enzyme Research Labs (South Bend, IN, USA). Thrombin was purchased from Enzyme Research Laboratories ( South Bend, IN). Adenosine diphosphate (ADP) was purchased from Roche (Mannheim, Germany). Cross-linked collagen related peptide (CRP) was a generous gift of R. Farndale (Cambridge, UK). Arachidonic acid (AA) was purchased from Bio/Data Corporation (Horsham, PA, USA). Alex Fluor 488-conjugated fibrinogen was purchased from Molecular Probes Inc (Eugene, OR, USA). Alex

Erythrocyte-platelet interaction Erythrocyte-platelet Fluor 546-conjugated fibrinogen was bought from Life Technologies Europe BV (Bleiswijk, the Netherlands). The RGD containing peptide D-Arginyl-Glycyl-L- Aspartyl-L-Tryptophan (dRGDW) was synthesized at the department of Membrane Enzymology, Faculty of Chemistry, University of Utrecht, the Netherlands. Collagen Type I (Collagen reagen Horm Suspension) was purchased from NYCOMED (Austria GmbH, Linz, Austria). DiOC6 dye for platelet labeling was bought from Life Technologies (Carlsbad, CA, USA). Cell isolation

Blood from healthy volunteers, who claimed not to have used aspirin or other nonsteroidal anti-inflammatory drugs for the preceding 10 days, was drawn into one-tenth volume of 3.2% sodium citrate. Washed platelets were prepared as described previously.30 In brief, the blood was centrifuged at 156g for 15 min at room temperature. The platelet-rich plasma (PRP) was collected and acidified by addition of one-tenth volume of acid citrate dextrose (ACD, 2.5% trisodium citrate, 1.5% citric acid, and 2% D-glucose). Platelets were centrifuged (330g, 15 min) and the platelet pellet was resuspended in Hepes-Tyrode buffer at pH 6.5 (10 mM Hepes [N-2-hydroxyethylpiperazine-N’-2ethanesulfonic acid], 137 mM NaCl, 2.68 mMKCl, 0.42 mM NaH2PO4, 1.7 mM MgSO4, 5 mM D-glucose). Prostacyclin (PGI2, 10 ng/ml) was added to prevent platelet activation during the subsequent washing step. The platelet suspension was centrifuged (330g, 15 min) and resuspended in a small volume of HEPES-Tyrode buffer at pH 7.3. Erythrocytes were isolated from the same blood sample as washed platelets by using a PEGG elution column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and cellulose.31 Briefly, an equal weight of α-cellulose and cellulose type 50 were suspended in excessive saline (0.9% NaCl). The cellulose suspension was kept in 4 °C overnight, and the supernatant was discarded until final cellulose volume and the supernatant volume was approximately 1:1. This cellulose suspension was poured into elution columns. After saline was drained from the column, 3 to 5 ml whole blood was applied on top of the cellulose. The column was placed in a clean 50 ml tube and centrifuged at room temperature for 5 min (50g). 5 ml saline was added and the column was centrifuged again at 50g for 5min to elute the erythrocytes. 3 Chapter The column was removed and eluted erythrocytes were washed with saline and 53 centrifuged at 1000g for 5 min (room temperature). Erythrocytes were washed again with Ringer solution (32 mM HEPES, 125 mM NaCl, 5 mM KCl, 1 mM MgSO4, 5 mM glucose, 1 mM CaCl2, pH=7.4) and finally resuspended in Ringer solution. Perfusion experiments Protein coating

Glass coverslips (24×50 mm, MenzelGläser) were pre-cleaned overnight with chromosulfuric acid (2% chromium trioxide) solution and rinsed with distilled water before coating. Platelet adhesive proteins (collagen type I, fibrinogen or VWF) were immobilized on coverslips by a 1.5 hour incubation with 300 µl 100 µg/ml collagen interaction Erythrocyte-platelet type I, 300 µl 100 µg/ml fibrinogen or 300 µl 30 µg/ml VWF. After coating, the coverslips were blocked with 1.5% human albumin in phosphate-buffered saline (PBS; 10 mM sodium phosphate, 150 mM NaCl, pH 7.4) for at least one hour at room temperature. Perfusion system

Perfusions were carried out in a single-pass perfusion chamber as described previously.32 The chamber contained an inlet, outlet, a vacuum channel, and on the top, a silicone rubber gasket. A flow hole was bored in this silicone gasket to create a perfusion slit (slit width: 2 mm; length: 3 cm; height = thickness of sheet: 0.125 mm) connecting the inlet with the outlet. On both sides, a slit was cut in the silicon gasket next to the flow area to obtain two vacuum compartments. The coverslip was mounted on this silicone sheet by a vacuum force through the vacuum channel. The chamber with a protein coated coverslip was put upside down on an inverted optical microscope (Carl Zeiss Axioobserver. Z1, Oberkochen, Germany) with a CCD camera attached. A Harvard infusion pump (pump 22, model 2400-004; Natick, MA, USA) was used to flow samples through the perfusion chamber with different shear rates. Perfusion with erythrocyte-platelet cell suspensions and whole blood

Washed platelets and washed erythrocytes were mixed in Ringer solution to reach platelet counts of 150 x 109/l and hematocrit of 5%. Samples of erythrocyte-platelet suspensions or whole blood were pre-warmed to 37°C and were drawn through the perfusion chamber over collagen- or VWF-coated surfaces for 5 min. After 5 minutes perfusion, no less than 8 pictures were taken under flow at different field views under differential interference contrast (DIC) settings. In indicated experiments, platelet stimuli, such as CRP, thrombin, ADP or arachidonic acid, were added directly prior to perfusion at the indicated concentrations. In other experiments, vehicle (PBS buffer) or reagents (dRGDW, anti-CD 61, anti-CD41, anti-ICAM4 or ICAM-4 peptide) were preincubated in the samples for 3 min at 37°C before perfusion. Images were Chapter 3 Chapter quantified by counting erythrocyte numbers on each picture by Image J software. 54 Erythrocyte number per mm2 (erythrocyte density) on the cover glass surface was calculated from the mean erythrocyte count per picture. Fold increase of erythrocyte binding was calculated by dividing erythrocyte density in each condition by that in vehicle control. Perfusion on endothelial cells

Human umbilical vein endothelial cells (HUVECs) from healthy newborns were isolated as described before,33 and cultured in endothelial basal medium (EBM-2) supplemented with EGM-2 bullet kits (Lonza). HUVECs at passage number 2 or 3 were grown on glass coverslips (24×50 mm, Menzel Gläser) until confluence.

Erythrocyte-platelet interaction Erythrocyte-platelet Prior to perfusion, coverslips were washed once in EBM medium. Platelets and erythrocytes were isolated and suspended in physiological buffer as described above. Platelet suspensions (200 x 109/L) together with histamine 10 µM were perfused over HUVEC-coated coverslips for 5 min at 100 s-1, followed by perfusion of erythrocyte suspensions at 100 s-1 for another 5 min. Images were taken under DIC microscopic settings at the end of the 5-min platelet perfusion as well as at the end of the erythrocyte perfusion. Perfusion with recalcified whole blood

Collagen type I was coated on glass coverslips as described above. Blood from healthy volunteers was drawn into one-tenth volume of 3.2% sodium citrate. DiOC6- (1 µM, to label platelets) and Alexa Fluor 546-labeled fibrinogen (6.2 µg/mL) was added into citrated whole blood. Whole blood samples with fluorescent dyes were then -1 perfused on collagen-coated coverslips for 10 min at a shear rate of 100 s . CaCl2 (50mM in 0.9% NaCl) was continuously injected into the blood flow (against the flow direction) at shear rate of 50 -1s during whole blood perfusion. The process of thrombus formation was monitored in real time under an inverted microscope (Carl Zeiss Axio observer. Z1) with DIC settings. After 10 min perfusion, pictures were taken under both fluorescent microscopic settings and DIC settings. PBS buffer (as vehicle control) or ICAM-4 peptide (10 µg/ml) was added to whole blood samples directly prior to perfusion. Flow cytometric analysis

The effect of an ICAM-4 peptide on fibrinogen binding to platelets and P-selectin expression under ADP stimulation was evaluated by flow cytometry. Serial dilutions of ADP (125 µM, 31 µM, 8 µM, 2 µM, 490 nM, 120 nM, 31nM and 8 nM) were prepared in 50 µl HEPES buffered saline (HBS;10 mM HEPES, 150 mM NaCl, 1 mM MgSO4, 5 mM KCl, pH 7.4). 2 µl PE-labeled mouse anti-human P-selectin antibody (dilution ratio 1:25) and 0.5 μL Alex Fluor 488-conjugated fibrinogen (dilution ratio 1:100, final concentration 15 µg/ml) were added into the ADP solutions. PE- conjugated mouse IgG1 was used as isotype control for P-selectin staining. Five µl Chapter 3 Chapter fresh, citrate-anticoagulated whole blood was added to each sample of the above serial dilutions and to the isotype control sample. After 20 minutes of incubation 55 at room temperature, the samples were fixed with 800 µl 0.2% formyl saline (0.2% formaldehyde in 0.9% NaCl). Samples were then analyzed on a FACS Canto II flow cytometer from BD Biosciences (Franklin Lakes, NJ, USA). Platelets were gated based on forward and side scatter properties. The mean fluorescence intensity (MFI) in PE and Alex Fluor 488 channels in the platelet gate was used as output for platelet P-selectin expression and fibrinogen binding respectively. Data were analyzed with BD FACS Diva software. Whole blood thrombin generation

34 Whole blood thrombin generation was executed as recently described. Paper interaction Erythrocyte-platelet discs were placed in flat bottom wells of a 96-wells microplate (Thermo Electron Corporation, Milford, MA, USA). Separately, 30 µL of citrated whole blood was mixed with 10 µL P2Rho (1.8 mM) and 20 µL TF and CaCl2 (1.5 pM and 50 mM, respectively). Immediately after mixing, a sample of 5 µL was pipetted on paper disks and covered with 40 µL of mineral oil (USB Corporation, Cleveland, OH, USA).

The final concentrations were: 50% whole blood or PPP, 0.5 pM TF, 16.7 mM CaCl2, and 300 mM P2Rho. In a parallel calibration experiment the TF containing solution was replaced by 20 µL human thrombin calibrator (alpha-2-macroglobulin-bound thrombin, α2M-T, 300 nM thrombin activity). Fluorescence signal was recorded with a Fluoroskan Ascent microplate fluorometer with exl =485 nm and lem=538 nm (Thermolabsystems, Helsinki, Finland). Samples were run at least in triplicate and the calibrated TG curves were calculated as previously described.35 All procedures were performed at 37°C. Scanning electron microscopy (SEM) analysis After perfusion of erythrocyte-platelet suspensions or recalcified blood, 1% glutardehyde in PBS was perfused into the perfusion chamber at 300 s-1 for 10 min to fix the platelet aggregates/clots formed on the coverslips. After fixation, the coverslips were rinsed with 80% ethanol and were kept in ethanol until scanning electron microscope analysis. The platelet aggregate samples collected after perfusion with erythrocyte-platelet suspensions on VWF coated surfaces were sputter-coated with a thin layer of 6.5 platinum (spurt density: 21.45) in a sputter coater and viewed with a scanning electron microscope (Philips XL30, Philips, Netherlands) with backscatter electron detector and secondary electron detector. The samples with A B Chapter 3 Chapter

56

C D Erythrocyte-platelet interaction Erythrocyte-platelet

Fig. 1. Erythrocyte binding to platelets under flow. (A) Representative image showing erythrocyte binding to platelets under conditions of low shear flow in whole blood. Citrated whole blood was perfused on collagen coated coverslips for 5 min at shear rate of 100 s-1. Lower panel is a magnification of the rectangular area depicted in the upper panel. White arrowheads indicate platelets, white arrows indicate erythrocytes. (B) Representative image showing erythrocyte binding to activated platelets in isolated cell suspension. Isolated erythrocyte and platelet cell suspensions in physiological buffer were perfused over Von Willebrand Factor (VWF) coated coverslips for 5 min at shear rate of 100 s-1. Collagen-related peptides (CRP, 500 ng/ml) were added into the cell suspension before perfusion. (C-D) Representative scanning electronic microscope (SEM) images of erythrocyte binding to platelet aggregates. Isolated erythrocyte and platelet mixture was perfused on VWF coated coverslips for 10 min at a shear rate of 100 s-1in the presence of 500 ng/ml CRP. After perfusion, samples were fixed with 1% glutheraldehyde and applied for SEM analysis. fibrin clot collected after purfusing recalcified whole blood on collagen surface were coated with gold and viewed with a desktop scanning microscope (Phenom-World BV, Eindhoven, The Netherlands). Tail-bleeding in mice

Each group consisted of 5 to 10 male imprinting control region (ICR) derived mice weighing 23 +/- 3 grams. Mice were either injected once with aspirin (30 mg/kg body weight, 5 min before tail bleeding test) intravenously, or continuously infused with vehicle control (5% DMSO/PBS), an ICAM-4 peptide resembling a region in domain 1 of murine ICAM-4 or a control peptide resembling a different part of ICAM-4. Continuous infusion took 10 minutes with a rate of 10 µL/min (peptide concentration 2 mg/mL). Immediately after continuous infusion was stopped; the tail of the mice was transacted (3 mm). The mice were immediately placed in holders vertically with 2 cm of the distal tail immersed in saline of 37°C. Maximum cut-off time was 180 seconds. Chapter 3 Chapter

Statistical analysis 57 Statistical analysis was performed using GraphPad Prism 5 software. All data are A B

C interaction Erythrocyte-platelet

Fig. 2. Representative images of erythrocyte binding to platelets, adhering to ultra large Von Willebrand Factor (ULVWF) newly secreted from endothelial cells. Human umbilical vein endothelial cells (HUVECs) from healthy newborns were isolated and grown on glass coverslips till confluence. Platelets and erythrocytes were isolated and suspended in physiological buffer. Platelet suspensions (200 x 109/l) were perfused over HUVEC coated coverslips for 5 min at 100 s-1, followed by perfusion of erythrocyte suspensions at 100 s-1 for another 5 min.(A) Representative image of platelets binding to ULVWF secreted from activated HUVECs, resulting in formation of platelet decorated strings. Image was taken directly after platelet perfusion and before erythrocyte perfusion. (B) Erythrocytes bind to platelet decorated strings and form erythrocyte/platelet aggregates (white ellipses). (C) Representative image showing a single erythrocyte attaching to a platelet decorated string after erythrocyte perfusion. White arrowheads indicate platelets, white arrow indicate erythrocyte. expressed as mean ± SEM, unless stated otherwise. Between-group variations were examined using paired student t-test. A p-value of less than 0.05 was considered statistically significant. Results Erythrocyte binding to platelets under flow

To study erythrocyte platelet interactions, we performed in vitro flow experiments. Whole blood was perfused over collagen at a shear rate of 100s-1 for 5 min. Platelets started to adhere to collagen after a few seconds of perfusion. About one minute later, erythrocytes began to attach to platelets resulting in a tear-drop shape, indicating a focal adhesion point (Fig. 1A). To investigate the interaction between platelets and erythrocytes in more detail and without interference of other cells or soluble factors, we performed perfusion experiments with isolated platelets and erythrocytes in physiological buffer. The perfusion of this cell suspension over a VWF-coated surface Chapter 3 Chapter in the presence of collagen-related peptides gave the same pattern of erythrocyte 58 binding to platelets as observed in whole blood (Fig. 1B). When we applied the latter coverslips to Scanning electron microscopy (SEM) analysis, we observed a direct erythrocytes binding to platelets and integrating in platelet aggregates (Fig. 1C-D). The duration of the direct contact between erythrocytes and platelets applying low shear flow during perfusions with whole blood or isolated cell suspensions varied A Erythrocyte-platelet interaction Erythrocyte-platelet

Fig. 3. Influence of platelet stimuli and RGD peptide on B erythrocyte binding to platelets under flow.(A) Repre- sentative images of erythrocyte binding to platelets un- der various platelet agonists. Erythrocytes and platelets were isolated separately and re-suspended together in ringer buffer to reach a final platelet count of 150 X 106/L and a 5% hematocrit. Erythrocyte-platelet cell suspensions were then perfused on VWF coated cover- slips for 5 minutes at 100 s-1 in the presence of vehicle (PBS buffer) or different platelet agonists—thrombin (1

U/ml), CRP (500 ng/ml), AA (10 µM) and ADP (10 µM). (B) Fold increase of erythrocyte binding to platelets as compared to vehicle control under different stimuli and in the presence or absence of Arg-Gly-Asp-containing pep- tide (RGD peptide, 100 µM). Experimental settings are the same as in Panel a. For each condition, no less than 8 images were applied to ImageJ software for counting bound erythrocytes. Erythrocyte number per mm2 on the coverslip surface was calculated for each condition. Bar graph indicates the fold increase of erythrocyte deposition onto adhered platelets as compared to the corresponding vehicle controls (without RGD peptide). Data are from 4 independent experiments (mean±SEM). *, P<0.05 comparing RGD treated samples with corresponding non-RGD treated samples by paired student t test. from a few seconds to several minutes. In addition, we observed an inverse relationship between the number of adhered erythrocytes and the shear rate (Suppl. Fig. 1). Under situations of damaged/activated endothelial cells (ECs), platelets can adhere to ECs via binding to secreted ultra large (UL) VWF and form aggregates. We used an in vitro perfusion system to mimic situations of EC activation and investigated whether erythrocyte binding to platelets can occur under such conditions. Perfusion of isolated platelets on activated Perfusion of platelets over HUVECs led to the formation of platelet decorated strings caused by platelet adhesion to endothelial derived ULVWF (Fig. 2A). Subsequently, perfusion of isolated erythrocytes over activated HUVECs with platelet strings formed resulted in the binding of erythroycte to the platelet decorated strings (Fig. 2B) and the formation of erythrocyte/platelet aggregates (Fig. 2C). Identification of platelet receptor responsible for binding erythrocytes Chapter 3 Chapter To analyse whether the platelet activation status has an effect on erythrocyte binding, various platelet agonists (thrombin 1 U/mL, collagen related peptide 500 59 ng/mL, arachidonic acid 10 µM or adenosine diphosphate 10 µM) were added to the erythrocyte-platelet cell suspension before perfusion. Subsequently, the samples were perfused over a VWF-coated surface for 5 minutes at a shear rate of 100s- 1. As expected, we observed an increase in erythrocyte binding to platelets in the presence of all four platelet agonists as compared to the vehicle control (Fig. 3A). The agonists induced 3 to 7 fold increase in the binding of erythrocyte to platelets (Fig. 3B white bars). These results indicate that platelets in an increased activation state are better capable to capture erythrocytes from the blood flow.

Since the conformational change of platelet integrin αIIbβ3 from an inactive form to an active form (fibrinogen binding form) upon stimulation is the pivotal step interaction Erythrocyte-platelet in platelet activation and aggregation,36 we investigated the involvement of this integrin in erythrocyte-platelet contact. An Arg-Gly-Asp (RGD) containing peptide

(d-RGDW), known to inhibit platelet aggregation by binding to the αIIbβ3 integrin, was added to the erythrocyte-platelet suspension together with platelet agonists prior to perfusion.37 Samples were then perfused over a VWF-coated survace for 5 min at a shear rate of 100 s-1. Our results show that the RGD peptide inhibited the binding of erythrocyte to platelets, irrespective of the agonists used (29% to 72% decrease, depending on the agonist used, p < 0.05 for all agonists, Figure 3B grey bars), indicating the involvement of αIIbβ3 in erythrocyte-platelet interaction. Erythrocyte-platelet interaction under flow is dependent on the interaction between ICAM-4 and integrin αIIbβ3

As ICAM-4 on erythrocytes has been reported to be a ligand for αIIbβ3 integrin on 38;39 platelets, we tested whether this ICAM-4 -αIIbβ3 interaction is responsible for the erythrocyte-platelet adhesion. Therefore, inhibitory antibody to ICAM-4 and αIIbβ3 A B

C D Chapter 3 Chapter

60

Fig. 4. Influence of antibodies directed against integrinIIb α β3 or ICAM-4 and ICAM-4 mimetic peptide on erythrocyte binding to activated platelets. (A) Erythrocyte binding to activated platelets in the presence of indicated antibodies or peptide. Erythrocytes and platelets were isolated separately and re-suspended together in ringer buffer to reach a final platelet count of 150 X 109/L and a hematocrit of 5%. Erythrocyte-platelet cell suspensions were perfused over VWF-coated coverslips for 5 minutes at a shear rate of 100 s-1. Anti-CD61 (6.25 µg/mL), anti-ICAM-4 (10 µg/mL), ICAM-4 P (10 µg/mL) and combinations of anti-CD61 with anti-ICAM-4 or ICAM-4 P were added to cell suspensions and incubated at 37 °C for 3 minutes. Collagen related peptide (CRP, 500 ng/mL) was added to cell suspensions directly prior to perfusion to activate platelets. The results are expressed as fold increase of bound erythrocytes per mm2 compared to vehicle control from 8 independent experiments. Data are indicated as mean ± SEM. **, p < 0.01, ***, p < 0.001 comparing antibody or peptide treated samples to vehicle controls by paired student t test. (B-D) Dose dependent inhibitory effect of ICAM-4P, anti-ICAM-4, and anti-CD61 on Erythrocyte-platelet interaction Erythrocyte-platelet erythrocyte binding to activated platelets. ICAM-4 peptide, anti-ICAM-4 or anti-CD61 were added to erythrocyte/ platelet mixtures at indicated concentrations and incubated at 37 °C for 3 minutes. CRP (500 ng/mL) was added to the cell suspensions directly prior to perfusion to activate platelets. CRP alone without other reagents was taken as vehicle controls.

(anti-CD61 (anti-β3) and an ICAM-4 peptide resembling the extracellular domain of human ICAM-4 were added to the erythrocyte-platelet mixture samples together with CRP before perfusion. Samples were perfused over a VWF-coated surface for 5 minutes. Our data revealed that the erythrocyte-platelet adhesion under conditions of flow was inhibited by both anti-ICAM-4 (40%,p < 0.01, n=8) and anti-CD61 (28%, p < 0.01, n=8) (Fig. 4A). In addition, the ICAM-4 peptide also reduced the binding of erythrocyte to platelets (46%, p=0.001, n=8). Combining anti-CD61 with either anti-ICAM-4 or ICAM-4 peptide further reduced erythrocyte adhesion as compared to the anti-CD61 alone (22% further reduction when combined with anti-ICAM-4, p < 0.05, n=8; 30% further reduction with ICAM-4 peptide, p < 0.05, n=8). ICAM- 4 peptide, anti-CD61 and anti-ICAM-4 showed a dose dependent inhibitory effect on the erythrocyte-platelet interaction (Fig. 4B-D). In order to confirm that above mentioned inhibitory effect of the ICAM-4 peptide is specific, we constructed a scrambled peptide with the same length and similar amino acid composition as the ICAM-4 peptide. We applied this scrambled peptide in the above described flow experiments. Our results show that the scrambled peptide shows no inhibitory effect (Suppl. Fig. 2) indicating the effect of the ICAM-4 peptide on erythrocyte-platelet interaction is specific. An ICAM-4 peptide promotes platelet P-selectin expression and blocks fibrinogen binding to integrin αIIbβ3 on platelets In an attempt to further explore the downstream events of ICAM-4 binding to integrin αIIbβ3, we tested the same ICAM-4 peptide that showed inhibitory effects on erythrocyte-platelet adhesion in the perfusion experiments in flow cytometric analyses. Blood samples were treated with either vehicle or ICAM-4 peptide in the Chapter 3 Chapter

A B 61

Fig. 5.Effect of ICAM-4 peptide on platelet fibrinogen binding and P-selectin expression under ADP stimulation. Gradient concentrations of adenosine diphosphate (ADP) were added into diluted whole blood samples (1:100 in PBS) in presence or absence of ICAM-4 peptide (10 µg/ml). Alex Fluor 488-conjugated fibrinogen (15 µg/ml) and PE-labeled anti-P-selectin (1:25 dilution) were used to analyse fibrinogen binding and P-selectin expression by flow

cytometry. Platelets were gated according to forward and side scatter. The median fluorescence intensity (MFI) interaction Erythrocyte-platelet of platelet population in two fluorescence channels was used as parameters for fibrinogen binding and P-selectin expression respectively. Experiments were performed on a BD FACS Canto II flow cytometer and analyzed with BD FACSDiva software. Data are indicated as mean ± SEM. *, p < 0.05, **, p < 0.01 comparing ICAM-4 peptide treated samples to control samples at the same ADP concentration by paired student t test. (A) Effect of ICAM-4 peptide on platelet fibrinogen binding. (B) Effect of ICAM-4 peptide on platelet P-selectin expression. presence of gradient concentrations of ADP. Platelet P-selectin as well as platelet bound fibrinogen were stained with PE-conjugated anti-P-selectin and Alexa 488-conjugated fibrinogen respectively. Treatment with ICAM-4 peptide led toa decreased fibrinogen binding to platelets (43% at ADP concentration of 125 µM, p < 0.05, n = 5) and an increased P-selectin expression (60% at ADP concentration of 125 µM, p < 0.01, n = 5) on platelets (Fig. 5A-B). The decrease in fibrinogen binding in the presence of ICAM-4 peptide indicates that the peptide may bind to αIIbβ3 in competition with fibrinogen. The increase in P-selectin expression in the presence of

ICAM-4 peptide suggests that binding of ICAM-4 peptide to αIIbβ3 results in outside- in signalling and further activates platelets. Blockage of erythrocyte-platelet interaction leads to reduced thrombus formation, altered fibrin structure and reduced thrombin generation

To investigate the influence of the ICAM-4 peptide in thrombus formation, we performed in vitro perfusion experiments with re-calcified whole blood to mimic in

A

Chapter 3 Chapter B 62 Erythrocyte-platelet interaction Erythrocyte-platelet

Fig. 6. Effect of ICAM-4 peptide on thrombus formation in vitro. (A) Effect of ICAM-4 peptide on fibrin formation in time during whole blood perfusion in vitro. DiOC6 (1 µM) and Alexa Fluor 546-labeled fibrinogen (6.2 µg/ml) was added into citrated whole blood. Whole blood samples with fluorescent dyes were then perfused on collagen- coated coverslips for 15 min at shear rate of 100 s-1. PBS buffer (as vehicle control) or ICAM-4 peptide (10 µg/ml)

were added to whole blood directly prior to perfusion. CaCl2 (50 mM in 0.9% NaCl) was continuously injected into the blood flow (against the flow direction) at shear rate of 50 s-1 during whole blood perfusion. Images were taken under two fluorescence channels at an interval of 2 seconds for a duration of 15 min. Green color indicates platelets, while red/orange color indicates fibrin. Images are representatives of 4 independent experiments. (B) Effect of ICAM-4 peptide on fibrin formation after 15 min perfusion under both fluorescence microscopy (colour images on the left panel) and differential interference contrast (DIC) microscopy (gray scale images on the right panel). Green colour represents platelet aggregates and red/orange colour represents fibrin. Images are representatives of 4independent experiments. vivo thrombus formation processes under venous shear rate. Alexa 546-conjugated fibrinogen and DiOC6 dye (for platelet labeling) were pre-incubated with citrated whole blood for 5 min prior to perfusion. The whole blood sample together with CaCl2 was then perfused over a collagen type I-coated surface at 100 s-1 for 15 minutes. The process of thrombus formation was monitored in real time under an inverted fluorescence microscope. After perfusion, pictures were taken under fluorescent microscopic settings and DIC settings at different view fields. During perfusion, single platelets were first seen to adhere to collagen and form small aggregates. Erythrocytes were observed to bind to platelets or platelet aggregates. After 6 to 8 minutes of perfusion, fibrin deposition would occur and form visible fibers under the microscope. As evident from Fig. 6A (which are representative images from 4 independent experiments), the onset of fibrin formation is delayed in ICAM-4 treated sample comparing to the vehicle control. At the end of a 15-min perfusion, vehicle samples demonstrated high numbers of platelets (inpicted by green color)

adhered to the collagen-coated surface and a dense homogeneous fibrin network 3 Chapter (red/orange color). In contrast, the samples with ICAM-4 peptide in this system 63 resulted in lower amounts of platelet deposition and a less dense fibrin network, which mainly concentrated around the bound platelets (Fig. 6B). When analyzing the fibrin network by SEM, clots formed from ICAM-4 peptide treated blood samples demonstrated a different fibrin network structure than those formed from the control samples. In the clot formed from ICAM-4 treated samples, there are less fibrin braches, and the fibrin fibers seem to be thicker (Fig. 7A). Quantification of the fiber thickness from the SEM images revealed that fibers were indeed thicker in thrombi formed from ICAM-4 peptide-treated samples (Fig. 7B). These results indicate that ICAM-4 mediated erythrocyte-platelet interaction is necessary to obtain a suitable microenvironment for optimal fibrin network Erythrocyte-platelet interaction Erythrocyte-platelet formation. To further characterize the role of the erythrocyte-platelet interaction in thrombosis, we investigated the effect of the ICAM-4 peptide on thrombin generation. Citrated whole blood samples were treated with either vehicle (PBS buffer) or ICAM-4- peptide (50 ug/ml final concentration), and then were directly applied to a fluorometer designed to measure thrombin generation. Our results demonstrated a prolonged lag time, lower thrombin peak and lower endogenous thrombin potential (ETP) in samples treated with ICAM-4 peptide (Fig. 7C). Thrombin generation in plasma was not influenced by ICAM-4 peptide (data not shown). ICAM-4 peptide increases tail-bleeding time in a mice model

To investigate whether the interaction between erythrocytes and platelets play a crucial role on thrombus formation we applied tail bleeding time in ICR mice. As peptides are cleared rapidly from circulation, mice were either injected once with aspirin or continuously infused with vehicle control, a peptide resembling domain 1 of murine ICAM-4 or a control peptide from a different part of ICAM-4. We found A Chapter 3 Chapter

64 B C

Fig. 7. Effect of ICAM-4 peptide on fibrin network structure and thrombin generation in whole blood. (A) Scanning electron microscope (SEM) images showing the effect of Erythrocyte-platelet interaction Erythrocyte-platelet ICAM-4 peptide on fibrin structure. Whole blood samples were perfused on collagen surface with or without ICAM-4 peptide as described above. At the end of the perfusion, blood clots on the coverslips were fixed with 1% glutaraldehyde and applied to SEM analysis. (B) Fiber thickness quantified from SEM images by Phenom software .The thickness of the fiber were measured in more than 500 fibers in each image. (C) Effect of ICAM-4 peptide on thrombin generation in whole blood. Vehicle (PBS buffer) or ICAM-4-peptide (50 µg/mL final concentration) was added to citrated whole blood prior totrigging

thrombin generation cascade. Thrombin generation was triggered by 0.5 pmol/L TF and 16.7 mM CaCl2. Samples were run in quadruplicate and the calibrated thrombin generation curves were calculated with method described by Hemker et al.

that both the ICAM-4 peptide and aspirin prolonged tail-bleeding significantly compared to mice treated with either vehicle or aspirin (Fig. 8). Discussion Erythrocytes are thought to be passively entrapped in the fibrin network as they flow through a developing thrombus. Our current study has provided indications for an active role of erythrocytes in thrombus formation. We demonstrated a direct interaction of erythrocytes with activated platelets and explored its physiological relevance. Using blood from healthy individuals we observed the capture and adhesion of erythrocytes to platelets with a focal adhesion point attached to platelet membranes under flow. This observation is similar to what has been reported by Goel et al., who perfused washed platelets and washed erythrocytes sequentially over collagen- or fibrinogen-coated surfaces and observed erythrocyte adhesion to platelets under flow shear below 100 -1s .38 With this two-step perfusion method, they reported a 4-fold increase in erythrocyte binding to collagen-adherent compared to fibrinogen-adherent platelets. In our study, we adopted a one-step perfusion method by perfusing washed platelets and erythrocytes together over a platelet adhesive protein (VWF or fibrinogen) coated surface. This one-step perfusion method enabled us to observe the erythrocyte-platelet interaction closer to physiology and enabled us to study the effect of platelet agonists and various other reagents on

erythrocyte adhesion to platelets. While the study of Goel et al. proposed that the 3 Chapter adhesive interactions of erythrocytes with platelets was induced by platelet receptor 65 glycoprotein VI-mediated platelet activation signaling, we here provide evidence that such erythrocyte-platelet adhesion could be induced by various platelet agonists (such as thrombin, ADP, AA and collagen related peptide), and thus is a phenomenon dependent on platelet activation. We consider the erythrocyte-platelet adhesion to be physiologically relevant since erythrocytes were able to bind platelets not only in physiological buffer but also in a whole blood environment under venous shear rates. Furthermore, we observed erythrocyte adhesion to platelet decorated strings formed over activated endothelial cell surfaces in vitro, which simulates the erythrocyte- platelet interaction in vivo in situations of endothelial activation or dysfunction. Moreover, additional evidence favors a physiological role for the erythrocyte-platelet interaction as erythrocytes present in platelet aggregates formed from whole blood interaction Erythrocyte-platelet 40;41 were shown to enhance platelet reactivity.34;42 In addition, erythrocyte membranes are able to support thrombin generation20;34 and erythrocyte-platelet aggregates could be observed in patients with sickle cell anemia and end-stage renal diseases.43;44 The molecular basis of the erythrocyte-platelet adhesion is intriguing. We showed that platelet activation is a prerequisite for erythrocyte-platelet adhesion under venous flow conditions. Since the conformational change of platelet integrin IIbα β3 from low affinity to high affinity for fibrinogen binding is a pivotal step in platelet 45 activation, integrin αIIbβ3 seemed to be a candidate involved in this interaction. Indeed, our results showed that both RGD peptide (d-RGDW) known to block the fibrinogen binding site of activated IIbα β3 and an antibody against integrin β3 chain of the integrin complex (anti-CD61) inhibited erythrocyte binding to platelets.37 These findings support the involvement of platelet integrin αIIbβ3 in erythrocyte-platelet adhesion under flow. As erythrocytes are known to possess adhesion molecules which can bind to multiple integrins on various cell types,46 erythrocyte adhesion molecules binding to integrin αIIbβ3 may be the link between erythrocytes and Fig. 8. Effect of ICAM-4 peptide on mice tail bleeding timein vivo. Mice were either injected once with aspirin (30 mg/kg body weight, 5 min before tail bleeding test) intravenously, or continuously infused with vehicle control (5% DMSO/ PBS), an ICAM-4 peptide resembling the domain 1 of murine ICAM-4 or a control peptide resembling a different part of ICAM-4. Continuous infusion took 10 minutes with a rate of 10 µL/min (peptide concentration 2 mg/mL). Immediately after continuous infusion was stopped; the tail of the mice was transacted (3 mm from tail top). The mice were immediately placed in holders vertically with 2 cm of the distal tail immersed in saline of 37°C. Maximum cut-off time was 180 seconds. Each condition was tested in a minimum of 5 mice. Horizontal line indicates mean, *, p < 0.05, **, p < 0.01. platelets. With an in vitro cell adhesion assay, Hermand et al. identified ICAM-4 is a 22 Chapter 3 Chapter ligand for platelet activated integrin αIIbβ3. Our flow experiments with anti-ICAM-4 antibody and an ICAM-4 mimetic peptide confirmed that ICAM-4 was indeed 66 involved in erythrocyte-platelet adhesion under flow conditions. The exact ICAM-4

binding site on integrin αIIbβ3 is still unclear. We hypothesize that the binding site is

likely to be located on the β3 chain based on the following findings. First, our data showed that d-RGDW inhibited erythrocyte binding to platelets. The binding site 47 of a RGD peptide is known to be located on the β3 chain. Secondly, we showed

that monoclonal anti-CD61 (antibody against β3) significantly reduced erythrocyte

binding to platelets. Our findings demonstrating3 β chain possesses the ICAM- 4 binding site(s) are consistent with those of Hermand et al.. ICAM-4 consists of two Ig-like type C2 domains.24;48 Hermand et al. showed that both of the 2 Ig-like 39 domains are required for αIIbβ3 interaction. Erythrocyte-platelet interaction Erythrocyte-platelet Our results showed that anti-CD61, anti-ICAM-4, and ICAM-4 peptide alone or in combination can only reach a maximum reduction of erythrocyte-platelet adhesion by 50%. Other mechanisms might also be involved in erythrocyte-platelet adhesion.

ICAM-4 are known to interact with various integrins. Besides integrin αIIbβ3, it may 28;49 also interact with αvβ3 on platelets. In addition to the direct ligand/receptor mediated interactions, the binding of erythrocyte to platelets under low shear rates could also be mediated by bridging proteins. One example would be fibrinogen. Fibrinogen has been shown to be able to attach to erythrocyte membranes by binding

to several erythrocyte surface molecules, of which an αIIbβ3-like integrin and CD47 were proposed to be the fibrinogen receptors on erythrocyte.50-52 It is therefore possible that fibrinogen could form bridges between activated platelets and erythrocytes. In addition to ICAM-4, erythrocytes are known to possess other adhesion molecules such as CD47 (receptor for thrombospondin and fibronectin), CD36 (receptor for VWF, thrombospondin, and fibronectin) and sulfated glycolipids (receptors for VWF, thrombospodin and laminin).53 As VWF and thrombospondin (TSP) are known to bind to the platelet surface via GPIb and CD36/CD47 respectively.54 VWF and thrombospondin might also serve as bridging proteins between erythrocytes and platelets. It is worth to note that although targeting the ICAM-4-αIIbβ3 can not fully abolish erythrocyte-platelet adhesion in vitro, this ICAM-4 mediated cell- cell interaction is likely to play an imprtant role in hemostasis as indicated by the results from the tail bleeding experiments in mice. We found that when we block the ICAM-4-αIIbβ3 interaction in mice, the tail bleeding time was significantly prolonged.

The downstream events following the ICAM-4-αIIbβ3 interaction requires futher scrutiny. By applying the ICAM-4 mimetic peptide, we were able to demonstrate that the binding of the ICAM-4 peptide to high affinity IIbα β3 can increase P-selectin exposure on platelets. P-selectin exposure is a measure of platelet-granule release in activated platelets.55 It is worth mentioning that the ICAM-4 peptide alone showed no direct effect on platelet P-selectin exposure. However, it could induce higher P-selectin exposure when platelet activation was primed by ADP. Altogether, these Chapter 3 Chapter data indicate that binding of ICAM-4 peptide to high active αIIbβ3 triggers signaling transductions (both outside-in signaling and inside-out signaling) in platelets. 67 Assuming that intact ICAM-4 on erythrocyte membranes interacts with αIIbβ3 in the same way as the ICAM-4 peptide, erythrocyte/platelet interactions would lead to signaling events promoting platelet activation. In fact, the ability of erythrocytes to influence platelet activation was also supported by a study of Valles et al., who, with an entirely different method, demonstrated that the presence of metabolically active erythrocytes in platelet suspensions led to enhanced platelet reactivity, 56 αIIbβ3 activation and P-selectin exposure. Since ICAM-4 and fibrinogen can both bind to activated αIIbβ3 and induce signaling events, future studies comparing the characteristics of signaling transduction induced by these two molecules would broaden our understanding of erythrocyte-platelet interactions. The effect of ICAM- Erythrocyte-platelet interaction Erythrocyte-platelet 4-αIIbβ3 interaction on erythrocyte biology is still unknown and requires further investigation.

As ICAM-4 and integrin αIIbβ3 have been identified as the molecular basis for erythrocyte- platelet interaction and considering the evidence supporting an active role of erythrocytes in thrombus formation, we suspected that ICAM-4 might be a molecular target in the erythrocyte-related thrombosis. Our experiments with a modified whole blood thrombin generation assay under static conditions demonstrated that the ICAM-4 peptide reduced thrombin generation. With an in vitro thrombus formation model, we found reduced fibrin deposition, thicker fibers and less fiber branches in the clot formed from whole blood treated with ICAM-4 peptide. These data suggest that ICAM-4 on erythrocyte membranes is likely to be able to influence local thrombin generation and thrombus formation. The mechanisms by which ICAM-4 mediated erythrocyte-platelet interaction could influence thrombus formation are still unclear. We think that erythrocytes may influence trhombus formation via the following pathways. Firstly, the ICAM-4-αIIbβ3 interaction may cause signal transduction in platelets and further promotes platelet activation. Secondly, by binding to platelets, erythrocytes might be recruited at the site of a developing thrombus at low shear conditions. The presence of the erythrocytes in a thrombus might increase the efficiency in initiating and propagating the coagulation cascade. It is known that the ICAM-4 surface antigen level is heterogeneous among individuals. The ICAM-4 angen level is phenotypically but not fundamentally related to the rhesus blood group antigen.57 Future research focusing on the relationship between ICAM-4 and thrombosis would shed light on the function of ICAM-4. In conclusion, our in vitro and in vivo data indicated an active role of erythrocytes in thrombus formation. We observed a direct adhesion of erythrocytes to platelets under venous shear rates. This direct erythrocyte-platelet adhesion required platelet activation. The adhesion was at least partially mediated via ICAM-4 on erythrocyte

and active αIIbβ3 on platelet. The binding of ICAM-4 to αIIbβ3 may induce signal transduction inside platelets and promote further platelet activation. Blocking ICAM-4 function with ICAM-4 mimetic peptide and anti-ICAM-4 led to reduced

Chapter 3 Chapter thrombin generation and fibrin deposition. ICAM-4 targeting approaches (e.g. 68 ICAM-4 antibodies and/ or ICAM-4 mimetic peptides) may hold promises as future therapeutic strategies in treating diseases with thrombotic complications. Acknowledgements We thank the Dutch Heart Foundation (NHS 2006T5301, BdL) and Sanquin Blood Supply Foundation (BdL) for financial support. We thank Dr. Y. Wu for the scanning electron microscopic analysis, Dr. E. Schurgers for proof reading, Dr. H. kelchtermans for help with whole blood thrombin generation assays, L. Pelkmans for help with fiber thickness analysis, Dr. C. Maas for helpful discussions, and G. Andringafor technical support. Erythrocyte-platelet interaction Erythrocyte-platelet References red cell-endothelial interaction, sickling and altered microvascular response to oxygen in 1. Furie B, Furie BC. Mechanisms of thrombus the sickle transgenic mouse. J.Clin.Invest formation. 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Erythrocyte-platelet interaction Erythrocyte-platelet 1981;2:114-115. 2008;6:615-621. 8. Carallo C, Pujia A, Irace C et al. Whole blood 18. Valles J, Santos MT, Aznar J et al. viscosity and haematocrit are associated Erythrocytes metabolically enhance with internal carotid atherosclerosis in men. collagen-induced platelet responsiveness Coron.Artery Dis. 1998;9:113-117. via increased thromboxane production, adenosine diphosphate release, and 9. Braekkan SK, Mathiesen EB, Njolstad recruitment. Blood 1991;78:154-162. I, Wilsgaard T, Hansen JB. Hematocrit and risk of venous thromboembolism in 19. Barr JD, Chauhan AK, Schaeffer GV, Hansen a general population. The Tromso study. JK, Motto DG. Red blood cells mediate the Haematologica 2010;95:270-275. onset of thrombosis in the ferric chloride murine model. Blood 2013;121:3733-3741. 10. Kaul DK, Nagel RL, Chen D, Tsai HM. Sickle erythrocyte-endothelial interactions in 20. Whelihan MF, Zachary V, Orfeo T, Mann microcirculation: the role of von Willebrand KG. Prothrombin activation in blood factor and implications for vasoocclusion. coagulation: the erythrocyte contribution to Blood 1993;81:2429-2438. thrombin generation. Blood 2012;120:3837- 3845. 11. Kaul DK, Fabry ME, Costantini F, Rubin EM, Nagel RL. In vivo demonstration of 21. Whelihan MF, Mann KG. The role of the red cell membrane in thrombin generation. with congenital or acquired alpha IIb beta Thromb.Res. 2013 3 deficiency to endothelial cell matrix and collagen under conditions of flow via tissue 22. Hermand P, Gane P, Huet M et al. Red factor-independent thrombin generation. cell ICAM-4 is a novel ligand for platelet- Blood 2003;101:1864-1870. activated alpha IIbbeta 3 integrin. J.Biol. Chem. 2003;278:4892-4898. 31. Beutler E, Gelbart T. The mechanism of removal of leukocytes by cellulose columns. 23. Ihanus E, Uotila LM, Toivanen A, Varis Blood Cells 1986;12:57-64. M, Gahmberg CG. Red-cell ICAM-4 is a ligand for the monocyte/macrophage 32. Sixma JJ, de Groot PG, van ZH, IJsseldijk integrin CD11c/CD18: characterization M. A new perfusion chamber to detect of the binding sites on ICAM-4. Blood platelet adhesion using a small volume of 2007;109:802-810. blood. Thromb.Res. 1998;92:S43-S46. 24. Bailly P, Hermand P, Callebaut I et al. The 33. Jaffe EA, Nachman RL, Becker CG, Minick LW blood group glycoprotein is homologous CR. Culture of human endothelial cells to intercellular adhesion molecules. Proc. derived from umbilical veins. Identification Natl.Acad.Sci.U.S.A 1994;91:5306-5310. by morphologic and immunologic criteria. Chapter 3 Chapter J.Clin.Invest 1973;52:2745-2756. 25. Hermand P, Le Pennec PY, Rouger P, 70 Cartron JP, Bailly P. Characterization of the 34. Ninivaggi M, Apitz-Castro R, Dargaud Y gene encoding the human LW blood group et al. Whole-blood thrombin generation protein in LW+ and LW- phenotypes. Blood monitored with a calibrated automated 1996;87:2962-2967. thrombogram-based assay. Clin.Chem. 2012;58:1252-1259. 26. Toivanen A, Ihanus E, Mattila M, Lutz HU, Gahmberg CG. Importance of molecular 35. Hemker HC, Giesen P, AlDieri R et al. The studies on major blood groups--intercellular calibrated automated thrombogram (CAT): adhesion molecule-4, a blood group antigen a universal routine test for hyper- and involved in multiple cellular interactions. hypocoagulability. Pathophysiol.Haemost. Biochim.Biophys.Acta 2008;1780:456-466. Thromb. 2002;32:249-253. 27. Bailly P, Tontti E, Hermand P, Cartron JP, 36. Bennett JS. Structure and function of the Erythrocyte-platelet interaction Erythrocyte-platelet Gahmberg CG. The red cell LW blood group platelet integrin alphaIIbbeta3. J.Clin.Invest protein is an intercellular adhesion molecule 2005;115:3363-3369. which binds to CD11/CD18 leukocyte 37. Saelman EU, Hese KM, Nieuwenhuis HK integrins. Eur.J.Immunol. 1995;25:3316- et al. Aggregate formation is more strongly 3320. inhibited at high shear rates by dRGDW, 28. Zennadi R, Moeller BJ, Whalen EJ et al. a synthetic RGD-containing peptide. Epinephrine-induced activation of LW- Arterioscler.Thromb. 1993;13:1164-1170. mediated sickle cell adhesion and vaso- 38. Goel MS, Diamond SL. Adhesion of normal occlusion in vivo. Blood 2007;110:2708- erythrocytes at depressed venous shear rates 2717. to activated neutrophils, activated platelets, 29. Huizinga EG, Tsuji S, Romijn RA et al. and fibrin polymerized from plasma. Blood Structures of glycoprotein Ibalpha and its 2002;100:3797-3803. complex with von Willebrand factor A1 39. Hermand P, Gane P, Callebaut I et al. domain. Science 2002;297:1176-1179. Integrin receptor specificity for human 30. Lisman T, Moschatsis S, Adelmeijer J, red cell ICAM-4 ligand. Critical residues Nieuwenhuis HK, de Groot PG. Recombinant for alphaIIbeta3 binding. Eur.J.Biochem. factor VIIa enhances deposition of platelets 2004;271:3729-3740. 40. Joseph R, Welch KM, D’Andrea G, Riddle 50. Carvalho FA, Connell S, Miltenberger- JM. Evidence for the presence of red and Miltenyi G et al. Atomic force microscopy- white cells within “platelet” aggregates based molecular recognition of a fibrinogen formed in whole-blood. Thromb.Res. receptor on human erythrocytes. ACS Nano. 1989;53:485-491. 2010;4:4609-4620. 41. Saniabadi AR, Lowe GD, Barbenel JC, 51. de Oliveira S., Vitorino dA, V, Calado A, Forbes CD. Further studies on the role Rosario HS, Saldanha C. Integrin-associated of red blood cells in spontaneous platelet protein (CD47) is a putative mediator for aggregation. Thromb.Res. 1985;38:225-232. soluble fibrinogen interaction with human red blood cells membrane. Biochim. 42. Santos MT, Valles J, Aznar J, Perez-Requejo Biophys.Acta 2012;1818:481-490. JL. Role of red blood cells in the early stages of platelet activation by collagen. Thromb. 52. Lominadze D, Dean WL. Involvement of Haemost. 1986;56:376-381. fibrinogen specific binding in erythrocyte aggregation. FEBS Lett. 2002;517:41-44. 43. Sirolli V, Strizzi L, Di SS et al. Platelet activation and platelet-erythrocyte 53. Telen MJ. Erythrocyte adhesion receptors: aggregates in end-stage renal disease blood group antigens and related molecules. Chapter 3 Chapter patients on hemodialysis. Thromb.Haemost. Transfus.Med.Rev. 2005;19:32-44. 2001;86:834-839. 71 54. Lagadec P, Dejoux O, Ticchioni M et 44. Wun T, Paglieroni T, Tablin F et al. al. Involvement of a CD47-dependent Platelet activation and platelet-erythrocyte pathway in platelet adhesion on inflamed aggregates in patients with sickle cell vascular endothelium under flow. Blood anemia. J.Lab Clin.Med. 1997;129:507-516. 2003;101:4836-4843. 45. Sims PJ, Ginsberg MH, Plow EF, Shattil 55. Kamath S, Blann AD, Lip GY. Platelet SJ. Effect of platelet activation on the activation: assessment and quantification. conformation of the plasma membrane Eur.Heart J. 2001;22:1561-1571. glycoprotein IIb-IIIa complex. J.Biol.Chem. 56. Valles J, Santos MT, Aznar J et al. Platelet- 1991;266:7345-7352. erythrocyte interactions enhance alpha(IIb) 46. Zennadi R, De CL, Eyler C et al. Role and beta(3) integrin receptor activation and regulation of sickle red cell interactions with P-selectin expression during platelet interaction Erythrocyte-platelet other cells: ICAM-4 and other adhesion recruitment: down-regulation by aspirin ex receptors. Transfus.Clin.Biol. 2008;15:23- vivo. Blood 2002;99:3978-3984. 28. 57. Storry JR. Review: the LW blood group 47. D’Souza SE, Ginsberg MH, Burke TA, system. Immunohematology. 1992;8:87-93. Lam SC, Plow EF. Localization of an Arg- Gly-Asp recognition site within an integrin adhesion receptor. Science 1988;242:91-93. 48. Hermand P, Gane P, Mattei MG et al. Molecular basis and expression of the LWa/ LWb blood group polymorphism. Blood 1995;86:1590-1594. 49. Litvinov RI, Vilaire G, Shuman H, Bennett JS, Weisel JW. Quantitative analysis of platelet alpha v beta 3 binding to osteopontin using laser tweezers. J.Biol.Chem. 2003;278:51285-51290. Supplementary figures Chapter 3 Chapter

72 Suppl. Fig. 1. Influence of shear rate on erythrocyte binding to adhered platelets. Erythrocytes and platelets were isolated separately and re-suspended together in ringer buffer to reach a final platelet count of 150 X 910 /L and a hematocrit of 5%. Erythrocyte-platelet cell suspensions were perfused on Von Willebrand factor coated surfacesfor 5 min at flow shear rates of 100s-1, 200 s-1 and 300 s-1 respectively. Collagen related peptide (500 ng/mL) was added into the cell suspension before perfusion to activate platelets. Erythrocyte deposition on to the adhered platelets were expressed as the number of adhered erythrocytes in an area of 1 mm2.Data are indicated as mean ± SEM, n = 5.

Suppl. Fig. 2. Specific inhibitory effect of ICAM- 4 peptide on erythrocyte binding to platelets.

Erythrocyte-platelet interaction Erythrocyte-platelet Erythrocytes and platelets were isolated separately and re-suspended together in ringer buffer to reach a final platelet count of 150 X 910 /L and a hematocrit of 5%. Erythrocyte-platelet cell suspensions were perfused over von Willebrand factor coated surfaces at 100 s-1 for 5 min. Collagen related peptide alone (CRP, serve as positive control), or CRP together with either scrambled peptide (20 µg/mL) or ICAM-4 peptide (20 µg/ mL) were added into the cell suspensions before perfusion. Data are indicated as mean ± SEM. n = 4; *, P < 0.05 comparing ICAM-4 peptide to scrambled peptide by paired student t-test. 4

unctional characterization of the ICAM-4-α β F IIb 3 interaction on platelet function

Vivian X.Du1,2, Coen Maas1, Philip G. de Groot1, Zaverio M. Ruggeri 3 and Bas de Chapter 4 Chapter Laat1,2,3,4,5 73

1Clinical Chemistry and Haematology, University Medical Center, Utrecht, the Netherlands 2Sanquin Research, Sanquin Blood Supply Foundation, Amsterdam, the Netherlands 3Roon Research Center for Arteriosclerosis and Thrombosis, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla , CA, USA ICAM-4 and platelet function ICAM-4 and platelet 4Laboratory for pathophysiology of thrombin generation, Department of Biochemistry, CARIM, Maastricht University Medical Center, Maastricht, the Netherlands; 5Synapse BV, Maastricht, the Netherlands.

Manuscript prepared for sumbission Abstract Introduction: Erythrocytes are generally considered as passive bystanders in hemostasis predominantly based on their rheological functions. In the last decades, evidence is piling up for a more active role of erythrocytes in thrombus formation. Our previous study revealed a direct interaction between erythrocyte and platelets under venous flow conditions. We have shown that this interaction is mediated

via erythrocyte intercellular molecule 4 (ICAM-4) and platelet integrin αIIbβ3. In the present study, we further characterized the effect of the ICAM-4 mediated erythrocyte-platelet interaction on platelet function. Methods: We designed a peptide library containing 24 peptides covering the 2 Ig- like domains of ICAM-4 protein. Each peptide is 15 amino acid long and 9 amino acid overlapping. We tested the influence of these peptides on different aspects of platelet function: 1) platelet aggregation by light transmission aggregometry, 2)

Chapter 4 Chapter P-selectin expression (as a marker for platelet activation) and binding of fibrinogen 74 to activated platelets by flow cytometry, and 3) p38 MAP kinase phosphorylation by western blot. Results: After testing the 24 individual peptides, we have found 5 peptides that showed significant effects on platelet function. These peptides are located at region 64P90K on domain I and 112P138L on domain II of the ICAM-4 protein. One peptide on the upper region of 64P90K and one on the lower region of 112P138L showed significant inhibitory effect on binding of fibrinogen to activated platelets. One peptide on the lower region of 64P90K and upper region of 112P138L induced aggregation in unstimulated fresh platelet samples, increased P-selectin surface expression, increased the binding of fibrinogen to platelets, and induced outside- ICAM-4 and platelet function ICAM-4 and platelet in signaling demonstrated by increased p38MAPK phosphorylation. Aggregation induced by these two peptides was shown to be insensitive to iloprost, a platelet function inhibitor. The fifth peptide located in the middle region of 112P138L induced platelet aggregation but slightly reduced fibrinogen binding to platelets. Conclusion: We have, for the first time, characterized the binding of ICAM-4 to

αIIbβ3 on platelet activity. We identified the binding sites within ICAM-4 for αIIbβ3 that are involved in modulation of platelet function. In concordance with Hermand

et al., our results indicate that ICAM-4 interacts with αIIbβ3 with both of its Ig-like domains. We found that region 64P90K on domain I and 112P138L on domain II are the crucial regions on ICAM-4 to mediate erythrocyte-platelet interaction. Two

peptides located on both regions of ICAM-4 modulate αIIbβ3 conformational change from an inactive form to an active form (high affinity for fibrinogen binding) on

non-activated platelets. Binding of ICAM-4 to αIIbβ3 induces signaling in platelets resulting in activation of platelets. Introduction The main function of erythrocytes is to ensure the respiratory gas transport throughout the body. Although erythrocytes have been known to influence the bleeding and thrombosis through their impact on hemorheology,1-3 they are generally considered as innocent bystanders in hemostasis. Besides its rheological function, erythrocytes are thought to be passively entrapped inside a developing thrombus as they flow through the vasculature. However, several studies indicated that erythrocytes may also be actively involved in maintaining the hemostatic balance. Erythrocytes are recently identified as an important source for pro-coagulant surfaces thereby supporting coagulation.4-6 Interestingly, mature erythrocytes are found to interact with platelets—the major cell type in thrombus formation. In the presence of erythrocytes, platelets become more responsive to agonist simulations and release their α-granule content (such as ADP and thromboxane A2) to recruit more platelets to form aggregates. 7-9 In addition, it has been shown that erythrocytes induce platelet Chapter 4 Chapter integrin αIIbβ3 activation and P-selectin expression on platelet surface in an in vitro biochemical study.10 Our previous study has revealed a direct erythrocyte-platelet 75 interaction. We demonstrated that erythrocytes can bind to surface-immobilized platelets under conditions of venous flow. By using blocking antibodies, we have shown that this direct erythrocyte-platelet interaction is mediated by intercellular adhesion molecule-4 (ICAM-4) on erythrocytes and the αIIbβ3 integrin on platelets. Erythrocytes are known to express numerous adhesion molecules (such as CD 44, Lu, CD47, CD147 and ICAM-4), many of which exhibit blood group specificities.11;12 ICAM-4 is previously named as Landsteiner-Wiener (LW) blood group antigen that shares a phenotypic relationship with Rhesus D (RhD) antigen.13 It is a glycoprotein

that is explicitly expressed in erythroid cells. Like other adhesion molecules, ICAM-4 function ICAM-4 and platelet protein possesses domains characteristic of the immunoglobulin superfamily (IgSF) proteins, suggesting some recognition function. The extracellular part of the protein contains two IgSF domains (domain I and domain II) and is structurally related to ICAM-2.13 Members of the ICAM family are ligands for integrins. Although the other ICAM family members are fairly specific for a single integrin, ICAM-4 appears 14 to be able to interact with a variety of integrins: αLβ2, αXβ2, αVβ1, αVβ3 and αIIbβ3.

The initial identification of ICAM-4 as the ligand for platelet integrin IIbα β3 is based on evidence provided by Hermand et al: 1) the adhesion of platelets to immobilized ICAM-4 (LW) negative erythrocytes is less than that to the ICAM-4 positive erythrocytes; 2) monoclonal antibodies against αIIbβ3 and those against ICAM-4 reduced the adhesion of platelets to erythrocytes; 3) platelets from Glanzmann’s thrombasthenia patients which are known to have the functional defect in αIIbβ3 failed to adhere to erythrocytes; and 4) αIIbβ3 expressing Chinese hamster ovary (CHO) cells are able to bind to immobilized recombinant ICAM-4 Fc fusion proteins.15 Later on, the same research group revealed that both domain I and domain II of the ICAM-

4 protein are involved in the binding to αIIbβ3. Our previous study has confirmed Chapter 4 Chapter

76 ICAM-4 and platelet function ICAM-4 and platelet that the interactions between ICAM-4 and αIIbβ3 under physiological flow conditions indeed require platelet activation. Moreover, we found the association of erythrocyte with single platelets or platelet aggregates when perfused over endothelial cells in vitro. This finding indicates that the ICAM-4 mediated direct erythrocyte-platelet interaction may play an important role in hemostasis. Although ICAM-4 has been shown to be the ligand to mediate erythrocyte-platelet interaction by binding to αIIbβ3, the effect of this interaction on platelet function remains unknown. To characterize the effect of ICAM-4- αIIbβ3 interaction on platelet function, we constructed an ICAM-4 peptide library and investigated the influence of each peptide of the library on platelet function and activity. Materials and Methods Materials

ICAM-4 peptide library with 24 peptides was produced by CASLO ApS (Lyngby Denmark). R-phycoerythrin (RPE) labeled mouse anti-human P-selectin antibody 4 Chapter was purchased from BD biosciences (Franklin Lakes, NJ, U.S.A). Alexa-488 77 conjugated fibrinogen was bought from Invitrogen Molecular Probes (Eugene, OR, USA). Collagen Type I (Horm Suspension) was purchased from NYCOMED (Austria GmbH, Linz, Austria). Adenosine diphosphate (ADP) was purchased from Roche (Almere, The Netherlands). Cross-linked Collagen Related Peptide (CRP) was a generous gift of R. Thrombin receptor agonist peptide (TRAP)-6 was bought from Farndale (Cambridge, UK). Rabbit anti-p38 mitogen-activated protein kinase (p38 MAPK) and rabbit anti-phosphor p38 MAPK (T180/Y182) were bought from Cell Signaling Technology (Danvers, MA, USA). Alexa Fluo 680 conjugated goat- anti-rabbit IgG was bought from Invitrogen Molecular Probes (Carlsbad, CA, USA). Protease inhibitor cocktail was bought from Sigma Life Science (St. Louis, MO, function ICAM-4 and platelet USA). Construction of ICAM-4 peptide library

Twenty-four peptides covering the 2 Ig-like domains of ICAM-4 protein were produced. Each peptide is 15 amino acid long and there was a 9-amino-acid overlapping region. The amino acid sequence of each peptide is listed in table 1. The location of the peptide on the ICAM-4 protein secondary structure and the solubility is summarized in table 2. Platelet isolation

Freshly drawn venous blood from healthy volunteers was collected into 0.1 volume 3.2% trisodium citrate. Blood donors claimed not to have taken any nonsteroidal anti-inflammatory drugs for the preceding 10 days. The blood was centrifuged at 156 x g for 15 min at room temperature. The platelet-rich plasma (PRP) was collected and acidified by addition of one-tenth volume of acid citrate dextrose (ACD, 2.5% trisodium citrate, 1.5% citric acid, and 2% D-glucose). Platelets were centrifuged Table 2 location of ICAM-4 peptides on ICAM-4 protein structure name peptide sequence disolution medium position on protein structure D1,1 A28-->S42 DMSO D1/A-B loop+B D1,2 S34-->N48 DMSO D1/B+B-C loop D1,3 S40-->P54 PBS D1/B-C loop+C+C-Dloop D1,4 P46-->T60 PBS D1/B-C loop+C+ C-D loop + D D1,5 R52-->W66 PBS D1/C-D loop + D +D-E loop D1,6 G58-->L72 PBS D1/ D+D-E loop + E D1,7 P64-S78 DMSO D1/D-E loop + E + E-F loop D1,8 Q70-->H84 DMSO D1/E +E-F loop + F D1,9 A76-->K90 DMSO D1/E-F loop + F+F-G loop D1,10 H82-->S96 PBS D1/F+F-G loop+G D1,11 A88-->K102 PBS D1/F-G loop+G D1,12 A94-->I108 PBS D1/G+D1G-D2A loop

Chapter 4 Chapter D2,1 P112-->T126 PBS D2/A-B loop +B D2,2 K118-->G132 PBS D2/B+B-C loop 78 D2,3 H124-->L138 DMSO D2/B-C loop+C D2,4 P130-->P144 PBS D2/B-C loop+C+C-C' loop D2,5 V136-->L150 PBS D2/C+C-C' loop+C' D2,6 S142-->L156 DMSO D2/C'+C'-E loop D2,7 E148-->T162 PBS D2/C'+C'-E loop+E D2,8 T154-->A168 PBS D2/C'-E loop+E D2,9 N160-->F174 DMSO D2/E+E-F loop D2,10 E166-->C180 PBS D2/E+ E-F loop+F D2,11 R172-->L186 DMSO D2/E-F loop+F D2,12 V178-->R192 DMSO D2/F+F-G loop + G ICAM-4 and platelet function ICAM-4 and platelet (330 x g, 15 min) and the platelet pellet was resuspended in Hepes-Tyrode buffer at pH 6.5 (10 mMHepes [N-2-hydroxyethylpiperazine-N’-2ethanesulfonic acid], 137

mMNaCl, 2.68 mMKCl, 0.42 mM NaH2PO4, 1.7 mM MgSO4, 5 mM D-glucose).

Prostacyclin (PGI2, 10 ng/ml) was added to prevent platelet activation during the subsequent washing step. The platelet suspension was centrifuged (330 x g, 15 min) again and resuspended in HEPES-Tyrode buffer at pH 7.3. Aggregometry

Platelet concentration was adjusted to 200 x 109 /L with HEPES-Tyrode buffer (pH=7.3). Fibrinogen was added to the platelet suspension to a final concentration of 100 µg/mL. Platelets (with fibrinogen) were divided into transparent cuvettes (300 µL) and placed in aggregometer (Chronolog, model 700, Harvertown, PA, USA) which has been pre-warmed at 37 °C. Individual peptides (at indicated concentration) were added to platelet samples to start aggregation. Aggregation was measured by light-transmission aggregometry at 37 °C at a stirring speed of 900 rpm. Changes in light transmission in the platelet suspension were recorded. Flow cytometry

The effect of individual ICAM-4 peptides on fibrinogen binding to platelets and P-selectin expression was evaluated by flow cytometry. 50 µl HEPES buffered saline (HBS;10 mM HEPES, 150 mM NaCl, 1 mM MgSO4, 5 mM KCl, pH 7.4) was added into each tube. 2 µl PE-labeled mouse anti-human P-selectin antibody (dilution ratio 1:25) and 0.5 μL Alex Fluor 488-conjugated fibrinogen (dilution ratio 1:100, final concentration 15 µg/ml) were added into the HBS. PE-conjugated mouse IgG1 was used as isotype control for P-selectin staining. ICAM-4 peptides (40 µg/ml) alone or together with one of the three platelet agonists—ADP, CRP, and TRAP— were added into the HBS solution. Platelet suspension (5 µl) was added to each tube containing the above mentioned reagents in HBS buffer. After 20 minutes of incubation at room temperature, the samples were fixed with 800 µl 0.2% formyl saline (0.2% formaldehyde in 0.9% NaCl). Samples were then analyzed on a FACS Canto II flow cytometer from BD Biosciences (Franklin Lakes, NJ, USA). Platelets were gated based on forward and side scatter properties. Percentage of P-selectin 4 Chapter positive platelets was used as the parameter for P-selectin expression on platelets 79 (thresh hold was set by using the measurement in the iso-type control sample). Mean fluorescence intensity (MFI) in the Alex Fluor 488 channel in the platelet gate was used as the parameter to indicate the binding of fibrinogen to platelets. Data were analyzed with BD FACSDiva software. Western Blot

Platelets in suspension or platelet aggregates after aggregometry experiments were lysed with RIPA buffer (10X) supplemented with protease inhibitor cocktail. Platelet lysates were applied to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to separate the proteins. Proteins were transferred function ICAM-4 and platelet to Millipore ImmobilonTM-FL PVDF membranes and subsequently blocked with Odyssey blocking buffer. Phosphorylated p38 MAPK was detected a rabbit anti- Phosphor p38MAPK (primary antibody) and an Alexa Fluo 680 conjugated goat- anti-rabbit IgG (secondary antibody). As loading control, P38 MAPK was detected by a rabbit anti-p38 MAPK and an Alexa Fluo 680 conjugated goat-anti-rabbit IgG. Protein bands were visualized with an Odyssey Infrared Imaging system (LI-COR Biosciences, Lincoln, NE). Quantification was performed using Odyssey application software version 2.1. Statistics

Data presented in all graphs are means ± standard error of the mean (SEM). Statistical analysis was performed with GraphPad Prism 5 (San Diego, CA, USA) software. Statistical differences between vehicle (or blank) control and peptides treatments were analysed by paired Student t test. Differences were considered significant at p < 0.05. A B

C Chapter 4 Chapter

80

D ICAM-4 and platelet function ICAM-4 and platelet

Fig.1. Effect of ICAM-4 peptides on platelet aggregation and P-selectin expression. Individual peptide (80 µg/mL) from an ICAM-4 peptide library was added into platelet suspension supplemented with fibrinogen (100 µg/mL) to induce aggregation. Platelet aggregation was measured by an aggregometor by light transmission. (A) Examples of aggregation curves in platelets treated with D2.1, D2.2 and D2.4 with PBS as vehicle control. (B) Examples of aggregation curves in platelets treated with D1.7, D1.9 and D2.3 with DMSO (0.8%) as vehicle control. (C) Quantified platelet aggregation in the presence of individual peptides expressed as the fold increase of the maximum light transmission comparing to that of the corresponding vehicle control. * p<0.05, ** p<0.01, *** p<0.001 vs vehicle control, N= 2 to 8 for each peptide. (D) Effect of the ICAM-4 peptides on platelet P-selectin expression. P-selectin expression on platelets treated with individual peptides (40 µg/mL) or vehicle control (PBS or DMSO 0.4%) was measured by flow cytometry. Bars with # indicate results from peptides dissolved in DMSO. Bars without # are results from peptides dissolved in PBS. * p<0.05, ** p<0.01 vs corresponding vehicle control, N = 4. Results A sub-category of peptides induced platelet aggregation and P-selectin externalization on platelets.

To investigate the influence of ICAM-4 peptides on platelet aggregation or agglutination, we performed aggregometry experiments with washed platelets in Hepes-Tyrode buffer in the presence of individual peptides. Our results showed that peptide D 1.7 and D 1.9 located on domain I and peptide D2.1, D2.2, D2.3 and D2.4 located on domain II were able to induce platelet aggregation at a concentration of 80 µg/mL (Fig 1A-B). The above mentioned peptides induced platelet aggregation (measured by light transmission) ranging from 40% upto 90% (Fig 1C). To confirm the above findings, we investigated the effect of each peptide of the library on platelet surface P-selectin expression by flow cytometry. Individual peptides (40 µg/ml) or corresponding vehicle (either PBS or 0.4% DMSO) and a PE-conjugated

anti-P-selectin antibody were added into platelet suspensions. The samples were 4 Chapter then incubated at room temperature for 20 minutes and subsequently fixed with 81 paraformaldehyde (0.02% in saline). P-selectin positive platelets were detected by a flow cytometer. In consistent with the results from the aggregometry experiments, we foud a significant increase of P-selectin expression in samples treated with peptide D 1.9, D2.1, and D2.3 (Figure 1D) ranging from 67% upto 480% increase comparing to vehicle control. The other peptides in the library showed no influence on either platelet aggregation or P-selectin expression (Fig. 1CD). These data indicate that the two clusters on ICAM-4, Region P64K90 on Domain I (correspond to D1.7 to D1.9) and the P112P138L (correspond to D2.1 to D2.3) on Domain II are the active regions for ICAM-4 mediated erythrocyte-platelet interaction.

Peptide D1.7 and D2.3 inhibited fibrinogen binding to platelets function ICAM-4 and platelet

Since ICAM-4 has been shown to be a ligand for platelet activated integrin αIIbβ3, we hypothesize that some of the ICAM-4 peptides in our peptide library may be able to interfere with the fibrinogen binding to IIbα β3. To test this hypothesis, we measured the binding of fibrinogen to platelets stimulated by 3 different agonists—ADP, CRP and TRAP—in the presence of individual peptides by flow cytometry. Peptide D1.2, D1.7, D2.2 and D2.3 significantly inhibited fibrinogen binding to platelets (Fig. 2A- D). Peptide D1.7 reduced binding of fibrinogen to activated platelets by 30, 44, and 40% in the presence of ADP, CRP and TRAP respectively. D2.3 reduced fibrinogen binding to platelets by 27% (ADP), 71% (CRP), and 80% (TRAP). Interestingly, Peptide D1.9 and D2.1 influenced fibrinogen binding in the opposite way. Both peptide slightly enhanced agonist-induced fibrinogen binding especially when platelets were exposed to ADP which is known to be a weak agonist (Fig. 2A). Since the effect of D1.9 and D2.1 on platelet fibrinogen binding can be masked by the agonist stimulation, we added D1.9 and D2.1 as well as D1.7 and D2.3 separately to unstimulated platelets and measured the platelet fibrinogen binding. As expected, A

B Chapter 4 Chapter

82

C ICAM-4 and platelet function ICAM-4 and platelet

D Fig. 2. Effect of ICAM-4 peptides on binding of fibrinogen to E activated platelets. Individual peptide (40 µg/mL) from an ICAM-4 peptide library was added into platelet suspension together with platelet agonists and Alexa-488 conjugated fibrinogen (100 µg/ml). Fibrinogen surface binding on platelets was measured by flow cytometry. Median fluorescence intensity (MFI) of the platelet population was used as the parameter to indicate levels of fibrinogen binding. Bars with # indicate results from peptides dissolved in DMSO. Bars without # are results from peptides dissolved in PBS. (A) Effect of ICAM- 4 peptides on binding of fibrinogen to platelets stimulated by ADP (125 µM). (B) Effect of ICAM-4 peptides on binding of fibrinogen to platelets stimulated by collagen related peptide (CRP, 250 ng/mL). (C) Effect of peptides on binding of fibrinogen to platelets stimulated by thrombin receptor agonist peptide (TRAP, 50 µM). (D) Pooled data from panel A-B indicating the effect of ICAM-4 peptides on binding of fibrinogen to activated platelets in general. ** p<0.01 vs corresponding vehicle control, N = 9. (E) Peptide D1.9 and D2.1 induced platelet fibrinogen binding in unstimulated platelet samples. * p<0.05 vs corresponding vehicle control, N = 8.

D1.9 or D2.1 significantly increased binding of fibrinogen to unstimulated platelets 4 Chapter compared to their controls (Fig. 2E). The other peptides of the library showed no 83 significant effect on platelet fibrinogen binding (Fig. A-D). These data suggest that the binding sites for peptide D1.7 and D2.3 on integrin αIIbβ3 may overlap with the fibrinogen binding site, while those for D1.9 and D2.1 probably shares no overlap with the fibrinogen binding site. The increase in platelet P-selectin expression in the presence of these peptides (D1.7, D1.9, D2.1 and D2.3) indicates that the binding of the peptides to integrin αIIbβ3 (whether on the fibrinogen binding site or not) triggers the outside-in signaling and activates platelets. D1.9 and D2.1 induce platelet aggregation/agglutination in the presence

of iloprost function ICAM-4 and platelet

In order to know whether the peptide-induced aggregation can be abolished by platelet function inhibition, we tested the ability of D1.9, D2.1 and D2.2 to induce aggregation in the presence of iloprost. Freshly isolated platelets in HT buffer (pH=7.3) were divided into two portions, one of which was incubated with iloprost (0.1 mg/mL) for half hour at room temperature. Collagen (as a positive control) or peptides were added into either fresh platelets or iloprost inhibited platelets to induce aggregation which was measured by an aggregometer. As expected, collagen induced aggregation was completely abolished in platelet samples treated with iloprost. Peptide D2.2 induced aggregation was also significantly reduced in iloprost treated platelets. In contrast, the D1.9 and D2.1 inducted aggregation was almost the same in iloprost treated platelets as in the fresh platelets (Fig. 3A). To confirm the ability of D2.1 in inducing aggregation of iloprost treated platelets, we added increasing concentrations of D2.1 (from 2.5 µg/mL to 80 µg/mL) to either fresh or iloprost inhibited platelets and observed the aggregation. Our results showed that D2.1 induced platelet aggregation in both fresh and iloprost inhibited platelets in a dose- dependent manner (Fig. 3B). However, the aggregation in iloprost treated samples reached a maximum at D2.1 concentration of 30 µg/mL, while the aggregation in fresh platelet samples seem to give further response to higher concentrations of D2.1 peptide. Since the low concentrations of D2.1 can already induce aggregation in both

iloprost treated and fresh platelets, binding of D2.1 might be able to change αIIbβ3 from an inactive to an active fibrinogen binding conformation. The same explanation may apply to D1.9 to explain the presence of aggregation in iloprost treated platelet samples. On the other hand, results from our flow cytometry experiments clearly showed that D1.9 and D2.1 are strong platelet agonists demonstrated by their ability to induce P-selectin expression. It is known that a strong stimulus can bypass the prostacyclin inhibition in activating platelets. Altogether, these data indicate that D1.9 and D2.1 are strong platelet agonists and they may directly influence the

conformational change of fibrinogen receptor IIbα β3. To further investigate the effect of D1.9, D2.1, D2.2 and D2.3 on platelet activation, we measured the phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) 16 Chapter 4 Chapter in platelets. P38 MAPK is a key molecule in platelet signaling pathways. When 84 platelets are exposed to agonists, phosphorylation of p38 MAPK occurs within minutes.16-19 For our experiments, collagen (as a positive control) or individual peptides were added to either fresh or iloprost treated platelets to induce aggregation monitored by an aggregometor. After stirring at 900 rpm/min with magnetic beads for 10 min in the presence of indicated reagents, platelet samples were lysed with 10 X RIPA buffer supplemented with protease inhibitor cocktail. Platelet lysate was mixed with sample buffer and loaded on SDS-gel for western blot. Comparing to blank control, collagen induced a near 5-fold increase in phosphorylated p38 MAPK (pho-p38MAPK) in freshly isolated platelets. D1.9, D2.1 and D2.2 induced a 2 to 3 fold increase, while D2.3 showed no effect in p38MAPK phosphorylation (Fig. ICAM-4 and platelet function ICAM-4 and platelet 4A). Collagen induced phosphorylation of p38MAPK was reduced by 70% in iloprost inhibited platelets comparing to fresh platelets. While D1.9, D2.1 and D2.2 induced p38 MAPK phosphorylation was reduced by 40% to 50% in iloprost treated platelets (Fig 4B). Comparing the effect of the peptide on platelet aggregation and the phosphorylation of p38MAPK, we can appreciate that peptide D2.2 activates platelets in a similar way as collagen, both of which activates p38 MAPK and are sensitive to prostacyclin inhibition. The influence of D1.9 and D2.1 on platelet activation is however more complicated. On the one hand, they were shown to induce platelet aggregation in the presence of iloprost; on the other hand, they were able to induce p38 MAPK phosphorylation in platelets. Altogether, these data indicate that

D1.9 and D2.1 are able to directly modulate the confirmation of IIbα β3 as well as induce out-side-in signaling to active platelets. Discussion In this study, we have characterized the influence of an ICAM-4 peptide library on platelet function. Of the 24 peptides that cover the two extracellular domains of ICAM-4 protein, we have found 5 peptides (D1.7, D1.9, D2.1, D2.2 and D2.3) A B

Fig. 3. ICAM-4 peptides induced aggregation in fresh and iloprost inhibited platelets. Isolated platelets were divided into two portions, one of which was incubated with iloprost (0.1 ug/mL) at room temperature for 30 min. Aggregation induced by collagen or ICAM-4 peptides in fresh and iloprost treated platelets was measured with an aggregometor by light transmission. (A) Comparison of aggregation induced by collagen (1 µg/mL), D1.9 (80 µg/mL), D2.1 (80 µg/mL), and D2.2 (80 µg/mL) in fresh and iloprost treated platelets. * p<0.05, *** p<0.001 comparing aggregation in fresh platelets and that in iloprost treated platelets, N = 3-9 for each condition. (B) Platelet aggregation in fresh and iloprost treated platelets in response to serial concentrations of D2.1. Chapter 4 Chapter that demonstrated significant stimulating effect on platelet aggregation. Although the 85 total effect of these peptides on platelet aggregation is positive, the detailed influence of these peptides on platelet function—demonstrated by P-selectin expression, fibrinogen binding and signaling transduction—varies depending on the individual peptide (Table 3). Peptide D1.9 and D2.1 significantly increased platelet aggregation, P-selectin expression, and fibrinogen binding. D1.7 and D2.3 significantly decreased fibrinogen binding to activated platelets, indicating that these two peptides may bind to αIIbβ3 at the same site as its natural ligand fibrinogen. Interestingly, peptide D1.9 and D2.1 induced platelet aggregation was insensitive to platelet function inhibitor iloprost, while the aggregation induced by D2.2 and D2.3 was not. Moreover, our data indicated that D1.9, D2.1 and D2.2 were able to trigger platelet signaling pathways. function ICAM-4 and platelet ICAM-4 protein has two Ig-like domains. Based on the results from our platelet function assays, we think that ICAM-4-αIIbβ3 interaction involves both domains of ICAM-4 protein. The region 64P90K on domain I (contains D1.7, D1.8 and D1.9) and 112P138L (contains D2.1, D2.2 and D2.3) on domain II are likely to be the  crucial region in ICAM-4- αIIbβ3 interaction (Table 1). In fact, region 64P 90K on domain I has been proposed to be an active region in ICAM-4 mediated interactions with several integrins. Point mutations in the E-F loop of ICAM-4 (D1.7, region 64P90K, Table 2) caused reduced ICAM-4 interaction with multiple β integrins, 20 such as αLβ2 (LFA-1, CD11a/CD18 on leukocytes), αMβ2 (Mac-1, CD11b/CD18 21 22 on leukocytes), αVβ3 (on endothelial cells, platelets and tumor cells), αVβ5 (on 23 22 endothelial cells, epithelial cells and fibroblasts) as well as αIIbβ3 on platelets. Replacement of the aromatic residue (W) at position 77 by a small apolar residue (A) reduced adhesion of αIIbβ3-expressing CHO cells to a recombinant ICAM-4 mutant protein.22 In our study, both D1.7 (contains 77W) and D1.9 in region 64P90K are shown to be able to influence platelet function. While D1.7 reduced recruitment of Fig. 4. ICAM-4 peptide induced platelet signaling A measured by phosphorylation of p38MAPK. Collagen (as a positive control) or indicated ICAM- 4 peptides were added to either fresh or iloprost treated platelets to induce aggregation monitored by an aggregometor. After stirring at 900rpm/min with magnetic beads for 10 min in the presence of indicated reagents, platelet samples were lysed with 10 X RIPA buffer supplemented with protease inhibitor cocktail. Platelet lysate was mixed with sample buffer and loaded on SDS-gel for western blot. Phosphorylated P38MAPK (pho-P38MAK) and P38MAPK (as loading control) were detected with rabbit antibodies. Protein bands were visualized B by Odyssey Imaging system and quantified by Odyssey application software. (A) ICAM-4 peptides induced P38MAPK phosphorylation. (B) Reduction

Chapter 4 Chapter of collagen and ICAM-4 peptide induced P38 MAPK phosphorylation in platelets treated with 86 iloprost.

fibrinogen to activated platelets (Fig. 2), D1.9 alone increased platelet fibrinogen binding on unstimulated platelets and further activated platelets (Fig. 3 and Fig. 4). The reduction of platelet fibrinogen binding in the presence of D1.7 indicates that ICAM-4 and platelet function ICAM-4 and platelet D1.7 binds to αIIbβ3 at the fibrinogen binding site thus blocking fibrinogen binding to its receptor. This finding is in agreement with what has been found by Hermand et al. who has demonstrated that a synthetic peptide 65G74V reduced the adhesion

of αIIbβ3-expressing CHO cells to recombinant ICAM-4 proteins. It is worth to note that both our peptide D1.7 (64P78S, Table 1) and that in the study of Hermand et al. (65G74V) harbour a Gln (Q)-X-X-Asp (D)-Val(V) motif which is one of the

αIIbβ3 binding motif on fibrinogen. D1.7 thus might bind to the fibrinogen binding site

on αIIbβ3 as evidenced by our flow cytometry experiment. The binding site of D1.9

on αIIbβ3 however does not seem to be related to that of the fibrinogen (Fig 1D). Point mutations in the D1.9 covered region (76A90K) in ICAM-4 Fc proteins, however,

did shown reduced interactions between ICAM-4 and platelet integrin αIIbβ3 as well as 22  αvβ3, which implicates the involvement of this region (76A 90K) in the interaction with the integrins. The fact that D1.9 can spontaneously induce aggregation in fresh

platelets and in iloprost inhibited platelets indicates that upon interaction with αIIbβ3 it may actively modulate the conformation of the integrin resulting in the high affinity

state of αIIbβ3 for fibrinogen binding. It has been shown that ligand binding itself

induces conformational changes in αIIbβ3. Such ligand-induced changes have been +, promotive effect or effective; - inhibitory effect; ±, no effect; NA, data not available proposed to be responsible for the immune-mediated thrombocytopenia associated 24 with the clinical use of αIIbβ3 antagonists (tirofiban and eptifibatide). Peptide D2.1 corresponds to the A-B loop and B strand of ICAM4 domain II (P112T126). The effect of this peptide on platelet function is almost identical to that of D1.9. Within the amino acid region covered by D2.1, 148K has been shown 22 to be a crucial residue in ICAM-4 interaction with αVβ1 and αVβ5, but not with αIIbβ3. Peptide D2.3 on domain II resembles peptide D1.7 on domain I in platelet function assays. D2.3 blocked the binding of fibrinogen to activated platelets indicating that  4 Chapter H124 L138 (D2.3 covered region) on ICAM-4 may interact with αIIbβ3 by binding 87 to the fibrinogen binding site on IIbα β3. Peptide D2.2 appears to be peculiar in the way that it affects platelet function. It possesses the characteristics of its adjacent peptides –D2.1 and D2.3. It induces platelet aggregation (so does D2.1) and slightly decreased platelet fibrinogen binding (so does D2.3). But D2.2 differs from D2.1 in that the aggregation induced by this peptide was sensitive to prostacyclin (iloprost) inhibition while D2.1 is not. Peptide D2.2 activates platelets in a similar way as collagen, both of which activates p38 MAPK and is sensitive to prostacyclin inhibition (Fig.3 & 4). The influence of D1.9 and D2.1 on platelet activation seems to be more difficult to comprehend. On the one hand these two peptides induced platelet aggregation in

spite of iloprost inhibition, which indicates an activation independent agglutination function ICAM-4 and platelet among platelets, on the hand they also induced signaling transduction and the externalization of P-selectin. We think that this paradox could be explained by the nature of the interaction between the peptides and the integrin. D1.9 and D2.1 interact with αIIbβ3 at non-fibrinogen binding sites, which bring about conformational change of αIIbβ3 and subsequently leading to platelet agglutination due to the binding of fibrinogen to the active αIIbβ3. In the meanwhile, the binding of the peptides to

αIIbβ3 triggers platelet outside-in siganaling. We showed that this signaling is indeed partially reduced under prostacyclin inhibition (Fig 4B). Comparing to D2.1 and

D1.9, the interaction of D2.2 with αIIbβ3 seems to slightly reduce platelet fibrinogen binding. The modulation it brings about in the integrin is strong enough to induce platelet activation in fresh platelets but not enough to bypass the prostacyclin inhibition. Given the potent stimulatory effects on platelet function, the D1.9 and D2.1 covered regions (76A90K and P112T126) are not likely to be exposed in the ICAM-4 protein on erythrocytes in a normal physiological environment. However, under pathological conditions, it is possible that these encrypted regions may become exposed. In this report, we revealed two pairs of paradoxes regarding the ICAM-4 peptide library and platelet function. Firstly, the effect of D1.7 and D2.3 on platelet function appears to be paradoxical. On the one hand, these two peptides blocks fibrinogen binding to activated platelets (Fig. 2D), on the other hand they induce platelet aggregation in unstimulated fresh platelets (Fig. 1A-C). Platelet aggregation

requires the binding of fibrinogen to IIbα β3 in its active confirmation. In Glanzmann thrombasthenia, platelets fail to aggregate under stimulation due to the abnormalities 25 in integrin αIIbβ3. It is possible that D1.7 and D2.3 bind to αIIbβ3 at both fibrinogen binding site and the non-fibrinogen binding site. However, the exact mechanism underlying the interaction between the peptides and the integrin needs further study. Secondly, the induction of aggregation in unstimulated fresh platelets by the peptides contradicts with the finding of the earlier studies which showed ICAM-4 interact

only with αIIbβ3 in its active confirmation. Study from Hermand et al demonstrated that unactivated platelets did not bind to immobilized recombinant ICAM-4 protein while those activated platelets did. 15 Our previous study also showed that the direct Chapter 4 Chapter binding of erythrocyte to platelets under low shear flow requires platelet activation. 88 The difference between the peptide and the protein in interacting with αIIbβ3 may well lie in the distinct 3D structure of the two substances. It is logical to assume that the much smaller and structurally simpler peptides can recognize more bondable sites

and hidden epitopes on αIIbβ3 than the whole ICAM-4 protein. In conclusion, this study with an ICAM-4 peptide library provides insights in

understanding the influence of ICAM-4-αIIbβ3 interaction on platelet function. Our results indicate that 64P90K on domain I and 112P138L on domain II of ICAM-

4 are the crucial regions in interacting with platelet integrin αIIbβ3.. The peptides located within these regions were able to induce platelet aggregation. Peptides

ICAM-4 and platelet function ICAM-4 and platelet   covering P64 S78 (D1.7) and P124 L138 (D2.3) bind to αIIbβ3 on the fibrinogen binding site, while those covering the rest of the two regions (D1.9, D2.1 and D2.2) bind to the non-fibrinogen binding sites on the integrin. Peptide D1.9 and D2.1 can

modulate αIIbβ3 conformational change from an inactive form to an active form on non-activated platelets. Erythrocyte-platelet interaction via binding of ICAM-

4 to αIIbβ3 is like to regulate the hemostatic balance by affecting platelet function. Modified peptides that blocks the erythrocyte-platelet interaction may be used as tools for future therapeutic strategies. More studies are needed to unravel the ICAM-

4-αIIbβ3 interaction in the functional level by using recombinant ICAM-4 proteins. Acknowledgement We thank the Dutch Heart Foundation (NHS 2006T5301, BdL), Sanquin Blood Supply Foundation (BdL) and the Netherlands Organization of Health Research and Development (ZonMW, CM) for financial support. References P-selectin expression during platelet recruitment: down-regulation by aspirin ex 1. HELLEM AJ, BORCHGREVINK vivo. Blood 2002;99:3978-3984. CF, AMES SB. The role of red cells 11. Spring FA, Parsons SF. Erythroid cell in haemostasis: the relation between adhesion molecules. Transfus.Med.Rev. haematocrit, bleeding time and platelet 2000;14:351-363. adhesiveness. Br.J.Haematol. 1961;7:42-50. 12. Telen MJ. Red blood cell surface adhesion 2. Ho CH. The hemostatic effect of adequate molecules: their possible roles in normal red cell transfusion in patients with anemia human physiology and disease. Semin. and thrombocytopenia. 1996 Hematol. 2000;37:130-142. 3. Ho CH. Increase of red blood cells can 13. Bailly P, Hermand P, Callebaut I et al. The shorten the bleeding time in patients with LW blood group glycoprotein is homologous iron deficiency anemia. 1998 to intercellular adhesion molecules. Proc. 4. Ninivaggi M, Apitz-Castro R, Dargaud Y Natl.Acad.Sci.U.S.A 1994;91:5306-5310. et al. Whole-blood thrombin generation 14. Delahunty M, Zennadi R, Telen MJ. LW monitored with a calibrated automated protein: a promiscuous integrin receptor 4 Chapter thrombogram-based assay. Clin.Chem. activated by adrenergic signaling. Transfus. 2012;58:1252-1259. 89 Clin.Biol. 2006;13:44-49. 5. Whelihan MF, Zachary V, Orfeo T, Mann 15. Hermand P, Gane P, Huet M et al. Red KG. Prothrombin activation in blood cell ICAM-4 is a novel ligand for platelet- coagulation: the erythrocyte contribution to activated alpha IIbbeta 3 integrin. J.Biol. thrombin generation. Blood 2012;120:3837- Chem. 2003;278:4892-4898. 3845. 16. Li Z, Xi X, Du X. A mitogen-activated 6. Whelihan MF, Mann KG. The role of the protein kinase-dependent signaling pathway red cell membrane in thrombin generation. in the activation of platelet integrin alpha Thromb.Res. 2013 IIbbeta3. J.Biol.Chem. 2001;276:42226- 7. Santos MT, Valles J, Aznar J et al. 42232. Prothrombotic effects of erythrocytes on function ICAM-4 and platelet 17. Kramer RM, Roberts EF, Strifler BA, platelet reactivity. Reduction by aspirin. Johnstone EM. Thrombin induces activation Circulation 1997;95:63-68. of p38 MAP kinase in human platelets. 8. Santos MT, Valles J, Lago A et al. Residual J.Biol.Chem. 1995;270:27395-27398. platelet thromboxane A2 and prothrombotic 18. Li Z, Ajdic J, Eigenthaler M, Du X. A effects of erythrocytes are important predominant role for cAMP-dependent determinants of aspirin resistance in patients protein kinase in the cGMP-induced with vascular disease. J.Thromb.Haemost. phosphorylation of vasodilator-stimulated 2008;6:615-621. phosphoprotein and platelet inhibition in 9. Valles J, Santos MT, Aznar J et al. humans. Blood 2003;101:4423-4429. Erythrocytes metabolically enhance 19. Saklatvala J, Rawlinson L, Waller RJ et collagen-induced platelet responsiveness al. Role for p38 mitogen-activated protein via increased thromboxane production, kinase in platelet aggregation caused by adenosine diphosphate release, and collagen or a thromboxane analogue. J.Biol. recruitment. Blood 1991;78:154-162. Chem. 1996;271:6586-6589. 10. Valles J, Santos MT, Aznar J et al. Platelet- 20. Hermand P, Huet M, Callebaut I et al. erythrocyte interactions enhance alpha(IIb) Binding sites of leukocyte beta 2 integrins beta(3) integrin receptor activation and (LFA-1, Mac-1) on the human ICAM-4/ LW blood group protein. J.Biol.Chem. 2000;275:26002-26010. 21. Casasnovas JM, Springer TA, Liu JH, Harrison SC, Wang JH. Crystal structure of ICAM-2 reveals a distinctive integrin recognition surface. Nature 1997;387:312- 315. 22. Hermand P, Gane P, Callebaut I et al. Integrin receptor specificity for human red cell ICAM-4 ligand. Critical residues for alphaIIbeta3 binding. Eur.J.Biochem. 2004;271:3729-3740. 23. Mankelow TJ, Spring FA, Parsons SF et al. Identification of critical amino-acid residues on the erythroid intercellular adhesion molecule-4 (ICAM-4) mediating adhesion Chapter 4 Chapter to alpha V integrins. Blood 2004;103:1503- 90 1508. 24. Bougie DW, Wilker PR, Wuitschick ED et al. Acute thrombocytopenia after treatment with tirofiban or eptifibatide is associated with antibodies specific for ligand-occupied GPIIb/IIIa. Blood 2002;100:2071-2076. 25. Nurden AT. Glanzmann thrombasthenia. Orphanet.J.Rare.Dis. 2006;1:10. ICAM-4 and platelet function ICAM-4 and platelet 5

Platelet apoptosis by cold-induced glycoprotein Ibα clustering

Dianne E. van der Wal1, Vivian X. Du1, Kimberly S.L. Lo1, Jan T. Rasmussen2, San- dra Verhoef1 and Jan Willem N. Akkerman1 Chapter 5 Chapter

91

1Department of Clinical Chemistry and Haematology , University Medical Centre Utrecht, Utrecht, the Netherlands 2Protein Chemistry Laboratory, Department of Molecular Biology, Aarhus University, Aarhus, Denmark GP1b Clustering in platelet apoptosis in platelet GP1b Clustering

J Thromb Haemost. 2010; 8: 2554-2562 Abstract Background: Cold-storage of platelets followed by rewarming induces changes in Glycoprotein (GP) Iba-distribution indicative for receptor clustering and initiates

thromboxane A2-formation. GPIbα is associated with 14-3-3 proteins, which contribute to GPIbα-signaling and in nucleated cells take part in apoptosis regulation. Objectives and methods: We investigated whether GPIbα-clustering induces platelet apoptosis through 14-3-3 proteins during cold (4 hours 0ºC)-rewarming (1 hour 37ºC). Results: During cold-rewarming, 14-3-3 proteins associate with GPIbα and dissociate from Bad inducing Bad-dephosphorylation and activation. This initiates

pro-apoptotic changes in Bax/Bcl-xL and Bax-translocation to the mitochondria inducing cytochrome c release. The result is activation of caspase-9 which triggers phosphatidylserine exposure and platelet phagocytosis by macrophages. Responses Chapter 5 Chapter are prevented by N-acetyl-D-glucosamine (GN), which blocks GPIbα-clustering and 92 by O-sialoglycoprotein endopeptidase, which removes extracellular GPIbα. Conclusions: Cold-rewarming triggers apoptosis through a GN-sensitive GPIbα- change indicative for receptor clustering. Attempts to improve platelet transfusion by cold-storage should focus on prevention of the GPIbα-change. GP1b Clustering in platelet apoptosis in platelet GP1b Clustering Introduction Glycoprotein (GP) Iba is associated with GPIbβ, GPV and GPIX in a 2:4:1:2 stoichiometry. GPIbα mediates primary platelet adhesion at high shear by binding von Willebrand factor (VWF) attached to the injured vessel wall. VWF-ligation initiates multiple reactions e.g. phospholipase Cg-mediated activation of aIIbb3 leading to aggregation, GPIbα-release from the membrane skeleton inducing GPIbα- clusters and macrophage recognition1,2, and a conformational change in Bax inducing apoptosis.3 GPIbα is highly glycosylated with N-linked carbohydrate chains located at leucine- rich repeats 2 and 4 covered by sialic acid. Incubation at 0-4°C exposes underlying galactose- and β-N-acetyl-D-glucosamine (βGN)-residues and disrupts anchorage to the membrane cytoskeleton thereby inducing GPIbα-clusters.1,4 The clusters then bind to the glycan recognition sites of MAC-1 receptors on macrophages triggering 5 platelet destruction in vitro and clearance from the circulation in vivo. The damage 5 Chapter of GPIbα by low-temperature incubation is the major obstacle for wide-scale application of cold-storage of platelet concentrates prepared for transfusion. 93 The interference with platelet-macrophage interaction by added N-acetyl-D- glucosamine (GN) is a strong indication that clusters of damaged GPIbα mediate the destruction of cold-stored platelets.5 We found earlier that in addition to inhibiting MAC-1 binding to damaged GPIbα, GN might interfere with the formation of GPIbα-clusters before it acts as an inhibitor of platelet-macrophage interaction.1,6 Evidence was based on the binding of antibody AN51 directed against amino acids (aa) 1-35 of the N-terminus of GPIbα which changes in a reversible manner upon cold-rewarming in the absence but not in the presence of GN.1 The aa 1-35 region is located in the NH2-terminal hairpin called β-finger. This domain is about 200 aa’s separated from the β-switch (aa 227-241), which is a flexible loop that changes upon GPIbα-binding to the VWF A1-domain.7,8 apoptosis in platelet GP1b Clustering GPIbα-perturbations by VWF/ristocetin (VWF/risto) and cold-storage have in common de-attachment of the membrane skeleton, formation of clusters and generation of thromboxane A2 (TxA2). Cold-induced responses primarily show up during subsequent incubation at 37°C, indicating that metabolic processes expose the damage inflicted to GPIbα at low temperature. The finding that VWF-binding to GPIbα affects key intermediates in the apoptotic pathway3 implies that similar changes might occur during cold-rewarming. If this were true, the possibility of cold-storage of platelets for transfusion would become less attractive. In the present study we applied incubation at 0ºC to cluster GPIbα followed by incubation at 37°C to reveal cold-induced changes in apoptosis regulation. Special focus was on the scaffolding protein 14-3-3ζ, which in addition to contributing to GPIbα-signaling to phosphatidylinositol 3-kinase during VWF-ligation, also controls the de-phosphorylation of Bad, which is an upstream element in the intrinsic apoptotic pathway. Materials and methods We used the following products (with sources): N-Acetyl-D-Glucosamine (GN,

Merck, Darmstadt, Germany), prostacyclin (PGI2, Cayman Chemical, Ann Arbor, MI), cantharidin and the InnoCyte Flow cytometric cytochrome c release kit (Calbiochem, Darmstadt, Germany), 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide JC-1 (Sigma, St. Louis, MO), Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE), Cytofix/Cytoperm and Perm/Wash buffer (Becton Dickinson, San Diego, CA), mitochondria isolation kit (Pierce Biotechnology, Rockford, IL), Human VWF was purified as described.7 The GN- and galactose binding lectin ricinus communis agglutinin-1 (RCA-1) was from Vector Laboratories, Burlingame, CA. Sources of other chemicals have been published1 or are given in the Supplement. Platelet isolation and incubations Chapter 5 Chapter Platelets were isolated while maximally preventing their activation using free-flow 94 blood collection,9 discarding the first 2 mL of blood and all collections that showed micro-aggregates as determined by particle sizing. Procedures were approved by the Medical Ethical Committee of our hospital. Platelets were resuspended in Hepes- Tyrode’s (pH 7.2) to 2x1011 cells/L. Incubations were without stirring at 0°C for 4 hours in the absence and presence of GN (100 mM) to prevent GPIbα-clustering and O-sialoglycoprotein endopeptidase (osge; 80 μg/mL, pre-incubation 30 minutes, 22°C) to cleave the N-terminal part of GPIbα.10 The 0ºC-treatment was followed by 1 hour incubation at 37°C to mimic cold-storage and post-transfusion conditions (0/37°, in short). Concurrently, fresh platelets from the same donor were stimulated with VWF/ristocetin (5 minutes, 37°C, without stirring) at suboptimal concentrations (3 μg/mL VWF; 0.3 mg/mL ristocetin) to keep VWF-induced responses in the range induced by 0/37°-incubation.1 GP1b Clustering in platelet apoptosis in platelet GP1b Clustering Western blots and immunoprecipitations

Platelet suspensions were added to lysis buffer supplemented with 10 μM cantharidin.1 Proteins were separated by SDS-PAGE. After blocking with Odyssey Blocking buffer, membranes were incubated with primary antibodies (1 μg/mL) and protein bands visualized with an Odyssey Imaging system (LI-COR Biosciences, Lincoln, NE). Quantification was performed with Image-J software (NIH, Bethesda, MD). For immunoprecipitations, 450 μL washed platelets (5x1011 platelets/L) was lysed, (15 minutes, 0°C), centrifuged (10 000g, 10 minutes, 4ºC) to remove cell debris and mixed with 55 μL (10% vol/vol) protein G beads together with antibody (1 μg/mL, 30 minutes, 4ºC, rotating). Mitochondria were isolated (3x108 platelets/sample) using the Mitochondria Isolation Kit, according to the manufacturer’s protocol (Pierce Biotechnology, Rockford, IL), with extra addition of PMSF (1 μM), leupeptin (1 μg/mL) and cantharidin (10 μM. all f.c.) to reagents A and C. As a control for equal loading, blots were incubated with antibody against cytochrome c oxidase IV. Flow cytometric analysis

For analysis of 0/37°-induced desialylation of GPIbα, platelets were incubated with RCA-1 (0.5 µg/mL, 30 minutes, RT) and binding to exposed GN- and galactose- residues was measured by FACS. 11 FITC-conjugated RCA-1 binding was expressed as ratio of 0º and 0/37º-platelets over fresh platelets. For measurement of Bax and

Bcl-xL, 500 μL platelet suspension was centrifuged (330g, no-brake, 10 minutes,

RT) in the presence of 10 ng/mL PGI2. Functionality was recovered by 30 minutes incubation (RT) before the pellet was permeabilized with Cytofix/Cytoperm (30 minutes, 4°C), washed with Perm/Wash buffer, incubated with primary (2 µg/mL, 1 hour, 4°C) and secondary antibodies (2 hours, 4°C). PBS was added and 10 000 events were measured on a FACS Calibur (BD Biosciences, San Jose, CA).

For determination of the mitochondrial membrane potential ΔYm, 100 mL platelet suspension was incubated with the JC-1 (0.5 μM, 30 minutes, 37°C). In viable cells, 5 Chapter the high ΔY promotes a directional uptake of JC-1 into the matrix where JC-1 forms m 95 J-aggregates (λex 490 nm, λem 570-610 nm). In apoptotic cells, the low ΔYm preserves 12,13 the monomeric form (λex 490 nm, λem 535 nm) . Changes in ΔYm were expressed as the ratio of platelets in lower- over upper-right quartiles.14,15 Cytochrome c release was measured in 500 mL platelet suspension with the InnoCyte Cytochrome c kit, according to the manufacture’s protocol. Retained cytochrome c in fresh platelets was set at 100%. Statistics

Data are means ± SEM (n=3,4), as indicated. Statistical analysis was performed using GraphPad InStat (San Diego, CA) software. Differences between fresh and treated samples were considered significant at p < 0.05 (*). Results apoptosis in platelet GP1b Clustering GPIbα-clustering causes redistribution of 14-3-3 proteins

Earlier work showed that cold-storage of platelets induces GPIbα-clusters that become recognition sites for macrophages.5 Subsequent incubation at 37°C, starts 1 the production of TxA2. The cause of the GPIbα-change is desialylation of GPIbα as illustrated by an increase in RCA-1 binding, confirming earlier observations11 (Fig. 1A). GPIbα is associated with 14-3-3 proteins which in nucleated cells are known to mediate signaling to cell death.16 To investigate whether the 0/37°-induced GPIbα-change triggers apoptosis, we measured GPIbα-association with 14-3-3 proteins. Fresh platelets showed a slight association between GPIbα and 14-3-3ζ which increased 6-fold after stimulation with VWF/risto, such in agreement with recent literature.3 Also incubation at 0°C induced formation of the [GPIbα-14-3- 3ζ] complex which increased further during subsequent incubation at 37°C (Fig. 1B). Importantly, GN prevented complex formation, illustrating its dependence on GPIbα-clusters. A similar GPIbα-association was observed with 14-3-3b (not shown) and 14-3-3γ (Fig. 1C). Much smaller associations were found between 14-3-3ζ and GPIX and GPIbβ (Suppl. Fig. 1). GPIbα-clustering initiates dephosphorylation of Bad

In nucleated cells, 14-3-3ζ is an upstream regulator of the intrinsic pathway for apoptosis, where it sequesters cytosolic Bad thereby protecting its phosphorylated, dormant state against activation. Since association of 14-3-3ζ with GPIbα might disturb its association with Bad, we investigated immunoprecipitates of total Bad. Cold-incubation induced a 40% fall in 14-3-3ζ-associated Bad, which decreased further during subsequent incubation at 37°C (Fig. 2A). GN prevented the decrease, illustrating its dependence on the cold-induced GPIbα-change. VWF/risto induced a similar fall in [14-3-3ζ-Bad] complex as induced by cold-incubation. Analysis of Chapter 5 Chapter

96 A

B C GP1b Clustering in platelet apoptosis in platelet GP1b Clustering

Figure 1: GPIbα-clustering causes redistribution of 14-3-3 proteins. Fresh platelets and platelets incubated at 0° (4 hours) - and 0/37° (4 hours/1 hour) were studied in the absence and presence of GN (100 mM N-acetyl-D- glucosamine, added at start of incubations). Stimulation of fresh platelets with VWF/ristocetin (3 μg/ml VWF, 0.3 mg/ml ristocetin, 5 min, 37°C, without stirring) served as control for GPIbα activation. A) RCA-1 binding to exposed βGN- and galactose-residues. B) Association between GPIbα and 14-3-3ζ. C) Association between GPIbα and 14-3- 3γ. Data are means ± SEM (n=3) with significant difference p < 0.05 (*). A B

Fig. 2. GPIbα-clustering initiates apoptosis through dephosphorylation of Bad. Platelets were treated as indicated in the legend to Figure 1. In addition, extracellular GPIbα was removed by osge-treatment (80 μg/ml, 30 min, 37°C) prior to the 0/37°-incubation. A) Dissociation of the [14-3-3ζ-Bad] complex. B) Dephosphorylation of Ser- phosphorylated Bad. Data are means ± SEM (n=3) with significant difference p < 0.05 (*). Chapter 5 Chapter Ser-phosphorylated Bad confirmed that loss of 14-3-3ζ induced dephosphorylation. 97 Again, GN reduced this response and also treatment with osge interfered with the fall in phosphorylated Bad (Fig. 2B). GPIbα-clustering induces changes in Bcl-2 family proteins

To confirm that Bad Ser-dephosphorylation led to its activation, two Bcl-2 family members were investigated known to undergo a conformational change induced by active Bad. FACS-analysis with antibodies against the active epitope of Bax and the active conformation of Bcl-xL revealed that Bax and Bcl-xL changed in parallel with the phosphorylation of Bad (Fig. 3). Cold-incubation induced a 2-fold increase in active Bax and a similar fall in active Bcl-xL. Antibody binding to Bax was further increased during subsequent incubation at 37°C, while Bcl-xL remained unchanged. Prevention of GPIbα-clustering by GN and by GPIbα-removal with osge apoptosis in platelet GP1b Clustering opposed these changes and again, VWF/risto-stimulation induced similar changes as 0/37°-incubation.Thus, the change in GPIbα-distribution induced by cold-storage is a potent inducer of platelet apoptosis. GPIbα-clustering activates apoptosis markers in mitochondria

A next step in apoptosis induction is the translocation of Bax to the mitochondria in a first step in triggering release of cytochrome c. To address this issue, mitochondria were isolated and the presence of Bax was measured by immunoblotting. Mitochondria from fresh platelets contained a small amount of Bax and this level increased 2-fold upon cold-incubation and slightly decreased during subsequent 37°C-treatment (Fig. 4A). GN completely prevented Bax-translocation to the mitochondria. In contrast to upstream responses, VWF/risto had little effect on the amount of mitochondria-bound Bax. Binding of Bax is known to disrupt the outer mitochondrial membrane, inducing a collapse in electrochemical gradient over the inner mitochondrial membrane. To investigate whether Bax-binding to the mitochondria induced a similar change, platelets were incubated with the JC-1 dye. FACS-analysis showed that cold-treatment induced depolarization of the inner mitochondrial membrane and that subsequent 37°C-incubation greatly augmented this effect (Fig. 4B). Importantly, the presence of GN and treatment with osge completely prevented the depolarization induced by 0/37°-treatment. The effect of VWF/risto-stimulation

on ΔYm was much smaller than that induced by cold-rewarming. Thus, the GPIbα- change induced by 0/37°-treatment triggers translocation of Bax to the mitochondria and depolarization of the inner mitochondrial membrane. Mitochondrial-bound Bax is thought to form oligomers which form transition pores

A Chapter 5 Chapter

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B GP1b Clustering in platelet apoptosis in platelet GP1b Clustering

Fig. 3. GPIbα-clustering induces changes in Bcl-2 family proteins. Platelets were treated as indicated in the legend to Figure 1. Binding of antibodies directed against the Bcl-2 proteins pro-apoptotic Bax (to activation epitope of Bax, A) and pro-survival Bcl-xL (fall in antibody-binding due to de-activation, B) Data show fluorescence above 1% of background signal. The right panels show representative FACS plots of fresh and 0/37°-treated platelets. Data are means ± SEM (n=4). in the outer mitochondrial membrane thereby releasing cytochrome c from the intermembrane space.17 Indeed, the 0/37°-treatment led to a 30% fall in mitochondrial bound cytochrome c (Fig. 4C). As observed with upstream apoptosis regulators, the presence of GN and treatment with osge prevented the fall in mitochondrial cytochrome c. Again, in fresh platelets VWF/risto induced a similar change in cytochrome c localization as 0/37°-treatment.

A B Chapter 5 Chapter

99

C GP1b Clustering in platelet apoptosis in platelet GP1b Clustering

Fig. 4. GPIbα-clustering activates apoptosis markers in mitochondria. Platelets were treated as indicated in the legend to Figure 1. (A) Binding of Bax to mitochondria, as measured by western blotting. Equal protein loading was obtained using adjustments based on the BCA-assay and illustrated by expression of cytochrome c oxidase IV. (B) Δψm measured by JC-1 binding on the FACS (upper panel). Data are expressed as the ratio of fluorescent platelets in the lower-right (green fluorescence) over those in upper-right (green/orange fluorescence) quartiles. (C) Cytochrome c release was measured by binding of anti-cytochrome c antibody. Retained cytochrome c in fresh platelets was set at 100%. The left panel shows retained cytochrome c at the different incubation conditions. The right panel shows a representative FACS-plot after 0/37°-incubation with an without GN. Data are means ± SEM (n=4). A B

C Fig. 5. GPIbα-clustering induces caspase-9 activity, PS- exposure and phagocytosis. Platelets were treated as indicated in the legend to Figure 2 and with Pan-caspase inhibitor Q-VD-Oph (50 μM). (A) Caspase-9 activity was measured by cleavage of AFC-conjugated LEHD and expressed as % of fresh platelets. (B) PS-exposure was Chapter 5 Chapter measured by binding of lactadherin, anti- bovine lactadherin 100 and secondary anti-rabbit-FITC antibodies by FACS. The right panel shows a representative FACS-histogram of fresh and 0/37°-treated platelets. Data show fluorescence above 1% of background signal. (C) Phagocytosis was measured after incubation of CFMDA-labelled platelets to matured THP-1 cells. Data are means ± SEM (n=4).

GPIbα-clustering activates caspase-9 and phosphatidylserine surface expression

To investigate whether the changes in mitochondrial apoptosis markers that accompanied GPIbα-clustering affected downstream elements in the apoptotic pathway, caspase-9 was measured, an initiator caspase that controls the effector caspase-3. Cold-incubation led to a 50% increase in active caspase-9 which remained unchanged during rewarming. GN and osge-treatment opposed the activation GP1b Clustering in platelet apoptosis in platelet GP1b Clustering confirming their dependence on GPIbα-clustering. As expected, the caspase-inhibitor induced full inhibition and VWF/risto induced a similar activation as cold-incubation (Fig. 5A). Cold-incubation also induced surface expression of phosphatidylserine (PS), a marker of apoptotic cells. GN- and osge-treatment blocked PS-surface expression completely. Importantly, the caspase-inhibitor induced 100% suppression of cold-induced PS-expression. Stimulation with VWF/risto used as positive control also induced PS-expression in agreement with earlier observations (Figure 5B and right panel).3 PS-surface expression is a recognition signal for macrophage binding and platelet phagocytosis.6 Indeed, increases in surface-PS translated into similar increases in binding (data not shown) and phagocytosis (Figure 5C) of cold-treated platelets by matured THP-1 cells and inhibition of GPIbα-clustering abolished platelet interaction with macrophages.

1 We demonstrated earlier that 0/37°-incubation triggers the formation of TxA2. To

clarify whether this pathway contributed to apoptosis regulation, we measured TxA2 together with the apoptosis markers Bax and ΔYm and with P-selectin expression as a marker for platelet activation. Cold-incubation alone induced little TxA2-formation and the markers remained unchanged in the presence of indomethacin (data not shown). Subsequent rewarming started production of TxA2, a further increase in

Bax and ΔYm and triggered P-selectin expression. These responses were strongly inhibited by indomethacin, illustrating that rewarming induced a 2nd wave of apoptosis induction through TxA2, which was accompanied by secretion of a-granules (Suppl. Fig. 2). Discussion The present study shows that 0/37°-treatment of platelets induces apoptosis through a sequence involving (i) a change in GPIbα possibly caused by receptor clustering, (ii) an increase in GPIbα-association with the adapter protein 14-3-3ζ/β/γ, (iii) the release of 14-3-3ζ from Ser-phosphorylated Bad inducing its dephosphorylation and activation, (iv) a conformational change in Bax and Bcl-x in favor of apoptosis L 5 Chapter induction, (v) translocation of Bax to the mitochondria, (vi) depolarization of the inner mitochondrial membrane and release of cytochrome c, (vi) activation of 101 caspase-9 inducing PS-exposure and binding to matured THP-1 cells followed by their phagocytosis. These are defined steps in the intrinsic apoptotic pathway of nucleated cells leading to formation of the apoptosome complex, activation of caspases and cell death.18 Extending the cold-incubation to 48 hours leads to more

GPIbα-clustering [6], an increase in ΔΨm-change from 0.14±0.04 (4 hrs 0/37º) to 0.20±0.05 (48 hrs 0/37º) and an increase in exposed PS from 22±4 to 42±4%, illustrating that apoptosis increases at longer periods of cold-incubation. The change in GPIbα-conformation is caused by extracellular exposure of bGN- and galactose-residues and the intracellular de-attachment from the membrane skeleton.1 These findings suggest that the glycosylation state of the extracellular domain of

GPIbα affects the signalling properties of its intracellular domain. A similar effect apoptosis in platelet GP1b Clustering is seen with platelet PECAM-1, where a deficiency in α2,6-linked sialic acid leads to decreased tyrosine-phosphorylation of the cytosolic tail.19 The present data show that GN also blocks GPIbα-induced apoptosis, emphasizing the importance of the GPIbα-change in control of platelet survival. For simplicity, this GPIbα-change has been termed clustering but the molecular mechanisms involved remain to be elucidated. A similar change in GPIbα is seen after prolonged platelet storage at RT and following exposure to a shear of 1600 s-1.1 If these conditions also initiate apoptosis, this property would seriously affect the survival of RT-stored platelets after transfusion and circulating platelets that roll over VWF bound in a damaged vessel wall. In line with these conclusions are earlier observations showing a ΔYm- change and caspase-3 activation in platelets exposed to high shear stress and together illustrate that GPIbα is extremely sensitive to mechanic stress.14 Our observation that a poor blood collection alone activates the apoptosis markers identified in this study also supports this reasoning (data not shown). A next step in GPIbα-induced apoptosis induction is the association with cytosolic 14-3-3- adapter proteins. 14-3-3 denotes a family of ~30 kDa acidic proteins that plays a role in a wide range of cellular functions.20 The family consists of nine members, but only the b, g, ε, η, θ and z isotypes are present in platelets.21,22 All bind to GPIbα at residues 580-590 and 606.22 The 0/37º-treatment induces 14-3-3ζ-binding to GPIbα in a similar way as VWF/risto-treatment in fresh platelets and also initiates GPIbα-association with 14-3-3β and 14-3-3γ. Apart from GPIbα, also GPIbβ, GPIX and GPV contain binding sites for 14-3-3ζ.23,24 However, 14-3-3ζ binding to GPIbβ and GPIX was low (Suppl. Fig. 1) and absent to GPV (not shown). The difference might be caused by the high number of 14-3-3ζ-binding sites on GPIbα and the short cytoplasmic domains of GPIbβ and GPIX. The 14-3-3 family consist of phosphoserine binding proteins and control the activity of pro-apoptotic Bad.25,26 This BH3-only protein belongs to the B-cell lymphoma-2 (Bcl-2) superfamily, which contains pro-survival and pro-apoptotic proteins.18 In 27 Chapter 5 Chapter pro-survival conditions, Bad is phosphorylated on at least five serine residues. 27,28 102 Phosphorylation at Ser-75 and -99 is essential for binding to 14-3-3 proteins. Our data show that association of 14-3-3 proteins with GPIbα is accompanied by dephosphorylation of Bad, which is a crucial step in intrinsic apoptosis induction.28

Dephosphorylated-Bad replaces Bax on the [Bax-Bcl-xL] complex liberating active 29 Bax. The ratio of Bak over Bcl-xL has been proposed as an important determinant of platelet life span. 29,30,31 The activation of Bax following 0/37°-treatment is in agreement with findings in RT-stored platelets reported earlier.32,33 The increase in antibody-binding to Bax reflects the exposure of the cryptic N-terminal after its activation.34 Its translocation to the mitochondria is accompanied by depolarization of the inner membrane and release of cytochrome c. Together these findings reveal a pathway which starts with GPIbα-clustering and signals through 14-3-3 proteins, Bad and Bax to cytochrome c release, which is a key step in the GP1b Clustering in platelet apoptosis in platelet GP1b Clustering induction of later steps in intrinsic apoptosis. In GPIbα-transfected CHO-cells, deletion of the 14-3-3ζ-binding site in GPIbα (aa 551-610) reduced VWF/risto-

induced Δψm-change, caspase-3 activity and PS-exposure, which strongly support the GPIbα-induced apoptosis induction found in the present report.3 A more detailed analysis of 0/37°-induced apoptosis demonstrates that most activation signals are generated during incubation at 0°C. Prior to rewarming, there is an increase in GPIbα-14-3-3 association, a fall in 14-3-3ζ-Bad association, dephosphorylation of

phosphorylated-Bad, an increase in active Bax with concomitant decrease in Bcl-xL, an increase in mitochondrial-bound Bax and membrane depolarization and a fall in mitochondrial cytochrome c. These changes lead to the activation of caspase-9, PS-surface expression ultimately inducing binding of platelets to matured THP- 1 cells followed by their phagocytosis. The finding that caspase inhibition aborts PS-expression and platelet-macrophage interaction clearly places these events downstream of caspase activation. Most changes in apoptosis markers remain intact during subsequent rewarming and a few show slight reversibility (mitochondrial Bax, PS-exposure). In contrast, GPIbα-14-3-3 association, 14-3-3ζ-Bad, Bax activation and Δψm-change increase further during 37°C-incubation. Incubation with indomethacin show that these changes are triggered by TxA2 which starts when cold- stored platelets enter the 37°C-incubation.1 Also other platelet activators such as thrombin, VWF/risto,12 epinephrine, U46619 and ADP trigger apoptosis inducing 35,36,37 a change in ΔYm and activation of caspases. The apoptosis induction at 0°C is not accompanied by TxA2-formation or P-selectin expression. This is a first demonstration of apoptosis induction without concurrent platelet activation in the absence of pharmacological blockade. In conclusion, cold-storage induces a change in GPIbα that makes the receptor an initiation site for platelet activatio,1 apoptosis (this report) and macrophage recognition.5 Attempts to improve platelet transfusions by cold-storage should focus on the regulation of GPIbα. Acknowledgements 5 Chapter 103 This study was supported by a grant from the Landsteiner Foundation of Blood transfusion Research (LSBR grant nr. 0510). Prof. Dr. J.W. N. Akkerman is supported by the Netherlands Thrombosis Foundation. GP1b Clustering in platelet apoptosis in platelet GP1b Clustering References scavenger receptor-A. Arterioscler Thromb Vasc Biol 2007;27:2476-83. 1. van der Wal DE, Du V, Verhoef S, Akkerman 10. Bergmeier W, Bouvard D, Eble JA, Mokhtari- JW. Role of glycoprotein Ibalpha mobility Nejad R, Schulte V, Zirngibl H, Brakebusch in platelet function. Thromb Haemost C, Fässler R, Nieswandt B. Rhodocytin 2010;103:1033-43. (aggretin) activates platelets lacking alpha(2) 2. Englund GD, Bodnar RJ, Li Z, Ruggeri ZM, beta(1) integrin, glycoprotein VI, and the Du X. Regulation of von Willebrand factor ligand-binding domain of glycoprotein binding to the platelet glycoprotein Ib-IX by Ibalpha. J Biol Chem 2001;276:25121-6. a membrane skeleton-dependent inside-out 11. Rumjantseva V, Grewal PK, Wandall HH, signal. J Biol Chem 2001;276:16952-9. Josefsson EC, Sorensen AL, Larson G, 3. Li S, Wang Z, Liao Y, Zhang W, Shi Q, Yan Marth JD, Hartwig JH, Hoffmeister KM. R, Ruan C, Dai K. The glycoprotein Ibalpha- Dual roles for hepatic lectin receptors in von Willebrand factor interaction induces the clearance of chilled platelets. Nat Med platelet apoptosis. J Thromb Haemost 2009;15:1273-80. 2010;8:341-50. 12. Lopez JJ, Salido GM, Pariente JA, Rosado Chapter 5 Chapter 4. Hoffmeister KM, Josefsson EC, Isaac JA. Thrombin induces activation and 104 NA, Clausen H, Hartwig JH, Stossel TP. translocation of Bid, Bax and Bak to the Glycosylation restores survival of chilled mitochondria in human platelets. J Thromb blood platelets. Science 2003;301:1531-4. Haemost 2008;6:1780-8. 5. Hoffmeister KM, Felbinger TW, Falet H, 13. Smiley ST, Reers M, Mottola-Hartshorn Denis CV, Bergmeier W, Mayadas TN, C, Lin M, Chen A, Smith TW, Steele GD von Adrian UH, Wagner DD, Stossel TP, Jr, Chen LB. Intracellular heterogeneity Hartwig JH. The clearance mechanism of in mitochondrial membrane potentials chilled blood platelets. Cell 2003;112:87-97. revealed by a J-aggregate-forming lipophilic cation JC-1. Proc Natl Acad Sci USA 6. Badlou BA, Spierenburg G, Ulrichts H, 1991;88:3671-5. Deckmyn H, Smid WM, Akkerman JW. Role of glycoprotein Ibalpha in phagocytosis 14. Leytin V, Allen DJ, Mykhaylov S, Mis of platelets by macrophages. Transfusion L, Lyubimov EV, Garvey B, Freedman 2006;46:2090-9. J. Pathologic high shear stress induces GP1b Clustering in platelet apoptosis in platelet GP1b Clustering apoptosis events in human platelets. Biochem 7. Huizinga EG, Tsuji S, Romijn RA, Biophys Res Commun 2004;320:303-10. Schiphorst ME, de Groot PG, Sixma JJ, Gros P. Structures of glycoprotein Ibalpha 15. Albanyan AM, Harrison P, Murphy MF. and its complex with von Willebrand factor Markers of platelet activation and apoptosis A1 domain. Science 2002;297:1176-9. during storage of apheresis- and buffy coat- derived platelet concentrates for 7 days. 8. Uff S, Clemetson JM, Harrison T, Clemetson Transfusion 2009;49:108-17. KJ, Emsley J. Crystal structure of the platelet glycoprotein Ib(alpha) N-terminal 16. Morrison DK. The 14-3-3 proteins: domain reveals an unmasking mechanism integrators of diverse signaling cues that for receptor activation. J Biol Chem impact cell fate and cancer development. 2002;277:35657-63. Trends Cell Biol 2009;19:16-23. 9. Korporaal SJ, Van Eck M, Adelmeijer J, 17. Kim BJ, Ryu SW, Song BJ. JNK- and p38 IJsseldijk M, Out R, Lisman T, Lenting kinase-mediated phosphorylation of Bax PJ, Van Berkel TJ, Akkerman JW. Platelet leads to its activation and mitochondrial activation by oxidized low density translocation and to apoptosis of human lipoprotein is mediated by CD36 and hepatoma HepG2 cells. J Biol Chem 2006;281:21256-65. Fischer A, Schmitz W, Zahedi RP, Sickmann A, Metz R, Albert S, Benz R, 18. Youle RJ, Strasser A. The BCL-2 protein Hekman M, Rapp UR. Identification of family: opposing activities that mediate cell novel in vivo phosphorylation sites of the death. Nat Rev Mol Cell Biol 2008;9:47-59. human proapoptotic protein BAD: pore- 19. Kitazume S, Imamaki R, Ogawa K, Komi Y, forming activity of BAD is regulated Futakawa S, Kojima S, Hashimoto Y, Marth by phosphorylation. J Biol Chem JD, Paulson JC, Taniguchi N. {alpha}2,6- 2009;284:28004-20. sialic acid on platelet endothelial cell 28. Downward J. How BAD phosphorylation adhesion molecule (PECAM) regulates its is good for survival. Nat Cell Biol 1999; homophilic interactions and downstream 1:E33-E35. antiapoptotic signaling. J Biol Chem 2010;285:6515-6521. 29. Mason KD, Carpinelli MR, Fletcher JI, Collinge JE, Hilton AA, Ellis S, Kelly 20. Yaffe MB. How do 14-3-3 proteins work? PN, Ekert PG, Metcalf D, Roberts AW, Gatekeeper phosphorylation and the Huang DC, Kile BT. Programmed anuclear molecular anvil hypothesis. FEBS Lett cell death delimits platelet life span. Cell 2002;513:53-7. 2007;128:1173-86. 5 Chapter 21. Wheeler-Jones CP, Learmonth MP, Martin H, 30. Oltersdorf T, Elmore SW, Shoemaker AR, Aitken A. Identification of 14-3-3 proteins in 105 et al. An inhibitor of Bcl-2 family proteins human platelets: effects of synthetic peptides induces regression of solid tumours. Nature on protein kinase C activation. Biochem J 2005;435:677-81. 1996;315:41-7. 31. Schoenwaelder SM, Yuan Y, Josefsson EC, 22. Mangin PH, Receveur N, Wurtz V, David White MJ, Yao Y, Mason KD, O’Reilly LA, T, Gachet C, Lanza F. Identification of five Henley KJ, Ono A, Hsiao S, Willcox A, novel 14-3-3 isoforms interacting with the Roberts AW, Huang DC, Salem HH, Kile BT, GPIb-IX complex in platelets. J Thromb Jackson SP. Two distinct pathways regulate Haemost 2009;7:1550-5. platelet phosphatidylserine exposure and 23. Calverley DC, Kavanagh TJ, Roth GJ. procoagulant function. Blood 2009;114:663- Human signaling protein 14-3-3zeta interacts 6. with platelet glycoprotein Ib subunits Ibalpha 32. Vanags DM, Orrenius S, guilar-Santelises

and Ibbeta. Blood 1998;91:1295-303. apoptosis in platelet GP1b Clustering M. Alterations in Bcl-2/Bax protein levels 24. Andrews RK, Harris SJ, McNally T, in platelets form part of an ionomycin- Berndt MC. Binding of purified 14-3-3 zeta induced process that resembles apoptosis. Br signaling protein to discrete amino acid J Haematol 1997;99:824-31. sequences within the cytoplasmic domain of 33. Brown SB, Clarke MC, Magowan L, the platelet membrane glycoprotein Ib-IX-V Sanderson H, Savill J. Constitutive death complex. Biochemistry 1998;37:638-47. of platelets leading to scavenger receptor- 25. Masters SC, Fu H. 14-3-3 proteins mediate mediated phagocytosis. A caspase- an essential anti-apoptotic signal. J Biol independent cell clearance program. J Biol Chem 2001;276:45193-200. Chem 2000;275:5987-96. 26. Zha J, Harada H, Yang E, Jockel J, 34. Upton JP, Valentijn AJ, Zhang L, Gilmore Korsmeyer SJ. Serine phosphorylation of AP. The N-terminal conformation of Bax death agonist BAD in response to survival regulates cell commitment to apoptosis. Cell factor results in binding to 14-3-3 not BCL- Death Differ 2007;14:932-42. X(L). Cell 1996;87:619-28. 35. Verhoeven AJ, Verhaar R, Gouwerok EG, de 27. Polzien L, Baljuls A, Rennefahrt UE, KD. The mitochondrial membrane potential in human platelets: a sensitive parameter for platelet quality. Transfusion 2005;45:82-9. 36. Leytin V, Allen DJ, Mutlu A, Mykhaylov S, Lyubimov E, Freedman J. Platelet activation and apoptosis are different phenomena: evidence from the sequential dynamics and the magnitude of responses during platelet storage. Br J Haematol 2008;142:494-7. 37. Tonon G, Luo X, Greco NJ, Chen W, Shi Y, Jamieson GA. Weak platelet agonists and U46619 induce apoptosis-like events in platelets, in the absence of phosphatidylserine exposure. Thromb Res 2002;107:345-50. Chapter 5 Chapter

106 GP1b Clustering in platelet apoptosis in platelet GP1b Clustering Supplementary Materials Materials and Methods

We used the following products (with sources): 5-chloromethyl fluorescein diacetate (Cell tracker green\CFMDA, Molecular Probes, Invitrogen, Carlsbad, CA), THP- 1 monocytic cells (ATCC/LGC Standards GmBH, Wesel, Germany), phorbal

12-myristate 13-acetate (PMA, MP Biochemicals, Illkirch, France), vitamin D3, trypan blue and anti- rabbit- FITC-conjugated Fab fragments (Sigma, St. Louis, MO), (platelet derived) human TGF-β1 and Pan-caspase-inhibitor Q-VD-Oph (R&D systems, Minneapolis, MN), anti-human P-selectin antibody (BD Pharmingen, San

Diego, CA) and TxA2 Enzyme Immuno Assay (EIA) kit (Assay Designs, Ann Arbor, MI). Lactadherin (PAS-6/7 or MFG-E8) and anti-bovine lactadherin antibody were from own sources.1 Antibodies used for western blotting were directed against 14- 3-3ζ (C-16), 14-3-3γ (C-16), 14-3-3β (A-15), GPIX, (all Santa Cruz Biotechnology;

Santa Cruz, CA), GPIbα (SZ2, Beckman Coulter, Fullerton, CA), GPIbβ, total Bad 5 Chapter (Zymed, Invitrogen, Carlsbad, CA), phospho-Ser proteins (Millipore, Bedford, 107 MA), Bax (3/Bax, BD Transduction, San Diego, CA), cytochrome c oxidase IV (Abcam, Cambridge, UK) and secondary antibodies Alexa-680 (Molecular Probes, Invitrogen, Carlsbad, CA) and IRDYe 800CW (LI-COR Biosciences, Lincoln, NE). Antibodies for immunoprecipitation (IP) were against GPIbα (AK2), 14-3-3ζ (V-16), (both Santa Cruz Biotechnology), total Bad (Cell signaling Technology, Danvers,

MA); for FACS against active Bax (6A7, Sigma, St. Louis, MO) and Bcl-xL (2H12, BD Pharmingen, San Diego, CA). Caspase-9 activity

For determination of caspase-9 activity, 1 mL platelet suspension (2x1011 cells/L) was centrifuged (330g, no-brake, 10 minutes, RT) in the presence of 10 ng/mL PGI2 and the pellet was permeabilized and lysed in 100 μL lysis buffer (10 minutes, 0°C) apoptosis in platelet GP1b Clustering provided by the caspase-9 fluorimetric activity kit (R&D systems, Minneapolis, MN). 50 μL lysate was used to measure cleavage of the 7-amino-4-trifluoromethylcoumarin (AFC)-conjugated substrate peptide (LEHD). Caspase-9 activity was measured (2 hours, 37°C) on the Spectramax M2 (Molecular Devices, Sunnyvale, CA, λex 400 nm, λem 505 nm) and expressed as % of fresh platelets. Phosphatidylserine-exposure

Phosphatidylserine (PS)-exposure was measured using the PS-binding protein lactadherin [2]. Bovine lactadherin was isolated from milk. Platelets were incubated with 5.6 μg/mL lactadherin (30 minutes, RT). Rabbit anti-bovine lactadherin antibody (1:100 dilution, 15 minutes, RT) and secondary anti-rabbit-FITC (7 μg/mL, 30 minutes, RT) were added and antibody binding was measured on a FACS Calibur (BD Biosciences, San Jose, CA). Binding and Phagocytosis Platelets were resuspended in HEPES-tyrode’s, pH 6.5 (4x1011 cells/L), labelled with CFMDA (20 μM, 1 hour, RT) and centrifuged (330g, no-brake, 10 minutes, RT) in the presence of 10 ng/mL and resuspended in tyrode’s, pH 7.2. Functionality was recovered by 30 minutes incubation (RT). For measurement of combined binding and phagocytosis, THP-1 cells were cultured in a 96-wells plate (5x104 cells/well)

and stimulated with 50 nM vitamin D3 and 1 ng/mL TGF-β1 (12 hours, 37°C) and

250 nM of PMA (2 hours, 37°C). CaCl2 and MgCl2 (1 mM each, f.c.) were added and 2x106 platelets were incubated with the maturated THP-1 cells (90 minutes, 37°C). Unbound platelets were removed and fluorescence was measured on a Fluorstar Galaxy (BMG LABTECH GmbH, Offenburg, Germany). Trypan blue was added, which quenches fluorescence of bound platelets to separate fluorescence from phagocytozed and bound + phagocytozed platelets.2-4 Phagocytosis of fresh platelets was set at 100%.

P-selectin expression and formation of TxA2 Chapter 5 Chapter To measure P-selectin expression, 100 μL platelet suspension (2x1011 cells/L) was 108 A B

GP1b Clustering in platelet apoptosis in platelet GP1b Clustering C

Suppl. Fig. 1. GPIbα-clustering initiates redistribution of 14-3-3 proteins.Fresh platelets and platelets incubated at 0° (4 hours) - and 0/37° (4 hours/1 hour) were incubated in the absence and presence of GN (100 mM N-acetyl-D- glucosamine, added at start of incubations). Stimulation of fresh platelets with VWF/ristocetin (3 μg/ml VWF, 0.3 mg/ml ristocetin, 5 min, 37°C, without stirring) served as control for GPIbα activation. A) Association of 14-3-3ζ and GPIX (n=2). B) Association of 14-3-3ζ and GPIbβ (n=1). Suppl. Fig. 2. Apoptosis induction and platelet activation during 0/37°-incubation. Platelets were treated as indicated in the legend to Figure

S1. Bax, Δψm, TxA2-formation and P-selectin (P-sel) expression were measured after 0ºC and 0/37°C-incubation. Incubations were without (open bars) and with (grey bars) indomethacin (30 μM). Data found after 0/37°C-incubation were expressed as percentage of those obtained at 0°C. Data are means ± SEM (n=3).

incubated with anti-human antibody against P-selectin (1:20 diluted, 15 minutes, 37°C) and 10 000 platelets were measured on a FACS Calibur.5

For measurement of TxA2-formation, suspensions were incubated without stirring. At the end of incubations, samples were rapidly mixed with indomethacin (30 μM, 5 Chapter final concentration) to halt COX-1 activity. Platelets were pelleted (15 000g, 30 sec, 109

22°C) and TxA2 was measured in the supernatant by EIA. Data were expressed as 11 5 ng TxA2 formed/min/10 platelets.

assay. J Immunol Methods 1993; 162:1-7. Supplementary References 5. van der Wal DE, Verhoef S, Schutgens RE, Peters M, Wu Y, Akkerman JW. Role of 1. Waehrens LN, Heegaard CW, Gilbert GE, glycoprotein Ibalpha mobility in platelet Rasmussen JT. Bovine lactadherin as a function. Thromb Haemost 2010; 103:1033- calcium-independent imaging agent of 43. phosphatidylserine expressed on the surface of apoptotic HeLa cells. J Histochem apoptosis in platelet GP1b Clustering Cytochem 2009; 57: 907-14. 2. Nuutila J, Lilius EM. Flow cytometric quantitative determination of ingestion by phagocytes needs the distinguishing of overlapping populations of binding and ingesting cells. Cytometry A 2005; 65:93- 102. 3. Van Amersfoort ES, Van Strijp JA. Evaluation of a flow cytometric fluorescence quenching assay of phagocytosis of sensitized sheep erythrocytes by polymorphonuclear leukocytes. Cytometry 1994;17: 294-301. 4. Wan CP, Park CS, Lau BH. A rapid and simple microfluorometric phagocytosis Chapter 5 Chapter

110 GP1b Clustering in platelet apoptosis in platelet GP1b Clustering 6

Accelerated uptake of VWF/platelet complexes in macrophages contribute to VWD-type 2B associated thrombocytopenia

Caterina Casari1.2, Vivian Du3, Ya-Ping Wu3, Alexandre Kauskot1,2, Philip G. de Chapter 6 Chapter Groot3, Olivier D. Christophe1,2, Cécile V. Denis1,2, Bas de Laat3, Peter J. Lenting1,2 111

1Department of Clinical Chemistry and Hematology, University Medical Center Utrecht, Utrecht, The Netherlands 2Sanquin Research, Amsterdam, The Netherlands 3Departement of Biochemistry, CARIM, University of Maastricht, Maastricht, The Netherlands 4Synapse BV, Maastricht, The Netherlands Clearance of VWF/platelet complexes in VWD-type 2B complexes of VWF/platelet Clearance

Blood 2013;122: 2893-2902 Abstract Von Willebrand Disease (VWD)-type 2B is characterized by mutations causing enhanced binding of von Willebrand factor (VWF) to platelets. Bleeding tendency is associated with heterogeneous clinical manifestations, including moderate to severe thrombocytopenia. The underlying mechanism of the thrombocytopenia has remained unclear. Here, a mouse model of VWD-type 2B was used to investigate pathways contributing to thrombocytopenia. Immuno-histochemical analysis of blood smears revealed that mutant VWF was exclusively detected on platelets of thrombocytopenic VWD-type 2B mice, suggesting that VWF binding to platelets is needed to induce thrombocytopenia. Platelet counts in thrombocytopenic VWD- type 2B mice were elevated 2-3-fold upon chemical macrophage depletion. Co- localization of platelets with CD68-positive Kupffer cells and CD168-positive marginal macrophages in liver and spleen, respectively, confirmed the involvement of macrophages in the removal of VWF/platelet complexes. Significantly more Chapter 6 Chapter platelets were found in liver and spleen of VWD-type 2B mice compared to control 112 mice. Finally, platelet survival was significantly shorter in VWD-type 2B mice compared to control mice, providing a rationale for lower platelet counts in VWD- type 2B mice. In conclusion, our data indicate that VWF-type 2B binds to platelets and this is a signal for clearance by macrophages, which could contribute to the thrombocytopenia in patients with VWD-type 2B. Clearance of VWF/platelet complexes in VWD-type 2B complexes of VWF/platelet Clearance Introduction Von Willebrand factor (VWF) is particularly known for its role in haemostasis, where it functions as a chaperone-protein for coagulation factor VIII and is essential for the recruitment of platelets to the growing thrombus under conditions of high shear stress.1 Its importance for the haemostatic system is illustrated by the bleeding tendency that is associated with the functional deficiency of VWF, a disorder known as von Willebrand disease (VWD). VWD can originate from a quantitative deficiency (type 1 and type 3 for mild and severe deficiencies, respectively) and or qualitative abnormalities (type 2).2 VWD- type 2 is classified according to the functional defect and of particular relevance to this study is VWD-type 2B, which is caused by gain-of-function mutations that increase the affinity of VWF for platelets.2 These gain-of-function mutations are clustered in exon 28 of the VWF gene encoding the VWF A1 domain, which harbors

the binding site for its platelet receptor Glycoprotein (GP) Ibα. The increased affinity 6 Chapter for platelets is often detected via enhanced ristocetin-induced platelet aggregation.3 113 Besides their bleeding tendency, VWD-type 2B patients frequently manifest a decrease of high molecular weight (HMW)-VWF multimers in their plasma and a moderate to severe thrombocytopenia.4 Blood smear analysis further revealed that the thrombocytopenia is associated with the presence of giant platelets and spontaneous platelet aggregates.4 Interestingly, a large study involving 67 VWD-type 2B patients revealed a marked mutation-dependent variety in the degree of thrombocytopenia and the clinical severity of the disease.4 Importantly, a strong correlation between the extent of thrombocytopenia and the bleeding score of VWD-type 2B patients was observed. Despite the progress in the diagnosis of VWF-type 2B, there are still a number of unknowns in our understanding of the disease. For instance, the molecular mechanism underlying the thrombocytopenia has not yet been identified. Several mechanisms can be considered in this regard. First, Nurden et al. have reported that megakaryocytopoiesis is disturbed in VWD-type 2B, due to continuous interactions between VWF and GPIbα at the surface of the .5-7 This impaired megakaryocytopoiesis may not only explain the occurrence of giant platelets, but could also contribute to a lower platelet count in VWD-type 2B patients. A second in VWD-type 2B complexes of VWF/platelet Clearance possibility was proposed already three decades ago, in which the thrombocytopenia is explained by an accelerated clearance of VWF/platelet complexes.8,9 This possibility is still being referred to in the literature10 ,11, although no experimental evidence in support of this possibility has been provided so far. It should also be noted that both abovementioned possibilities are not mutually exclusive. Recently, we and others have developed a murine model for VWD-type 2B, in which the clinical phenotype of human patients is faithfully reproduced.12,13 VWD- type 2B mice are characterized by a severe bleeding phenotype, thrombocytopenia, circulating aggregates, giant platelets and an increased clearance of the VWF-type 2B mutant protein. Interestingly, the loss of HMW-VWF multimers was observed in but 50% of the mice.12 Importantly, the severity of the thrombocytopenia was dependent on the mutation tested, with the p.V1316M mutation being more severe than the p.R1306Q mutations, similar to the observations in human patients.4,12,13 In the present study, we have used this mouse model to show that VWD-type 2B platelets have a shorter circulatory half-life than wt-platelets, which could contribute to the lower platelet counts in VWD-type 2B mice. Further analysis revealed that VWF-type 2B is present at the surface of platelets of thrombocytopenic VWD- type 2B mice, and that these VWF/platelet complexes are taken up efficiently by macrophages in liver and spleen. We thus provide direct evidence that part of the thrombocytopenia in VWD-type 2B can be explained by an increased clearance of VWF/platelet complexes by macrophages in liver and spleen. Materials and Methods Chapter 6 Chapter

114 An extensive description of materials and methods are included in the supplementry materials, a brief summary of which is given below. Mice

VWF-deficient mice on a C57BL/6 background were used throughout the study.14 Both male and females were used, and bodyweight varied between 20 and 25 g. All experimental procedures were performed in accordance with European legislation concerning the use of laboratory animals and approved by the local animal care and ethical committee (CEEA 26) under the authorization 2012-039. Perfusion experiments

Perfusions experiments were performed using phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 macrophages that were grown on glass coverslips. All perfusions were executed using a shear stress of 5 dynes/cm2. Following perfusion with VWF (5 mg/ml) in the absence or presence of botrocetin (1.6 U/ml), cells were fixed and stained for VWF as described in the Supplemental Methods. Scanning electron microscopy Clearance of VWF/platelet complexes in VWD-type 2B complexes of VWF/platelet Clearance

Following incubation of THP-1 macrophages with VWF-coated microspheres, cells were fixed using glutaraldehyde and osmium tetroxide, dehydrated using successive ethanol incubations and finally coated with gold alloy. Images were acquired using a Hitachi S-2500 scanning electron microscope. Plasmids and hydrodynamic injection

Plasmids encoding full-length wild-type murine VWF (wt-mVWF) or VWD-type 2B mutants p.V1316M (p.V1316M-mVWF) and p.R1306Q (p.R1306Q-mVWF) have been described previously and were used to induce in vivo expression following hydrodynamic gene-transfer.12 All experiments were performed or initiated 4 days after hydrodynamic gene-transfer. For each mouse included in the study, VWF antigen (VWF : Ag) and platelet counts were determined at day 4 after hydrodynamic injection and only mice with the antigen being within the 200-1200% range were included in the study. VWF:Ag levels were quantified as described 15, and antigen levels were found to be 607±34% (mean ± SEM; n=77), 627 ± 28% (n=90) and 597 ± 50% (n=37) for wt-mVWF, mVWF/p.V1316M and mVWF/p. R1306Q, respectively. In some cases experiments were performed over several days (e.g. clearance experiments and effects of liposome-clodronate). In accordance with our previous reports12,16, antigen levels remained stable throughout the duration of the experiments. For platelet counts, blood was collected on EDTA and counts were performed with an automated animal blood-cell counter (Scil Vet ABC, Scil, Gurnee, IL). Since antigen levels were above the normal range (200-1200%), we checked how antigen levels affected platelet counts. For both wt-mVWF and mVWF/p. R1306Q, no correlation between antigen levels and platelet counts were observed (r2 Chapter 6 Chapter = 0.022 [P = .20] and r2 = 0.04 [P = .23], respectively; Suppl. Fig. 1). For mVWF/p. V1316M, a poor but statistically significant correlation was observed (r2 = 0.06, p = 115 0.02). It should be noted that platelet counts in mice within the lower antigen range (200-200% VWF : Ag) were already thrombocytopenic (212 ± 29; mean ± SEM) and just slightly higher than platelet counts in mice within the higher antigen range (1100-1200% VWF:Ag: platelet counts: 134 ± 25). Quantitative analysis of platelet biodistribution

Washed platelets were fluorescently labeled with CMTMR-Orange and infused in the tail-vein of wt-mVWF or mutants mVWF-expressing mice. Two hours after injection, liver and spleen were isolated and used for cryo-section preparation. Accumulation of platelets in liver and spleen sections was determined using NanoZoomer 2.0-HT equipment, which allows quantitative whole-slide assessment (for details supplementary material). Qualitative analysis of platelet biodistribution

For quantitative analysis of platelet biodistribution, liver and spleen cryo-sections were stained for specific subpopulations of macrophages. Immunofluorescence Clearance of VWF/platelet complexes in VWD-type 2B complexes of VWF/platelet Clearance microsopic analysis was then performed as described in detail in the supplementary material to detect co-localization between platelets and macrophages. Platelet survival and flow cytometry

Platelet survival was essentially performed as described previously.17 More detailed information is provided in the supplementary material. Results VWF activation facilitates its uptake by macrophages Chapter 6 Chapter

116 Clearance of VWF/platelet complexes in VWD-type 2B complexes of VWF/platelet Clearance

Fig. 1. VWF active conformation enhances endocytosis by macrophage.A-H) ( wt-rVWF (5 µg/ml) was perfused over PMA-stimulated THP-1 macrophages in the absence (A-D) or presence (E-H) of botrocetin (1.6 U/ml). Perfusion was performed for 15 minutes at 5 dyne/cm2. Nuclei were stained with DAPI (blue), actin was stained with AlexaFluor 488-conjugated phalloidin (green) and VWF was detected with primary rabbit anti-human VWF antibody followed by TRITC-conjugated goat anti-rat IgG antibody (red). Original magnification is X 100. (I) VWF internalization was quantified with ImageJ as the percentage of surface covered by red spots (VWF staining) per number of cells per field. Data represent mean±SEM. *P < 0.05. Previously, we have reported that macrophages bind and endocytose VWF18, and recently it was found that activation of VWF via exposure to shear stress enhances this process.19,20 VWF may also be forced in its active conformation via VWD-type 2B mutations or via modulators such as botrocetin. Therefore, we first addressed the possibility that botrocetin enhances uptake of VWF by macrophages. To this end, VWF was perfused over PMA-stimulated THP-1 cells at low shear stress (5 dynes/ cm2) for 15 min in the absence or presence of botrocetin. Cells were then washed to remove extracellular VWF, fixed and stained for VWF via immunofluorescence. The number of VWF-positive cells was similar in the absence or presence of botrocetin (16/29 and 17/27, respectively). However, it appeared that more VWF was taken up per cell by macrophages in the presence of botrocetin compared to in its absence (Fig. 1A-H). Quantitative analysis using ImageJ, confirmed that botrocetin enhanced VWF endocytosis by 7-fold (Fig. 1I). Apparently, VWF is endocytosed by macrophages more efficiently when it is in its active, platelet-binding conformation. Chapter 6 Chapter

117 Clearance of VWF/platelet complexes in VWD-type 2B complexes of VWF/platelet Clearance Fig. 2. Uptake of p.R1306Q-coated beads by macrophages. Fluoresbrite-YG microspheres (1 X 104 beads/well) coated with HSA, wt-rVWF or p.R1306Q-rVWF (25 µg/1 X 104 beads) were incubated with PMA-stimulated THP1 cells adhered to glass coverslips for 1 h at 37˚C. Subsequently, cells were thoroughly washed to remove non- digested beads. The number of phagocytosed beads was quantified via microscopical analysis and is expressed as beads/cell (A). Data represent the mean ± SD of two independent experiments, in which at least three microscopic fields were analyzed. (B-C) Representative images of wt-rVWF-coated and p.R1306Q-coated beads phagocytosed by THP1-macrophages. (D-F) Scanning electron images of macrophages phagocytosing microspheres coated with p.R1306Q-rVWF. (D) The first moment of capturing a VWF-coated bead. (E) A 3 µm hole through which a bead has been phagocytosed. (F) Macrophage that has phagocytosed multiple beads. Chapter 6 Chapter

118 Clearance of VWF/platelet complexes in VWD-type 2B complexes of VWF/platelet Clearance

Fig. 3. VWF/platelet complexes circulate in blood of VWD-type 2B mice. Blood smears prepared with blood col- lected mice expressing either wt-mVWF (A), p.R1306Q-mVWF (B-C) or p.V1316M-mVWF (D). Mice had normal or slightly low platelet counts (A-B) or were thrombocytopenic (C-D). Platelets were detected by staining for the CD41-surface marker (red) and VWF (green) was detected with primary rabbit anti-human VWF antibody followed by a secondary AlexaFluor 488-goat anti-rabbit antibody. Original magnification is X100. (A-B) single platelets can be observed and no VWF at the platelet surface could be detected. (C-D) Thrombocytopenic mice display circu- lating single platelets and platelet aggregates, both of which being characterized by the presence of VWF at the platelet surface. VWD-type 2B mutations enhance VWF internalization by macrophages

Next, we tested how the active VWD-type 2B configuration affects uptake of VWF by macrophages. Fluorescent beads were coated with human albumin (HSA), wt- rVWF or the p.R1306Q-rVWF type 2B mutant, and subsequently incubated with THP-1 macrophages. Cells were then washed to remove extracellular microspheres. More wt-rVWF-coated than HSA-coated beads were phagocytosed (2.0 ± 0.9 versus 0.4 ± 0.2 beads/cell; p < 0.05; Fig. 2A), further validating that macrophages bind and internalize VWF. Interestingly, the uptake was even further increased for p.R1306Q- rVWF-coated beads (3.6 ± 1.1 beads/cell; p < 0.05 compared to wt-rVWF-coated beads; Fig. 2A-C). The process of internalization of p.R1306Q-rVWF-coated beads was analyzed in more detail via scanning electron microscopy. Several stages could be distinguished: the beginning of the formation of ruffles around a coated bead (stage 1; Fig. 2D), engulfment in an advanced stadium (stage 2; Fig. 2E) and complete internalization of multiple beads (stage 3; Fig. 2F). These results indicate that VWD-type 2B mutations promote the uptake of VWF by macrophages. 6 Chapter VWF-covered platelets circulate in vivo in VWD-type 2B mice 119

The observation that VWD-type 2B mutations promote phagocytosis of micropheres by macrophages may be extended to platelets likely to absorb VWD-type 2B mutants to their surface. Indeed, electron microscopical analysis has previously revealed the presence of VWF at the surface of platelets derived from VWD-type 2B patients21, showing that VWF-coated platelets may be present in the circulation of VWD- type 2B patients. We therefore tested whether VWF is also present at the surface of platelets in VWD-type 2B mice and if this correlates with the presence or absence of thrombocytopenia. Different sets of mice were considered: mice expressing (1) wt-mVWF, which have normal platelet counts (652 ± 196 X 103 platelets/ml; n=18); (2) p.V1316M-mVWF, which have severe thrombocytopenia (177± 113 X 103 platelets/ml; n = 26); or (3) p.R1306Q, which have either mild thrombocytopenia (485 ± 93 X103 platelets/ml; n = 37) or severe thrombocytopenia (221 ± 65 X 103 platelets/ml; n = 14). Blood smears were prepared with EDTA-anticoagulated blood and immunofluorescence staining was performed for VWF together with the platelet surface-marker CD41. In non-thrombocytopenic mice or mildly thrombocytopenic mice, we observed numerous single CD41-positive platelets. However, no VWF in VWD-type 2B complexes of VWF/platelet Clearance could be detected at the surface of these platelets (Fig. 3A & 3B). As expected, blood smears of thrombocytopenic mice contained fewer platelets, which were present as single platelets or assembled in platelet aggregates. Interestingly, numerous platelets (both isolated and assembled in aggregates) stained strongly positive for VWF, irrespective whether thrombocytopenic mice were expressing mutant p.V1316M- mVWF or p.R1306Q-mVWF (Fig. 3C & 3D). These data indicate that VWF mutants carrying type 2B mutations may adsorb onto circulating platelets and that platelet- binding seems to be associated with the occurrence of thrombocytopenia. Macrophage depletion relieves thrombocytopenia in VWD-type 2B mice

Having established that (a) microspheres coated with a VWD-type 2B mutant are taken up efficiently by macrophages, (b) such mutants adsorb to the surface of circulating platelets, and (c) adsorption to platelets seems to be associated with thrombocytopenia, we further studied the role of macrophages in vivo in the removal of VWF/platelet complexes from the circulation. Mice expressing wt-mVWF, p.R1306Q-mVWF or p.V1316M-mVWF were treated with a single-dose infusion of the macrophage-destructing agent liposome-clodronate or the control liposome- PBS.22 Blood was collected one hour before and 24 & 48 hours after liposome- treatment and analyzed for platelet count and VWF antigen levels. VWF antigen was used as a measure for the efficacy of macrophage depletion.18 Administration of control liposome-PBS did not affect VWF levels in mVWF expressing mice (Fig. 4A). In contrast, VWF levels were increased 3- to 5-fold after liposome-clodronate treatment in mice expressing either wt-mVWF or mutant m-VWF (Fig. 4A), Chapter 6 Chapter indicating that macrophage depletion was successful in these mice. As for platelet 120 Clearance of VWF/platelet complexes in VWD-type 2B complexes of VWF/platelet Clearance

Fig. 4. Liposome-clodronate macrophage depletion relieves VWD-type 2B-associated thrombocytopenia. (A) VWF antigen levels were measured in mice before and 24 h after treatment with liposome-PBS or liposome- clodronate. VWF antigen levels after treatment were compared to those before treatment. Antigen levels before treatment were arbitrarily set at 100%. (B-D) Platelet counts were determined in mice expressing wt-mVWF (B), p.V1316M-mVWF (C) or p.R1306Q-mVWF (D) before and 24 & 48 h after injection with liposome-PBS or liposome- clodronate. Data represent mean±SEM of three injected mice/treatment. counts, these remained unchanged in wt-mVWF mice after injection of either the control liposome-PBS or liposome-clodronate, suggesting that macrophages play a minor role in the clearance of normal platelets in mice (Fig. 4B). As for VWD- type 2B mice, severe thrombocytopenia remained stable after control liposome-PBS treatment (Figs. 4C and 4D). In contrast, the infusion of liposome-clodronate in these mice resulted in a 2-3 fold increase in platelet counts (from 180 ± 24 X 103 platelets/ ml to 405 ± 34 X 103 platelets/ml after 48 h; p = 0.0015 for p.V1316M-mVWF & 326 ± 88 X 103 platelets/ml to 608 ± 21 X 103 platelets/ml after 48 h; p = 0.0028 for p.R1306Q-mVWF), thereby relieving the severe thrombocytopenic state (Figs. 4C and 4D). These results point to a dominant role of macrophages in the uptake of VWF/platelet complexes that circulate in VWD-type 2B. Platelets are targeted to macrophages in liver and spleen of VWD-type 2B mice

Next, we determined whether VWF/platelet complexes are indeed targeted to Chapter 6 Chapter macrophages in liver and/or spleen of VWD-type 2B mice. To this end, platelets isolated from p.V1316M-mVWF expressing mice were isolated, stained with 121 fluorescent CMTMR-cell tracker and re-infused in mice expressing p.V1316M- mVWF. Two hours after platelet infusion, liver and spleen were harvested, snap- frozen and organ cryo-sections were prepared. To identify the cellular destination of VWF/platelet complexes in liver and spleen of these mice, cryo-sections were stained for organ specific macrophage subpopulations via immunofluorescence. In liver, the vast majority (78 ± 9%) of platelets co-localized with resident Kupffer cells, identified via a monoclonal anti-mouse CD68 antibody (Fig. 5A). A more dispersed picture was obtained for the spleen. We observed some platelets co-localizing with F4/80- positive macrophages in the red pulp, and with MARCO-positive macrophages in the marginal zone (not shown). However, most of the platelets present in the spleen (68 ± 6%) co-localized with CD169-positive marginal metallophilic macrophages (Fig. 5B). Increased uptake of platelets in spleen and liver of VWD-type 2B mice

To compare the quantitative distribution of platelets in wt-mVWF- expressing and p.V1316M-mVWF-expressing mice, organs were isolated 2 h after injection of Clearance of VWF/platelet complexes in VWD-type 2B complexes of VWF/platelet Clearance fluorescent platelets Subsequently, liver and spleen non-consecutive whole cryo- section slides (n= 6 and 9 for liver and spleen, respectively) were scanned using NanoZoomer-equipment, and fluorescence was subsequently quantified with ImageJ software. Representative images of liver and spleen sections of wt-mVWF and p.V1316M-mVWF-expressing mice are shown in figure 6. Quantitative analysis revealed that both liver and spleen of wt-mVWF-expressing mice contained significantly less fluorescent spots, and thus platelets, compared to organs taken from p.V1316M-mVWF-expressing mice (p = 0.0098 and p = 0.0002 for liver and spleen, respectively; Fig. 6C & 6F). Thus, our results indicate that there is an Chapter 6 Chapter

122 Clearance of VWF/platelet complexes in VWD-type 2B complexes of VWF/platelet Clearance

Fig. 5. Platelets co-localize with macrophages in liver and spleen of VWD-type 2B mice. Platelets were isolated from wt-mVWF or p.V1316M-mVWF expressing mice, stained with CMTMR cell tracker and re-infused (8 X 107 platelet/mouse) in wt-mVWF or p.V1316M-mVWF expressing mice, respectively. Two hours after platelet infusion, liver and spleen were isolated to prepare cryo-sections. Macrophage subpopulations were identified via immuno- fluorescence: CD68-positive resident macrophages (Kupffer cells) in liver (A), CD169-positive marginal metallophilic macrophages in spleen (B). Macrophages are in red, CMTMR-fluorescent platelets are in green and nuclei in blue. Original magnification is X 20. accumulation of VWF/platelet complexes in liver and spleen of VWD-type 2B mice and that macrophages are implicated in this process. Platelet clearance is accelerated in VWD-type 2B

The finding that VWF/platelet complexes in VWD-type 2B mice are takenup efficiently by macrophages in liver and spleen may suggest that the platelets have a shorter circulatory lifespan in these mice compared to normal mice. We therefore decided to measure platelet survival in mice expressing wt-mVWF or mutant mVWF. Four days after onset of VWF-expression, mice were intravenously infused with NHS-biotin allowing biotinylation of > 90% of the platelets.17 It has previously been demonstrated that this approach does not interfere with platelet function or lifespan in vivo.17 The disappearance of biotinylated platelets was followed daily as percentage of residual biotinylated platelets relative to the total CD41-positive platelet population. A more rapid disappearance of platelets in p.V1316M-mVWF or p.R1306Q-mVWF expressing mice compared to wt-mVWF expressing mice Chapter 6 Chapter was observed (Fig. 7). For instance, residual platelet numbers 48 h after injection were 29.6±8.4% (p.V1316M) and 34.9 ± 8.8% 9p.R1306Q) versus 47.3 ±7.1 % 123 in VWD-type 2B and wt-mVWF-expressing mice, respectively (p < 0.001 and p = 0.02). Interestingly, the p.R1306Q seems to have an intermediate clearance rate between p.V1316M-mVWF and wt-mVWF: similar platelet counts as wt-mVWF were observed after 24 h, while similar to p.V1316M-mVWF no platelet could be detected after 72 h. Of note, a similar disappearance rate of platelets was found for wt-mVWF-expressing mice and for control wt-C57B6 mice (49.5 ± 6.2% residual platelets after 48 h; not shown). The observation that platelet lifespan is reduced in VWD-type 2B mice, suggests that the presence of mutated VWF at the platelet surface might accelerate their clearance. Discussion VWD-type 2B is a paradoxical example where the assembly of platelets into aggregates is associated with bleeding rather than thrombosis. Interestingly, the bleeding tendency in VWD-type 2B patients correlates strongly with their platelet count, as became apparent in a cohort study involving 67 patients.4 The bleeding risk is indeed 5-fold higher in patients having platelets counts below 140´103 Clearance of VWF/platelet complexes in VWD-type 2B complexes of VWF/platelet Clearance platelets/ml compared to those with 140´103 platelets/ml or more.4 The degree of thrombocytopenia is also of relevance for the clinical management of the patients in that it may affect the choice of treatment. Desmopressin is usually contra-indicated for use in VWD-type 2B patients, but it has been used safely in several patients with a mildly reduced platelet count.11,23-26 In contrast, replacement-therapy is preferred for VWD-type 2B patients that manifest severe thrombocytopenia, eventually in combination with platelet transfusion. Alternatively, agents blocking VWF-platelet interactions may be used to correct desmopresin-induced thrombocytopenia.27 The molecular basis of the thrombocytopenia in VWD-type 2B has remained obscure and may originate from a disturbed megakaryocytopoiesis.5-7 However, it has originally been proposed that the thrombocytopenia would result from the removal of VWF/platelet aggregates from the circulation.8-10 By using a mouse model for VWD-type 2B, we provide direct evidence that the formation of VWF/ platelet aggregates is indeed associated with an enhanced uptake of such complexes Chapter 6 Chapter

124 Clearance of VWF/platelet complexes in VWD-type 2B complexes of VWF/platelet Clearance

Fig. 6. Quantification of wt- or V1316M-platelet-recovery in liver and spleen of wt- and p.V1316M-mVWF- expressing mice. Liver (A-B) and spleen (panels D-E) cryo-sections were prepared as described in the legend of Fig. 5 and scanned with a NanoZoomer. Images comprising whole sections were generated via Virtual Microscopy- technology using NDPview software. Quantification (C, F) was performed using ImageJ-software and is expressed as the percentage of surface covered by fluorescent spots (corresponding to fluorescent platelets) per field. Data represent mean±SEM. The number of fields analyzed is indicated in the figure (**P = 0.0098, ***P = 0.0002). Representative images for cryo-sections of liver (A-B) and spleen (D-E) obtained from wt-mVWF-expressing mice (A-D) or p.V1316M-mVWF-expressing mice (B-E) are shown. Arrows point to fluorescent spots. in macrophages in liver and spleen (Figs. 5 & 6). In addition, the lifespan of platelets in VWD-type 2B mice is significantly shorter compared to the lifespan of platelets in control mice (Fig. 7). Together, these mechanisms may contribute to the VWD-type 2B related thrombocytopenia observed in these mice. Evidence for the involvement of macrophages in the clearance of the VWF/platelet complexes became apparent from different experimental approaches. First, we tested the effect of chemical macrophage depletion. The efficacy of macrophage-depletion was assessed via the measurement of VWF antigen levels, which were increased following liposome-clodronate infusions but not after control liposome infusion

(Fig. 4). These data are in agreement with our previous observation in which GdCl3- mediated depletion of macrophages resulted in increased endogenous VWF antigen levels.18 Macrophage depletion did not affect platelet counts in mice expressing wt- mVWF within the 48-h observation period (Fig. 4). This observation is in line with a previous study that reported no effect on platelet numbers in control mice within 28 the first 48 hours after liposome-clodronate injection. Interestingly, we observed a 6 Chapter remarkable 2-3 fold increase in platelet counts in thrombocytopenic mice expressing 125 p.V1316M-mVWF: from 157±62 ´103 platelets/ml to 420±30 ´103 platelets/ml after 24 h and 405±60 ´103 platelets/ml after 48 h (Fig. 4). Thus, macrophage depletion relieves the thrombocytopenic state in VWD-type 2B mice. Similarly, in a mouse model for immune thrombocytopenia, a comparable increase in platelet counts following macrophage depletion was described Alves-Rosa and colleagues.28 More direct evidence for the role of macrophages in the scavenging of VWF/platelet complexes came from immuno-histochemical analysis of liver and spleen sections of mice injected with fluorescently labeled platelets. In liver, near 80% of fluorescent platelets co-localized with CD68-positive macrophages, i.e. Kupffer cells (Fig. 5). In spleen, the majority of platelets (68%) co-localized with CD169-positive marginal metallophilic macrophages (Fig. 5), although some platelets were found in association with F4/80-positive macrophages in the red pulp, and with MARCO- positive macrophages in the marginal zone. This selective targeting of VWF/platelets complexes to CD169-positive marginal metallophilic macrophages rather than F4/80- positive or MARCO-positive macrophages is unexpected, and additional studies are needed to decipher the mechanism behind this selective targeting. CD169-positive marginal macrophages are known for their role in homeostasis, clearance in VWD-type 2B complexes of VWF/platelet Clearance of apoptotic cells and the suppression of immune-responses towards these apoptotic cells.29,30 In contrast to CD169-positive marginal macrophages, the role of liver Kupffer cells in the clearance of platelets is less surprising. It has previously been shown that these cells mediate the uptake of platelets in immune thrombocytopenia.31 In addition, liver Kupffer cells also mediate the uptake of short-term refrigerated platelets.32 The mechanism that explains the enhanced phagocytosis of platelets by macrophages in VWD-type 2B mice remains to be elucidated. Our observation that Fig. 7. Platelet life span is reduced in VWD-type 2B mice with respect to wt-mVWF expressing mice. Mice expressing wt-mVWF (A-B, closed circle), p.V1316M-mVWF (A, open circles) or p.R1306Q-mVWF (B, open squares) were infused with NHS-biotin that allows platelet biotinylation. Residual biotinylated platelets were quantified

Chapter 6 Chapter by flow cytometry. Data are expressed as percentage of biotinylated platelets relative to the total CD41-positive platelet population with t = 0 being arbitrarily set at 100%. Data represent mean ± SD. N = 3 to 13 mice. 126 thrombocytopenia only occurs in those mice where circulating platelets are covered by VWF mutants (Fig. 3) seems to indicate that increased phagocytosis is limited to VWF/platelet complexes. It is of interest to mention that it has been assumed that in particular HMW-multimers adsorb to the platelet surface, since it is those multimers that are lacking in VWD-type 2B. However, we have reported that HMW-multimers are selectively lacking in VWD-type 2B mice that express ADAMTS13, whereas the full multimer range is present in VWD-type 2B mice that are deficient for this VWF-cleaving protease.12 This strongly suggests that the lack of HMW-multimers is mainly due to ADAMTS13-mediated degradation rather than adsorption on platelets. In contrast, adsorption of either large or small multimers onto platelets is more likely to be the signal that provokes uptake of VWF/platelet complexes by macrophages. The notion that both VWF and platelets are potential ligands for macrophages complicates the elucidation of the exact phagocytosis mechanism. Previously, we and others have shown that shear stress enhances the LRP1-dependent uptake of VWF by macrophages.18-20 In the present study, we found that conversion of VWF in its active, platelet-binding conformation (either by botrocetin or via a gain-of-function Clearance of VWF/platelet complexes in VWD-type 2B complexes of VWF/platelet Clearance VWD-type 2B mutation) also promotes the uptake of VWF by macrophages (Figs. 1 & 2). It is possible therefore that platelets are a bycatch when macrophages bind and endocytose VWF-type 2B mutants that are at the platelet surface. Alternatively, binding of mutant VWF to the platelet GPIbα-receptor may modulate platelets in such a manner that the platelet is more easily phagocytosed by macrophages. For instance, VWF binding may mimic exposure of platelets to cold and induce clustering of the VWF-receptor GPIbα, a process known to promote platelet uptake by macrophages.33 Additional studies are needed to distinguish between the various possibilities. In conclusion, we have now identified at least one of the mechanisms underlying thrombocytopenia in VWD-type 2B. Although long suspected, the increased removal of VWF/platelet aggregates from the circulation had never been formally demonstrated. It is becoming clear that in severe VWD-type 2B such as in patients with the p.V1316M mutations, other mechanisms are also involved, in particular with relation to megakaryocytopoiesis. Our results with mice expressing the p.R1306Q mutation are also of particular interest since they suggest that the situation can be fluctuating: under specific circumstances, the mutant can start accumulating on platelets, a situation associated with more severe thrombocytopenia. In a clinical setting, such potential variations should be closely monitored, for instance during pregnancy. Our findings may also be of relevance for other pathological conditions where the presence of active VWF is associated with thrombocytopenia, such as thrombotic thrombocytopenic purpura or malaria.34,35. Indeed, both disorders are associated with an increased uptake of platelets by macrophages36,37, which could be due to the adsorption of active VWF to the platelet surface under these conditions. Chapter 6 Chapter It is tempting to speculate that this process may be a mechanism to minimize the presence of circulating VWF/platelet complexes, which have the potential to occlude 127 the microvasculature. Acknowledgements The authors would like to acknowledge Dr. Nico van Rooijen of the Foundation Clodronate Liposomes (Haarlem, the Netherlands) for generously providing the liposomes clodronate, Dr. Olivier Trassard for help with microscopic analysis and Hamamatsu (Massy, France) for making the NanoZoomer 2.0-HT available for analysis. Financial support from the Fondation pour la Recherche Médicale (FRM- SPF20101220866; CC & PJL), from Agence Nationale de la Recherche (ANR-11- BSV1-010-01; CVD), from the Dutch Heart Foundation (NHS 2006T5301; BdL) and from Sanquin Blood Supply Foundation (BdL) is gratefully acknowledged. Clearance of VWF/platelet complexes in VWD-type 2B complexes of VWF/platelet Clearance References type IIB von Willebrand’s disease. Blood. 1987;69(3):786-789. 1. Ruggeri ZM. The role of von Willebrand 10. Mathew P, Greist A, Maahs JA, Lichtenberg factor in thrombus formation. Thromb Res. EC, Shapiro AD. Type 2B vWD: the varied 2007;120 Suppl 1:S5-9. clinical manifestations in two kindreds. 2. Sadler JE, Budde U, Eikenboom JC, et Haemophilia. 2003;9(1):137-144. al. Update on the pathophysiology and 11. De Meyer SF, Deckmyn H, Vanhoorelbeke classification of von Willebrand disease: K. von Willebrand factor to the rescue. a report of the Subcommittee on von Blood. 2009;113(21):5049-5057. Willebrand Factor. J Thromb Haemost. 2006;4(10):2103-2114. 12. Rayes J, Hollestelle MJ, Legendre P, et al. Mutation and ADAMTS13-dependent 3. Federici AB, Canciani MT. Clinical and modulation of disease severity in a mouse laboratory versus molecular markers for model for von Willebrand disease type 2B. a correct classification of von Willebrand Blood. 2010;115(23):4870-4877. disease. Haematologica. 2009;94(5):610- 615. 13. Golder M, Pruss CM, Hegadorn C, et al.

Chapter 6 Chapter Mutation-specific hemostatic variability 4. Federici AB, Mannucci PM, Castaman G, in mice expressing common type 2B von 128 et al. Clinical and molecular predictors of Willebrand disease substitutions. Blood. thrombocytopenia and risk of bleeding in 2010;115(23):4862-4869. patients with von Willebrand disease type 2B: a cohort study of 67 patients. Blood. 14. Denis C, Methia N, Frenette PS, et al. A 2009;113(3):526-534. mouse model of severe von Willebrand disease: defects in hemostasis and 5. Nurden P, Debili N, Vainchenker W, thrombosis. Proc Natl Acad Sci U S A. et al. Impaired megakaryocytopoiesis 1998;95(16):9524-9529. in type 2B von Willebrand disease with severe thrombocytopenia. Blood. 15. Lenting PJ, Westein E, Terraube V, et al. 2006;108(8):2587-2595. An experimental model to study the in vivo survival of von Willebrand factor. Basic 6. Nurden AT, Federici AB, Nurden P. Altered aspects and application to the R1205H megakaryocytopoiesis in von Willebrand mutation. J Biol Chem. 2004;279(13):12102- type 2B disease. J Thromb Haemost. 2009;7 12109. Suppl 1:277-281. 16. Marx I, Christophe OD, Lenting PJ, et 7. Nurden P, Gobbi G, Nurden A, et al. Abnormal al. Altered thrombus formation in von VWF modifies megakaryocytopoiesis: Willebrand factor-deficient mice expressing studies of platelets and megakaryocyte von Willebrand factor variants with defective cultures from patients with von Willebrand binding to collagen or GPIIbIIIa. Blood. Clearance of VWF/platelet complexes in VWD-type 2B complexes of VWF/platelet Clearance disease type 2B. Blood. 2010;115(13):2649- 2008;112(3):603-609. 2656. 17. Berger G, Hartwell DW, Wagner DD. 8. Saba HI, Saba SR, Dent J, Ruggeri ZM, P-Selectin and platelet clearance. Blood. Zimmerman TS. Type IIB Tampa: a variant 1998;92(11):4446-4452. of von Willebrand disease with chronic thrombocytopenia, circulating platelet 18. van Schooten CJ, Shahbazi S, Groot E, et aggregates, and spontaneous platelet al. Macrophages contribute to the cellular aggregation. Blood. 1985;66(2):282-286. uptake of von Willebrand factor and factor VIII in vivo. Blood. 2008;112(5):1704-1712. 9. 9. Rick ME, Williams SB, Sacher RA, McKeown LP. Thrombocytopenia 19. Rastegarlari G, Pegon JN, Casari C, et associated with pregnancy in a patient with al. Macrophage LRP1 contributes to the clearance of von Willebrand factor. Blood. purpura in a mouse model. Blood. 2012;119(9):2126-2134. 2000;96(8):2834-2840. 20. Castro-Nunez L, Dienava-Verdoold I, 29. Gordy C, Pua H, Sempowski GD, He Herczenik E, Mertens K, Meijer AB. YW. Regulation of steady-state neutrophil Shear stress is required for the endocytic homeostasis by macrophages. Blood. uptake of the factor VIII-von Willebrand 2011;117(2):618-629. factor complex by macrophages. J Thromb 30. Miyake Y, Asano K, Kaise H, Uemura Haemost. 2012;10(9):1929-1937. M, Nakayama M, Tanaka M. Critical role 21. Nurden P, Chretien F, Poujol C, Winckler of macrophages in the marginal zone in J, Borel-Derlon A, Nurden A. Platelet the suppression of immune responses to ultrastructural abnormalities in three patients apoptotic cell-associated antigens. J Clin with type 2B von Willebrand disease. Br J Invest. 2007;117(8):2268-2278. Haematol. 2000;110(3):704-714. 31. Neiman JC, Mant MJ, Shnitka TK. 22. van Rooijen N. Liposome-mediated Phagocytosis of platelets by Kupffer cells in elimination of macrophages. Res Immunol. immune thrombocytopenia. Arch Pathol Lab 1992;143(2):215-219. Med. 1987;111(6):563-565. Chapter 6 Chapter 23. Mannucci PM. How I treat patients with von 32. Hoffmeister KM, Felbinger TW, Falet H, 129 Willebrand disease. Blood. 2001;97(7):1915- et al. The clearance mechanism of chilled 1919. blood platelets. Cell. 2003;112(1):87-97. 24. McKeown LP, Connaghan G, Wilson 33. Josefsson EC, Gebhard HH, Stossel O, Hansmann K, Merryman P, Gralnick TP, Hartwig JH, Hoffmeister KM. The HR. 1-Desamino-8-arginine-vasopressin macrophage alphaMbeta2 integrin alphaM corrects the hemostatic defects in type 2B lectin domain mediates the phagocytosis von Willebrand’s disease. Am J Hematol. of chilled platelets. J Biol Chem. 1996;51(2):158-163. 2005;280(18):18025-18032. 25. Mauz-Korholz C, Budde U, Kruck H, 34. Hulstein JJ, de Groot PG, Silence K, Korholz D, Gobel U. Management of Veyradier A, Fijnheer R, Lenting PJ. A novel severe chronic thrombocytopenia in von nanobody that detects the gain-of-function Willebrand’s disease type 2B. Arch Dis phenotype of von Willebrand factor in Child. 1998;78(3):257-260. ADAMTS13 deficiency and von Willebrand disease type 2B. Blood. 2005;106(9):3035- 26. Schneppenheim R. The pathophysiology 3042. of von Willebrand disease: therapeutic implications. Thromb Res. 2011;128 Suppl 35. de Mast Q, Groot E, Lenting PJ, et al. 1:S3-7. Thrombocytopenia and release of activated von Willebrand Factor during early

27. Jilma-Stohlawetz P, Knobl P, Gilbert JC, in VWD-type 2B complexes of VWF/platelet Clearance Plasmodium falciparum malaria. J Infect Jilma B. The anti-von Willebrand factor Dis. 2007;196(4):622-628. aptamer ARC1779 increases von Willebrand factor levels and platelet counts in patients 36. Jaff MS, McKenna D, McCann SR. Platelet with type 2B von Willebrand disease. phagocytosis: a probable mechanism of Thromb Haemost. 2012;108(2):284-290. thrombocytopenia in Plasmodium falciparum infection. J Clin Pathol. 1985;38(11):1318- 28. Alves-Rosa F, Stanganelli C, Cabrera J, 1319. van Rooijen N, Palermo MS, Isturiz MA. Treatment with liposome-encapsulated 37. Kadri A, Noinuddin M, de Leeuw clodronate as a new strategic approach in the NK. Phagocytosis of blood cells by management of immune thrombocytopenic splenic macrophages in thrombotic thrombocytopenic purpura. Ann Intern Med. 1975;82(6):799-802. Chapter 6 Chapter

130 Clearance of VWF/platelet complexes in VWD-type 2B complexes of VWF/platelet Clearance Supplementary Materials Materials and Methods Reagents Monoclonal rat anti-mouse CD41 antibody (clone MWReg30; unlabeled & FITC- labeled) and FITC-labeled rat IgG1κ-isotype control were from BD Biosciences (Le Pont de Claix, France). Polyclonal rabbit anti–Human VWF (unlabeled & peroxidase-conjugated) were obtained from Dako (Trappes, France). Apyrase and Prostaglandin E1 were from Sigma-Aldrich (Saint-Quentin, France). CellTracker

CMTMR-Orange, purified mouse IgG, AlexaFluor 488 F(ab’)2 fragment of goat anti-rabbit IgG (H+L), AlexaFluor 488-conjugated phalloidin, DAPI and ProLong Gold-antifade reagent were from Invitrogen (Saint Aubin, France). Monoclonal rat anti-mouse CD68 antibody (clone FA-11) and monoclonal rat anti-mouse CD169 antibody (clone 3D6.112) were from AbD Serotec (Colmar, France). AlexaFluor

647-conjugated AffiniPure goat anti-rat IgG was from Jackson ImmunoResearch 6 Chapter (Marseille, France). TRITC-conjugated goat anti-rat IgG was from SouthernBiotech 131 (Nanterre, France). N-hydroxysuccinimide (NHS)-biotin and Phycoerythrin (PE)- conjugated streptavidin were from Calbiochem (Darmstadt, Germany). Fluoresbrite- YG microspheres (3.0 micron) were from Polysciences Inc (Warrington PA, USA). Human TGF-β1 was from R&D Systems (Minneapolis MN, USA). RPMI-1640 medium was obtained from Gibco (Paisley, UK). Phorbol 12-myristate 13-acetate (PMA) was from BP-Biochemical (St. Louis MO, USA). Purified plasma-derived VWF and recombinant VWF were prepared as described.1,2 Perfusion experiments THP-1 cells were cultured in RPMI-1640 medium supplemented with fetal calf serum (10%), β-mercaptoethanol (50 μM), penicillin/streptomycin (1%). Differentiation into macrophages was performed by culturing THP-1 cells on glass coverslips in RPMI-1640 medium supplemented with TGF-β1 (1 ng/ml) and PMA (250 nM) for 48 hours. Subsequently, glass coverslips with matured THP-1 macrophages were transferred to the perfusion system. VWF (5 μg/ml) was perfused over the THP-1 macrophages in the absence or presence of botrocetin (1.6 U/ml) at a shear stress of 5 dynes/cm2. After 15 minutes, the perfusion solution was changed for a phosphate buffered saline (PBS) solution containing para-formaldehyde (4%), which was in VWD-type 2B complexes of VWF/platelet Clearance perfused over the cells for 15 min at a shear stress of 1 dyne/cm2. Glass coverslips were then collected, rinsed with PBS and incubated with PBS/ bovine serum albumin (BSA, 3%) to block non-occupied sites and cells were permeabilized by the addition of PBS/Triton X-100 (0.5%). Cells were stained for VWF, the cell membrane and the nucleus using polyclonal anti-VWF antibodies & TRITC-conjugated goat anti- rat IgG, AlexaFluor 488-conjugated phalloidin and DAPI, respectively. Images were acquired using a Zeiss Observer Z1 fluorescence microscope equipped with an EC Plan-Neofluor 100×/1.4 NA oil objective. Scanning electron microscopy THP-1 cells were differentiated into macrophages as described above, while cultured on glass coverslips in 6-wells plates. Before incubation with VWF-coated microspheres, cells were washed 4 times with PBS and subsequently kept in RPMI- 1640 medium without supplements. VWF-coated microspheres were prepared by the incubation of Fluoresbrite-YG (3 μm) with purified recombinant wt-VWF, VWF/p. R1306Q or human serum albumin (HSA) (25 μg/1×104 beads) as described.3 Adsorption of VWF to microspheres does not coincide with the exposure of the shear stress-dependent LRP1-binding site,3 suggesting that this procedure is associated with minimal activation of VWF. Beads were diluted in RPMI-1640 medium, added to the cells (1×104 beads/well) and incubated for 1 hour at 37˚C. After washing using PBS, images were acquired using a Zeiss Observer Z1 fluorescence microscope equipped with an EC Plan-Neofluor 100×/1.4 NA oil objective. For scanning electron microscopy analysis, cells were fixated in glutaraldehyde (2.5%) for 1 hour at 4˚C.

Chapter 6 Chapter Cells were then washed 5 times using PBS and fixed using osmium tetroxide (1%). After another washing step, cells were dehydrated via subsequent 5-min incubations 132 in ethanol (25%, 50%, 75% and 100%, respectively). Finally, samples were coated with gold alloy. Images were acquired using a Hitachi S-2500 scanning electron microscope. Platelet preparation Mice were anesthetized with tribromoethanol 2.5% (0.15 ml/10 g body weight) and blood was obtained by retro-orbital bleeding in 10% (vol/vol) ACD-C buffer (124mM tri-sodium citrate, 130mM citric acid, 110mM dextrose, pH6.5). Platelet rich plasma (PRP), obtained by centrifugation for 7 minutes at 160g, was further centrifuged for 10 minutes at 670g and washed in 140mM NaCl, 5mM KCl, 12mM tri-sodium citrate, 10mM glucose and 12.5mM saccharose (pH 6.5) supplemented with apyrase (100 mU/mL) and prostaglandin E1 (1mM). Platelets were finally diluted in HEPES-Tyrode buffer (10mM HEPES, 140mM NaCl, 3mM KCl, 5mM NaHCO3, 0.5mM MgCl2, and 10mM glucose; pH7.4). Quantitative analysis of platelet biodistribution Washed platelets were fluorescently labeled with CMTMR-Orange (10μM for 30

Clearance of VWF/platelet complexes in VWD-type 2B complexes of VWF/platelet Clearance minutes at 37°C). Fluorescent platelets were centrifuged to remove non-incorporated dye and diluted in HEPES-Tyrode buffer to final concentration of 4×105 platelets/ μl. Platelet preparations (200 μl) were infused in the tail vein. Subsequently, liver and spleen were harvested two hours after injection and snap-frozen in Tissue-Tek OCT-compound (Sajura Finetek, Zoeterwoude, the Netherlands) for cryosection preparation. Liver and spleen sections (3μm) were mounted with glycerol (90%) and scanned usingNanoZoomer 2.0-T equipped with a Fluorescence Module (Hamamatsu, Massy, France), which is a high-speed and high-resolution digital slide scanner system that acquires whole-slide digital images using time-delayed integration (TDI)-technology. TDI-technology allows scanning large area of whole slides without sacrificing scanning speed, image resolution, or quality.4 Whole-slide images were generated by NDP.Scan 2.5.80 (Hamamatsu) using 1x DAPI, 2x FITC, 2x TRITC exposure and 40x magnification. Virtual images (20x magnification) covering the whole organ area were generated with the NDP.view 1.2.46 software (Hamamatsu) and finally examined with ImageJ 1.45s software for quantitative analysis. Immunofluorescence microscopy Liver and spleen cryosections were fixed in 3.7% formaldehyde (liver) or ice-cold acetone (spleen) and subsequently blocked in PBS/BSA 3% solution in the presence of mouse IgG (2.5 μg/ml). Primary antibodies recognizing specific subpopulations of macrophages were incubated over night at 4°C. After incubation for 1 hour with appropriate secondary antibodies, sections were dried in the dark and mounted with ProLong Gold-antifade reagent with the addition of DAPI (1μg/ml). Blood smears were prepared with 5μl EDTA-anticoagulated blood on a glass slide and dried for several hours. After fixation in 3.7% formaldehyde, primary antibodies were applied 6 Chapter for 30 minutes at 37°C followed by incubation with secondary antibodies (30 min at 133 37˚C). Finally, slides were carefully washed and mounted with ProLong Gold-antifade reagent. Images were acquired with an AxioImager A1 (Carl Zeiss, Gottingen, DE) equipped with EC Plan-Neofluor 20x/0.5 or 40x/0.75 air objective for organ section exploitation and with 100x/1.4 oil objective for blood smear analysis. Platelet survival and flow cytometry Mouse platelets were biotinylated in vivo by tail vein infusion of 500μl NHS‐biotin (1.25 mg/ml) diluted in physiological serum. At indicated time points, mice were anesthetized by isofluorane and two drops of blood (60μl) were collected by retro- orbital bleeding in 500 μl Hanks’ Balanced Salt solution (HBSS) supplemented with ACD-C buffer (9% vol/vol). Platelets were isolated by subsequent centrifugation (100g for 7 minutes followed by 800g for 12 minutes) and finally resuspended in 500μl HBSS. Biotinylated platelets were identified by PE-Streptavin (5 μg) as a percentage of total platelet population identified with a FITC-conjugated rat anti- mouse CD41 antibody (2 μg/ml). Flow cytometric analysis was conducted using a Beckman Coulter EPICS XL flow cytometer and data analyzed with CellQuest

software. in VWD-type 2B complexes of VWF/platelet Clearance Liposome clodronateinduced macrophage depletion Liposome-encapsulated clodronate (containing 5 mg/ml of clodronate) and control liposome-PBS were generously provided the Foundation Clodronate Liposomes (Molecular Cell-Biology and Immunology, Free University Medical Center, Amsterdam, The Netherlands). Liposome suspensions (0.1 ml/10 gram of bodyweight) were applied via a single tail-vein injection. Mice were bled 24 & 48 hours after injection and platelet counts were determined with an automated animal blood-cell counter (Scil Vet ABC). Supp. Fig. 1. Effect of VWF:Ag levels on platelet counts. Mice were subjected to hydrodynamic gene transfer to induce expression of wt‐mVWF (green squares), mVWF/p.R1306Q (blue circles) or mVWF/p. 1316M (red circles). Chapter 6 Chapter Four days after the hydrodynamic injection, blood samples were taken and VWF:Ag and platelet counts were 134 determined. In the presence study, only mice with antigen levels between 200% and 1200% were included. Platelet counts appeared to be independent of VWF:Ag levels for both wt‐mVWF and mVWF/p.R1306Q, while a poor but statistically significant correlation was found for mutant mVWF/p.V1316M. Of note, even at the lower range of antigen levels for mVWF/p.V1316M a profound thrombocytopenia was observed, with platelets counts being dramatically lower compared to wt‐mVWF or mVWF/p.R1306Q.

Supplementary References R. High-throughput profiling of tissue and tissue model microarrays: 1. Sodetz JM, Pizzo SV, McKee PA. Combined transmitted light and 3-color Relationship of sialic acid to function fluorescence digital pathology. J Pathol and in vivo survival of human factor Inform. 2011;2:50. VIII/von Willebrand factor protein. J Biol Chem. 1977;252(15):5538-5546. 2. Lankhof H, Damas C, Schiphorst ME, et al. Recombinant vWF type 2A mutants R834Q and R834W show a defect in mediating platelet adhesion to collagen,

Clearance of VWF/platelet complexes in VWD-type 2B complexes of VWF/platelet Clearance independent of enhanced sensitivity to a plasma protease. Thromb Haemost. 1999;81(6):976-983. 3. Rastegarlari G, Pegon JN, Casari C, et al. Macrophage LRP1 contributes to the clearance of von Willebrand factor. Blood. 2012;119(9):2126-2134. 4. Nederlof M, Watanabe S, Burnip B, Taylor DL, Critchley-Thorne 7 From antibody to clinical phenotype, the black box of the antiphospholipid syndrome: pathogenic mechanisms of antiphospholipid syndrome Chapter 7 Chapter Vivian X.Du,1 ,2 Hilde Kelchtermans,3 Philip.G. de Groot1 and Bas de Laat1,2,3,4 135

1Department of Clinical Chemistry and Hematology, University Medical Center Utrecht, Utrecht, The Netherlands 2Sanquin Research, Amsterdam, The Netherlands 3Departement of Biochemistry, CARIM, University of Maastricht, Maastricht, The Netherlands

4 Synapse BV, Maastricht, The Netherlands syndrome aPLs in antiphospholipid

Thromb Res. 2013; 132: 319-326 Abstract The antiphospholipid syndrome (APS) is diagnosed by the combination of vascular thrombosis and/or pregnancy morbidity and the detection of antiphospholipid antibodies (aPLs) in plasma. In the last few years, a great effort has been made to unravel the mechanism by which aPLs cause thrombosis and a vast amount of mechanisms have been proposed. aPLs were proposed to induce a prothrombotic state by influencing the cellular blood compartment, the plasma compartment, the vascular wall and even metabolic pathways beyond the hemostatic system. However, due to the diversity in the mechanisms and the differences in the methodology, the focus of the mechanistical studies in this field seems to be largely diffused. It is hard to imagine that aPLs can exert such a diversity of effects, resulting in either thrombosis and/or pregnancy morbidity and the relationship between aPLs and the clinical manifestations remains to be a mysterious “black box”. In an attempt to get

Chapter 7 Chapter insight in what takes place inside the black box, we have analyzed 124 mechanistical studies on aPLs and discussed differences in the type of antibodies that were used, 136 the involvement of beta2-glycoprotein I (β2GPI), and the criteria used to diagnose APS patients. aPLs in antiphospholipid syndrome aPLs in antiphospholipid Introduction Vascular thrombosis and pregnancy morbidity are major health issues due to their high incidence in society and their life-threatening character. Although both clinical manifestations might have a different pathophysiology, patients suffering from the antiphospholipid syndrome (APS) can have a history of both thrombosis and/or pregnancy morbidity, indicating clinical heterogeneity 1. A patient is diagnosed with APS when he/she has a history of one of the former mentioned clinical symptoms (thrombosis and/or pregnancy morbidity) with the detection of antiphospholipid antibodies (aPLs) in the blood 2. It is now well-accepted that, although the name “antiphospholipid syndrome” implies direct binding to phospholipids, aPLs bind phospholipid-binding plasma proteins, of which beta2-glycoprotein I (β2GPI) is regarded as most relevant 3. At present, aPLs are detected by 3 different assays; anti-β2GPI IgM/IgG antibody assay, anticardiolipin IgM/IgG antibody assay and prolongation of phospholipid-dependent coagulation assays (lupus anticoagulant Chapter 7 Chapter (LAC)). Both the anti-β2GPI and the anticardiolipin ELISA are based on the 2;4 binding of auto-antibodies to plasma protein β2GPI . Prolongation of phospholipid- 137 dependent coagulation assays (known as LAC) can be caused by aPLs without further restriction to antibody specificity. However, it has been shown that LAC- causing antibodies with affinity towards β2GPI are clinically superior to antibodies 5;6 with specificity towards other plasma proteins . Apart from β2GPI, several other cofactors have been shown to relate to either vascular thrombosis or pregnancy morbidity. However, none of these plasma proteins have made it to the serological criteria of APS due to either controversy results, small sample size or the absence of confirmatory studies7 . As the name “syndrome” implies, a patient is diagnosed with APS by the association of aPLs in blood in combination with thrombosis and/or pregnancy morbidity and not by a causative relation between the clinical and serological criteria. In the last syndrome aPLs in antiphospholipid decade, numerous studies have tried to find the mechanism explaining the association between the serological and clinical criteria of APS 8. aPLs have been shown to exert a range of activities, including the promotion of coagulation, the activation of blood cells and of the complement system. However, so far there is no consensus about the pathophysiological mechanisms causing the thrombotic and obstetric events. A possible explanation for this heterogeneity is either that antibodies do have multiple actions leading to thrombotic events or that in these studies different type of antibodies have been used leading to different results. In order to find a possible reason for the potpourri of pathogenic mechanisms and to lift the “black box” effect, we have analyzed 126 studies focused on the mode of action of the auto-antibodies published between February 2006 (since the introduction of the revised ISTH ‘Sydney’ criteria ) and March 2012 2;4. Papers studying mechanistical actions of aPLs leading to clinical manifestations were selected and could be found on Pubmed using the terms “antiphospholipid”, “anti-phospholipid”, “beta2-glycoprotein I” or “apolipoprotein H”. Studies investigating predominantly associations between clinical complications and the presence of antibodies were excluded. In an attempt to understand the heterogeneity in the proposed mechanisms of aPLs, we investigated a possible correlation between the pathogenic mechanism

and (1), the source of (anti-β2GPI) antibodies (mouse/human); (2), character of the antibodies (monoclonal or polyclonal); (3) the criteria used to diagnose the APS

patients from which the antibodies were derived; and (4), the involvement of β2GPI. Proposed actions of antiphospholipid antibodies In the last decade, multiple hypotheses have been formulated to explain a possible prothrombotic character of aPLs. As Giannakopoulos et al. and de Groot et al. have published extensive reviews on this subject we will not go into details 8;9. In our study we have selected 126 publications, of which 76 focused on mechanisms regarding thrombosis, 18 focused on fetal loss, 5 on aPLs-related arthrosclerosis, 7 on central nervous system (CNS) pathology, 11 on the immunogenic mechanisms of Chapter 7 Chapter aPLs, and 7 on the possible new therapeutic approaches harvested from the proposed 138 mechanisms (Table 1). Interaction of aPLs with cells

Table 1 clearly shows that the main focus of studies from the last 6 years have been the effects of aPLs on cellular components of the blood or vessel wall, i.e. monocytes,platelets, and endothelial cells (ECs). aPLs have been demonstrated to cause increased tissue factor (TF) expression 10-13 and cytokine release on monocytes 14;15, leading to a prothrombotic status in APS patients. This increase in TF by aPLs was shown to be dependent on the activation of NF-kappaB and p38 MAPK and MEK-1/ ERK pathways 16-19. Multiple receptors and mediators have been demonstrated to be involved in this aPLs-mediated monocyte activation, such as annexin A2 20, toll-like aPLs in antiphospholipid syndrome aPLs in antiphospholipid receptor (TLR) 4 21, TLR2 22, TLR8 14, vascular endothelial growth factor 23, lipid raft 24, oxidative stress 25, and interleukin-1 receptor-associated kinases and tumour necrosis factor receptor-associated factors 26. In addition, several groups have shown the capacity for aPLs to activate platelets 27-29. The group of de Groot et al. has shown

that certain β2GPI-recognizing aPLs are able to interact with the Glycoprotein Ib (GP Ib)-apolipoprotein E receptor 2 (ApoER2) complex, leading to outside in signaling and sequential platelet activation 30-32. Infusion of mice with domain I of ApoER2 inhibits thrombus formation caused by 33 anti-β2GPI antibodies . In addition, platelet GP Ib alpha and Ia/IIa polymorphisms were found to correlate with a higher risk of thrombosis in APS. Platelet activation was indicated to be a better parameter than endothelial injury to identify risks of thrombosis in subjects positive for aPLs 29. In addition to ApoER2, platelet factor 34 4 was also proposed as a receptor for the β2GPI /anti-β2GPI complex . Besides dysfunction of monocytes and platelets, endothelial dysfunction is also a well- studied feature in APS. aPLs were shown to be able to induce a pro-inflammatory phenotype 35;36 and procoagulant activity in EC 37-39. In vitro stimulation of ECs with aPLs lead to time-dependent upregulation of interleukin (IL)-8 and intracellular adhesion molecule-1 (ICAM-1) mRNA and IL-8 expression 40. The group of Erwig et al. demonstrated an inhibitory effect of aPLs on apoptotic EC clearance. The delayed apoptotic EC clearance may provoke inflammatory and prothrombotic changes in professional phagocytes 41. TLR2, TLR4 and annexin A2 were proposed to be the receptor/mediator 42-44, Kruppel-like transcription factors and Vitamin D the modulators 45;46, and NF-kb/p38 MK the signaling pathways 47;48 in the aPLs- mediated EC dysfunction. aPLs and the coagulation system

Actions of aPLs on the coagulation system, such as the inhibition of the anticoagulant properties of activated protein C (APC) 49;50, impairment of the fibrinolysis 38;51-54, 55;56 reduction of the tissue factor pathway inhibitor (TFPI) activity , β2GPI-thrombin interaction 57-59, and disruption of annexin A5 anticoagulant shield have been Chapter 7 Chapter 60-64 extensively studied . β2GPI was demonstrated to be able to attenuate thrombin generation by inhibition of FXI activation. And the aPLs-induced dysregulation of 139 the thrombin inhibitory effect of β2GPI was insinuated to be an important mechanism for thrombosis in APS 65. Increased APC resistance can be caused by antibodies with different specificity such as2 β GPI, prothrombin and protein C itself. It is thought that anti-β2GPI antibodies induce increased APC resistance by competing for phospholipids with APC. Interestingly enough it was shown that increased APC 49 resistance is associated with a β2GPI-dependent LAC . This β2GPI-dependent LAC is highly related with thrombosis suggesting the same correlation for increased APC resistance. Other groups have found that anti-prothrombin antibodies also cause increased APC resistance 50. Besides APC resistance, disruption of an annexin A5 anticoagulant shield is also known to be an important thrombogenic mechanism.

60;61 syndrome aPLs in antiphospholipid Recently β2GPI has been found to possess properties of a regulator of the 66 human complement system . Upon binding apoptotic cells, β2GPI changes its conformation into an elongated shape. This J-shaped β2GPI was shown to exert C3 binding properties resulting in a conformational change in C3. Factor H is then able to bind and induce degradation of C3 by protease factor I. It has been demonstrated by several groups that by binding of β2GPI on the cellular surface of apoptotic cells, aPLs inhibit the regulatory function of β2GPI on the complement system and thereby the clearance of apoptotic cells. Effect of aPLs on fetal loss, arthrosclerosis, and central nervous system pathology

The number one mechanism in aPLs-mediated fetal loss is thought to be trophoblast perturbation. Possible effects of aPLs on trophoblasts include: modulating of trophoblast angiogenic factor secretion 67, limiting trophoblast migration 68;69, promoting non-apoptotic trophoblast shedding 70, and suppressing placenta growth Chapter 7 Chapter

140 aPLs in antiphospholipid syndrome aPLs in antiphospholipid factor production 71. Other mechanisms such as inflammation 72;73, neutrophil activation 74;75, impaired fibrinolysis76;77 , impaired endometrial angiogenesis 76, direct placenta damage 78, and disruption of the annexin A5 shield 79 were also proposed. Having aPLs in the circulation induces a risk factor for venous and arterial thrombosis and may contribute to the development of atherosclerosis although this latter statement was challenged 80. The mechanism of aPLs involvement in arthrosclerosis is poorly understood. It was published that IgG anti-β2GPI autoantibodies were able to affect lipid metabolism 81. Additionally, aPLs are associated with CNS pathology. Experimental APS models have been developed to study aPLs-related CNS 82-84. However, the underling mechanisms are still rather vague. Heterogeneity in methodology used to identify the mechanism of action of aPLs Type of antibodies Chapter 7 Chapter Recently a debate has been started about which antibodies should be used ideally to study the effect of aPLs on thrombosis or pregnancy morbidity. Main topics 141 in these discussions include the use of monoclonal versus polyclonal antibodies, human versus murine antibodies and total patient-derived versus affinity-purified IgG antibodies. We did not elaborate on the use of IgG versus IgM antibodies as studies using purely IgM antibodies are rare. It is known that patient-derived antibodies in general have a significantly lower affinity compared to murine antibodies. This difference in affinity may influence their effects on target organs. Furthermore, most murine antibodies have been raised by immunizing mice in the presence of adjuvants for increased production of antibodies. The resulting mixture of antibodies most likely recognizes different epitopes of

β2GPI and do not per se resemble the antibody profile in patients suffering from aPLs in antiphospholipid syndrome aPLs in antiphospholipid APS. In humans, several specific epitopes have been identified to be recognized by thrombosis-related antibodies with epitope G40-R43 as dominant one 85. Additional research is necessary to verify if epitope G40-R43 is the only important target of thrombosis-related antibodies. Another important difference among the studies we selected was the use of either purified total IgG or affinity purified anti-β2GPIIgG. It is known that only a small part of all the IgGs in the purified total IgG fraction recognizes β2GPI. Therefore, by using total IgG fractions in experiments, the result of a mixture of different types of antibodies is studied instead of purely anti-β2GPI antibodies. This problem can be avoided by using affinity-purified anti-β2GPI antibodies. On the other hand, this purification introduces the risk to miss potential (low-affinity) pathogenic antibodies.

Furthermore, the method used to couple β2GPI in affinity chromatography may impact the exposure of (cryptic) epitopes and thereby introduce a selection bias. Figure 1 shows that the type of antibodies used in the studies can be assigned to 4 categories: monoclonal murine antibodies, monoclonal human antibodies, patient- derived total IgG antibodies and affinity-purified patient-derived IgG antibodies. Some studies could not be assigned to any of these categories, as they used dimer-

β2GPI as a model for antibodies, goat-anti-human β2GPI antibodies, or induced antiphospholipid auto-antibodies in mice. These studies were put together in a separate category NA/different. Most of the selected studies used either patient- derived total IgG antibodies (45%) or monoclonal (human or murine) antibodies (both 18%) (Figure 1A). Studies that did not use either of these antibodies predominantly

used goat anti-human β2GPI antibodies, patient-derived IgM antibodies or mutants

of β2GPI mimicking the actions of antiphospholipid antibodies (19%). Subsequently, we verified if these differences in antibody usage could explain the differences in proposed pathogenic mechanisms of aPLs. Studies investigating coagulation pathways, monocytes and endothelial cells resemble the overall pattern of study

Chapter 7 Chapter A 142

B aPLs in antiphospholipid syndrome aPLs in antiphospholipid

Figure 1. Antibody usage in the aPLs-mediated mechanism studies. (A) The types of antibodies used in the mechanism studies could be assigned to 4 categories: monoclonal murine antibodies, monoclonal human antibodies, patient-derived total IgG antibodies and affinity-purified patient-derived IgG antibodies. Studies that could not be assigned to any of these categories are classified in the category NA/different. Panel A shows the general profile of antibody usage in the mechanism studies. (B) The antibody usage profile in the subcategories of aPLs-mediated mechanisms, i.e. the effect of aPLs on endothelial cells (ECs), platelets, monocytes, and the coagulation system. distribution between the different antibody categories. Interestingly, studies exploring the role of platelets in APS show an aberrant pattern with a significant reduction in the use of patient-derived total IgG. Instead, predominantly murine monoclonal antibodies and mutants of β2GPI were used. Criteria used to diagnose APS patients

82% of all the studies included in this review used patient-derived antibodies. Although APS classification criteria were published as early as 1999 (Sapporo criteria) and revised in 2006 (Sydney criteria), more than half of the studies (54%) did not mention the criteria used to diagnose the patients the antibodies were derived from. Additionally, 22% of the studies used the old Sapporo criteria and 23% the new Sydney criteria (Figure 2A). Comparing the old and new criteria, the clinical characteristics did not differ much, except that transient cerebral ischemia and stroke were included in the revised Sydney criteria. However, major differences exist in the serological criteria that need to be fulfilled for the diagnosis of APS. Firstly, Chapter 7 Chapter the detection of anti-β2GPI antibodies of either IgM or IgG class was introduced in 2006 as an official criterion. Secondly, the 6 weeks interval between two positive 143 laboratory tests was expanded to 12 weeks interval with 5 years as maximum. In addition, the new serological criteria are based on the following laboratory findings:

I) more than one aPL, IIa) LAC alone, IIb) aCL alone, IIc) anti-β2GPI alone. Taken together, the revised Sydney criteria are more strict and specific in diagnosing APS. In 2007, Kaul et al. investigated the clinical implication of the changes in the criteria. Their results showed that from the 81 patients diagnosed with APS according to the Sapporo criteria, only 47 patients met the revised 2006 Sydney criteria 86. These differences between the old and new revised criteria may result in different patient populations selected for antibody purification in mechanistic studies using the old versus new APS criteria. In the 4 subcategories of aPLs-mediated mechanisms, a different application profile of diagnostic criteria is observed (Figure 2B). As most syndrome aPLs in antiphospholipid of the studies observing an effect of aPLs on platelets used murine antibodies, no clear profile can be concluded. Studies showing an effect of aPLs on monocytes predominantly selected patients with the Sydney criteria, while the Sapporo criteria prevail in studies investigating the effect of aPLs on endothelial cells and coagulation. It should be mentioned that even if the updated criteria are used for the diagnosis of the patients, correct diagnosis of the syndrome remains extremely difficult. To prevent over- or under-diagnosis of the syndrome, it is of utmost importance that both clinical and diagnostic criteria are well-defined. Clinical symptoms have a high prevalence in the western society. Despite the attempts to increase specificity of the clinical diagnosis, it remains almost impossible to discriminate between APS-related thrombosis and pregnancy morbidity and the same symptoms caused by different pathologies. As a result, diagnosis of APS relies predominantly on the serological criteria. However, up to this moment, serological diagnosis is impeded mainly because of the questionable correlation with clinical symptoms and the lack of standardization of the different available antibody assays. External quality Control of diagnostic Assays and Tests Disagreement reports have shown that laboratories obtain good results for negative or clearly positive samples. However, a wide variability in sample classification exists when the sample is weakly positive. Various studies tried to find the origin of this high variability, in the different ways to calculate cut- offs (using the 99th percentile instead of the mean and standard deviation), lack of reference calibrators, different plates used for the coating, different sources of the antigen, and different buffers. However, so far no simple explanation nor solution for the high variability was found. Taken together, the differences in diagnostic criteria together with the mediocre applications of the updated criteria, will most likely result in a mixture of APS- related and non-related antibodies and thus add up to the huge variability of the proposed mechanism of action of aPLs.

Chapter 7 Chapter A 144

B aPLs in antiphospholipid syndrome aPLs in antiphospholipid

Figure 2. The application of classification criteria in aPLs mechanism studies using patient-derived antibodies. (A) General profile of the application of diagnostic criteria. Among all the studies analyzed in this manuscript, 18% (category “Not applicable”) did not use patient-derived antibodies, while the vast majority (82%) has used patient- derived antibodies. More than half of the studies (54%) that have used patient-derived antibodies did not mention the diagnostic criteria, 22% used the old Sapporo criteria and 23% used the new Sydney criteria. (B) Detailed profile of the application of diagnostic criteria in the subcategories of aPLs-mediated mechanisms, i.e. the effect of aPLs on endothelial cells (ECs), platelets, monocytes, and coagulation system, which indicates the great inconsistency in diagnostic criteria application in all subcategories. A

B Chapter 7 Chapter

145

Figure 3. The involvement of β2GPI in aPLs mediated APS mechanism studies. (A) Profile of the involvement of β2GPI in the aPL-mediated mechanisms. Most of the studies investigated the effect of aPLs alone (no2 β GPI was involvement) (55%); some studies demonstrated the effect of anti-β2GPI /β2GPI complex (10%); some studies demonstrated the direct effect of β2GPI and an indirect effect of anti- β2GPI (10%); and a small portion of studies either injected purified human aPLs into animal models (no β2GPI involvement) (7.5%) or used human aPL positive serum (involvement of β GPI was not excluded) as the action taker (4%). (B) Profile of the involvement of β GPI in 2 2 syndrome aPLs in antiphospholipid the aPL-mediated mechanisms in different subcategories of the studies.

Involvement of β2GPI in the aPLs mechanism

β2GPI has been shown to be the major co-factor involved in the APS and is so far the only antigen included in the official Sydney criteria. As mentioned before, the 2β GPI/ anti-β2GPI complex is able to bind to and activate multiple cell types, influence and disturb the complement system and inhibit clearance of apoptotic cells by binding 87 to β2GPI . Although indications of direct β2GPI involvement have been popping up here and there, the solid evidence and the consensus on this issue has yet not been sorted out. In an attempt to unravel the involvement of β2GPI in the aPLs- mediated mechanisms, we analyzed 80 publications focused on the effect of aPLs on afore mentioned 4 important sub-categories, i.e. effect of aPLs on endothelial cells, monocytes, coagulation, platelets and fetal loss. As deducted from the methodology part of the studies, 10% of the studies used anti-β2GPI together with β2GPI in the in vitro experiments; 10% used β2GPI as the

primary reagent and revealed a direct effect of β2GPI and an indirect effect of aPLs;

while 55% used purified human aPLs in the in vitro experiments (no 2β GPI was involved) and demonstrated a direct effect of aPLs alone. Yet some other studies

injected human aPLs into animal models (they proposed that murine β2GPI could

replace humanβ2GPI) (7.5%); and a small portion of studies used human aPLs

positive serum as primary reagent (involvement of β2GPI was not excluded) (4%) (Figure 3A). The subcategories of aPLs-mediated mechanisms demonstrated

different profiles regarding aPLs and 2β GPI usage (Figure 3B). The involvement of

β2GPI seems to be studied the most in the platelets and coagulation subcategories. From “bench” to “bedside”: recommendations for further research In the past 6 years, over 100 papers have been published investigating the aPLs- mediated mechanisms resulting in the APS. So far, only 4 new therapeutic approaches Chapter 7 Chapter

have been proposed and tested in animal models, which include β2GPI domain V/ 146 domain I mimics, recombinant human Fc fragment, and B cell reduction by BAFF (a tumor necrosis factor–like cytokine) blockage. Therefore, the vast majority of research lines did not find its way into a possible clinical application. Toour opinion, this huge distance between the bench and bedside may result in part from the differences in auto-antibodies used by different studies to unravel the mechanism of action of aPLs. We hereby suggest that, by finding a consensus on the optimal methodology to study the mechanism of aPLs, efforts can be combined to hopefully identify the pivotal chain in the APS machinery and speed up new therapeutical approaches. To get to this optimal methodology, we would like to propose some recommendations based on the current knowledge of the APS. At first, the patients used as source of the aPLs in antiphospholipid syndrome aPLs in antiphospholipid auto-antibodies should be diagnosed with APS without doubt. To reach this, efforts should be made to improve current diagnosis of APS by increasing the specificity and sensitivity of current serologic assays. This will not only avoid the exposure of falsely-diagnosed patients to a bleeding risk without benefit of such anticoagulant treatment, but also assure the isolation of ‘real’ APS-related aPLs from these patients. Secondly, since it is now well-recognized that the main antigenic target of these aPLs

is β2GPI, all studies should be focused on the combined effects of β2GPI and affinity-

purified anti-β2GPI antibodies. Importantly, our group, among others, already

demonstrated that antibodies recognizing epitope G40-R43 on domain I of β2GPI correlate much better with the occurrence of thrombosis 85. Although it may be short- sighted to think this subpopulation is the only important antibody population in APS, it makes sense to combine our efforts to test these proven, thrombosis/pregnancy morbidity-related, antibodies for their effect on different metabolic pathways, at least until more convincing data on other subpopulations of antiphospholipid antibodies that strongly correlate with clinical manifestations are available. In this way, we can compare the observed effects of these aPLs obtained in different laboratories with different model systems. Hopefully, by following a consensus methodology based on these and other recommendations, the main target of these aPLs can be identified, ultimately leading to a strategy tackling the actual cause instead of the peripheral mechanisms. Chapter 7 Chapter

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signaling. J.Thromb.Haemost. 2007;5:1538- syndrome aPLs in antiphospholipid pathways involving lipid rafts. Arthritis 1544. Rheum. 2007;56:2687-2697. 33. Romay-Penabad Z, guilar-Valenzuela R, 25. Perez-Sanchez C, Ruiz-Limon P, Aguirre Urbanus RT et al. Apolipoprotein E receptor MA et al. Mitochondrial dysfunction in 2 is involved in the thrombotic complications antiphospholipid syndrome: implications in a murine model of the antiphospholipid in the pathogenesis of the disease and syndrome. Blood 2011;117:1408-1414. effects of coenzyme Q(10) treatment. Blood 2012;119:5859-5870. 34. Sikara MP, Routsias JG, Samiotaki M et al. {beta}2 Glycoprotein I ({beta}2GPI) binds 26. Xu G, Wen H, Zhou H et al. Involvement platelet factor 4 (PF4): implications for the of IRAKs and TRAFs in anti-beta(2)GPI/ pathogenesis of antiphospholipid syndrome. beta(2)GPI-induced tissue factor expression Blood 2010;115:713-723. in THP-1 cells. Thromb.Haemost. 2011;106:1158-1169. 35. Hamid C, Norgate K, D’Cruz DP et al. Anti- beta2GPI-antibody-induced endothelial cell 27. Shi T, Giannakopoulos B, Yan X et al. gene expression profiling reveals induction Anti-beta2-glycoprotein I antibodies in of novel pro-inflammatory genes potentially complex with beta2-glycoprotein I can involved in primary antiphospholipid syndrome. Ann.Rheum.Dis. 2007;66:1000- in antiphospholipid antibody-mediated 1007. pathogenic effects in vitro and in vivo. Blood 2009;114:3074-3083. 36. Soltesz P, Der H, Veres K et al. Immunological features of primary anti- 45. Allen KL, Hamik A, Jain MK, McCrae phospholipid syndrome in connection with KR. Endothelial cell activation by endothelial dysfunction. Rheumatology. antiphospholipid antibodies is modulated (Oxford) 2008;47:1628-1634. by Kruppel-like transcription factors. Blood 2011;117:6383-6391. 37. Belizna C, Lartigue A, Favre J et al. Antiphospholipid antibodies induce vascular 46. gmon-Levin N, Blank M, Zandman- functional changes in mice: a mechanism Goddard G et al. Vitamin D: an instrumental of vascular lesions in antiphospholipid factor in the anti-phospholipid syndrome by syndrome? Lupus 2008;17:185-194. inhibition of tissue factor expression. Ann. Rheum.Dis. 2011;70:145-150. 38. Patterson AM, Ford I, Graham A, Booth NA, Greaves M. The influence of anti- 47. Montiel-Manzano G, Romay-Penabad endothelial/antiphospholipid antibodies on Z, Papalardo De ME et al. In vivo fibrin formation and lysis on endothelial effects of an inhibitor of nuclear factor- Chapter 7 Chapter cells. Br.J.Haematol. 2006;133:323-330. kappa B on thrombogenic properties of 150 antiphospholipid antibodies. Ann.N.Y.Acad. 39. Vega-Ostertag M, Liu X, Kwan-Ki H, Sci. 2007;1108:540-553. Chen P, Pierangeli S. A human monoclonal antiprothrombin antibody is thrombogenic 48. Vega-Ostertag ME, Ferrara DE, Romay- in vivo and upregulates expression of tissue Penabad Z et al. Role of p38 mitogen- factor and E-selectin on endothelial cells. activated protein kinase in antiphospholipid Br.J.Haematol. 2006;135:214-219. antibody-mediated thrombosis and endothelial cell activation. J.Thromb. 40. Clemens N, Frauenknecht K, Katzav A, Haemost. 2007;5:1828-1834. Sommer C, von LP. In vitro effects of antiphospholipid syndrome-IgG fractions 49. de Laat B, Eckmann CM, van SM et al. and human monoclonal antiphospholipid Correlation between the potency of a IgG antibody on human umbilical beta2-glycoprotein I-dependent lupus vein endothelial cells and monocytes. anticoagulant and the level of resistance

aPLs in antiphospholipid syndrome aPLs in antiphospholipid Ann.N.Y.Acad.Sci. 2009;1173:805-813. to activated protein C. Blood Coagul. Fibrinolysis 2008;19:757-764. 41. Graham A, Ford I, Morrison R et al. Anti- endothelial antibodies interfere in apoptotic 50. Galli M, Willems GM, Rosing J et al. cell clearance and promote thrombosis in Anti-prothrombin IgG from patients with patients with antiphospholipid syndrome. anti-phospholipid antibodies inhibits the J.Immunol. 2009;182:1756-1762. inactivation of factor Va by activated protein C. Br.J.Haematol. 2005;129:240-247. 42. Alard JE, Gaillard F, Daridon C et al. TLR2 is one of the endothelial receptors 51. Bu C, Gao L, Xie W et al. beta2-glycoprotein for beta 2-glycoprotein I. J.Immunol. i is a cofactor for tissue plasminogen 2010;185:1550-1557. activator-mediated plasminogen activation. Arthritis Rheum. 2009;60:559-568. 43. Pierangeli SS, Vega-Ostertag ME, Raschi E et al. Toll-like receptor and antiphospholipid 52. Ieko M, Yoshida M, Naito S et al. mediated thrombosis: in vivo studies. Ann. Increase in plasma thrombin-activatable Rheum.Dis. 2007;66:1327-1333. fibrinolysis inhibitor may not contribute to thrombotic tendency in antiphospholipid 44. Romay-Penabad Z, Montiel-Manzano MG, syndrome because of inhibitory potential Shilagard T et al. Annexin A2 is involved of antiphospholipid antibodies toward TAFI activation. Int.J.Hematol. 2010;91:776-783. 61. Hunt BJ, Wu XX, de LB et al. Resistance to annexin A5 anticoagulant activity in women 53. Gombas J, Tanka-Salamon A, Skopal with histories for obstetric antiphospholipid J et al. Modulation of fibrinolysis by syndrome. Am.J.Obstet.Gynecol. the combined action of phospholipids 2011;205:485-23. and immunoglobulins. Blood Coagul. Fibrinolysis 2008;19:82-88. 62. Rand JH, Wu XX, Quinn AS et al. Hydroxychloroquine directly reduces the 54. Lopez-Lira F, Rosales-Leon L, Martinez binding of antiphospholipid antibody-beta2- VM, Ruiz Ordaz BH. The role of beta2- glycoprotein I complexes to phospholipid glycoprotein I (beta2GPI) in the activation bilayers. Blood 2008;112:1687-1695. of plasminogen. Biochim.Biophys.Acta 2006;1764:815-823. 63. Rand JH, Wu XX, Quinn AS et al. Hydroxychloroquine protects the annexin 55. Lean SY, Ellery P, Ivey L et al. The effects A5 anticoagulant shield from disruption by of tissue factor pathway inhibitor and anti- antiphospholipid antibodies: evidence for beta-2-glycoprotein-I IgG on thrombin a novel effect for an old antimalarial drug. generation. Haematologica 2006;91:1360- Blood 2010;115:2292-2299. 1366. Chapter 7 Chapter 64. Wu XX, Guller S, Rand JH. 56. Liestol S, Sandset PM, Jacobsen EM, Hydroxychloroquine reduces binding Mowinckel MC, Wisloff F. Decreased 151 of antiphospholipid antibodies to anticoagulant response to tissue factor syncytiotrophoblasts and restores annexin pathway inhibitor type 1 in plasmas A5 expression. Am.J.Obstet.Gynecol. from patients with lupus anticoagulants. 2011;205:576-14. Br.J.Haematol. 2007;136:131-137. 65. Shi T, Iverson GM, Qi JC et al. Beta 57. Giles I, Pericleous C, Liu X et al. 2-Glycoprotein I binds factor XI and Thrombin binding predicts the effects of inhibits its activation by thrombin and factor sequence changes in a human monoclonal XIIa: loss of inhibition by clipped beta antiphospholipid antibody on its in vivo 2-glycoprotein I. Proc.Natl.Acad.Sci.U.S.A biologic actions. J.Immunol. 2009;182:4836- 2004;101:3939-3944. 4843. 66. Gropp K, Weber N, Reuter M et al. 58. Rahgozar S, Yang Q, Giannakopoulos B

beta(2)-glycoprotein I, the major target in syndrome aPLs in antiphospholipid et al. Beta2-glycoprotein I binds thrombin antiphospholipid syndrome, is a special via exosite I and exosite II: anti-beta2- human complement regulator. Blood glycoprotein I antibodies potentiate the 2011;118:2774-2783. inhibitory effect of beta2-glycoprotein I on thrombin-mediated factor XIa generation. 67. Carroll TY, Mulla MJ, Han CS et al. Arthritis Rheum. 2007;56:605-613. Modulation of trophoblast angiogenic factor secretion by antiphospholipid antibodies 59. Rahgozar S, Giannakopoulos B, Yan X et is not reversed by heparin. Am.J.Reprod. al. Beta2-glycoprotein I protects thrombin Immunol. 2011;66:286-296. from inhibition by heparin cofactor II: potentiation of this effect in the presence 68. Jovanovic M, Bozic M, Kovacevic T et al. of anti-beta2-glycoprotein I autoantibodies. Effects of anti-phospholipid antibodies on a Arthritis Rheum. 2008;58:1146-1155. human trophoblast cell line (HTR-8/SVneo). Acta Histochem. 2010;112:34-41. 60. de Laat B, Wu XX, van LM et al. Correlation between antiphospholipid antibodies that 69. Mulla MJ, Brosens JJ, Chamley LW et al. recognize domain I of beta2-glycoprotein I Antiphospholipid antibodies induce a pro- and a reduction in the anticoagulant activity inflammatory response in first trimester of annexin A5. Blood 2007;109:1490-1494. trophoblast via the TLR4/MyD88 pathway. Am.J.Reprod.Immunol. 2009;62:96-111. 78. Schwartz N, Shoenfeld Y, Barzilai O et al. Reduced placental growth and hCG secretion 70. Chen Q, Viall C, Kang Y et al. Anti- in vitro induced by antiphospholipid phospholipid antibodies increase non- antibodies but not by anti-Ro or anti-La: apoptotic trophoblast shedding: a studies on sera from women with SLE/ contribution to the pathogenesis of pre- PAPS. Lupus 2007;16:110-120. eclampsia in affected women? Placenta 2009;30:767-773. 79. Poindron V, Berat R, Knapp AM et al. Evidence for heterogeneity of the obstetric 71. Ichikawa G, Yamamoto T, Chishima F et al. antiphospholipid syndrome: thrombosis can Effects of anti-beta2-glycoprotein I antibody be critical for antiphospholipid-induced on PlGF, VEGF and sVEGFR1 production pregnancy loss. J.Thromb.Haemost. from cultured choriocarcinoma cell line. 2011;9:1937-1947. J.Obstet.Gynaecol.Res. 2011;37:1076-1083. 80. Petri M. Update on anti-phospholipid 72. Iwasawa Y, Kawana K, Fujii T et al. antibodies in SLE: the Hopkins’ Lupus A possible coagulation-independent Cohort. Lupus 2010;19:419-423. mechanism for pregnancy loss involving

Chapter 7 Chapter beta(2) glycoprotein 1-dependent 81. Kobayashi K, Tada K, Itabe H et al. antiphospholipid antibodies and CD1d. Distinguished effects of antiphospholipid 152 Am.J.Reprod.Immunol. 2012;67:54-65. antibodies and anti-oxidized LDL antibodies on oxidized LDL uptake by macrophages. 73. Martinez de la TY, Buracchi C, Borroni Lupus 2007;16:929-938. EM et al. Protection against inflammation- and autoantibody-caused fetal loss by the 82. Kahn P, Ramanujam M, Bethunaickan R et chemokine decoy receptor D6. Proc.Natl. al. Prevention of murine antiphospholipid Acad.Sci.U.S.A 2007;104:2319-2324. syndrome by BAFF blockade. Arthritis Rheum. 2008;58:2824-2834. 74. Martinez-Zamora MA, Tassies D, Carmona F et al. Clot lysis time and thrombin 83. Katzav A, Litvinjuk Y, Pick CG et al. activatable fibrinolysis inhibitor in severe Genetic and immunological factors interact preeclampsia with or without associated in a mouse model of CNS antiphospholipid antiphospholipid antibodies. J.Reprod. syndrome. Behav.Brain Res. 2006;169:289- Immunol. 2010;86:133-140. 293. aPLs in antiphospholipid syndrome aPLs in antiphospholipid 75. Redecha P, Franzke CW, Ruf W, Mackman 84. Menachem A, Chapman J, Katzav A. N, Girardi G. Neutrophil activation by Hyperactivity induced by antiphospholipid the tissue factor/Factor VIIa/PAR2 axis syndrome serum. Ann.N.Y.Acad.Sci. mediates fetal death in a mouse model of 2009;1173:422-426. antiphospholipid syndrome. J.Clin.Invest 85. de Laat B, Derksen RH, van LM, Pennings 2008;118:3453-3461. MT, de Groot PG. Pathogenic anti-beta2- 76. Carmona F, Lazaro I, Reverter JC et al. glycoprotein I antibodies recognize Impaired factor XIIa-dependent activation domain I of beta2-glycoprotein I only of fibrinolysis in treated antiphospholipid after a conformational change. Blood syndrome gestations developing late- 2006;107:1916-1924. pregnancy complications. Am.J.Obstet. 86. Kaul M, Erkan D, Sammaritano L, Lockshin Gynecol. 2006;194:457-465. MD. Assessment of the 2006 revised 77. Di SN, Di NF, D’Ippolito S et al. antiphospholipid syndrome classification Antiphospholipid antibodies affect human criteria. Ann.Rheum.Dis. 2007;66:927-930. endometrial angiogenesis. Biol.Reprod. 87. Gropp K, Weber N, Reuter M et al. 2010;83:212-219. beta(2)-glycoprotein I, the major target in antiphospholipid syndrome, is a special human complement regulator. Blood 2011;118:2774-2783. Chapter 7 Chapter

153 aPLs in antiphospholipid syndrome aPLs in antiphospholipid Supplementary data Publications analyzed for the antiphospholipid antibody mediated pathogenic mechanisms in the antiphospholipid syndrome*

Studies investigating mechanistical action of antiphospholipid antibodies were selected. To be included in this analysis, the studies need to be published between 2006 (publication of the revised ISTH criteria for APS) and March 2012 (end of data collection). Effect of aPL on monocytes1-20 Effect of aPL on endothelial cells3;21-35 Effect of aPL oncoagulation system36-51 Effect of aPL on platelets52-61

Chapter 7 Chapter Disruption of annexin V shield62-69 154 Complement activation70-75 aPL-mediated fetal loss76-93 Immunopathology of aPL94-104 aPLs and arthrosclerosis105-109 aPLs and CNS110-116 Novel therapeutic approaches112;117-122 Others123-126 * Reference number 3 was assigned into both “monocytes” and “endothelial cells”

aPLs in antiphospholipid syndrome aPLs in antiphospholipid subcategories and number 110 was assigned into both “CNS” and “Novel therapeutic approaches”.

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Resistance to Hemost. 2012;18:59-65. annexin A5 anticoagulant activity in women with histories for obstetric antiphospholipid 60. Membre A, Wahl D, Latger-Cannard V syndrome. Am.J.Obstet.Gynecol. et al. The effect of platelet activation on 2011;205:485-23. the hypercoagulability induced by murine monoclonal antiphospholipid antibodies. 69. de Laat B, Wu XX, van LM et al. Correlation Haematologica 2008;93:566-573. between antiphospholipid antibodies that recognize domain I of beta2-glycoprotein I 61. Jy W, Tiede M, Bidot CJ et al. Platelet aPLs in antiphospholipid syndrome aPLs in antiphospholipid and a reduction in the anticoagulant activity activation rather than endothelial injury of annexin A5. Blood 2007;109:1490-1494. identifies risk of thrombosis in subjects positive for antiphospholipid antibodies. 70. Ritis K, Doumas M, Mastellos D et al. Thromb.Res. 2007;121:319-325. A novel C5a receptor-tissue factor cross- talk in neutrophils links innate immunity 62. Gaspersic N, Ambrozic A, Bozic B et al. to coagulation pathways. J.Immunol. Annexin A5 binding to giant phospholipid 2006;177:4794-4802. vesicles is differentially affected by anti- beta2-glycoprotein I and anti-annexin 71. Gropp K, Weber N, Reuter M et al. A5 antibodies. Rheumatology.(Oxford) beta(2)-glycoprotein I, the major target in 2007;46:81-86. antiphospholipid syndrome, is a special human complement regulator. Blood 63. Frostegard AG, Su J, von LP, Frostegard J. 2011;118:2774-2783. Effects of anti-cardiolipin antibodies and IVIg on annexin A5 binding to endothelial 72. Romay-Penabad Z, Liu XX, Montiel- cells: implications for cardiovascular Manzano G, Papalardo De ME, Pierangeli disease. Scand.J.Rheumatol. 2010;39:77-83. SS. C5a receptor-deficient mice are protected from thrombophilia and 64. Rand JH, Wu XX, Quinn AS et al. endothelial cell activation induced by some antiphospholipid antibodies. Ann.N.Y.Acad. Pathomorphologic and immunohistochemical Sci. 2007;1108:554-566. study on the devastation of rat embryos by antiphospholipid antibody positive serum. 73. Cohen D, Buurma A, Goemaere NN Am.J.Reprod.Immunol. 2008;60:523-528. et al. Classical complement activation as a footprint for murine and human 82. Ichikawa G, Yamamoto T, Chishima F et al. antiphospholipid antibody-induced fetal Effects of anti-beta2-glycoprotein I antibody loss. J.Pathol. 2011;225:502-511. on PlGF, VEGF and sVEGFR1 production from cultured choriocarcinoma cell line. 74. Shamonki JM, Salmon JE, Hyjek E, Baergen J.Obstet.Gynaecol.Res. 2011;37:1076-1083. RN. Excessive complement activation is associated with placental injury in 83. Iwasawa Y, Kawana K, Fujii T et al. patients with antiphospholipid antibodies. A possible coagulation-independent Am.J.Obstet.Gynecol. 2007;196:167. e1-5. mechanism for pregnancy loss involving beta(2) glycoprotein 1-dependent 75. Seshan SV, Franzke CW, Redecha P et antiphospholipid antibodies and CD1d. al. Role of tissue factor in a mouse model Am.J.Reprod.Immunol. 2012;67:54-65. of thrombotic microangiopathy induced

by antiphospholipid antibodies. Blood 84. Jovanovic M, Bozic M, Kovacevic T et al. 7 Chapter 2009;114:1675-1683. Effects of anti-phospholipid antibodies on a human trophoblast cell line (HTR-8/SVneo). 159 76. Carroll TY, Mulla MJ, Han CS et al. Acta Histochem. 2010;112:34-41. Modulation of trophoblast angiogenic factor secretion by antiphospholipid antibodies 85. Lazaro I, Carmona F, Reverter JC et al. is not reversed by heparin. Am.J.Reprod. Antiphospholipid antibodies may impair Immunol. 2011;66:286-296. factor XIIa-dependent activation of fibrinolysis in pregnancy: in vitro evidence 77. Chen Q, Viall C, Kang Y et al. Anti- with human endothelial cells in culture phospholipid antibodies increase non- and monoclonal anticardiolipin antibodies. apoptotic trophoblast shedding: a Am.J.Obstet.Gynecol. 2009;201:87-6. contribution to the pathogenesis of pre- eclampsia in affected women? Placenta 86. Martinez-Zamora MA, Tassies D, Carmona 2009;30:767-773. F et al. Clot lysis time and thrombin activatable fibrinolysis inhibitor in severe 78. Di Simone N, Castellani R, Raschi E et al. preeclampsia with or without associated syndrome aPLs in antiphospholipid Anti-beta-2 glycoprotein I antibodies affect antiphospholipid antibodies. J.Reprod. Bcl-2 and Bax trophoblast expression without Immunol. 2010;86:133-140. evidence of apoptosis. Ann.N.Y.Acad.Sci. 2006;1069:364-376. 87. Martinez de la TY, Buracchi C, Borroni EM et al. Protection against inflammation- 79. Di Simone N, Marana R, Castellani R et al. and autoantibody-caused fetal loss by the Decreased expression of heparin-binding chemokine decoy receptor D6. Proc.Natl. epidermal growth factor-like growth Acad.Sci.U.S.A 2007;104:2319-2324. factor as a newly identified pathogenic mechanism of antiphospholipid-mediated 88. Mulla MJ, Brosens JJ, Chamley LW et al. defective placentation. Arthritis Rheum. Antiphospholipid antibodies induce a pro- 2010;62:1504-1512. inflammatory response in first trimester trophoblast via the TLR4/MyD88 pathway. 80. Di Simone N, Di Nicuolo F, D’Ippolito S Am.J.Reprod.Immunol. 2009;62:96-111. et al. Antiphospholipid antibodies affect human endometrial angiogenesis. Biol. 89. Poindron V, Berat R, Knapp AM et al. Reprod. 2010;83:212-219. Evidence for heterogeneity of the obstetric antiphospholipid syndrome: thrombosis can 81. Halperin R, Elhayany A, Ben-Hur H et al. be critical for antiphospholipid-induced pregnancy loss. J.Thromb.Haemost. 98. Inic-Kanada A, Stojanovic M, Zivkovic I et 2011;9:1937-1947. al. Murine monoclonal antibody 26 raised against tetanus toxoid cross-reacts with 90. Redecha P, Tilley R, Tencati M et al. Tissue beta2-glycoprotein I: its characteristics and factor: a link between C5a and neutrophil role in molecular mimicry. Am.J.Reprod. activation in antiphospholipid antibody Immunol. 2009;61:39-51. induced fetal injury. Blood 2007;110:2423- 2431. 99. Tzang BS, Lee YJ, Yang TP et al. Induction of antiphospholipid antibodies 91. Redecha P, Franzke CW, Ruf W, Mackman and antiphospholipid syndrome-like N, Girardi G. Neutrophil activation by autoimmunity in naive mice with antibody the tissue factor/Factor VIIa/PAR2 axis against human parvovirus B19 VP1 mediates fetal death in a mouse model of unique region protein. Clin.Chim.Acta antiphospholipid syndrome. J.Clin.Invest 2007;382:31-36. 2008;118:3453-3461. 100. Van OS, G, Meijers JC, Agar C et al. 92. Schwartz N, Shoenfeld Y, Barzilai O et al. Induction of anti-beta2 -glycoprotein Reduced placental growth and hCG secretion I autoantibodies in mice by protein H in vitro induced by antiphospholipid Chapter 7 Chapter of Streptococcus pyogenes. J.Thromb. antibodies but not by anti-Ro or anti-La: Haemost. 2011;9:2447-2456. 160 studies on sera from women with SLE/ PAPS. Lupus 2007;16:110-120. 101. Velayuthaprabhu S, Matsubayashi H, Sugi T et al. A unique preliminary study on placental 93. Mulla MJ, Myrtolli K, Brosens JJ et al. apoptosis in mice with passive immunization Antiphospholipid antibodies limit trophoblast of anti-phosphatidylethanolamine antibodies migration by reducing IL-6 production and and anti-factor XII antibodies. Am.J.Reprod. STAT3 activity. Am.J.Reprod.Immunol. Immunol. 2011;66:373-384. 2010;63:339-348. 102. Yamaguchi Y, Seta N, Kaburaki J et al. 94. Alessandri C, Sorice M, Bombardieri M Excessive exposure to anionic surfaces et al. Antiphospholipid reactivity against maintains autoantibody response to cardiolipin metabolites occurring during beta(2)-glycoprotein I in patients with endothelial cell apoptosis. Arthritis Res. antiphospholipid syndrome. Blood Ther. 2006;8:R180. 2007;110:4312-4318. aPLs in antiphospholipid syndrome aPLs in antiphospholipid 95. Cesarman-Maus G, Rios-Luna NP, Deora AB 103. Chen XX, Gu YY, Li SJ et al. Some plasmin- et al. Autoantibodies against the fibrinolytic induced antibodies bind to cardiolipin, receptor, annexin 2, in antiphospholipid display lupus anticoagulant activity and syndrome. Blood 2006;107:4375-4382. induce fetal loss in mice. J.Immunol. 96. de Laat B, van Berkel M, Urbanus RT et 2007;178:5351-5356. al. Immune responses against domain I 104. Tzang BS, Tsai CC, Chiu CC, Shi JY, Hsu of beta(2)-glycoprotein I are driven by TC. Up-regulation of adhesion molecule conformational changes: domain I of expression and induction of TNF-alpha on beta(2)-glycoprotein I harbors a cryptic vascular endothelial cells by antibody against immunogenic epitope. Arthritis Rheum. human parvovirus B19 VP1 unique region 2011;63:3960-3968. protein. Clin.Chim.Acta 2008;395:77-83. 97. Ede K, Hwang KK, Wu CC et al. Plasmin 105. Adler A, Afek A, Levy Y, Keren G, George J. immunization preferentially induces Antiphospholipid antibodies from a patient potentially prothrombotic IgG anticardiolipin with primary antiphospholipid syndrome antibodies in MRL/MpJ mice. Arthritis enhance experimental atherosclerosis. Nat. Rheum. 2009;60:3108-3117. Clin.Pract.Cardiovasc.Med. 2009;6:215- 218. 114. Katzav A, Litvinjuk Y, Pick CG et al. Genetic and immunological factors interact 106. Kobayashi K, Tada K, Itabe H et al. in a mouse model of CNS antiphospholipid Distinguished effects of antiphospholipid syndrome. Behav.Brain Res. 2006;169:289- antibodies and anti-oxidized LDL antibodies 293. on oxidized LDL uptake by macrophages. Lupus 2007;16:929-938. 115. Menachem A, Chapman J, Katzav A. Hyperactivity induced by antiphospholipid 107. Stanic AK, Stein CM, Morgan AC et syndrome serum. Ann.N.Y.Acad.Sci. al. Immune dysregulation accelerates 2009;1173:422-426. atherosclerosis and modulates plaque composition in systemic lupus 116. Tanne D, Katzav A, Beilin O et al. erythematosus. Proc.Natl.Acad.Sci.U.S.A Interaction of inflammation, thrombosis, 2006;103:7018-7023. aspirin and enoxaparin in CNS experimental antiphospholipid syndrome. Neurobiol.Dis. 108. Dunoyer-Geindre S, Kwak BR, Pelli G et 2008;30:56-64. al. Immunization of LDL receptor-deficient mice with beta2-glycoprotein 1 or human 117. Blank M, Baraam L, Eisenstein M et al. serum albumin induces a more inflammatory beta2-Glycoprotein-I based peptide regulate Chapter 7 Chapter phenotype in atherosclerotic plaques. endothelial-cells tissue-factor expression Thromb.Haemost. 2007;97:129-138. via negative regulation of pGSK3beta 161 expression and reduces experimental- 109. Laplante P, Amireault P, Subang R et antiphospholipid-syndrome. J.Autoimmun. al. Interaction of beta2-glycoprotein I 2011;37:8-17. with lipopolysaccharide leads to Toll- like receptor 4 (TLR4)-dependent 118. de la Torre YM, Pregnolato F, D’Amelio activation of macrophages. J.Biol.Chem. F et al. Anti-phospholipid induced murine 2011;286:42494-42503. fetal loss: novel protective effect of a peptide targeting the beta2 glycoprotein 110. Dale RC, Yin K, Ding A et al. Antibody I phospholipid-binding site. Implications binding to neuronal surface in movement for human fetal loss. J.Autoimmun. disorders associated with lupus and 2012;38:J209-J215. antiphospholipid antibodies. Dev.Med.Child Neurol. 2011;53:522-528. 119. Halperin R, Shinnar N, Kronfeld-Schor

N, Hadas E. Human decidua-associated syndrome aPLs in antiphospholipid 111. Frauenknecht K, Bargiotas P, Bauer H et al. protein 200 neutralizes the detrimental Neuroprotective effect of Fn14 deficiency is effect of serum containing antiphospholipid associated with induction of the - antibodies on fetal survival in the rat. colony stimulating factor (G-CSF) pathway Am.J.Reprod.Immunol. 2006;55:246-250. in experimental stroke and enhanced by a pathogenic human antiphospholipid 120. Ioannou Y, Romay-Penabad Z, Pericleous C antibody. J.Neuroimmunol. 2010;227:1-9. et al. In vivo inhibition of antiphospholipid antibody-induced pathogenicity utilizing the 112. Kahn P, Ramanujam M, Bethunaickan R et antigenic target peptide domain I of beta2- al. Prevention of murine antiphospholipid glycoprotein I: proof of concept. J.Thromb. syndrome by BAFF blockade. Arthritis Haemost. 2009;7:833-842. Rheum. 2008;58:2824-2834. 121. Ostertag MV, Liu X, Henderson V, 113. Kajiwara T, Yasuda T, Matsuura E. Pierangeli SS. A peptide that mimics the Intracellular trafficking of beta2- Vth region of beta-2-glycoprotein I reverses glycoprotein I complexes with lipid vesicles antiphospholipid-mediated thrombosis in in macrophages: implications on the mice. Lupus 2006;15:358-365. development of antiphospholipid syndrome. J.Autoimmun. 2007;29:164-173. 122. Xie W, Zhang Y, Bu C et al. Anti-coagulation effect of Fc fragment against anti-beta2-GP1 antibodies in mouse models with APS. Int. Immunopharmacol. 2011;11:136-140. 123. Hulstein JJ, Lenting PJ, de LB et al. beta2- Glycoprotein I inhibits von Willebrand factor dependent platelet adhesion and aggregation. Blood 2007;110:1483-1491. 124. Andrade D, Redecha PB, Vukelic M et al. Engraftment of peripheral blood mononuclear cells from systemic lupus erythematosus and antiphospholipid syndrome patient donors into BALB- RAG-2-/- IL-2Rgamma-/- mice: a promising model for studying human disease. Arthritis Rheum. 2011;63:2764-2773.

Chapter 7 Chapter 125. Ioannou Y, Zhang JY, Qi M et al. Novel assays of thrombogenic pathogenicity in 162 the antiphospholipid syndrome based on the detection of molecular oxidative modification of the major autoantigen beta2-glycoprotein I. Arthritis Rheum. 2011;63:2774-2782. 126. Lin WS Chen PC, Yang CD et al. Some antiphospholipid antibodies recognize conformational epitopes shared by beta2- glycoprotein I and the homologous catalytic domains of several serine proteases. Arthritis Rheum. 2007;56:1638-1647. aPLs in antiphospholipid syndrome aPLs in antiphospholipid 8

Indications for a protective function of beta2- glycoprotein I in thrombotic thrombocytopenic purpura

Vivian X. Du1,2, Gwen van Os1,3, Johanna A. Kremer Hovinga4, Ilze Dienava- Verdoold2, Jacques Wollersheim2, Zaverio M. Ruggeri5, Rob Fijnheer1, Philip G de Groot1, and Bas de Laat1,2,5,6,7 8 Chapter 163

1Department of clinical Chemistry and Haematology, University Medical Center, GPI in TTP Utrecht, the Netherlands 2 β 2Sanquin Research, Sanquin Blood Supply Foundation, Amsterdam, the Netherlands 3Department of Experimental Vascular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands 4University Clinic of Haematology and Central Haematology Laboratory, Bern University Hospital and the University of Bern, Bern, Switzerland 5Roon Research Center for Arteriosclerosis and Thrombosis, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California,USA 6Laboratory for pathophysiology of thrombin generation, Department of Biochemistry, CARIM, Maastricht University Medical Center, Maastricht, the Netherlands; 7Synapse BV, Maastricht, the Netherlands.

Br J Haematol. 2012; 159: 94-103 Abstract

It has been shown that beta2-glycoprotein I (β2GPI) interacts with von Willebrand factor (VWF) at the glycoprotein (GP) Ibα-binding site. Given the presenceof active VWF multimers in thrombotic thrombocytopenic purpura (TTP), we speculated that

β2GPI might play a role in TTP. We found that β2GPI plasma levels were significantly lower in acute and remission TTP patients than in normal controls, showing a direct correlation with ADAMTS 13 levels and an inverse correlation with the extent of

VWF activation. In vitro flow experiments demonstrated that 2β GPI can block platelet adhesion to endothelial cell-derived VWF strings. We confirmed the direct binding

of β2GPI to VWF by surface plasmon resonance, and determined that domain I of

β2GPI is the binding site of VWF A1 domain. Adhesion of β2GPI to erythrocytes and

platelets was increased in the presence of active VWF, indicating that β2GPI may be cleared from the circulation during TTP episodes together with blood cells. Our

Chapter 8 Chapter findings suggest that 2β GPI may protect from the effects of hyper-functional VWF by inhibiting its interaction with platelets. 164 GPI in TTP 2 β Introduction Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disease characterized by thrombocytopenia, microangiopathic hemolytic anaemia, fever, renal and neurological manifestations.1 In the majority of cases, the disease is the result of a severe deficiency of ADAMTS13 activity due to the presence of circulating inhibitory autoantibodies.2;3ADAMTS13 is the metalloproteinase that cleaves ultra large (UL) von Willebrand Factor (VWF) multimers as they are released from the Weibel-Palade bodies of endothelial cells, where they are stored after synthesis. A deficiency of ADAMTS13 function results in increased levels of circulating UL VWF that can bind to platelets spontaneously via the A1 domain. Our knowledge of TTP and its association with the absence of ADAMTS13 activity has increased significantly over the last years, but the underlying pathophysiologic mechanisms are still incompletely understood. Although severe ADAMTS13 deficiency is found in the majority of patients with clinically diagnosed TTP, the absence of ADAMTS13 activity does not necessarily result in TTP-like symptoms. On the other hand, clinical 8 Chapter remission can be achieved in patients suffering from an acute TTP episode despite 165 persistently severe ADAMTS13 deficiency.4 These observations suggest that other factors besides ADAMTS 13 may regulate VWF activity in TTP.

Beta2-glycoprotein I (β2GPI) is a highly abundant (3-6 µM) plasma protein, best GPI in TTP

5 2 known as the antigen against which anti-phospholipid antibodies are directed. β Several of its biochemical properties have been described and recent evidence suggested a role in clearance of unwanted components in our circulation.6;7,8 We have shown previously that β2GPI interacts with the VWF A1 domain competing with glycoprotein (GP) Ibα-binding and, thus, inhibiting platelet adhesion.9 Furthermore,

β2GPI appears to bind with approximately 100-fold higher affinity to VWF in the active conformation (i.e. the GPIbα-binding conformation) than to native VWF.9

In this study, we investigated whether β2GPI could influence UL VWF function exerting a regulatory role in TTP. To test this hypothesis, we measured plasma β2GPI levels in TTP patients during acute and remission phases to evaluate how variations might influence platelet interactions with VWF and/or reflect mechanisms for2 β GPI clearance from the circulation. Our results delineate a mechanism through which domain I of β2GPI may contribute to limiting the prothrombotic effects of circulating UL VWF. Patients, Materials and Methods Patients In this study we investigated two patient groups, one individual patient with distinctive characteristics and one control group of healthy individuals. Plasma containing trisodium citrate (0.0129 M final concentration) was collected from 38 patients diagnosed with an acute episode of acquired TTP in the University Clinic of Haematology and Central Haematology Laboratory, Inselspital, Bern, Switzerland. All patients had severe ADAMTS13 deficiency (<5% of the normal), thrombocytopenia (platelet count <150x109/L), and microangiopathic hemolytic anaemia with fragmented erythrocytes in blood smears. Plasma samples (0.0109 M trisodium citrate final concentration) were also collected from 34 patients who were in TTP remission phase after a first acute episode at University Medical Center (UMC), Utrecht, the Netherlands. Seven of the above patients had plasma samples drawn and stored during their previous acute episode. One patient (diagnosed at UMC, Utrecht), who was a regular blood donor, had his plasma samples collected before, during and after his first acute TTP episode. Healthy blood donors (n=54) were included in this study as controls. Antibodies and proteins

Murine monoclonal antibody 2B2 directed against β2GPI was a kind gift of Prof. A. Tincani, Brescia, Italy. Polyclonal rabbit-anti human β GPI antibody was purchased Chapter 8 Chapter 2

from Kordia (Leiden, the Netherlands). Murine polyclonal antibody β2GPI 3B7 166 was produced by immunizing mice with human β2GPI. Mouse-anti-human VWF RAG201 directed against VWF A3 domain and RAG 35 directed against VWF A1 domain were produced as previously described.10 Mouse anti-human CD235a, as marker for erythrocytes, was purchased from BD Bioscience (Erembodegem-Aalst, GPI in TTP

2 10

β Belgium). β2GPI was purified as previously described. Recombinant mutants of

β2GPI domains (DI, DII, DI-II-III, DII-V, DI-V, DII-V, DIV-V) were a generous

gifts from La Jolla Pharmaceutical Company (La Jolla, CA, USA). Individual β2GPI domains (I, II, IV and V) were expressed in HEK293E cells.10;12 Plasma derived VWF was purified as previously described.13 The VWF-2B mutant (VWF-R1306W) was cloned into the expression vector PC-DNA 3.1 and expressed in HEK293T cells with furin activity. Expressed proteins were purified from serum-free medium using an affinity-column coupled with anti-human VWF RAG-201. Recombinant VWF A1-domain containing the R1306Q mutation was produced and purified as described before.10;14 Diagnostic assays

ADAMTS13 activity (FRETS-VWF73 assay), VWF antigen and VWF activity levels were determined as described before.3;15;16 Active VWF levels was measured by immunosorbent assays with llama antibodies AU/VWF-a11, which specifically bind to active VWF A1 domain. VWF activation factor was used to express the relative amount of VWF that circulates in its active conformation. The VWF activation factor of normal pool plasma (NPP) was referred to as 1. ADAMTS13 antibody titre was measured by a commercially available enzyme-linked immunosorbent assay (ELISA) according to manufacturer instructions (Technoclone, Vienna, Austria). Plasma

levels of β2GPI were measured with an in house ELISA. In short, murine monoclonal

antibody 2B2 directed against β2GPI was diluted in Tris-buffered saline (TBS, pH 7.4) and coated overnight at 4°C onto ELISA plates (Costar, New York, USA). The plates were washed 3 times with TBS/ 0.1% Tween, blocked with 3% bovine serum albumin (BSA)/ TBS solution for 1 hour at 37°C and washed again 3 times with TBS/ 0.1% Tween. Patient plasma diluted 1:1000 (v/v) in 3% BSA/TBS/0.1% Tween was added to the plates. Serial dilutions of normal pool plasma that has been calibrated against a purified plasma-derived 2β GPI standard were used as reference standard. After incubation for 1 hour at 37°C, the plates were washed and incubated further with polyclonal rabbit-anti human β2GPI antibody at final concentration of 5 μg/mL in 3% BSA/TBS/0.1% Tween. After extensive washing, plates were incubated with a peroxidase-labeled goat-anti-rabbit antibody (DAKO, Glostrup, Denmark) for 1 hour. Staining was performed using an ortho-phenylenediamine (OPD) solution (4 mg/mL OPD diluted in 0.1 M NaH2PO4.2H2O/0.1 M Na2HPO4•H2O) and the color reaction was stopped by addition of 1 M H2SO4. Absorbance was measured at 490 nm.

Endothelial and endothelial cell derived UL VWF 8 Chapter

Human umbilical vein endothelial cells (HUVECs) were used to produce UL VWF 167 and as a substrate surface in perfusion studies. HUVECs were isolated from the umbilical cords of healthy newborns as described17 and cultured in endothelial basal medium (EBM-2) supplemented with EGM-2 GingleQuots (Clonetics, San GPI in TTP Diego, CA, USA). UL VWF was obtained from cultured HUVECs as described 2 β previously.18;19 Briefly, confluent HUVECs in a 75 2cm culture bottle were washed with phosphate buffered saline (PBS) and incubated with 1 mL 100 µM histamine (Sigma Chemicals, St Louis, MO) dissolved in PBS for 10 minutes at room temperature (RT) to stimulate the release of UL VWF. After incubation, liquid in culture bottle was collected and centrifuged at 500 g for 3 minutes to remove cell debris. The supernatant was used as source of UL VWF. VWF antigen level in this supernatant was measured by ELISA. Blood reconstitution with blood cells from healthy donor and plasma from TTP patients

Blood from healthy donors, who claimed not to have used aspirin or other non- steroidal anti-inflammatory drugs for the preceding 10 days, was collected into trisodium citrate (0.0109 M final concentration). Erythrocytes were isolated by alpha-cellulose columns20 and concentrated in PBS containing 2.5% human albumin to reach a hematocrit of over 95%. Platelets were isolated according to a previously described method21 and suspended in PBS/albumin at a count of 1500 × 109 /L. Packed platelet/erythrocyte mixture was made by mixing 1.2 mL concentrated erythrocytes and 560 µL concentrated platelet suspension. Reconstituted blood was obtained by mixing 200 µL plasma from TTP patients with 100 µL packed platelet/ erythrocyte mixture, which resulted a hematocrit of 23% and platelet count of 160 × 109 /L in the end. Flow cytometric analysis (FACS)

Binding of β2GPI to erythrocytes and platelets in whole blood and in reconstituted blood was detected by FACS. Briefly, 1 µL whole blood or reconstituted blood was added to 100 µL PBS without or with other additions (plasma derived VWF 10

µg/mL; VWF-R1306W 10 µg/mL; UL VWF 430 ng/mL). Anti-β2GPI 3B7 labeled with Alexa-488 (Invitrogen, Eugene, Oregon, USA) and anti-CD235a conjugated to phycoerythrin were added to the above reaction system and incubated at RT for 20min. Samples were fixed and further diluted with 900 µL 0.2% formaldehyde. A total of 60,000 events were measured by FACScanto II (BD Bioscience, Franklin Lakes, New Jersey, USA). The erythrocyte population was selected based on fluorescence from PE-conjugated anti-CD235a, and the platelet population was selected based on distinctive forward and side scatter properties. Mouse IgG2b/PE (BD Bioscience) and mouse IgG1/FITC (DakoCytomation, Glostrup, Denmark) were used as isotype

control for the anti-CD235a and anti-β2GPI antibodies, respectively, to determine Chapter 8 Chapter nonspecific background fluorescence. 168 Platelet agglutination

Freshly drawn blood from healthy volunteers, denying intake of any medication known to interfere with platelet function, was collected into trisodium citrate (0.0109

GPI in TTP 21

2 M final concentration). Washed platelets were isolated as previously described. β Agglutination studies were performed on an optical aggregometer (Chrono-log Corporation, Havertown, PA, USA). Agglutination was initiated by the addition of ristocetin (1.5 mg/mL) in the presence of recombinant wild type (WT) VWF, various

domains of β2GPI or antibodies against VWF. Surface plasmon resonance (SPR) analysis

The interaction between β2GPI and VWF was studied by SPR analysis using Biacore T100 biosensor system (both from Biacore, Uppsala, Sweden) as described 22 before. Recombinant full-length β2GPI and domain I of β2GPI were immobilized on a CM5-sensor chip using the amine-coupling kit according to manufacturer instructions (Enzyme Research Laboratories, Swansea, UK). A control channel was routinely activated but without specific coating to measure nonspecific interactions.

SPR analysis of VWF-R1306W binding to immobilized full length β2GPI and

β2GPI domain I was performed in buffer with 100 mMNaCl, 2 mM CaCl2, 0.005% Tween-20 and 20 mM Hepes (pH 7.4) at a flow rate of 20μL/min for 5 minutes at 25°C. Similarly, VWF A1 domain containing the R1306Q mutation was immobilized

on a C1-sensor chip to assess its interactions with full-length β2GPI and its separate domains. Protein-coupling to latex-beads

To investigate binding of β2GPI to endothelial cell derived UL VWF (EC-UL VWF),

β2GPI or albumin was coupled to 1 µm-diameter carboxylated modified latex (CML) beads (Invitrogen). To do so, the beads were washed 3 times with 0.1 M phosphate buffer (pH 8.1) and re-suspended in 0.1 M phosphate buffer containing 2 mg/mL 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) (Invitrogen). After overnight incubation, the beads were centrifuged and re-suspended in 0.1 M phosphate buffer containing 1 mg/mL β2GPI or 1 mg/mL albumin and incubated for 24 hours. Subsequently, the mixture was centrifuged and the supernatant buffer was removed for protein measurement to determine coupling efficiency. The beads used in these studies showed coupling efficiency of at least 90%. Perfusion studies

HUVECs were grown on glass coverslips till confluence.23 Twenty-four hours prior to the experiments, the endothelial cells were washed three times with pre-warmed HEPES-buffered balanced salt (HBBS; 138.3 mMNaCl/ 25 mM HEPES/ 0.82 mM

CaCl2/ 5.36 mMKCl/ 0.61 mM MgCl2/ 1 mM α,D-glucose, pH 7.4) followed by overnight incubation with FCS-free medium (EBM-2, Clonetics, Walkersville, MD, Chapter 8 Chapter USA). Three minutes before perfusion, endothelial cells were activated by incubation with 25µM histamine. Platelets or protein-coated beads (in absence of erythrocytes) 169 in culture medium were perfused over the endothelial cell layer at a shear-stress of 15 dynes/cm2 for 3 minutes. Adhesion of platelets/beads to UL VWF strings was visualized under a bright field microscope (Zeiss, Jena, Germany). Perfusion GPI in TTP of platelets/beads on non-activated endothelial cell surface was used as a negative 2 β control. Images were captured by a digital camera attached to the microscope and were quantified by ImageJ software. Statistical analysis

Measurements are presented as means ± standard deviation. Differences between groups were calculated with t-tests. The Pearson/Spearman correlation coefficient was calculated to determine the relationship between two parameters. P values<0.05 were considered statistically significant. Analysis was performed with GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, California, USA). Results

Plasma β2GPI levels in TTP patients

The patient groups with acute and remission TTP had lower plasma β2GPI levels than the healthy control group (102 ±59 and163± 80 µg/mL, respectively, vs. 220 ± 98 µg/mL; p < 0.001; Figure 1A). TTP patients with acute episodes were divided into two groups based on the Bethesda score for anti-ADAMTS13 antibody titer.

Patients with Bethesda score ≥ 3 had significantly lower 2β GPI levels than those with scores between 1 and 2 (86 ± 36 vs. 119 ± 74 µg/mL; p < 0.05). Seven patients were enrolled in a paired study in which plasma β2GPI levels were measured during an acute episode and after remission. The level of β2GPI was significantly lower during the acute phase than after remission (95 ± 46 vs.155 ± 40 µg/mL; p < 0.05; Figure A B

Figure 1: β2GPI plasma levels in TTP patients and healthy controls. (A) Comparison of β2GPI plasma levels among TTP patient group in acute phase, TTP patient group in remission, and healthy control group. (B) Paired study of

plasma β2GPI levels in seven patients measured during acute TTP attack and after remission. Symbols represent significance of difference: p* < 0.05; *** p < 0.001 Chapter 8 Chapter

170

A B GPI in TTP 2 β

C

Figure 2. Associations among β2GPI levels, ADAMTS13 levels and relative VWF activation factor in TTP patients.

(A) Association between2 β GPI levels and ADAMTS 13 levels (Person, n = 34, r = 0.51, p < 0.01). (B) Inversed

association between β2GPI levels and relative VWF activation factor (Pearson, n=30, r = -0.37, p < 0.05). (C).

Chronological study of β2GPI and ADAMTS 13 levels before, during and after first acute TTP attack in one patient. Blood before the first episode was obtained as patient appeared to be a blood donor. 1B).

In TTP patients in remission, plasma β2GPI levels showed a positive correlation with ADAMTS13 activity (Pearson r = 0.51; p < 0.01; Figure 2A) but not with total VWF antigen levels; however, there was an inverse correlation between β2GPI levels and the VWF activation factor in plasma (Pearson r = − 0.37; p < 0.05;

Figure 2B). ADAMTS 13 activity and β2GPI levels were also measured in plasma samples collected from a patient before, during and after the first TTP episode. Both parameters decreased during the acute phase of the disease and started to increase back to normal levels after approximately two weeks (Figure 2C).

Binding of β2GPI to platelets and erythrocytes

The decrease of β2GPI plasma levels during acute TTP episodes cannot be explained simply by binding to VWF multimers because of the much lower molar concentration of the latter, suggesting the existence of alternative mechanisms for β2GPI clearance. Chapter 8 Chapter A B 171 GPI in TTP 2 β

Figure 3. Influence of active VWF on β2GPI binding to platelets (PLTs) and erythrocytes (RBCs). Binding of β2GPI to PLTs and RBCs was measured by flow cytometric analysis. (A-B) Binding of β2GPI to PLT and RBCs in presence of plasma derived VWF (10 µg/mL), VWF-R1306W (10 µg/mL) and UL VWF (400 ng/mL, obtained from HUVECs supernatant). Data are expressed as means ± SD of 8 replicated experiments. Symbols represent significance of difference comparing to plasma derived VWF, or significance of difference between two correlation coefficients: * p < 0.05; *** p < 0.001. Given that TTP is characterized by thrombocytopenia and anaemia, we hypothesized that β2GPI could be cleared from the circulation together with platelets and erythrocytes. To test this hypothesis, β2GPI binding to platelets and erythrocytes was measured by flow-cytometry. We found that the percentage of 2β GPI positive platelets and erythrocytes was greater in the presence of exogenously added gain of function mutant VWFR1306W or EC-derived UL VWF than plasma purified VWF, and the difference between the latter two was highly significant (p < 0.001; Figure 3A, B). To investigate whether these results might reflect in vivo alterations, we measured β2GPI binding to platelets and erythrocytes from healthy donors added into plasma collected from seven patients with TTP both during the acute episode and in remission. In this ex vivo test, although no significant differences with respect to β2GPI binding between acute and remission plasma samples were found, a weak correlation between β2GPI bound on erythrocytes and VWF activation factor was revealed in the presence of TTP plasma(Spearman r = 0.65, P=0.1). No significant correlation was found between the amount of β2GPI bound to platelets and the VWF activation factor (Spearman r=0.52, P = 0.06).

Assessment of β2GPIbinding to VWF by SPR

Previously, we have shown that β2GPI inhibits platelet-VWF interaction by binding 9 to the VWF A1 domain. Here we explored which domain of β2GPI is involved in binding to VWF using SPR. Firstly, we found that the gain of function mutant VWF R1306W but not WT VWF (data not shown) was able to interact with immobilized

β2GPI (Figure 4A). Secondly, separate domains of β2GPI (I, II, IV and V) were evaluated for binding to immobilized VWF A1 domain containing the gain-of- function-mutation R1306Q, and only domain I exhibited a significant interaction (Figure 4B). Thirdly, mutant VWF R1306Q but not WT VWF was able to interact

with immobilized β2GPI domain I (Figure 4C). Thus, only β2GPI domain I interacts with VWF A1 domain in active conformation.

Chapter 8 Chapter Inhibition of 2β GPI on VWF/ristocetin induced platelet agglutination 172 To evaluate further the inhibitory effects of β2GPI on the VWF-platelet interaction we measured ristocetin-induced agglutination of washed platelets. Ristocetin was added to platelet suspension containing recombinant VWF (10 µg/mL) in the GPI in TTP 2 β A B

C Figure 4. Direct interaction between β2GPI and VWF by surface plasmon resonance (SPR). After adjusting for binding to a blank channel, response at equilibrium was determined and plotted against the concentration applied. (A) Binding of VWF-R1306W to immobilized

β2GPI. (B) Binding of individual β2GPI domains (DI, DII, DIV and DV) to immobilized VWF A1 domain containing a gain-of-function mutation (R1306Q).

Left top corner are sensorgrams of binding ofβ2GPI Domain I to immobilized VWF A1-R1306Q. (C) Binding

of VWF-R1306W to immobilized Domain I of β2GPI. Figure 5. Influence of β2GPI and its individual domains on VWF/ristocetin induced platelet agglutination. Agglutination of washed platelets was initiated by the addition of VWF and ristocetin in presence ofRAG35

(antibody against VWF A1 domain,50 µg/ml), RAG201 (antibody against VWF A3 domain, 50 µg/ml), full length 8 Chapter

β2GPI (2 µM and 4 µM), and recombinant domains of β2GPI (1 µM). Symbols represent significance of difference comparing to VWF/ristocetin alone induced platelet agglutination. *p < 0.05, **p < 0.01, ***p <0.001. 173 presence of either β2GPI (2 and 4 µM), domain-deleted mutants of β2GPI (1 µM), or the antibodies RAG35 (50 µg/mL) and RAG201 (50 µg/mL). Full-length β2GPI and mutants containing domain I of β2GPI were able to inhibit VWF/ristocetin-induced GPI in TTP 2 platelet agglutination, whereas β2GPI mutants lacking domain I were not (Figure 5). β Even in the presence of β2GPI, a monoclonal antibody against the VWF A1 domain (RAG-35) - but not one against the A3 domain (RAG-201 - completely inhibited VWF/ristocetin mediated platelet agglutination (Figure 5).

Inhibition of 2β GPI on platelet–VWF interaction under conditions of flow

To mimic the role of β2GPI in regulating platelet-VWF interactions in vivo, an in vitro perfusion studies were performed. Dong et al22 have shown that platelets perfused over activated endothelial cells were able to form beads-on-a-string-like patterns which are thought to mimic the formation of VWF-platelet microthrombi in the absence of ADAMTS13. Endothelial cells were stimulated with 25 µM histamine for 3 minutes, followed by perfusion of platelets in the absence or presence of RAG-

35 (50 and 100 µg/mL, Figure 6A) or β2GPI (2 and 4 µM, Figure 6B). Both β2GPI and RAG-35 inhibited platelet binding to EC-UL VWF dose-dependently. Next, we investigated whether domain I of β2GPI was involved in inhibiting platelet binding to EC-UL VWF. Domain I (10 µM), but not domain II-V, was able to block platelet adhesion to EC-UL VWF strings (Figure 6C).

To investigate whether the above inhibitory effect was due to direct binding of β2GPI to the EC-UL VWF, we coated latex beads of similar size and weight as platelets with purified plasma 2β GPI or albumin. First we confirmed that beads-on-a-string patterns were reproduced by perfusing β2GPI coated latex beads on histamine stimulated endothelial cells (Figure 7A). The formation of these beads-on-a-string A B C

Figure 6. Inhibitory effectof 2β GPI and its individual domains on platelet adhesion to endothelial derived VWF under flow. Endothelial cells grown on glass coverslips were activated by 25 µM histamine 3 minutes before perfusion. Washed platelets were perfused over the endothelia monolayer at a shear-stress of 15 dynes/cm2 in presence of indicated antibodies and proteins for 3minutes under a bright field microscope. Platelets were counted at the end of the perfusion. Platelet count at histamine alone was used as positive control. (A) RAG35 (antibody

against VWF A1 domain), (B) β2GPI, (C) β2GPI domain I (10 µM) and domain II-V (10 µM). Symbols represent Chapter 8 Chapter significance of difference: **p < 0.01, ***p <0.001. 174

patterns were similar when perfusing β2GPI-coated latex beads or fixed platelets over activated endothelial cells (Figure 7B). Beads coated with only domain I of

β2GPI were also able to form strings on histamine stimulated EC surface (Figure GPI in TTP

2 7C). As a negative control, bovine serum albumin-coated beads failed to form strings β on endothelial cell surface. Discussion

In this study we identified different lines of evidence suggesting that 2β GPI may

interact with active VWF, a conclusion supported by the observation that β2GPI plasma levels are decreased in patients with acute TTP or, to a lesser extent, with

a history of TTP. The plasma levels of β2GPI are directly correlated to ADAMTS 13 activity and inversely correlated to the level of VWF activation. It is generally accepted that thrombotic microangiopathy, the hallmark of TTP, is caused by enhanced platelet interaction with UL VWF multimers, which are peculiar in that they appear to contain A1domain in the active - i.e. in GPIb binding – conformation.24 In physiologic conditions, UL VWF multimers are proteolytically processed by ADAMTS13 as they emerge onto the endothelial cell surface, thus are not detected in any appreciable amount in circulating blood. During acute episodes of TTP, in contrast, ADAMTS-13 activity can drop to less than 5% of normal and, consequently, UL VWF can escape the local check point at the site of cellular release and appear

in the circulation. If such a situation develops, our results now indicate β2GPI as an additional protective mechanism capable of directly controlling the adhesive function of UL VWF in circulating blood. Indeed, the evidence presented here that

β2GPI in solution or coated onto beads can block platelet adhesion to ULVWF under

flow conditions is in line with the known ability of 2β GPI to inhibit VWF-dependent 9 platelet aggregation. These effects have been associated with β2GPI binding to the A

B 8 Chapter 175 GPI in TTP 2 β C

Figure 7. Binding of β2GPI coated beads to endothelial cell derived UL VWF (EC- UL VWF). (A) beads-on-a-string- like patterns formed by perfusing β2GPI coated latex beads over histamine stimulated endothelial monolayer. (B)

Comparison of beads-on-a-string-like patterns formed by β2GPI coated beads (i) and fixed platelets (ii) on EC-UL

VWF. (C) Binding of β2GPI domain I coated beads to EC-ULVWF.

VWF A1 domain.9 Here we provide new evidence that the binding site for VWF is located within domain I of β2GPI, a conclusion supported both by functional inhibition experiments and direct evidence for a high affinity bimolecular interaction between β2GPI domain I and VWF A1 domain.

Beyond the demonstration that β2GPI is the target of antibodies in the antiphospholipid syndrome (APS), its physiologic function has remained undefined until recently. It 8 has been shown that β2GPI can act as a scavenger for unwanted components. Now we propose a new function for β2GPI modulating the potentially excessive adhesive potential of UL VWF multimers that have escaped into the circulation. It explains the marked (>50%) drop of β2GPI plasma levels during acute TTP episodes. Based on the demonstrated interaction between UL VWF and β2GPI, it is reasonable to assume that clearance of complexes formed in the circulation plays a role in the latter event. In this regard, however, it is worth noting that the VWF concentration in blood is approximately 50 nM (calculated on subunit molecular mass, thus directly

reflecting A1 domain molar concentration), while that of circulating 2β GPI is 100- fold greater. Thus, there is not enough VWF in plasma to account for the marked drop

of β2GPI in the blood of TTP patients on the basis of a 1:1 bimolecular interaction.

Rather, for β2GPI clearing to occur solely via complexing to VWF, the assumption must be made of a substantial release of UL VWF multimers from damaged endothelial cells in the microcirculation. Indeed, very large “strings” of ULVWF, each composed of a large number of subunits, can be visualized on the surface of endothelial cells following acute stimulation with histamine, and because of their active A1 domain conformation these may act as efficient sponges for circulating

β2GPI. An important component of the clearance mechanism may be association of

VWF-β2GPI complexes with blood cells. Such a concept is in agreement with the evidence provided here that active VWF - for example, VWF containing a gain- of-function type 2B mutation or endothelial cell-derived ULVWF multimers - can Chapter 8 Chapter

mediate β2GPIbinding to platelets and erythrocytes. On this basis - and in view of 176 the fact that acute TTP episodes are typically characterized by thrombocytopenia and anemia; and that platelets and erythrocytes are the most abundant blood cell

population -it seems plausible to envision that β2GPI may be cleared from the circulation in association with these blood cells. GPI in TTP 2 β The demonstration provided here that β2GPI in plasma decreases significantly during an acute TTP episode, as well as its inverse correlation with an index of VWF activation, suggest that this measurement might have diagnostic value and perhaps help in gauging the risk of thrombotic complications. However, the known

large variation of β2GPI plasma levels in the healthy population limit the possible usefulness of such an assay to cases of recurrent TTP in which baseline levels of

plasmaβ2GPI have been previously determined during a remission phase. With respect

to novel therapeutic approaches, the demonstrated ability of β2GPI and its domain I to inhibit VWF-dependent platelet aggregation and adhesion to UL VWF multimers, apparently through preferential binding to active and more prothrombotic VWF

species, suggest the possible development of β2GPI as a specific antithrombotic agent

in TTP. In line with this suggestion, it has been shown previously that β2GPI may effectively reduce thrombus formation by binding to VWF A1 and thus preventing the interaction with platelet GPIbα.9 Accordingly, a previous study demonstrated

a dose-dependent protective effect of increased β2GPI plasma levels on the risk of myocardial infarction in elderly men, a protection that persisted even in the presence 25 of high VWF levels. However, in spite of the findings indicating that 2β GPI may

counteract the prothrombotic effects of VWF, the potential value of β2GPI domain I as an antithrombotic agent is greatly reduced by the knowledge that injection into mice, even without adjuvant, induces the formation of auto-antibodies.26 Arad et al27 recently showed that purified auto-antibodies directed against the first domain of 25 β2GPI, one of the criteria defining APS, strongly enhance the response in a mouse thrombosis model. In this light,it has been suggested that using β2GPI domain I as soluble antigen to neutralize autoantibodies might yield an antithrombotic effect in 27 APS. Further investigations into the complex biological properties of β2GPI will help define more definitively its possible role as a specific physiologic modulator of VWF prothrombotic activities and, perhaps, identify strategies for its use as an antithrombotic agent avoiding undesired side effects. Acknowledgements This work was supported in part by a grant from the Netherlands Heart Foundation (Grant Nr 2006T053 to BdL), the Swiss National Science Foundation (Grant Nr 32003B-124892 to JAKH), the Netherlands organization for Scientific Research (ZonMW 91207002 to PdG). Chapter 8 Chapter

177 GPI in TTP 2 β References 9. Hulstein JJ, Lenting PJ, de Laat B et al. beta2-Glycoprotein I inhibits von Willebrand 1. Sadler JE, Moake JL, Miyata T, George factor dependent platelet adhesion and JN. Recent advances in thrombotic aggregation. Blood 2007;110:1483-1491. thrombocytopenic purpura. Hematology. 10. Stel HV, Sakariassen KS, Scholte BJ et Am.Soc.Hematol.Educ.Program. 2004407- al. Characterization of 25 monoclonal 423. antibodies to factor VIII-von Willebrand 2. Furlan M, Robles R, Galbusera M et al. factor: relationship between ristocetin- von Willebrand factor-cleaving protease induced platelet aggregation and platelet in thrombotic thrombocytopenic purpura adherence to subendothelium. Blood and the hemolytic-uremic syndrome. 1984;63:1408-1415. N.Engl.J.Med. 1998;339:1578-1584. 11. Oosting JD, Derksen RH, Hackeng TM et al. 3. Hulstein JJ, de Groot PG, Silence K et al. In vitro studies of antiphospholipid antibodies A novel nanobody that detects the gain- and its cofactor, beta 2-glycoprotein I, of-function phenotype of von Willebrand show negligible effects on endothelial cell factor in ADAMTS13 deficiency and mediated protein C activation. Thromb.

Chapter 8 Chapter von Willebrand disease type 2B. Blood Haemost. 1991;66:666-671. 2005;106:3035-3042. 178 12. Agar C, de Groot PG, Morgelin M et al. beta- 4. Groot E, Fijnheer R, Sebastian SA, de Groot glycoprotein I: a novel component of innate PG, Lenting PJ. The active conformation immunity. Blood 2011;117:6939-6947. of von Willebrand factor in patients with 13. Sodetz JM, Pizzo SV, McKee PA. thrombotic thrombocytopenic purpura in

GPI in TTP Relationship of sialic acid to function and 2

β remission. J.Thromb.Haemost. 2009;7:962- in vivo survival of human factor VIII/von 969. Willebrand factor protein. J.Biol.Chem. 5. McNeil HP, Simpson RJ, Chesterman CN, 1977;252:5538-5546. Krilis SA. Anti-phospholipid antibodies 14. Huizinga EG, Tsuji S, Romijn RA et al. are directed against a complex antigen Structures of glycoprotein Ibalpha and its that includes a lipid-binding inhibitor complex with von Willebrand factor A1 of coagulation: beta 2-glycoprotein I domain. Science 2002;297:1176-1179. (apolipoprotein H). Proc.Natl.Acad. Sci.U.S.A 1990;87:4120-4124. 15. Groot E, Fijnheer R, Sebastian SA, de Groot PG, Lenting PJ. The active conformation 6. Agar C, van Os GM, Morgelin M et al. Beta2- of von Willebrand factor in patients with glycoprotein I can exist in 2 conformations: thrombotic thrombocytopenic purpura in implications for our understanding of remission. J.Thromb.Haemost. 2009;7:962- the antiphospholipid syndrome. Blood 969. 2010;116:1336-1343. 16. Eckmann CM, De Laaf RT, Van Keulen 7. Agar C, de Groot PG, Levels JH, Marquart JM, van Mourik JA, de LB. Bilirubin JA, Meijers JC. Beta2-glycoprotein I oxidase as a solution for the interference is incorrectly named apolipoprotein H. of hyperbilirubinemia with ADAMTS-13 J.Thromb.Haemost. 2009;7:235-236. activity measurement by FRETS-VWF73 8. Agar C, de Groot PG, Mörgelin M, Monk assay. J.Thromb.Haemost. 2007;5:1330- SD, van Os G, Levels JH, de Laat B, 1331. Urbanus RT, Herwald H, van der Poll T, 17. Jaffe EA, Nachman RL, Becker CG, Minick Meijers JC. β -glycoprotein I: a novel 2 CR. Culture of human endothelial cells component of innate immunity. Blood. 2011 derived from umbilical veins. Identification Jun 23;117(25):6939-47. by morphologic and immunologic criteria. J.Clin.Invest 1973;52:2745-2756. 2011 18. Moake JL, Turner NA, Stathopoulos NA, 27. Arad A, Proulle V, Furie RA, Furie BC, Furie Nolasco LH, Hellums JD. Involvement of B. beta-Glycoprotein-1 autoantibodies from large plasma von Willebrand factor (vWF) patients with antiphospholipid syndrome multimers and unusually large vWF forms are sufficient to potentiate arterial thrombus derived from endothelial cells in shear formation in a mouse model. Blood stress-induced platelet aggregation. J.Clin. 2011;117:3453-3459. Invest 1986;78:1456-1461. 28. Ioannou Y, Romay-Penabad Z, Pericleous C 19. Ruggeri ZM, Zimmerman TS. The complex et al. In vivo inhibition of antiphospholipid multimeric composition of factor VIII/von antibody-induced pathogenicity utilizing the Willebrand factor. Blood 1981;57:1140- antigenic target peptide domain I of beta2- 1143. glycoprotein I: proof of concept. J.Thromb. Haemost. 2009;7:833-842. 20. Beutler E, Gelbart T. The mechanism of removal of leukocytes by cellulose columns. Blood Cells 1986;12:57-64.

21. Korporaal SJ, Van EM, Adelmeijer J et al. 8 Chapter Platelet activation by oxidized low density 179 lipoprotein is mediated by CD36 and scavenger receptor-A. Arterioscler.Thromb. Vasc.Biol. 2007;27:2476-2483. 22. Lenting PJ, Neels JG, van den Berg BM et GPI in TTP

al. The light chain of factor VIII comprises 2 a binding site for low density lipoprotein β receptor-related protein. J.Biol.Chem. 1999;274:23734-23739. 23. Dong JF, Moake JL, Nolasco L et al. ADAMTS-13 rapidly cleaves newly secreted ultralarge von Willebrand factor multimers on the endothelial surface under flowing conditions. Blood 2002;100:4033-4039. 24. Arya M, Anvari B, Romo GM et al. Ultralarge multimers of von Willebrand factor form spontaneous high-strength bonds with the platelet glycoprotein Ib-IX complex: studies using optical tweezers. Blood 2002;99:3971- 3977. 25. de Laat B, de Groot PG, Derksen RH et al. Association between beta2- glycoprotein I plasma levels and the risk of myocardial infarction in older men. Blood 2009;114:3656-3661. 26. de Laat B, van BM, Urbanus RT et al. Immune responses against domain I of beta(2) -glycoprotein I are driven by conformational changes. Arthritis Rheum. Chapter 8 Chapter

180 GPI in TTP 2 β 9

General discussion Chapter 9 Chapter

181 General discussion General Preface The balance of hemostasis is a tightly regulated process, which involves several different types of cells and plasma elements. While normal thrombus formation seals a leak in the vessel, an hyperresponsive hemostatic response in the vessel leads to thrombotic complications such as those demonstrated in stroke, myocardial infraction, arterial and venous thrombosis. Treatment of these thrombotic complications generally involves platelet inhibitors (e.g. asprin and clopidogrel), anti-coagulants (e.g. low molecular weight heparin, warfarin, thrombin inhibitor and factor Xa inhibitor) and thrombolytic agents (e.g. urokinase, streptokinase, and tissue plasminogen activators). Although new drugs in treating thrombotic complications keep emerging, there is still no ideal medicine that is both safe (no bleeding risk) and effective (no recurrence in thrombosis). A better understanding of the rather complicated mechanisms underlying bleeding and thrombosis would help us to find better strategies to cope with these clinical challenges. In the studies described in Chapter 9 Chapter this thesis we have focused on exploring the involvement of erythrocyte, platelet, 182 von Willebrand factor, and beta (2)-glycoprotein I (β2GPI) in bleeding and clotting. Erythrocyte-platelet interaction Of the subjects that we have studied and described in this thesis, platelets are obviously best studied and accepted as one the most important contributors in regulating bleeding and clotting. However, evidence indicating thrombosis as a multicellular event has been accumulating during the last century.1 Endothelial General discussion General cells and leukocytes have been shown to interact with platelets.2-4 Erythrocytes are the most abundant cells in our blood stream, comprising 99% of the cellular mass. Erythrocytes are thought be predominantly passively involved in hemostasis due to their rheological role by skinning platelets to the vessel wall. We thought it would be remarkable that such an abundant cell was not involved in the delicate balance between bleeding and thrombosis in an active way. In Chapter 2, we reviewed the available literature about the role of erythrocytes in thrombosis and hemostasis. In Chapter 3, we reported our own findings regarding a receptor/ligand mediated erythrocyte-platelet interaction. Erythrocyte-platelet interaction – the ‘how’ and ‘what then?’

In chapter 3 of this thesis, we have reported the binding of erythrocytes to platelets under low shear rate conditions. Erythrocytes were seen to attach to surface- immobilized platelets with a focal adhesion point under flow. In addition, we have for the first time found evidence indicating a physiological relevance of this phenomenon. Our results revealed that erythrocytes were able to bind to platelets not only in physiological buffer but also in a whole blood environment under venous shear rates. When perfused over endothelial cells in vitro, erythrocytes adhered to platelet decorated strings and forms platelet-erythrocyte aggregates. Since endothelial dysfunction is one of the underlying pathology of venous thrombosis,5;6 the adhesion of erythrocytes to platelets captured by newly secreted VWF strings from endothelial cells may contribute to the development of a venous thrombus. Our study with inhibitory antibodies and peptide indicates that the binding of erythrocyte to platelet under flow conditions is at least partially mediated by erythrocyte intercellular adhesion molecule 4 (ICAM-4) and platelet integrin αIIbβ3. We therefore propose that under low flow rates, erythrocytes and platelets can interact with each other via a receptor/ligand (ICAM-4 - αIIbβ3) mediated manner (Figure 1A). Moreover, we have for the first time demonstrated the physiological relevance of this direct erythrocyte-platelet adhesion. Our data showed that when perfused over endothelial cells in vitro, erythrocytes adhered to platelet decorated strings and forms platelet-erythrocyte aggregates. Since endothelial dysfunction is one of the underlying pathology of venous thrombosis,5;6 the adhesion of erythrocytes to platelets captured by newly secreted VWF strings from endothelial cells may contribute to the development of a venous thrombus. Chapter 9 Chapter Interestingly, our results described in chapter 3 indicate that the ICAM-4 mediated erythrocyte-platelet interaction may further promote platelet activation. With an 183 ICAM-4 mimetic peptide, we were able to demonstrate that the binding of this

ICAM-4 peptide to high affinity (or open confirmation)IIb α β3 induced by ADP stimulation can increase further P-selectin (a platelet activation marker) exposure on platelets, which indicates that the binding of ICAM-4 peptide to activated αIIbβ3 triggers signaling pathways (both outside-in signaling and inside-out signaling) inside platelets. Assuming that receptor ICAM-4 on the erythrocyte membrane General discussion General interacts with αIIbβ3 in the same way as the ICAM-4 peptide, the erythrocyte-platelet interaction would lead to signaling inside the platelet thereby further activating platelets. This finding is in line with that of two earlier studies.7;8 Since both ICAM-

4 and fibrinogen can bind to activatedIIb α β3 and induce signaling events, future studies comparing the characteristics of signaling transduction induced by these two molecules would broaden our understanding of erythrocyte-platelet interactions. We showed that the binding of erythrocytes to platelets requires platelet activation. However, the functional status of platelets may not be the only factor that influences the erythrocyte-platelet adhesion. The properties of erythrocytes, such as age and types/ levels of blood group antigens may also influence the interaction. A subpopulation of erythrocytes may interact with platelets more intensively than others. Moreover, it is worth to note that the anti-ICAM-4, anti-CD61 (antibody against β3 chain of the αIIbβ3 complex) or the ICAM-4 mimetic peptides in our study can only reduce erythrocyte-platelet adhesion by 50%, which indicates that it is likely that there are other mechanisms involved in the erythrocyte-platelet interaction. These possible alternative mechanisms may include the interactions between ICAM-4 and other 9;10 integrins on platelets (e.g. αvβ3), and plasma proteins forming fridges between erythrocyte and platelets (e.g. fibrinogen, VWF, and thrombospondin).11-13 Besides the receptor/ligand mediated cross-talking that we proposed in this thesis, erythrocytes and platelets may possibly interact with each other through other pathways – such as the chemical messenger model proposed in an earlier study (Figure 1B). 14 With a static two-step platelet recruitment assay, Santos and Valles et al showed that erythrocytes increase platelet reactivity to physiological agonists (such as collagen) by metabolically modify platelet arachidonic and eicosanoid formation.7;14 The presence of erythrocytes induced a two-fold increase in platelet

thromboxane B2 (TxB2) synthesis upon collagen stimulation. Cell-free releasates from stimulated platelet-erythrocyte suspensions contained 6.9 fold more ADP and A Chapter 9 Chapter

184

B General discussion General

Figure 1. The two proposed models of erythrocyte-platelet interaction. (A) The receptor/ligand model. In this model, erythrocyte bind to platelet via a receptor/ligand mediated manner, resulting in signaling transduction in platelets and possibly in erythrocytes as well. (B) The chemical massager model. In this model, ADP/ATP and thromboxane serve as chemical messengers to mediate the cross-talking between erythrocyte and platelets, modulating the erythrocyte and platelet function.

4.9 fold more ATP than that from stimulated platelets in the absence of erythrocytes.14 Later on, the same research group revealed that this effect of erythrocyte on platelet activation was enhanced in patients with insulin-dependent diabetes mellitus and was held accountable for the aspirin resistance in patients with vascular disease.15-19 Based on these findings, a chemical messenger model was proposed to explain the interactions between erythrocyte and platelets. In this model, the interaction between erythrocytes and platelets was thought to be carried out through components of platelet releasate upon stimulation (i.e. adenosine diphosphate ADP, adenosine triphosphate ATP, and thromboxane). These components act as extracellular messengers that bring about biochemical modifications generating prothrombotic activity on erythrocytes (Fig 1B).7;14 This chemical mediated cell- cell interaction—especially regarding eicosanoid synthesis—has also been found between platelets and endothelial cells, which has been termed as the ‘endoperoxide steal hypothesis’.20 Endothelium synthesize and secret prostacyclin to inhibit platelet function. In a perturbed mixture of endothelial cells and platelets, approximately half of the prostacyclin produced by the endothelial cells were shown to be originated from platelet endoperoxides.21 Platelet-erythrocyte interaction may adopt the same mechanism as the platelet-endothelial cell interaction. It is worth to note that besides ADP/ATP and thromboxane, Lysophosphatidic acid which is also a component of platelet releasate has been shown to induce erythrocyte aggregation in an in vitro static experimental setting.22 While the LPA induced modification on erythrocytes has been shown to be related to calcium in flux in erythrocytes,22 the effect of ADP/ ATP and thromboxane on erythrocytes remains to be elucidated.

ICAM-4-αIIbβ3 interaction on platelet function

23

Although previous biochemical study and our own in intro perfusion study (chapter 9 Chapter 3) clear showed that ICAM-4 is the ligand to mediate erythrocyte-platelet interaction 185 by binding to αIIbβ3, the effect of this interaction on platelet function remains unknown.

In the study described in chapter 4, we characterized the effect of ICAM-4-αIIbβ3 interaction on platelet function by testing the influence of ICAM-4 peptides on platelet aggregation, P-selectin surface expression (a marker for platelet activation), binding of fibrinogen to activated platelets and p38 MAPK phosphorylation (an indication for platelet signalling transduction). Our results showed that 64P90K

on domain I and 112P138L on domain II are likely to be the crucial regions in discussion General  ICAM-4-αIIbβ3 interaction. Our finding about 64P 90K on domain I as an active region in influencing platelet function is consistent with the finding of Hermand et al. who have shown that this region contains critical residuals for ICAM-4 binding 24 to integrin αIIbβ3. Our results showed that peptide D1.7 on domain I can bind to the fibrinogen binding site on αIIbβ3, which is in agreement with the results from the study of Hermand et al. However, while Hermand et al. identified E151 (within D2.6 in our ICAM-4 peptide libary) as the critical residual on domain II for ICAM-

4 binding to integrin αIIbβ3, our study indicates that the binding site on domain II of ICAM-4 lies within region covered by D2.3. This discrepancy could be derived from the difference in the methodology between our two studies. More studies are thus needed in order to further elucidate the ICAM-4- αIIbβ3 interaction in molecular level. Data from our study indicates that the interaction between ICAM and αIIbβ3 are likely to induce platelet signal transduction and further activates platelets. Another interesting message derived from this study is that ICAM-4 protein possesses encrypted epitopes on domain I (peptide D1.9 covered region) and domain II (peptide D2.1 covered region) which when exposed can induce platelet aggregation promptly. Future research on ICAM-4 conformational change and the corresponding consequence on physiology, especially thrombosis and hemostasis, would be of great interest in the sense of understanding the erythrocyte-platelet interaction and the intervention of thrombosis.

The effect of ICAM-4 – αIIbβ3 interaction on thrombosis and hemostasis Our data described in chapter 3 demonstrated that the ICAM-4 mimetic peptide reduced thrombin formation in whole blood samples.25 With an in vitro thrombus formation model, we found reduced fibrin deposition, thicker fibers and less fiber branches in the clot formed from whole blood treated with ICAM-4 peptide. These data suggest that erythrocyte ICAM-4 may influence local thrombin generation and thrombus formation. In addition, the ICAM-4 peptide prolonged tail bleeding time in mice, indicating the possible contribution of erythrocyte-platelet interaction in supporting hemostasis. Targeting the ICAM-4 mediated erythrocyte-platelet interaction might be a new strategy in developing anti-thrombotic medicines.

Additional studies scrutinizing the influence of ICAM-4 and αIIbβ3 interaction on platelet function and thrombus formation in animal models may provide further clues in pursuing this direction. Chapter 9 Chapter

186 Platelet clearance in transfusion medicine and in VWD type-2B Platelets are important in the maintenance of hemostasis. When a vessel is damaged, platelets adhere at the site of injury to the exposed subendothelium. The subsequent attachment of other platelets to the already adherent platelets results in the generation of a platelet plug that seals off the damaged vessel wall and arrests blood loss from the vessel. When the platelet count drops to a certain amount (severe thrombocytopenia, 9

General discussion General platelet count is < 10 x 10 /L), patients should receive prophylactic platelet transfusion to reduce the risk of bleeding. Platelet products from blood banks are currently stored at room temperature, which leads to a short shelf life and the risks of bacterial contamination in the products. Cold storage (0-4°C) would be better in preserving platelet hemostatic properties and reduce the risk of contamination, but the transferred platelets stored this way are known to be cleared rapidly from the circulation. Our own findings regarding the mechanisms of this fast clearance of the platelets stored at cold conditions was described in chapter 5. Another situation with possibly increased platelet clearance is in von Willebrand disease (VWD) type 2B, which is a disease with a gain-of-function mutation in VWF gene leading to hyper active VWF. VWF type-2B is characterized by thrombocytopenia and bleeding. Since the underlying mechanisms for the development of thrombocytopenia in VWF type-2B patients is unclear, we studied the clearance of platelets in a VWF type2B murine animal model, the results of which is described in Chapter 6. Cold induced platelet apoptosis and transfusion medicine

In chapter 5 of this thesis, we described our findings regarding the mechanisms by which transfused platelets stored at cold conditions are cleared rapidly from the circulation. Our data revealed that the cold (0-4°C, mimicking the in vitro storage condition of platelet concentrates) and rewarming (37°C, mimicking the in vivo condition after platelet transfusion) treatment induces apoptosis in platelets by inducing glycoprotein (GP) Ibα conformational change—clustering. The clustered GPIbα would then associate with cytosolic 14-3-3- adapter proteins, that control the activity of pro-apoptotic Bad.26;27 The association of GPIbα and 14-3-3 proteins is accompanied by dephosphorylation of Bad, which is a crucial step in intrinsic apoptosis pathway. Dephosphorylated-Bad replaces Bax on the [Bax-Bcl-xL] complex liberating active Bax.28 The ratio of Bak over Bcl-xL has been proposed as an important determinant of platelet life span.28-30 Activated Bax expose the cryptic N-terminal and translocate to mitochondria, leading to depolarization of the mitochondria inner membrane, release of cytochrome c, activation of caspase-9, and exposure of phosphatidylserine (PS) on the platelet surface.31 The externalized PS serves as “eat-me” signals for phagocytes leading to the clearance of platelets. The apoptosis process has already begun and is accelerating in time under cold storage prior to the rewarming stage. Although apoptotic process is present, the platelets stored at 0°C seems to remain in a resting state, demonstrated by the lacking of platelet Chapter 9 Chapter activation markers, such as TxA2-formation or P-selectin expression. However, the rewarming stage induces further apoptosis and initiates platelet activation. The 187 GPIbα-14-3-3 association, 14-3-3ζ-Bad association, Bax activation and the change of mitochondrial transmembrane potential increase further during 37°C-incubation. Incubation with indomethacin shows that these changes are triggered by thromboxane 32 A2 which starts when cold-stored platelets enter the 37°C-incubation. We have previously shown that GPIbα clustering is caused by extracellular exposure of β-acetyl-D-glucosamine (βGN) and galactose-residues as well as the intracellular discussion General de-attachment from the membrane skeleton.32 It is reasonable to assume that the glycosylation state of the extracellular domain of GPIbα affects the signalling properties of its intracellular domain. Our results showed that βGN indeed blocks GPIbα-induced apoptosis, emphasizing the importance of the GPIbα-change in control of platelet survival. Since our previous study and this study described in chapter 4 have clearly identified GP1bα as the receptor responsible for initiating apoptosis, we think that the attempts to improve platelet transfusion by cold-storage should focus on the regulation of GP1bα. Thrombocytopenia in VWD-type 2B

VWD-type 2 B is a qualitative VWF deficiency with a gain-of-function mutation within the VWF A1 domain. Patients with VWD-type 2B lack high molecular weight (HMW)-VWF multimers and have enhanced VWF-platelet interaction. Paradoxically, the increased platelet aggregation in VWD-type 2B is associated with bleeding rather than thrombosis. The bleeding tendency is strongly correlated with the severity of the thrombocytopenia in these patients.33 However, the molecular basis of the thrombocytopenia is still unclear. Nurden and colleagues have reported a family in which severe thrombocytopenia may be linked to abnormal megakaryocytopoiesis.34 Megakaryocytopiesis was shown to be inhibited by the enhanced VWF-GP1α interaction.35;36 Increased clearance of VWF/platelet complex has also been implicated as one of the mechanisms underlying the thrombocytopenia in VWD type 2B.37;38 But no experimental evidence in support of this possibility has been provided so far. In our study reported in Chapter 6, by using a mouse model for VWD-type 2B, we provided direct evidence that the formation of VWF/platelet aggregates is indeed associated with an enhanced uptake of such complexes in macrophages in liver and spleen. In addition, we showed that the lifespan of platelets in VWD-type 2B mice is significantly shorter compared to the lifespan of platelets in control mice. Our first line of evidence for the involvement of macrophages in the clearance of VWF/platelet complexes comes from mice experiments with macrophage depletion procedures. The depletion of macrophages resulted in an increase in endogenous VWF antigen levels, but did not affect platelet counts in mice expressing wild-type murine VWF. In contrast, the depletion of macrophages induced 2-3 fold increase

Chapter 9 Chapter in platelet counts in thrombocytopenic mice expressing VWD type 2B mutant VWF 188 (p.V1316M-mVWF). This finding is in line with that has been described by Alves- Rosa et al, who have shown an increase in platelet count following macrophage depletion in a mouse model for immune thrombocytopenia.39 Immune-histochemical analysis of liver and spleen sections of mice injected with fluorescence labeled platelets revealed that near 80% of fluorescent platelets co-localized with CD68- positive macrophages in liver (i.e. Kupffer cells), and 68% co-localized with CD169- positive marginal metallophilic macrophages. Interestingly, our data showed a specific General discussion General colocation of VWF/platelet complexes with CD169-positive marginal metallophilic macrophages but not with F4/80-positive or MARCO-positive macrophages. The mechanism behind this specific pattern warrants further scrutiny. Although we have clearly demonstrated an enhanced phagocytosis of platelet clearance in VWD-type 2B mice, the underlying mechanisms remain to be elucidated. Previously we, and others have shown that macrophages can bind to and uptake active VWF induced by shear stress via lipoprotein receptor-related protein - 1.40-42 In this study (Chapter 6) we found that conversion of VWF in its active, platelet- binding conformation (either by botrocetin or via a gain-of-function VWD-type 2B mutation) also promotes the uptake of VWF by macrophages. It is therefore possible that platelets are a bycatch when macrophages bind and endocytose VWF-type 2B mutants that are at the platelet surface. Alternatively, similar to the mechanisms underlying the cold induced platelet apoptosis described in Chapter 5, binding of mutant VWF to the platelet GpIbα-receptor may trigger the platelet apoptotic cascade leading to increased phagocytosis by macrophages.

Indications for a protective role ofβ 2GPI in TTP A third subject described in thesis, that is closely related to the previous part, is the

function of β2GPI. β2GPI is best known as the antigen against which antiphospholipid antibodies are developed. In the study described in chapter 7, we explored the mechanisms by which these antiphospholipid antibodies (including anti- β2GPI) lead to thrombotic complications in antiphospholipid syndrome. In chapter 8, we reported our findings indicating that 2β GPI has a protective function in in situations of hyper-active VWF, such as in patients with acute thrombotic thrombocytopenic purpura (TTP).

In addition to its role in antiphospholipid syndrome, β2GPI has been shown to possess other biological functions, such as inhibition of the contact system of the blood coagulation,43 and clearance of lipopolysaccharide (LPS, a major constituent of the outer membrane of Gram-negative bacteria) from our circulation. 44-46 Previously we have shown that β2GPI interacts with the VWF A1 domain competing with GPIbα- binding and, thus, inhibiting platelet adhesion.47 In the study described in chapter

8, we propose a new function for β2GPI. Our data suggest that β2GPI can modulate the potentially excessive adhesive potential of ultra large (UL) VWF multimers that have escaped into the circulation as in the situations of thrombotic thrombocytopenic purpura (TTP). 9 Chapter TTP is a life-threatening disease characterized by thrombocytopenia and thrombotic 189 microangiopathy.48 It is generally accepted that the thrombotic microangiopathy in TTP is caused by an enhanced platelet interaction (via GP1bα) with UL VWF multimers. The increase in UL VWF in circulation is in most cases the result of a severe deficiency of ADAMTS 13 activity due to the presence of autoantibodies 49 . We observed that β2GPI plasma levels are decreased in patients with acute TTP (50% drop) or, to a lesser extent, with a history of TTP. The plasma levels of β GPI

2 discussion General are directly correlated to ADAMTS 13 activity and inversely correlated to the level of VWF activation. During acute episodes of TTP, ADAMTS-13 activity can drop to less than 5% of normal and, consequently, UL VWF appears in the circulation.

Our results indicate that β2GPI as an additional protective mechanism may be able to directly influence the platelet-UL VWF interaction in circulating blood. Our finding that β2GPI in solution or coated onto beads can block platelet adhesion to ULVWF under flow conditions is supported by the known ability of β2GPI to inhibit VWF- 47 dependent platelet aggregation. It has been shown that the binding site of β2GPI on VWF lies within the A1 domain of VWF.47 In the study described in Chapter 7, we identified domain I ofβ 2GPI as the binding site for VWF.

It is worth noting that the concentration of β2GPI in circulation is 100-fold greater than that of circulating VWF. Thus, the clearance of β2GPI via UL VWF/ β2GPI complex pathway cannot be held accountable for the 50% drop of plasma β2GPI levels observed in acute TTP. We have shown that active VWF (either a gain-of-function type 2B mutation or UL VWF multimers) can mediate β2GPI binding to the surface of platelets and erythrocytes. Since acute TTP episodes are typically characterized by thrombocytopenia and anemia combined with the fact that erythrocyte and platelets are the most abundant cell population in blood, we think that β2GPI may be cleared from the circulation in association with these blood cells.

Although we have found indications for a protective role of β2GPI in TTP, such as

the drop of β2GPI levels in plasma as well as the binding of β2GPI to UL VWF, the

evidence for a cause and effect relationship between β2GPI levels and TTP pathology

still need to be proven, perhaps by investigating the effect of β2GPI depletion in TTP

animal models. If the relationship between an artificial reduction of β2GPI and the increased severity of TTP phenotype is established in the animal models, one may

suggest the possible development of β2GPI as a specific antithrombotic agent in TTP. The expanding horizons and future directions In the studies described in this thesis, we have observed new horizons in better understanding thrombosis and hemostasis. Firstly, we have found a direct interaction between erythrocyte and platelets under low flow rate conditions. This interaction is at least partially mediated by an adhesion molecule on the erythrocyte membrane Chapter 9 Chapter (ICAM-4) and the fibrinogen receptor (αIIbβ3) on platelets. Interruption of this 190 interaction leads to reduced thrombus formation in vitro and prolonged bleeding time in a mouse tail bleeding assay. Future studies should focus on deciphering the exact mechanism underlying erythrocyte-platelet interaction and on exploring the possibilities of targeting this interaction as a new therapeutic strategy for preventing thrombosis. Secondly, we have found explanations for the rapid clearance of cold stored platelets after transfusion as well as for the thrombocytopenia in VWD-type 2B. We found that the conformational change of GP1bα (also called clustering) is the General discussion General crucial and initial step in inducing apoptotic chain reactions in cold stored platelets. Since the glycosylation state of the extra cellular domain of GP1bα is an important regulator of its confirmation, combined with our own finding showing an inhibition of apoptosis with a sugar kind (βGN), we believe that future research should aim to improve platelet transfusion by cold-storage and focus on the regulation of the GP1bα, especially the glycosylation. Moreover, for the first time we found direct evidence that increased uptake of platelet/VWF complex by macrophages is one of the underlying mechanisms for thrombocytopenia in VWD-type 2B. Thirdly, we

have found indications for a protective function of β2GPI in situations of hyperactive

VWF (such as in TTP). Since the plasma levels of β2GPI drops dramatically in TTP acute phase and our in vitro experiments showed inhibition of VWF induced platelet

aggregation by β2GPI, we think that the binding of β2GPI to active VWF blocks at least partially the VWF-platelet binding, alleviating the thrombotic pathology in

TTP. Future research about the role of β2GPI in thrombosis animal models would

shed more light on the possibilities of applying β2GPI as an anti-thrombotic agent. References 10. Zennadi R, Moeller BJ, Whalen EJ et al. Epinephrine-induced activation of LW- 1. Marcus AJ, Safier LB. Thromboregulation: mediated sickle cell adhesion and vaso- multicellular modulation of platelet occlusion in vivo. Blood 2007;110:2708- reactivity in hemostasis and thrombosis. 2717. FASEB J. 1993;7:516-522. 11. Carvalho FA, Connell S, Miltenberger- 2. Maclouf J, Murphy RC, Henson PM. Miltenyi G et al. Atomic force microscopy- Transcellular sulfidopeptide leukotriene based molecular recognition of a fibrinogen biosynthetic capacity of vascular cells. receptor on human erythrocytes. ACS Nano. Blood 1989;74:703-707. 2010;4:4609-4620. 3. Marcus AJ, Safier LB, Ullman HL et 12. de Oliveira S., Vitorino dA, V, Calado A, al. Interactions between platelets and Rosario HS, Saldanha C. Integrin-associated neutrophils in the eicosanoid pathway. Adv. protein (CD47) is a putative mediator for Prostaglandin Thromboxane Leukot.Res. soluble fibrinogen interaction with human 1989;19:263-266. red blood cells membrane. Biochim. Biophys.Acta 2012;1818:481-490. 4. Palabrica T, Lobb R, Furie BC et al. Chapter 9 Chapter Leukocyte accumulation promoting fibrin 13. Lominadze D, Dean WL. Involvement of deposition is mediated in vivo by P-selectin fibrinogen specific binding in erythrocyte 191 on adherent platelets. Nature 1992;359:848- aggregation. FEBS Lett. 2002;517:41-44. 851. 14. Santos MT, Valles J, Marcus AJ et al. 5. Kleinegris MC, Ten Cate-Hoek AJ, Ten Enhancement of platelet reactivity and CH. Coagulation and the vessel wall in modulation of eicosanoid production by thrombosis and atherosclerosis. Pol.Arch. intact erythrocytes. A new approach to Med.Wewn. 2012;122:557-566. platelet activation and recruitment. J.Clin.

Invest 1991;87:571-580. discussion General 6. Migliacci R, Becattini C, Pesavento R et al. Endothelial dysfunction in patients with 15. Valles J, Santos MT, Aznar J et al. spontaneous venous thromboembolism. Modulatory effect of erythrocytes on the Haematologica 2007;92:812-818. platelet reactivity to collagen in IDDM patients. Diabetes 1997;46:1047-1053. 7. Valles J, Santos MT, Aznar J et al. Erythrocytes metabolically enhance 16. Valles J, Santos MT, Aznar J et al. Erythrocyte collagen-induced platelet responsiveness promotion of platelet reactivity decreases the via increased thromboxane production, effectiveness of aspirin as an antithrombotic adenosine diphosphate release, and therapeutic modality: the effect of low- recruitment. Blood 1991;78:154-162. dose aspirin is less than optimal in patients with vascular disease due to prothrombotic 8. Valles J, Santos MT, Aznar J et al. Platelet- effects of erythrocytes on platelet reactivity. erythrocyte interactions enhance alpha(IIb) Circulation 1998;97:350-355. beta(3) integrin receptor activation and P-selectin expression during platelet 17. Santos MT, Valles J, Aznar J et al. recruitment: down-regulation by aspirin ex Prothrombotic effects of erythrocytes on vivo. Blood 2002;99:3978-3984. platelet reactivity. Reduction by aspirin. Circulation 1997;95:63-68. 9. Litvinov RI, Vilaire G, Shuman H, Bennett JS, Weisel JW. Quantitative analysis of 18. Santos MT, Valles J, Aznar J et al. Aspirin platelet alpha v beta 3 binding to osteopontin therapy for inhibition of platelet reactivity using laser tweezers. J.Biol.Chem. in the presence of erythrocytes in patients 2003;278:51285-51290. with vascular disease. J.Lab Clin.Med. 2006;147:220-227. 19. Santos MT, Valles J, Lago A et al. Residual 28. Morrison DK. The 14-3-3 proteins: platelet thromboxane A2 and prothrombotic integrators of diverse signaling cues that effects of erythrocytes are important impact cell fate and cancer development. determinants of aspirin resistance in patients Trends Cell Biol. 2009;19:16-23. with vascular disease. J.Thromb.Haemost. 29. Oltersdorf T, Elmore SW, Shoemaker AR 2008;6:615-621. et al. An inhibitor of Bcl-2 family proteins 20. Fitzpatrick FA, Gorman RR. A comparison induces regression of solid tumours. Nature of imidazole and 9,11-azoprosta-5,13- 2005;435:677-681. dienoic acid. Two selective thromboxane 30. Schoenwaelder SM, Yuan Y, Josefsson synthetase inhibitors. Biochim.Biophys. EC et al. Two distinct pathways regulate Acta 1978;539:162-172. platelet phosphatidylserine exposure and 21. Marcus AJ, Weksler BB, Jaffe EA, Broekman procoagulant function. Blood 2009;114:663- MJ. Synthesis of prostacyclin from platelet- 666. derived endoperoxides by cultured human 31. Upton JP, Valentijn AJ, Zhang L, Gilmore endothelial cells. J.Clin.Invest 1980;66:979- AP. The N-terminal conformation of Bax 986. regulates cell commitment to apoptosis. Cell Chapter 9 Chapter 22. Kaestner L, Steffen P, Nguyen DB et al. Death.Differ. 2007;14:932-942. 192 Lysophosphatidic acid induced red blood cell 32. van der Wal DE, Verhoef S, Schutgens RE aggregation in vitro. Bioelectrochemistry. et al. Role of glycoprotein Ibalpha mobility 2012;87:89-95. in platelet function. Thromb.Haemost. 23. Hermand P, Gane P, Huet M et al. Red 2010;103:1033-1043. cell ICAM-4 is a novel ligand for platelet- 33. Federici AB, Mannucci PM, Castaman G activated alpha IIbbeta 3 integrin. J.Biol. et al. Clinical and molecular predictors of Chem. 2003;278:4892-4898. thrombocytopenia and risk of bleeding in General discussion General 24. Hermand P, Gane P, Callebaut I et al. patients with von Willebrand disease type Integrin receptor specificity for human 2B: a cohort study of 67 patients. Blood red cell ICAM-4 ligand. Critical residues 2009;113:526-534. for alphaIIbeta3 binding. Eur.J.Biochem. 34. Nurden P, Debili N, Vainchenker W et al. 2004;271:3729-3740. Impaired megakaryocytopoiesis in type 25. Du VX, de Groot PG, van Wijk R, Ruggeri 2B von Willebrand disease with severe ZM, de Laat B. Binding of erythrocyte thrombocytopenia. Blood 2006;108:2587- ICAM–4 to the platelet activated integrin 2595. α β leads to a direct erythrocyte-platelet IIb 3 35. Nurden AT, Federici AB, Nurden P. Altered adhesion under venous flow shear rate. megakaryocytopoiesis in von Willebrand Paper presented at:Annual Meeting of the type 2B disease. J.Thromb.Haemost. 2009;7 American Society of Hematology; December Suppl 1:277-281. 9, 2012; Atlanta, GA 36. Nurden P, Gobbi G, Nurden A et al. Abnormal 26. Masters SC, Fu H. 14-3-3 proteins mediate VWF modifies megakaryocytopoiesis: an essential anti-apoptotic signal. J.Biol. studies of platelets and megakaryocyte Chem. 2001;276:45193-45200. cultures from patients with von Willebrand 27. Zha J, Harada H, Yang E, Jockel J, disease type 2B. Blood 2010;115:2649- Korsmeyer SJ. Serine phosphorylation of 2656. death agonist BAD in response to survival 37. Saba HI, Saba SR, Dent J, Ruggeri ZM, factor results in binding to 14-3-3 not BCL- Zimmerman TS. Type IIB Tampa: a variant X(L). Cell 1996;87:619-628. of von Willebrand disease with chronic thrombocytopenia, circulating platelet 47. Hulstein JJ, Lenting PJ, de Laat B et al. aggregates, and spontaneous platelet beta2-Glycoprotein I inhibits von Willebrand aggregation. Blood 1985;66:282-286. factor dependent platelet adhesion and aggregation. Blood 2007;110:1483-1491. 38. Rick ME, Williams SB, Sacher RA, McKeown LP. Thrombocytopenia associated 48. Sadler JE, Moake JL, Miyata T, George with pregnancy in a patient with type IIB von JN. Recent advances in thrombotic Willebrand’s disease. Blood 1987;69:786- thrombocytopenic purpura. Hematology. 789. Am.Soc.Hematol.Educ.Program. 2004407- 423. 39. Alves-Rosa F, Stanganelli C, Cabrera J et al. Treatment with liposome-encapsulated 49. Arya M, Anvari B, Romo GM et al. Ultralarge clodronate as a new strategic approach in the multimers of von Willebrand factor form management of immune thrombocytopenic spontaneous high-strength bonds with the purpura in a mouse model. Blood platelet glycoprotein Ib-IX complex: studies 2000;96:2834-2840. using optical tweezers. Blood 2002;99:3971- 3977. 40. van Schooten CJ, Shahbazi S, Groot E et al. Macrophages contribute to the cellular Chapter 9 Chapter uptake of von Willebrand factor and factor VIII in vivo. Blood 2008;112:1704-1712. 193 41. Rastegarlari G, Pegon JN, Casari C et al. Macrophage LRP1 contributes to the clearance of von Willebrand factor. Blood 2012;119:2126-2134. 42. Castro-Nunez L, Dienava-Verdoold I, Herczenik E, Mertens K, Meijer AB. Shear stress is required for the endocytic uptake discussion General of the factor VIII-von Willebrand factor complex by macrophages. J.Thromb. Haemost. 2012;10:1929-1937. 43. Schousboe I. beta 2-Glycoprotein I: a plasma inhibitor of the contact activation of the intrinsic blood coagulation pathway. Blood 1985;66:1086-1091. 44. Agar C, de Groot PG, Levels JH, Marquart JA, Meijers JC. Beta2-glycoprotein I is incorrectly named apolipoprotein H. J.Thromb.Haemost. 2009;7:235-236. 45. Agar C, de Groot PG, Morgelin M et al. beta(2)-glycoprotein I: a novel component of innate immunity. Blood 2011;117:6939- 6947. 46. Agar C, van Os GM, Morgelin M et al. Beta2- glycoprotein I can exist in 2 conformations: implications for our understanding of the antiphospholipid syndrome. Blood 2010;116:1336-1343. Chapter 9 Chapter

194 General discussion General Summary/Samenvatting/概要 概要

195 Summary / Samenvatting / Summary / Samenvatting The Expanding Horizons in Thrombosis and Hemostasis: Summary for non-professionals Blood clotting

In the human body, blood is flowing through the blood vessels and is constantly circulating from the heart to the peripheral body tissues, back to the lungs, and finally back to the heart again. When blood vessels are cut or damaged, the loss of blood must be stopped (hemostasis) before the body goes into shock. This is accomplished by solidification of the blood around the wound, a process called coagulation or clotting. A blood clot consists of a plug of platelets enmeshed in a network of insoluble fibrin fibers. Clot formation is a very well regulated process. Normally, in healthy people, the clotting system is in an inactive state. However, when an injury

概要 occurs in the blood vessel, collagen and tissue factor—two proteins which normally lie beneath the vessel wall become exposed to blood. The collagen will activate the smallest blood cells, the sticky platelets to finally form a platelet plug. The tissue 196 factor will catalyze a biochemical reaction called coagulation in blood and finally lead to fibrin (an insoluble protein with fiber-like structure) formation. In the end, a clot composed of platelets and fibrin is formed to stop the bleeding. After the fiber network of the fibrin is formed, red blood cells can be trapped within the fibers and become part of a clot.

Summary / Samenvatting / Summary / Samenvatting The role of red blood cell in blood clotting

Red blood cells are the most abundant cell type in the blood. They take up around 40% of blood volume. The main function of red blood cells is to transport oxygen from the lungs to the peripheral tissues and carry Carbon Dioxide from peripheral tissue back to the lungs. In the studies described in this thesis (chapter 2 to 4), we have investigated the involvement of red blood cells in blood clotting. We have found that red blood cells are not only passively entrapped in a blood clot, but are also able to actively modulate blood clot formation. Our experimental data indicate that red blood cells can influence clotting by interacting with platelets. Red blood cells are able to bind to platelets under flow conditions. The binding of red blood cells to platelets is mediated by a protein called ICAM-4 on the red blood cell membrane

and its receptor protein called αIIbβ3 on the platelet membrane. When the binding of these proteins were blocked by using antibodies and/or synthetic peptides, the clot size was reduced. Based on the above findings, we think that the drugs that target the red blood cell-platelet interaction may become one of the new therapeutic strategies to treat or prevent hyper clotting conditions, such as in the situations of heart attack and stroke. Platelet clearance post-transfusion and in von Willebrand Disease

Platelets are important in maintaining hemostasis. When the platelet count drops to a certain amount (severe thrombocytopenia, platelet count is < 10 x 109/Liter), patients should receive prophylactic platelet transfusion to reduce the risk of bleeding. Platelet products from blood banks are currently stored at room temperature, which leads to a short shelf life (max 7 days) and the risks of bacterial contamination in the products. Cold storage (0-4°C) would be better in preserving the quality of platelet products. However, after transfusion, the cold-stored platelets are known to be cleared rapidly from the circulation. In our studies described in this thesis (chapter 5), we found new mechanisms by which the transfused platelets stored at cold conditions are cleared rapidly. Cold temperature can induce a structural change in an important protein called GP1b on the platelet surface. This change induces changes inside platelets which in the end lead to platelet death . The platelets with these “eat-me” markers of cell death will be eaten by macrophages – giant cells that clear dead cells and pathogens such as bacteria.

Von Willebrand factor (VWF) is a protein in the blood that helps to recruit platelets to 概要 the injured vessel wall. Deficiency of VWF leads to von Willebrand disease (VWD) which is characterized by defects in blood clotting at sites of vascular injury. Patients with VWD-type 2B have enhanced VWF-platelet interaction but with bleeding 197 tendency rather than hyper blood clotting. The bleeding tendency in these patients is related to the severity of thrombocytopenia (low platelet numbers). In our study (chapter 6), we further investigated the mechanism of the thrombocytopenia in VWD- type 2B. We found that the enhanced VWF-platelet interaction in these patients is

related to the increased uptake of the VWF/platelet complex by macrophages in both / Summary / Samenvatting the liver and spleen. Our mouse experiments clearly showed that the lifespan of platelets in VWD-type 2B mice is significantly shorter compared to the lifespan of platelets in control mice. Patients with VWD-type 2B have enhanced VWF-platelet interaction but with bleeding tendency rather than hyper blood clotting. The bleeding tendency in these patients is related to the severity of thrombocytopenia (low platelet numbers).

β2GP I in thrombotic thrombocytopenic Purpura

β2GP I (a protein in the blood) is best known as the antigen against which anti- phospholipid antibodies are developed. The detection of antiphospholipid antibodies in blood is one of the diagnostic criteria for the anti-phospholipid syndrome (APS)—a disease that is characterized by vascular thrombosis and pregnancy loss (chapter7).

In addition to its role in APS, β2GPI has been shown to possess other biological functions. In our study (chapter 8), we proposed a new function for β2GPI. Our data suggest that β2GPI can play a protective function in patients with thrombotic thrombocytopenic purpura (TTP) by binding to ultra large VWF. TTP is a life- threatening disease and patients with TTP have increased blood clotting in the small blood vessels. This enhanced blood clotting is caused by increased platelet binding to ultra large VWF. We found that β2GPI inhibits the platelet-VWF interaction, thus protects the patients from hyper blood coagulation. De Uitbreiding van de Horizon van Trombose en Hemostase: Nederlandse Samenvatting (voor niet-ingewijden) Bloedstolling

In het menselijk lichaam stroomt het bloed door de bloedvaten en circuleert voortdurend van het hart naar de perifere lichaamsdelen, en daarna weer terug naar de longen en het hart. Wanneer de bloedvaten beschadigd raken, moet het bloeden zo snel mogelijk gestopt worden (hemostase) voordat het lichaam in shock terecht komt. Daarvoor moet het bloed stollen rondom de wond, bloedstolling genoemd. Een bloedstolsel bestaat uit een bloedplaatjes prop, ingebed in een netwerk van onoplosbare fibrine vezels. De bloedstolling is zeer goed gereguleerd. In gezonde personen, is normaalgesproken de bloedstolling inactief. Maar wanneer er een

概要 wond ontstaat in een bloedvat, komen collageen en weefselfactor- twee eiwitten die normaalgesproken verborgen liggen- in de bloedvatwand, aan de oppervlakte en in aanraking met het bloed. Het collageen zal vervolgens de kleverige bloedplaatjes 198 (kleinste bloedcellen in het bloed) activeren om een bloedplaatjesprop te vormen. Het weefselfactor zal vervolgens de biochemische stollingsreactie aanzetten om uiteindelijk fibrine te vormen (onoplosbaar eiwit met vezelachtige structuur). Uiteindelijk zal een bloedstolsel bestaan uit bloedplaatjes en fibrine, gevormd om een bloeding te stelpen. In het klassieke stollingsmodel, nadat er een fibrine vezel

Summary / Samenvatting / Summary / Samenvatting netwerk gevormd is, kunnen rode bloedcellen hierin gevangen worden en op die manier een onderdeel worden van het stolsel. De rol van rode bloedcellen in bloedstolling

Bloed bestaat voor het grootste gedeelte uit rode bloedcellen. Dit is ongeveer 40% van het totale bloedvolume. De belangrijkste functie van de rode bloedcel is het transport van zuurstof van de longen naar de verschillende perifere organen. Ook transporteren ze koolstofdioxide van de perifere organen weer terug naar de longen. In onze studies hebben we een niet-eerder beschreven zeer belangrijke rol gevonden voor rode bloedcellen in de bloedstolling. We beschrijven in dit proefschrift dat rode bloedcellen niet alleen passief deel uitmaken van een stolsel maar ook actief de bloedstolling beïnvloeden. Onze experimenten laten zien dat rode bloedcellen een rol hebben in de bloedstolling door een interactie met bloedplaatjes aan te gaan. Rode bloedcellen kunnen zich vasthechten aan bloedplaatjes, alleen bij een bepaalde snelheid van bloedstroming. De binding van rode bloedcellen aan plaatjes vindt plaats door middel van het ICAM-4 eiwit wat zich op het oppervlak bevindt van de rode bloedcelmembraan en het receptor eiwit alpha2b/3a op de bloedplaatjesoppervlak. Wanneer we de binding van deze twee eiwitten blokkeren door specifieke antistoffen te gebruiken en/of kleine eiwitfragmenten, zien we dat de grootte van het bloedstolsel afneemt. Gebaseerd op deze bevindingen denken we dat bepaalde medicijnen die de rode bloedcel-bloedplaatjes interactie blokkeren, interessante nieuwe therapeutische toepassingen kunnen hebben in de behandeling van hyperbloedstollings condities, zoals het geval is bij een hartaanval of beroerte. De klaring van bloedplaatjes post-transfusie en in von Willebrandziekte

Bloedplaatjes zijn zeer belangrijk voor hemostase. Maar wanneer het bloedplaatjesaantal in het bloed daalt naar een bepaalde hoeveelheid (ernstige trombocytopenie, wanneer het plaatjesaantal <10x109/Liter is), krijgen patiënten een profylactische plaatjes transfusie om het bloedingsrisico te verkleinen. Bloedplaatjesproducten worden tegenwoordig in de bloedbanken bewaard op kamertemperatuur, maar dit leidt tot een korte levensduur (max 7 dagen) en risico op bacteriële besmetting in deze producten. Bewaren op koudere temperaturen (0-4°C) zou beter zijn om de kwaliteit van de plaatjes te verbeteren, maar het is bekend dat koud-bewaarde plaatjes sneller verdwijnen (geklaard) uit de circulatie. In onze studies, beschreven in dit proefschrift, hebben we nieuwe mechanismen 概要 gevonden hoe het komt dat de koud-bewaarde plaatjes sneller geklaard worden. Het koud bewaren kan structurele veranderingen veroorzaken in een belangrijk eiwit op de oppervlakte van bloedplaatjes, GP1b. Deze verandering zorgt voor signalen 199 binnenin de plaatjes wat uiteindelijk kan leiden tot celdood. De bloedplaatjes die deze “eet-mij” signalen op het oppervlakte hebben, worden vervolgens opgegeten door macrofagen-grote bloedcellen die helpen om dode cellen en ziekteverwekkers uit de weg te ruimen, zoals bacteriën.

Von Willebrand Factor (VWF) is een eiwit in het bloed welke helpt om bloedplaatjes / Summary / Samenvatting te binden aan de verwonde bloedvatwand. Een gebrek aan VWF leidt tot von Willebrandziekte (VWD), dit is een ziekte waarbij de bloedstolling verstoort. Patiënten met VWD-Type 2B hebben een verhoogde VWF-plaatjes interactie, maar met een hoge bloedingsneiging in plaats van een hyperactieve bloedstolling. De bloedingsneiging in deze patiënten komt door het lage aantal bloedplaatjes (ernstige trombocytopenie). In onze studies hebben we de mechanismen onderzocht achter VWD-type 2B. We hebben gevonden dat een hogere VWF-bloedplaatjes interactie zorgt voor een hogere opname van VWF-plaatjes complexen door macrofagen uit de lever en milt. Onze proeven die gedaan zijn in muizen laten heel duidelijk zien dat de levensduur van bloedplaatjes in VWD-type 2B muizen veel korter is dan in normale gezonde muizen. Een beschermende rol voor 2βGPI in trombotische trombocytopenische purpura

2βGP1 is een eiwit in het bloed die het meest bekend is als het antigen tegen anti- fosfolipiden antistoffen. Het aantonen van anti-fosfolipide antistoffen is een van de belangrijkste criteria in de ziekte anti-fosfolipiden syndroom (APS)-een ziekte waarbij er spontaan bloedstolsels in de bloedvaten kunnen ontstaan maar ook miskramen kan veroorzaken. Naast de rol in APS, heeft β2GP1 ook andere belangrijke biologische functies. In deze studies beschrijven we een nieuwe rol voor β2GP1. Onze bevindingen tonen aan dat β2GP1 een beschermende functie kan hebben door aan ultra-lang VWF te binden in patiënten met trombotische trombocytopenische purpura (TTP). TTP is een levensbedreigende ziekte en patiënten met TTP hebben een te hyperactieve bloedstolling in de kleine bloedvaten. De toename in bloedstolling wordt veroorzaakt doordat bloedplaatjes binden aan ultra-lang VWF. Wij hebben gevonden dat β2GP1 de bloedplaatjes-VWF interactie remt, dus patiënten beschermt tegen een hyperactieve bloedstolling. 概要

200 Summary / Samenvatting / Summary / Samenvatting 血栓与凝血新进展: 中文摘要 （针对非专业型读者） 止血的过程

血液在人体的心血管系统中持续不断的循环，从心脏流向肢体末梢，从肢体 末梢流向肺，再从肺流回心脏。当血管受到损害，血液就会从血管中流出， 称为出血。人体有一套精密的止血系统。血液可以通过在血管的伤口上形成 血栓来防止出血过度而导致的休克。血栓主要由血小板和纤维蛋白组成。血 管壁受损后，原本跟血液隔离的血管内膜下的成分—胶原蛋白和组织因子— 就会跟血液接触。胶原蛋白可以激活血小板—血液中体积最小的一种细胞。 被激活的血小板发生自身凝集，形成血栓的内核。组织因子跟血液的接触会 概要 引起一系列凝血因子的活化，最后形成蜘蛛网样的纤维蛋白网。纤维蛋白网 覆盖在血小板聚集物的周围，加固了血栓的止血作用。在血流缓慢的情况 下，大量红细胞（红血球）会被纤维蛋白网捕获，成为血栓的组成部分。 201

红细胞在血栓形成中的作用

红细胞是血液中数量最多的细胞。他们占据着血液40%的容积。红细胞的 Summary / Samenvatting / Summary / Samenvatting 主要功能是运输气体。红细胞在肺组织中吸收氧气，携带氧气的红细胞顺血 流流到周围组织，把氧气释放给周围组织细胞，同时吸收周围组织细胞产生 的二氧化碳。红细胞携带的二氧化碳将在肺组织中被氧气取代。尽管红细胞 是血栓的组成部分，它在血栓形成过程中的作用通常被认为是被动的、没有 重大影响的。本论文的第二至第四章探讨了红细胞在血栓形成中的作用。与 传统的观念不同，我们的研究结果表明红细胞不仅只是被动地参与血栓的形 成。红细胞可以通过与血小板相互作用进而调节血栓的形成。我们的体外血 栓形成模拟实验显示红细胞可以黏附在被激活的血小板表面上。红细胞和血 小板的结合是通过其表面的两种蛋白实现的—在红细胞表面的一种被称细胞 间黏附分子4（ICAM-4）的蛋白和一种在血小板上的被称为糖蛋白IIb/IIIa的 蛋白。红细胞与血小板的结合可以引起血小板的进一步活化，从而促进血栓 形成。体外及动物实验表明，阻断红细胞与血小板的结合将会使血栓明显减 小。针对红细胞和血小板相互作用的药物有希望成为新一代的抗血栓药物。 这些药物的研制和开发对于血栓性疾病（如动、静脉血栓）或血栓关联性疾 病（如冠心病，中风）的治疗有重要意义。

血小板的清除 血小板是血栓形成中的重要细胞。当血液中血小板的数量低于一定程度 （ 被称为血小板减少症），病人就需要接受预防性的输浓缩血小板治疗以防止 出血。目前，血库中制备的浓缩血小板等血小板产品都是室温保存的。室温 保存有助于细菌的生长，使血小板产品的污染几率增加。低温（0-4°C）保 存将有助于保存血小板的品质。但是，低温保存的一大缺点是经过低温保存 的血小板被输入到患者体内后会被很快地清除掉。本论文的第五章描述了低 温保存的血小板输入体内后被清除的机制。我们的研究发现血小板表面的一 种被称为糖蛋白1b的蛋白在低温状态下会发生结构的转化。糖蛋白1b的结构 转化导致血小板内部信号转导通路的激活，进而引起“吞噬我”信号（一种被 称为磷脂酰丝氨酸的膜磷脂）在血小板表面的表达。表面带有“吞噬我”信号 概要 的血小板将被巨噬细胞吞噬掉，从而从血液中消失。阻止糖蛋白1b的结构转 化可以阻断“吞噬我”信号的表达从而起到延长血小板生存期的作用。本论文 202 的第六章探讨了2B型血管性学友病中血小板的清除机制。血管性血友病是一 种常见的出血性遗传性疾病。其根本病理学机制在于VW因子—血液中一种 介导血小板凝集的蛋白—的异常。2B型血管性血友病患者的VW因子与血小 板的结合力增强，然而此类患者往往不具备异常高凝状态，而是多有出血倾 Summary / Samenvatting / Summary / Samenvatting 向。既往研究表明患者出血的严重程度与患者血小板减少的程度有关。我们 的研究结果表明由于血小板和VW因子的亲和力增加，肝和脾中的巨噬细胞 对VW因子/血小板结合物的吞噬作用也随之增加。我们的小鼠实验显示2B型 血管性血友病小鼠的血小板体内存活时间明显短于正常小鼠。

β2糖蛋白1的病理生理作用

β2糖蛋白1是血液中的一种磷脂蛋白。抗磷脂综合征患者体内的自身抗体就 是针对此蛋白产生的。抗磷脂综合征的诊断需要临床标准（血栓的发生和/或 习惯性流产）和实验室标准（抗磷脂蛋白抗体的出现）的结合。本论文的第 七章综述了抗磷脂综合征抗体在抗磷脂综合征的病理机制中的作用。第八章 探讨了β2糖蛋白1在血栓性血小板减少性紫癜中的作用。血栓性血小板减少 性紫癜是一种罕见，却可致命的血栓和出血混合性疾病。我们的研究发现β2 糖蛋白1可以通过阻断血小板与VW因子的结合进而减轻血栓性血小板减少性 紫癜患者的症状。 Acknowlegements/Dankwoord/致谢 致谢

203 Acknowledgements / Dankwoord / / Dankwoord Acknowledgements Just as the studies described in this book has broadened our understanding about thrombosis and hemostasis, the experience of working as a Ph.D student in the last years has no doubt broadened my own view of the world and myself. I have come a long way from a primary-school student in a small village to study medicine in Beijing and then to be a Ph.D student in the Netherlands. Along the way, I came across many teachers, mentors, and friends who helped me solving problems, inspired me to go forward, and nurtured me with their kind and warm encouragements. I take this opportunity to thank everyone who has supported me in one way or another in the past years. First of all, I would like to thank my promotor Flip de Groot and co-promotor Bas de Laat. Dear Flip, thank you for your support and guidance in all these years.

致谢 You gave me both the freedom in conducting our research pursuits as well as clear guidance. It is always a nice atmosphere during our work discussions. Thank you for always being there when I needed answers for difficult questions. Dear Bas, I couldn’t imagine that I am now already at the end of my Ph.D. From the moment you congratulated me for getting the job in lab 3 to the moment I received your email 204 congratulating me for finishing my Ph.D thesis, it feels like only days in between. When I look back, I see a consistently warm, optimistic, and generous mentor. In the last 3 years, you traveled from Maastricht to Utrecht almost every week for our work discussions. In the busy period, we even had extra Skype meetings in the Tuesday evenings. Your dedication to work is inspiring. I have learned a lot from you during

Acknowledgements / Dankwoord / / Dankwoord Acknowledgements my ph.d, from the crucial research skills to being a more flexible person. Thank you for your support and good advices for both my work and my personal development. Dear prof. Akkerman, dear Jan Willem, thank you for all the guidance during the year that I worked for you as a technician/research assistant. You are patient and systematic in supervising. I am grateful for what I have learned from you. From the first work discussion in your office until now, I am still wondering about one question：how does Jan Willem arrange his file cabinet? Dear Mark, you are my first supervisor in the Netherlands. I cannot thank you enough for your kindness in helping me settle down in this foreign country. You are a good teacher and always have a sincere heart to help the young students. I have learned a lot from you in the beginning of my career in the Netherlands. Thank you for all the jokes as well. It was nice laughing together with you, although I never quite got them. Dear Coen, you are a wonderful mentor, who is always open for a coffee and discussion. The ‘één + n gratis’ policy for advice is always attractive for a ph.d student. Thank you for the occasional special coffee treats as well as the stimulating ideas with the coffee scent. Dear Richard, thank you for your enthusiasm and advice in our erythrocyte work. It is always a pleasure to discuss erythrocyte research with you. And thank you for inviting me to the 19th Meeting of the European Red Cell Society held on the Forteiland. It was a pleasant and special experience. Dear Rob and Quirijn, thank you for being my group mates for the Monday work discussions. It was a nice experience to share our research stories together. Dear Rolf, I always admire your vast knowledge as well as your wisdom in all sorts of areas. Thank you for your advice and counseling. Dear Dr. Wu, Dear Yaping, I admire your tireless pursuit in doing research. After retirement, you are still active in leading a research group in China. Thank you for your support and advice all these years. Dear Harry, thank you for the discussions about platelet imaging. And thank you for my first paper in Blood. Dear Ray, thanks for the laugh, it is always nice and fun talking with you. Arjan, the prince of lab 4, I consider you as my first Dutch friend. You taught me the first technics that I use in research. You are patient and thorough in teaching. It was always a pleasure to chat with you. Thank you for all your help and support. Silvie, I have known you for six and half years, and I am still surprised to discover your multi-talents now and then. You are good at decorating our LKCH borrels, 致谢 good at dancing, good at experiments, good at mountain climbing,….and recently, I got to know that you are good at drawing as well. Thank you for being my group mate at the Monday work discussion, thank you for providing me with HUVECs, and helping me find VWF reagents now and then. Arjan and Silvie, thanks for being 205 my paranimfen. Arnold, our chief technician, I couldn’t imagine how our labs would be without you being around. You are always understanding and helpful. I consider myself lucky that I have the daily support from such a colleague. Grietje, thank you for teaching me red cell related technics and thank you for the chats now and then. Kim, thank you for the nice time together. Elske, thank you for providing me with

HUVECs when you were working here in lab 3. Brigit, thanks for the chats. Thank / / Dankwoord Acknowledgements you for the sharp observations and nice suggestions during my work presentations. You have a good sense for details. Thanks for the good time we had on the forteiland. Sandra, Annet and Tesy, it was nice talking with you about Children, which we share the mother feelings. Simone, thanks for the tips about ELISA, and good luck with your work and study. Dear fellow ph.d students in the ‘AIO Kamer’ of LKCH, it was an unforgettable experience to share a special way of life called ph.d with you in the same office. Since I have been in the AIO Kamer for two periods, I guess I know the most of the ph.d students at the end of my ph.d. Dianne, you were my supervisor when I was working for Jan Willem. I am glad that we kept contact and become good friends. Thank you for your advices and thank you for help me with the Dutch summary for this thesis. Anja, Cees, Eric, thank you for the old time’s memory, and wish you guys enjoy your current work. Gwen, thanks for the good advices and good time together. I appreciate your frankness and friendship. Thijs, it was nice chatting with you now and then in the aio room, or in front of the coffee machine. You are a good listener. Claudia and Agon, thanks for the practical tips now and then. Elo and Jerney, it must be a special experience for you two to work abroad together, wish you enjoy it. Esther, thank you for being so patient and enthusiastic in help me learning Dutch. Susan, it is always so nice to be around you. You can make anything into a good laugh. Thank you for the good time together during the red cell congress on the forteiland, and good luck with your promotion further. Hay, hi and hoi Sander, thanks for being the lunch reminder for me. It is indeed my loss when I miss the lunch with you and the others. Steven, what can I say to you, we always do Kongfu fight in the hallway, but still, thanks for being my desk neighbor, who would ask now and then ” are you okay? You made a big sigh!”. Holala, Bert, it is fun to be around you. Thanks for all the laugh, and thanks for organizing the thesis printing deal for ph.d students. Marije and Perter Paul, thank you for the chats and good luck in the clinic. Marco, thanks for bringing me back home after the NVTH, and thanks being my labmate. Attie, it was always nice talking with you. Thank you for the encouragements and share of feelings. Wish you enjoy the wonderful time with your baby and good luck with the work you love. Valentina, you are amazing in making friends, and you are

致谢 a worthy one yourself. Good luck in your career in Germany. Jessica and Rick, you are the future of the AIO room, good luck with your research. Krijn, Roy and Eszter, Marcel, thanks for the chats and advices. Dear Suus (Dr. Korporaal), thanks for the good time during the European platelet congress in Maastricht. And thanks for the tips as a mother. Marten, good luck with your new career in education. 206 Dear Joukje, thank you for your support all these years, from helping me mailing the reagents to taking care of the congress registrations and others. Beste Rosmina, bedankt voor het gezellige praatjes. During my phd study, I got help from 3 internship students. Khalid, I feel lucky to have you as my first internship student. On your first day, I taught you how to do Acknowledgements / Dankwoord / / Dankwoord Acknowledgements FACS, the next day, you can already perform the experiment by yourself. Thank you for the data you collected about ICAM-4 expression levels on erythrocytes. Salih, you are a special student. It is not easy to do an internship in a lab at your age. Everything in the lab must seem so fast and chaotic to you. But you tried to handle the lab work as the best as you can. I admire your courage and dream. I wish you good luck in the Netherlands. Anil, thanks a lot for your help with the vesicle and thrombin generation work as well as the nice time together. You are very quick in learning and very social and understanding. Actually, I have learned a lot from you. Munisia, Renske, Tiffany, Chantal and other internship students, who worked in LKCH, thank you for the chats and nice time together. Pavla and Livia, thank you for the culture education about Czech and Brizil, good luck with your research. Mirjam, thanks for being my bench neighbor in lab 3. Good luck with your promotion further. Dear colleagues in Synapse, it was a great pleasure to know you and work together with you. Hilde, thank you for help me with the APS paper. Evelyn thank you for correcting my manuscript about red blood cell and platelet interaction. Dana and Raed, it was nice to write the red blood cell review together with you, thank you for your input. Dear Prof. Hemker, Dear Coen, thank you for all the inspirations. Thank you for the nice time during the synapse social activities, such as the BBQ at your place and the traditional Christmas dinners. Iris, thank you for taking care of the declarations and other chores for me. And it is always nice talking with you about the girls. Ganesh, Evelien, and Adam, thanks for helping me and Anil with setting up the thrombin generation assay. Leonie, thank you for the companionship in Atlanta, and thanks for helping me with the try-out flow experiments in Maastricht as well as the SEM analysis. Good luck with your promotion further. Ilze, Romy, thanks for the good time in Paris and the tips about promotions. Joke, thank you very much for the mice study. Marisa, thanks for the nice chats. Marieke, Bas gave me small toys (such as the Hello Kitty) for Linna’s birthdays. I believe you prepared them. Thank you! Wish you enjoy your maternity leave. Andrea, it was a great pleasure working together with you for making the scientific drawings for the red cell review article as well as the front and back cover for this thesis. Dear honey Iwan, thank you for all the support for my work. In particular, thank you for all the patience and resilience in training me with the Dutch jokes. Statistics 致谢 predicted that you might have an improved chance in making pronouncing and maybe significant progress in the future. Little Linna, thanks for all the fun together with Mama. Beste Pa en Ma Fernhout, heel erg bedankt voor jullie ondersteuning van mijn promotie. In de afgelopen jaren heeft Ma heel veel op Linna gepast en Pa 207 is altijd enthousiast over (en geïnteresseerd naar) mijn onderzoek. Ik wens Pa succes met zijn eigen onderzoekproject naar de gevolgen van fosfaat. Zoals Pa Fernhout heeft geconcludeerd is te veel fosfaat in het alledaagse voedsel niet alleen slecht voor nierpatiënten, maar ook voor gezonde mensen. Beste Ilse, Theodoor, Jeroen, Danielle, Reinate en Willem, bedankt voor de gezelligheid bij alle feestjes. Jolien en

Fabian, veel succes met jullie studie. / / Dankwoord Acknowledgements

爸爸，大爷和大娘感谢你们多年来对我学业的大力支持。萍萍，建波，丁哥 和嫂子，谢谢你们对老人们的照料。洞洞和庭庭，祝你们学业有成。小韩和 高姝，谢谢你们多年来的友谊。北京朝阳医院的陈文明和李丽红老师，谢谢 你们对我的指导和照顾，祝你们工作愉快。 致谢

208 Acknowledgements / Dankwoord / / Dankwoord Acknowledgements Publications and Curriculum Vitae

209 Publications and Curriculum Vitae Publications List of Publications in Peer-reviewed Journals • Du VX, Huskens D, Maas C et al. New insights into the role of erythrocytes in thrombus formation. Semin.Thromb.Hemost. 2014;40:72-80. • Du VX, Kelchtermans H, de Groot PG, de LB. From antibody to clinical phenotype, the black box of the antiphospholipid syndrome: pathogenic mechanisms of the antiphospholipid syndrome. Thromb.Res. 2013;132:319-326. • Casari C, Du V, Wu YP et al. Accelerated uptake of VWF/platelet complexes in macrophages contributes to VWD type 2B-associated thrombocytopenia. Blood 2013;122:2893-2902. • Du VX, van OG, Kremer Hovinga JA et al. Indications for a protective function of beta2-glycoprotein I in thrombotic thrombocytopenic purpura. Br.J.Haematol. 2012;159:94-103. • Tournoij E, Koekman CA, Du VX et al. The platelet P2Y12 receptor contributes 210 to granule secretion through Ephrin A4 receptor. Platelets. 2012;23:617-625. • van der Wal DE, Gitz E, Du VX et al. Arachidonic acid depletion extends survival of cold-stored platelets by interfering with the [glycoprotein Ibalpha-- 14-3-3zeta] association. Haematologica 2012;97:1514-1522.

Publications and Curriculum Vitae Publications • van der Wal DE, Du VX, Lo KS et al. Platelet apoptosis by cold-induced glycoprotein Ibalpha clustering. J.Thromb.Haemost. 2010;8:2554-2562. • van Nispen Tot Pannerden HE, van Dijk SM, Du V, Heijnen HF. Platelet protein disulfide isomerase is localized in the dense tubular system and does not become surface expressed after activation. Blood 2009;114:4738-4740. List of Abstracts • Du VX, Heijnen HFG, de Groot PG, Barendracht A, and Roest M. Development of a model to study platelet function under conditions of flow. XXIICongress of the International Society on Thrombosis and Hemostasis. 2009. Poster presentation. Top third poster award. • Du VX, van OS. G, Kremer Hovinga JA, Dienava-Verdoold I, Wollersheim J, Ruggeri ZM, Fijnheer R, de Groot PhG and de Laat B. Indications for a protective function of β2GPI in thrombotic thrombocytopenic purpura. First European Platelet Network conference. 2012. Poster presentation. • Du VX, de Groot PG, van Wijk R, Ruggeri ZM and de Laat B. Binding of erythrocyte ICAM–4 to the platelet activated integrin αIIbβ3 leads to a direct erythrocyte-platelet adhesion under venous flow shear rate. 54th annual meeting of American Society of Hematology. 2012. Oral communication. Abstract achievement award and Dr. Eberhard Mammen Young Investigator awards for best presentation. • Du VX, de Groot PG, van Wijk R, Marchese P, Ruggeri ZM and de Laat B. Disruption of ICAM-4 mediated direct erythrocyte-platelet interaction leads to reduced thrombus formation. Congress of the Dutch Organization of Thrombosis and Hemostasis. 2013. Oral communication. Award for Scientific Excellence in Biochemical Research and “Dr. J. Stibbe bokaal” for best presentation. • Du VX, de Groot PG, van Wijk R, Ruggeri ZM and de Laat B. Disruption of ICAM-4 mediated direct erythrocyte-platelet interaction leads to reduced thrombus formation. XXIV Congress of the International society on Thrombosis and Hemostasis. 2013. Oral communication. • Du VX, Kelchtermans H, de Groot PG and de Laat B. A systematic review and meta-analysis of pathogenic mechanisms of the antiphospholipid syndrome XXIV Congress of the International society on Thrombosis and Hemostasis. 2013. Poster presentation. • Du VX, de Groot PG, van Wijk R, Ruggeri ZM and de Laat B. Disruption of ICAM-4 mediated direct erythrocyte-platelet interaction leads to reduced 211 thrombus formation. 19th Meeting of the European Red Cell Society. 2013. Oral communication. Publications and Curriculum Vitae Publications Curriculum Vitae The author of this thesis was born on September the 23rd, 1980 in Dongping County, Shandong Province, China. In 1999, she was admitted into a five-year clinical medical training program in Jining Medical University, China. After she obtained her bachelor diploma in clinical medicine in 2004, she continued her medical/ research training in Capital Medical University (China) following a master program. Her medical training specialized in internal medicine was carried out in Beijing Chaoyang Hospital. Her research project was about Multiple Myeloma. In 2007, she obtained her master diploma and moved to the Netherlands to be an exchanged ph.d student under the support of the Chinese Scholarship Council at the University Medical Center Utrecht, Department of Clinical Chemistry and Hematology. Her supervisors were Prof. Dr. Ph.G. de Groot and Dr. M. Roest. From June 2009, she worked as a research technician under the supervision of Prof. Dr. J.W. Akkerman at the same department for one year. In 2010, she started working as a Ph.D candidate under the supervision of Prof. Dr. Ph.G. de Groot and Dr. B. de Laat at the same 212 department. Publications and Curriculum Vitae Publications Abbreviations

AA arachidonic acid aCLs anticardiolipin antibodies ADP adenosine diphosphate AMP adenosine monophosphate aPLs antiphospholipid antibodies APS antiphospholipid syndrome ATP adenosine triphosphate Bad bcl-2 associated death promoter Bak bcl-2 homologous antagonist/killer Bax bcl-2-like protein 2 Bcl-2 B-cell lymphoma-2 BSA bovine serum albumin 213 CHO Chinese hamster overy

CNS central nervous system Abbreviations CRP collagen related peptide DIC differential interference contrast DMSO dimethyl sulfoxide dRGDW d-Arginyl-Glycyl-L-Aspartyl-L-Tryptophan EBM-2 endothelial basal medium EC endothelial cells EDAC carbodiimide ELISA enzyme-linked immunosorbent assay ETP endogenous thrombin potential FIX factor IX FIXa activated factor IX Fva activated factor V FVIIIa activated factor VIII FXII factor XII GN N-acetyl-D-glucosamine GPIbα glocyprotein Ibα HMW-VWF high molecular weight von Willebrand factor HUVECs human umbilical vein endothelial cells ICAM-4 intercellular adhesion molecule - 4 ICR imprinting control region IgSF immunoglobulin superfamily IL interleukin LAC lupus anticoagulant LPS lipopolysaccharide LW Landsteiner-Wiener MFI mean fluorescence intensity MPs microparticles mVWF murine von Willebrand factor NPP normal pool plasma p38 MAPK p38 mitogen-activated protein kinase PBS phosphate-buffered saline PCs platelet concentrates PE phosphatidylethanolamine PfEMP1 falciparum erythrocyte membrane protein 1 PPP platelet-poor plasma PRP platelet-rich plasma PS phosphatidylserine PSL platelet storage lesion 214 RBCs red blood cells or erythrocytes RhD Rhesus D Abbreviations RPE R-phycoerythrin SDS-PAGE sodium dedecyl sulfate polyacrylamide gel electrophoresis SEM scanning selectron microscopy SPR surface plasmon resonance TF tissue factor TLR Toll-like receptor TRAP thrombin receptor agonist peptide TSP thrombospondin TTP thrombotic thrombocytopenic purpura TxA2 thrombaxane A2 TXB2 thromboxane B2 UL VWF ultra large von Willebrand factor VDRL Venereal Disease Research Laboratory VLA-4 very late antigen-4 VWD von Willebrand disease VWF von Willebrand factor VWF:Ag VWF antigen wt-mVWF wild-type murine VWF β2GPI beta2-Glycoprotien I βGN β-N-Acetyl-D-Glucosamine