Investigating the Role of Von Willebrand Factor in Fibrin Formation and Fibrinolysis

INVESTIGATING THE ROLE OF VON WILLEBRAND FACTOR IN FIBRIN FORMATION AND FIBRINOLYSIS

by Max A Mendez Lopez

A thesis submitted in partial fulfilment of the requirements for the degree of Master of Science in Molecular Medicine

Supervisor Thomas McKinnon. BSc, PhD.

Imperial College of Science, Technology and Medicine September 2014

ACKNOWLEDGEMENTS

I want to thank Adriana and both of our families for their patience, advice and unconditional support.

I am especially grateful with Dr. McKinnon for his guidance and recommendations, these months have been really enjoyable. To Dr. Nowak, Prof. Laffan and the rest of the members of the lab thank you for let me be part of your group.

I would also like to acknowledge the Ministry of Science and Technology of Costa Rica, whose financial support has allowed me to undertake this degree.

Pura Vida!

ABBREVIATIONS

Abs Absorbance ADAMTS13 A disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) aPTT Activated partial thromboplastin time GP Glycoprotein HK High molecular weight kininogen kDa KiloDaltons kDapp Apparent dissociation constant MW Molecular weight PBS Phosphate buffer saline PK Prekallikrein PT Prothrombin time TF Tissue Factor TAFI Thrombin-Activated Fibrinolysis Inhibitor t-PA Tissue-type plasminogen activator u-PA urokinase-type plasminogen activator VWF Von Willebrand factor

TABLE OF CONTENTS

Title……………………………………..…………………………………………….…… 1 Acknowledgements……………………………………………………………………..… 2 Abbreviations………………………………………………………………………………3 Table of Contents…………………………………………………………………….…… 4 Abstract…………………………………………………………………………………… 6 List of Figures…………………………………………………………………..………… 7 List of Tables……………………………………………………………………………… 7

1. INTRODUCTION………………………..………………………………………..… 8 1.1 Haemostasis Overview……………………………………………………...…… 8 1.2 The Cell-Based Model of Coagulation…………………………………...……... 8 1.3 The Contact System and The Intrinsic Pathway…………………………..…… 10 1.4 The Extrinsic and Common Pathways……………………………………….… 12 1.5 Fibrinolysis…………………………………………………………………….. 16 1.6 The Von Willebrand Factor………………………………………………….… 17 1.6.1 Biology and Biosynthesis………………………………………….… 17 1.6.2 Structure and Functional Domains………………………..……….… 18 1.6.3 Von Willebrand Dynamics Under Flow…………………..……….… 20 1.7 The Role of Platelets…………………………………………………………… 23

2. AIMS………………………………………………………………………………... 25

3. MATERIALS AND METHODS………………………………………..…………. 26 3.1 Binding Assays……………………………………………………………….... 26 3.1.1 FXIIa-VWF Binding Assay………………………………………..… 26 3.1.2 FXIIIa-VWF Binding Assay………………………………………… 26 3.2 Turbidity Assays…………………………………………………………..…… 27 3.2.1 Fibrin Formation………………………………………………...…… 27 3.2.2 Fibrinolysis………………………………………………………..…. 27 3.3 Confocal Microscopy……………………………………………………..……. 28

4. RESULTS…………………………………………………………………………… 29 4.1 Turbidity Assays for Fibrin Generation………………………………………..... 29 4.2 The effects of VWF on Turbidity……………………………………………...… 31 4.3 The Role of Thrombin on Von Willebrand Factor-mediated Effects ………..…. 33 4.4 Von Willebrand Factor Enhances Fibrin Polymerisation……………………..… 34 4.5 Role of Von Willebrand Factor-FXIIa Complex and FXIIIa on Turbidity Assays…………. ...………………………………………………………..……35 4.5.1 Von Willebrand Factor Binds to FXIIa………………………………...…. 35 4.5.2 Von Willebrand Factor binds to FXIIIa………………………………...… 36 4.5.3 Effects of Von Willebrand Factor-FXIIa Complex and FXIIIa on Lag Times………………………………………………………….………….. 37 4.6 Turbidity Assays for Fibrinolysis……………………………………………….. 38 4.6.1 Effects of VWF on Fibrinolysis……………………………………….…… 38

5. DISCUSSION……………………………………….………………………..…….. 39 6. CONCLUSION AND FUTURE PERSPECTIVES…………………………….... 43 7. REFERENCES…………………………………………………………………...… 44

ABSTRACT

Von Willebrand Factor (VWF) is a large multimeric glycoprotein produced by endothelial cells and megakaryocytes that mediates platelet adhesion to damaged vessel walls under conditions of high shear. In humans, its absence produces Von Willebrand Disease, a potentially life threatening condition characterised by a varied degree of bleeding phenotypes. Conversely, high levels of VWF increase the risk of ischemic heart disease and stroke.

The majority of thrombotic events share a common clot phenotype characterised by the formation of a compact fibrin mesh-works with impaired lysability and permeability. Whilst the platelet capture activity of VWF and its influence in thrombus growth is well defined, the effects of VWF in fibrin formation have not been described; therefore, the interaction of VWF with fibrin, its influence in fibres formation and role in fibrinolysis must be investigated. For these purposes, optical end-point measurements of turbidity are used in this project to address the effects of VWF by the analysis of the shape of the turbidity curve and comparisons of lag times. In addition, the impact of VWF on fibrin structure is further examined by confocal microscopy imaging of clots formed in the presence and absence of VWF.

Data arising from this project provide new information on the effects of VWF acting as a fibrin polymerisation enhancer and fibrinolysis protector, expanding current knowledge in the area of thrombus formation and presenting a novel mechanism by which VWF regulates clot stabilization. The results presented here are particularly important because they explain at least partly the increased risk of thrombosis in patients with high levels of VWF and serve as the basis for further investigations in translational haematology regarding the possible development of drugs that modulated this activity.

LIST OF FIGURES 1.1 The Coagulation Cascade……………………………………….…….…………….. 9 1.2 Activation of The Contact System………………………………….…….….…….. 11 1.3 Formation of The Tissue Factor:FVIIa Complex………………………………...... 13 1.4 Fibrinogen Structure and Conversion to Fibrin……………………………………. 15 1.5 Fibrinolysis……………………………………………………………………….... 17 1.6 The Von Willebrand Factor Domains……………………………………...……… 19 1.7 Representation of Blood Flow in a Vessel………………………………………… 22

4.1 Thrombin Determines the Turbidity Curve in a Concentration-Dependent Manner………………………………………………………………………………30 4.2.1 Von Willebrand Factor Effects on Turbidity Curves………………………………. 31 4.2.2 The Von Willebrand Factor Delays Fibres Aggregation and Increases Lag Times…32 4.3. The Effects of Von Willebrand Factor on Turbidity Assays are Independent of Thrombin Concentration………………………………………………………...…. 33

4.4 Von Willebrand Factor Enhances Fibrin Polymerisation………………………….. 34

4.5.1 Factor XIIa Binds to Von Willebrand Factor…………………………………….… 35

4.5.2 Von Willebrand Factor Binds FXIIIa…………………………………………….… 36

4.5.3 VWF-FXIIa Interaction Enhances the Effect on Lag Times……………………….. 37

4.6 Von Willebrand Factor Delays Fibrinolysis……………………………………..… 38

LIST OF TABLES 1.1. Laboratory Diagnosis of Von Willebrand Disease……………………………..……. 21

1. INTRODUCTION

1.1 Haemostasis Overview In 1905, Paul Morawitz postulated a theory for the blood coagulation with the four factors so far discovered: fibrinogen, protrombin, thrombokinase and calcium (I-IV). In his model, prothrombin was converted to thrombin in presence of calcium and thrombokinase and fibrinogen was transformed into fibrin by thrombin. His model persisted for almost 40 years until Paul Owren found a patient in Oslo who suffered nosebleeds and menorrhagia with a prolonged thrombin time. He showed that the abnormality was not due to a deficiency in the coagulation factors so far discovered. Following Morawitz’ nomenclature, Owren named the “missing factor” as Factor V and called the disease “parahaemophilia” (Stormorken H, 2003). Owren published a paper with the results of his doctoral thesis in 1947 and a new series of proteins emerged and were named sequentially in the following years. In 1964, two groups independently but simultaneously presented a new theory of blood coagulation called the “Cascade” or “Waterfall” in which a protein (factor) was enzymatically cleaved, sequentially activating the proteins downstream (Macfarlane RG, 1964; Davie EW & Ratnoff OD, 1964). The models suggested the presence of an extrinsic and an intrinsic pathway that converged into a common pathway forming a “Y” with the final goal of cleaving prothrombin to form thrombin (Figure 1.1).

1.2 The Cell-based Model of Coagulation Despite being clinically useful and easy to understand, the Cascade was not initially described as an in vivo model of physiology. The differences between the phenotypes of FXII and FVIII deficiencies and the finding that the Tissue Factor:Factor VIIa complex (TF:FVIIa) was able to activate FIX raised the possibility that the two pathways were acting together and not separately (Osterud B, 1977). In addition, the Cascade was taken for many years as a predictive model of bleeding in patients with prolonged TP or aPTT, which erroneously caused many surgical procedures to be cancelled or suspended in patients with no risks (Hoffman M and Monroe D, 2007). Considering emerging information on the interaction of the two pathways and lack of clarity in some aspects (e.g. the role of FXI), a new model emerged in 2001 that integrated aspects such as the role of activated platelets and the tissue factor bearing cells in the Cell Based Model of Blood Coagulation (Hoffman M, Monroe D, 2001). This model consists of three overlapping

8 steps; the first one or initiation is where the TF-bearing cells interact with injured endothelium; it resembles the extrinsic pathway of the Cascade and is characterised by TF binding to and activating FVII, which subsequently activates FX and FIX. In the second step or amplification, VWF and FVIII play major roles in the generation of thrombin on the surface of activated platelets, a signal required to continue to the third step or “propagation” where FXa:FVa complex converts large amounts of prothrombin into thrombin, resulting in the production of fibrin by cleavage of fibrinogen by thrombin.

Both the protein centred model (Cascade) and the cell-based model are widely accepted and used for both clinical and research purposes.

Figure 1.1 The Coagulation Cascade The extrinsic and intrinsic pathways converge in the common pathway, where prothrombin is cleaved into thrombin. Both aPTT and PT are used on a daily basis to evaluate blood coagulation. aPTT: activated partial thromboplastin time, PT: prothrombin time.

1.3 The Contact System and Intrinsic Pathway In 1955 Ratnoff OD & Colopy JE studied a patient that was found to have prolonged clotting times in test tubes during routine preoperative screening. John Hageman had no history of bleeding, and this finding led to the hypothesis that something was missing in his blood or maybe proteins could circulate as inactive precursor (Schmaier A, 2008). A few years later, both scientists identified the missing protein as Factor XII (FXII), now recognized as part of the contact system in the intrinsic pathway. The contact system is formed by the High Molecular Weight Kininogen (HK) (MW: 110000), prekallikrein (PK) (MW: 88000), FXII (MW: 80000) and FXI (MW:143000). FXII is a single-chain glycoprotein composed of 596 amino acid residues. It is found in plasma at a concentration of 375nM (30µg/mL) and has a half-life of 50-70 hours (Colman RW & Schmaier AH, 1997). FXII becomes activated by cleavage of the peptide bond R353-V354. There is a growing list of activators (Maas et al, 2011), but binding to negatively charged surfaces and cleavage during autoactivation result in different conformational changes. Activated FXII (α-FXIIa) cleaves FXI and prekallikrein (PK), generating activated FXI (FXIa) and kallikrein (Müller F et al, 2009). α-FXIIa is the initiator of a series of proteolytic reactions resulting in thrombin generation. Also, PK is activated by α-FXIIa, forming plasma kallikrein that acts reciprocally activating more FXII and releasing bradykinin from HK. Bradykinin, the ligand of the G coupled receptor kinin B2, acts as a mediator of vasodilatation and increased vascular permeability by stimulating endothelial cell production of nitric oxide (Leeb-Lundberg et al, 2005). Cleavage of α-FXIIa by plasma kallikrein produces Factor βXIIa (β-FXIIa), a light chain enzymatic form which activates the macromolecular complex of the first component of complement, resulting in classic complement system activation and prekallikrein (Müller F & Renne T, 2008). The serpin C1 esterase inhibitor (C1-inh), is the major plasma inhibitor of PK and FXII (de Agostini et al, 1984). In addition, corn trypsin inhibitor (CTI) has been demonstrated to inhibit FXII in vitro (Nielsen VG, 2009). In summary, the consequences of FXII activation can be seen in three different pathways: coagulation via activation of FXI, inflammation via bradykinin release and complement activation (Figure 1.2).

Based on the death of John Hageman by a massive pulmonary embolism and other case reports, it was thought that FXII deficiency increased the risk of venous thrombosis because of impaired fibrinolyis (Cushman M et al, 2009); however, its role on the development of thrombotic events in homozygous patients has been shown to be minor, if

10 important at all (Girolami et al, 2004). In one of the largest studies addressing FXII deficiency prevalence, only 2.3% of patients were found to have low levels of FXII and none of them developed thrombotic events (Halbmayer WM et al, 1994). Because patients with FXII deficiency do not exhibit a bleeding phenotype, the relevance of the FXIIa in initiating coagulation via the contact activation system is still a matter of debate. In fact, for many years it was somehow contradictory why John Hageman died of a pulmonary embolism after a pelvis fracture having a clotting factor deficiency (Ratnoff OD et al, 1968). It is interesting to note that FXII-deficient mice models showed no FXII plasma activity, no thrombophilia or impaired fibrinolysis and no signs of haemorrhage, a clinical phenotype similar to FXII-deficient humans (Renne T et al 2005). Although it has an uncertain role in haemostasis, FXII is still used as a haemostatic test in routine laboratory analysis such as activated partial thromboplastin time.

Figure 1.2. Activation of The Contact System FXII becomes activated by proteolysis on cell membranes and by autoactivation upon exposure to negatively charged surfaces; both mechanisms produce α-FXII, which is further cleaved to β-FXIIa. Components of the contact system are represented in red. Interactions with the complement system (grey) and inflammatory pathway (blue) are also shown.

Another protein of the Contact System, FXI, was considered the activator of FIX for a long time but despite its role in the contact system it was difficult to explain the presence of an injury-related bleeding in FXI-deficient patients compared to the absence of symptoms in patients with FXII deficiency. Additionally, a prolonged aPTT may be the laboratory hint for FXI deficiency, whereas normal bleeding times are present in FXII-deficient patients. In 1991 it was demonstrated that thrombin was able to bypass the contact system to activate FXI (Gailani D & Broze GJ, 1991) and since then FXI has been considered a thrombin formation amplifier following its initial formation by the TF-driven pathway. The contact system is independent of TF but it leads to a series of proteolytic reactions that produce fibrin. It has been shown that there is an inverse relationship between TF concentration and contribution of FXI to fibrin formation (von dem Borne et al, 1995), leading to a model in which the first step of the coagulation system is independent of FXI and dependent of TF, but becomes dependent of FXI as the thrombus grows (Seligsohn U, 2007).

Factor IX and FX are activated by the TF:FVIIa complex, producing serine proteases that remain membrane-bound (FIXa and FXa). Activation of FX directly by TF:FVIIa complex bypasses the need for FVIII or FIX. Additionally, FVIII exists mostly as a non-covalent complex with VWF.

1.4 The Extrinsic and Common Pathways. For decades, the extrinsic pathway has been considered the principal in vivo initiator of the coagulation (Mackman N, 2008). The extrinsic pathway contains elements of the blood and vasculature. TF, the most important protein, is a glycoprotein that associates with phospholipids on the surface of fibroblasts and leukocytes and binds to FVII in the presence of calcium (Figure 1.3). The TF:FVII complex auto activates (TF:FVIIa) and acts in conjunction with FVIIIa converting FIX to FIXa, which then cleaves FX to FXa and proceed to the common pathway to produce thrombin. It has been shown that the interaction of the four components of the prothrombinase complex (FXa, FV, phospholipids and calcium) provide a markedly increased rate of thrombin activation than the achievable only with the interaction FXa-Prothrombin. Patients with levels below 1% of FVIIa present with bleeding phenotype similar to haemophilia, whereas TF deficient-

12 patients have not been identified, possibly because it is involved in embryogenesis and its deficiency may not be compatible with life (Mackman N et al, 2007; Carmeliet P et al, 1995).

Thrombin is a key protein of the coagulation system. As a serine protease, it is involved in a wide variety of activities, including embryonic development and angiogenesis (Imokawa Y & Brockes, 2003), inflammation, immunity (Rittirsh D et al, 2008) and metastasis (Nierodzik et al, 2006). Because it acts on multiple substrates in the coagulation system, thrombin influences clot formation, limitation, form, and its role is illustrated in patients with the F2 20210*A mutation, which increases plasma concentrations of thrombin and predisposes to thrombosis (Soria JM et al, 2000; Cattaneo M et al, 1999). In addition, the activity of thrombin eliciting responses similar to hormones can be explained by its signalling through G-protein coupled protease-activated receptors (PARs) (Coughlin SR, 2000).

Figure 1.3. Formation of the Tissue Factor:FVIIa complex. FVIIa represents only 1% of total circulating FVII. Both FVII and FVIIa bind reversible to TF with similar affinities. Formation of the TF:FVIIa complex is critical for the integrity of the coagulation system. Reproduced from Morrisey J, 2001.

Thrombin’s structure is homologous to other serine proteases, with a serine in the active site cleft (Bode W, 2005), being S195, H57 and D189 an essential part of it. In addition, the presence of exosites contributes to its specificity (Crawley J et al, 2007). Among its

13 functions in the coagulation system, thrombin catalyses the conversion of fibrinogen to fibrin (Mosesson MW, 2005), activates FV and FVIII (Mann KG, 2003), and also FXIII and FXI (Baglia FA, 2002). It also elicit anticoagulant effects by the formation of the thrombin:thrombomodulin complex (Esmon CT, 1993), which operates by activating Protein C and Thrombin-Activated Fibrinolysis Inhibitor (TAFI) (Nesheim M, 2003).

Fibrinogen is a 45nm-elongated protein of 340 kD. It is the product of three closely linked genes located within 50-kb region in chromosome 4, each one specifying the primary structure of the six polypeptide chains (Aα2,Bβ2,γ2) (Henschen A, 1983). The three chains are homologous but have differences which serve as basis for fibrinogen function in vivo, for example, FXIIIa-susceptible cross linking sites are present in the Aα and γ, but not the β chains. Its concentration in human plasma is 6-12 µM (2-4 mg/ml), making it the most abundant protein of the clotting system (Ariens R, 2013). A coiled-coil segment connects the E central domain with the two outer D domains. Upon cleavage by thrombin, fibrinopeptides A and B are released and fibrin is formed with the conformation α2,β2,γ2 exposing new binding sites termed “A” and “B” sites or knobs; in addition, double- stranded fibrils form through end-to-middle domain associations (D:E). The polymer growth by side-to-side approximation of the fibrin fibres, and the two stranded protofibrils aggregate laterally forming thin, long fibrin strands (Weisel J & Litvinov R, 2013).

Fibrinogen is involved in clot strength, fibrin content and resistance to lysis (Machlus KR et al, 2011). The process of transformation of fibrinogen into an insoluble polymer in vivo can be divided into three steps: cleavage of fibrinopeptides by thrombin, a second non- covalent assembly process, and the covalent stabilisation by FXIIIa (Bagoly et al, 2012). FXIII is the last protein in the coagulation system and most of it is not free in plasma but bound to fibrinogen. FXIIIa is a transglutaminase that cross-links fibrin and other proteins involved in the formation of the clot and fibrinolysis, playing a central role in substrate binding to fibrin (Figure 1.4) (Muzbek L et al, 2011; Smith et al, 2013).

Figure 1.4 Fibrinogen Structure and Conversion to Fibrin

Double-stranded fibrin fibrils are formed through end-to-middle D:E domain associations. Thrombin cleaves FXIII and once activated it cross-links fibrin to form an insoluble mesh. Reproduced from de Mosesson MW, 2005.

Patients with qualitative fibrinogen deficiencies (dysfibrinogenemia) as well as quantitative deficiencies (hypo- and a-fibrinogenemia) are asymptomatic, may present bleeding or even develop thrombosis (de Moerloose, 2013). Although the reason for this wide spectrum of clinical presentations is unclear, fibrinogen-deficient mice have shown to develop abundant thrombi that detach easily and cause downstream embolism in a model of ferric chloride-injured arterioles, suggesting that thrombosis may be caused by rupture and embolization of unstable clots (Ni H et al, 2000).

The structure of a clot can be characterised by the diameter of the fibre, which is largely determined by lateral aggregation and by the size of the pores of the fibrin network (Undas A, 2014). The process of formation of half-staggered and double stranded protofibrils involves binding of the knobs A and B in the central nodule of fibrin monomer to

15 complementary holes “a” and “b” in the γ and β nodule of another monomer (Lord ST, 2011). Recent studies have shown that a single fibrin fibre is formed of thousands of protofibrils aligned side by side (Hategan et al, 2013). In addition, it is thought that cleavage of fibrinopeptide B contributes to lateral aggregation of the fibres by releasing the αC region that becomes available to interact with fibrin (Ariens R, 2013).

1.5 Fibrinolysis

Fibrinolysis is regarded as the response to coagulation leading to fibrin clot breakdown (Rein-Smith, 2014). Plasminogen is a 92 kDa single chain glycoprotein consisting of 791 amino acids. Activation involves cleavage of R561-V562 peptide bond by two proteins: tissue-type plasminogen activator (t-PA), involved in the dissolution of circulating fibrin and urokinase-type plasminogen activator (u-PA), which binds to its receptor u-PAR and activates cell-bound plasminogen (Collen D & Lijnen HR, 1991). Plasminogen cleaves fibrinogen or fibrin, or both, into degradation products that inhibit thrombin action and fibrin polymerisation. The process is highly regulated and can be blocked by the action of plasminogen activator inhibitor (PAI-1) or TAFI (Rijken DC & Lijnen HR, 2009) (Figure 1.5).

The Plasminogen activator system plays an essential role in fibrinolysis initiation; however, it has been associated with cancer, metastasis and chemo-resistance by expression of the plasminogen receptors Annexin A2 and uPAR (Rao JS et al, 2013). PAI- 1 has been associated with poor clinical prognosis in several tumour types. Xenografts with a stable knockdown PAI-1 have smaller tumour volumes and cell proliferation (Giacoia EG et al, 2014). In addition, following myeloablative stem cell transplantation, PAI-1 expression negatively regulates t-PA dependent haematopoietic stem cell transplantation, suggesting it enhances tumorigenesis (Ibrahim AA et al, 2013). The role of plasminogen in thrombosis is controversial and surprisingly plasminogen-deficient patients do not have an increased risk of thrombosis (Tefs K et al, 2006; Okamoto et al, 2003). It is important to remember that a potent initiating mechanism was described that may account for the enhanced fibrinolysis that occurs with the activation of the plasminogen activator system: cleavage of HK release bradykinin, which in turn enhances the release of t-PA (Colman RW et al, 1997b). In addition, kallikrein release from the activation of prekallikrein by

FXIIa is known to activate urokinase, supporting the initiation of the u-PA pathway (Ichinose A et al, 1986).

Figure 1.5. Fibrinolysis Plasmin cleaves fibrin into soluble fibrin degradation products (FDP), a process inhibited by TAFI. T-PA and u-PA are regulated by PAI-1, both enzymes convert plasminogen to plasmin. Reproduced from Rijken DC & Lijnen HR, 2009.

1.6 Von Willebrand Factor

1.6.1 Biology and Biosynthesis Von Willebrand Factor (VWF) is a large multimeric glycoprotein produced by endothelial cells and megakaryocytes that plays a critical role in platelet-mediated coagulation (Schneppenheim R & Budde U, 2011). The VWF gene is located at the short arm of chromosome 12 and comprises 180kb. The protein is synthesised as Pre-Pro-VWF of 2813 amino acids with a signal peptide of 22 residues directing the transport to the endoplasmic reticulum. Here, two Pro-VWF monomers are disulphide-bonded to dimers of approximately 500 kDa at their carboxy-terminal cysteine knot like domains. Only dimers are transported to Golgi and Post-Golgi compartments, where they are disulphide-linked at their cysteine-rich D3 domains to multimers that can reach molecular masses up to 24000 kDa (Ruggeri ZM, 2007). Following synthesis, two pathways allow the release VWF molecules produced in endothelial cells. The first one is a constitutive pathway in which

17 molecules are secreted as soon as they are produced. The regulated pathway, on the other hand, involves storage of mature VWF in the Weibel-Palade bodies for secretion after stimulation (Ruggeri ZM, 2007). VWF multimerisation has been considered for a long time the triggering event for Weibel-Palade formation (Hop et al, 1997). The regulated pathway also involves a physiological reduction in multimer size through a proteolytic cleavage of the Tyr1605-Met1606 peptide bond by a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13). This process is modulated by the N-linked glycans of VWF (McKinnon TA et al, 2008) and enhanced under high shear flow conditions (Levy GG et al, 2001). As a result of the controlled cleavage by ADAMTS13, ultra-large VWF can only be detected in plasma transiently after induction with I-deamino-8-d-arginine vasopressin (DDAVP) or in pathological conditions such as Thrombotic Thrombocytopenic Purpura (Reininger AJ, 2008), in which patient’s symptoms are triggered by widespread thrombosis due to the presence of large uncleaved VWF multimers in the circulation.

1.6.2 Structure and Functional Domains Mature VWF is a 2050 residue subunit formed by a repetitive sequence of domains with specific function and binding partners (Figure 1.6). Structural domains are involved in the post-translational processing of VWF, most importantly the cysteine knot domain (CK) for VWF dimerisation and the D3 domain for multimerisation (Schneppenheim R & Budde U, 2011). The A1 domain contains the receptor for platelet glycoprotein (GP) Iba through which VWF binds to platelets. The D’ domain on the other hand binds FVIII (Ruggeri ZM, 2007). Unlike the A3 domain, both A1 and A2 do not have access to their ligands until their domain structure is changed by means of mutations, hydrodynamic forces, immobilisation on a surface or by ristocetin (Auton M et al, 2010).

It is known that the A2 domain plays a role in the exposure of the platelet-binding A1 domain (Martin C et al, 2007). In globular VWF, the A1 and A2 domains are in close proximity, interfering with platelet access to the A1 domain; however, under shear stress conditions, the A2 moves away from the A1 domain, allowing platelet binding. Recently, it was demonstrated that the formation of a disulphide bridge between cysteines 1494 and 1594 renders the VWF A2 domain resistant to proteolysis in vitro (Marioka Y, 2014). It was also shown that vimentin is an additional A2 domain-binding partner under flow (Da

Q et al, 2014); using a triple A domain protein, that acts similar to full length VWF and lacks the binding site for GPIIb/IIIa, they showed that VWF exposes its cleavage site for ADAMTS13 in the A2 domain upon a conformational change induced by ristocetin, providing evidence for the specific binding of vimentin to the active form of VWF. Vimentin is a ubiquitously expressed protein present in the platelet cytoskeleton and it is still unclear how it is translocated to the platelet surface (Pall T et al, 2011). Among all VWF interactions, the A1-GPIba has been shown to be the only sufficient source to initiate platelet adhesion under high shear conditions (Nesbitt et al, 2009; Huck V et al, 2014). This is the initial adhesive interaction that supports transient bonds.

Figure 1.6. The Von Willebrand Factor Domains. The classical (A) and the updated (B) annotations are represented. The signal peptide (SP) is cleaved in the endoplasmic reticulum after translation to generate pro-VWF. The propeptide (D1-D2 domains) catalyse multimerisation and is cleaved to generate mature VWF. Locations of the binding partners are shown. Modified from Yee A & Kretz ,2014.

The importance of the VWF in haemostasis is evident in patients with Von Willebrand Disease, where mutations in the coding region of the gene cause a disorder with clinical phenotypes of bleeding due to qualitative or quantitative defects in VWF. Qualitative subtypes of VWD encompass types 2A, 2B, 2M and 2N disease, characterised by VWF locus alteration, with the exception of platelet-type VWD where the causative mutations are located in the GPIBA gene (James P & Lillicrap D, 2012). Types 2A and 2M result in reduced platelet binding capacity. In addition, Type 2B results in enhanced platelet binding capability and type 3 is the most severe form of the disease, it is also the less frequent and is characterised by undetectable levels of VWF and FVIII levels <10% (Eikenboom JC, 2001) (Table 1.1).

1.6.3 Von Willebrand Factor Dynamics under Flow Blood vessels are constantly subjected to hemodynamic forces induced by the pulsatile blood pressure and flow. The monolayer of endothelial cells bears most of the stress because it is in contact with the flowing blood. The effects of flow on thrombus formation and growth were described previously (Baumgartner HR, 1973). Under normal circumstances, red blood cells represent most of the cellular mass of circulating blood and therefore move to areas of higher flow velocity (Ruggeri M, 2009). A Newtonian fluid is the one that has a constant viscosity at all shear rates. Any increase in viscosity at low shear rates (<200 s-1) deviates blood from a Newtonian behaviour, a phenomenon which is not seen at higher shear rates due to the interruption of interactions between red cells and fibrinogen (Aarts PA et al, 1988; Goldsmith HL, 1986). In addition, according to Pouiseille flow the velocity profile of a fluid moving in a long cylindrical tube is parabolic, that means, the velocity is maximal at the centre and decreased towards the wall (Figure 1.7).

Type Mutation VIII:C VWF:Ag RiCo Multimer Analysis and Consequence 1 VWF gene L/N L L • Normal multimer analysis mutations • Reduced platelet binding (63% of cases) 2A Missense L/N L/N L • Failure to form dimers/multimers due substitution to mutations in A1 domain (Group I) or in exon 28 VWF more susceptible to ADAMTS13 of VWF cleavage due to mutations in A2 gene domain conformation (Group II). (75% of • Reduced platelet binding cases)

2B Mutations in L/N L L/N • VWF multimers are missing but exon 28 of mutations mimic the physiologically VWF gene activated state of GPIb alpha binding domain. • Enhanced platelet binding with low dose ristocetin in platelet-rich plasma 2M Missense N N L • Normal multimer pattern substitution • Reduced platelet binding capabilities in exon 28 due to changes in A1 domain structure (90% of and configuration cases) 2N VWF gene L N N • Normal multimer pattern • Affect the structure of FVIII binding region, reducing FVIII binding. 3 Wide range L L L • Absent multimers of mutations • Severe Bleeding

Table 1.1. Laboratory Diagnosis of Von Willebrand Disease RCo: Ristocetin Cofactor. L: Low, N: Normal

Figure 1.7 Representation of Blood Flow in a vessel Shear rate (γ) is maximal at the centre of the vessel, whereas shear stress (τ) is greater towards the wall, which favours the representation of the flowing blood as a series of layers moving a different velocities one from another (laminar flow). Reproduced from Ruggeri ZM, 2009.

Nesbitt et al (2009) have demonstrated that shear micro gradients occurring at stenotic or injured vessels control initial platelet recruitment at high shear exclusively by interaction of VWF-GPIb. Zhang et al (2009) has also suggested that elongational flows are an important player in VWF regulation. In general, hydrodynamic forces can be applied to a protein under flow by at least two different mechanisms:

• Shear Stress (τ). The force derived from the friction of the blood on endothelial surfaces. It is expressed in units of force/area: N/m2 or dyne/cm2 and is the product of the shear rate at the wall and the blood viscosity (Chatzizisis Y et al, 2007). Shear rate (γ) is the gradient that describes how fast blood velocity increases from areas at the arterial wall to areas of the centre of the lumen. In physiological conditions, shear rate decreases at the centre of the lumen and increases towards the wall (Chatzizisis Y et al, 2007) and the relationship between shear stress and shear rate is linear. The proportionality constant (τ/γ) defines the viscosity of the fluid (µ) (Ruggeri M, 2009). In brief, shear rate refers to the velocity of a flow gradient and shear stress to the force that result from the interaction of the blood with the arterial wall.

• Elongational Stress. It is the result of changes in velocity due to changes in flow direction. Under flow, an acceleration force is created when the end of polymers experience a different velocity than the front. A rate of elongation can be defined that refers to a tensile force along the polymer. Interestingly, it has been suggested that elongational stresses are more effective in activating VWF than shear rate (Sing & Alexander-Katz, 2010). Vasoconstriction during vessel injury diminishes the radius of the lumen and induces local VWF elongation because of the creation of accelerating forces on the polymer, thus activating clotting response with a lower threshold than constant shear rate (Sing & Alexander-Katz, 2010)

The difference between the two mechanisms may be explained by the rotational nature of shear flow; the extension of polymers in shear is limited by a “tumbling behaviour” in which globular VWF continuously changes between collapsed and stretched state (Sing & Alexander-Katz, 2010).

Upon endothelial cell activation (by thrombin, histamine, fibrin or reperfusion), VWF is released both into the subendothelial matrix and the blood flow expanding its structure and exposing multiple binding sites for platelet GPIb (Schneppenheim & Budde, 2011). Once released into the blood flow, VWF is exposed to a change from acidic to physiological pH and to the previously mentioned shear stress that allows the elongation of the multimer. At the same time, the exposure of the A2 domain allows the VWF-ADAMTS13 interaction, providing a self-regulatory mechanism and avoiding excessive fibre formation under physiological conditions (Dong et al, 2002). Still anchored at the EC surface via the integrin αIIbβ3, P-selectin or lipid membranes, VWF becomes “activated” when going through a transition to VWF fibres, providing sufficient density of binding sites in the A1 domain for platelet adhesion (Schneider et al, 2007). After initial platelet arrest due to GPIb-VWF interaction, intracellular signals activate platelets.

1.7 The Role of platelets

Platelets are fundamental in haemostasis. They activate, aggregate and facilitate the surface for thrombin production; however, the mechanisms of adhesion to the damaged 23 endothelium depends on the shear conditions and local blood flow. Platelets secrete serotonin, calcium and ATP from the dense granules; they also secrete a large variety of mediators from the alpha granules, including platelet factor-4, TGF-beta, PDGF and VWF among others (Koseoglu S & Flaumenhaft R, 2013).

Under normal circumstances, platelets do not interact significantly with the vasculature; however, stable platelet adhesion to the extracellular matrix is seen after endothelial damage, due to exposure of binding sites for VWF through the receptor GPIb/IX, and fibrinogen through the GP IIb/IIIa (Ruggeri ZM et al, 2006). This process involves tethering, rolling, activation and firm adhesion. Platelets become activated upon binding of

VWF with GPIb or collagen with GPVI and/or α2β1 receptor which stimulates signalling through tyrosine kinase pathways (Broos, 2011). Another pathway mediated by G-coupled receptors produce stimulatory and inhibitory signals, a balance that results in platelet activation and aggregation. Platelet aggregation is influenced by shear (Jackson et al 2009). Low-intermediate shear rate (<1000 s-1), as found in veins and larger arteries favours aggregation mediated by integrin αIIbβ3 (GPIIb/IIIa); subsequent stimulation by local agonists favour integrin-collagen bonds. High shear rates (1000-10000 s-1) are found in arterial microcirculation or regions of moderate stenosis; here, platelet-platelet interactions become more dependent on VWF with a critical role for GPIba in the formation of initial aggregates of discoid platelets. Finally, pathological shear rate (>10000 s-1) is found in areas of critical stenosis or vessel narrowing by atherothrombosis, at these rates aggregation does not require platelet activation and is exclusively mediated by VWF- GPIb bonds. A shear rate of 1000 to 5000 s-1 has been established as sufficient to activate VWF under unbound (soluble) conditions (Huck et al 2014). Once platelets are adherent, aggregation is mediated by binding to fibrinogen, VWF, fibronectin and vitronectin (Reheman A et al, 2005). The process is calcium-dependent and receives a negative feedback signals by prostaglandins. The importance of these events is illustrated in Bernard- Soulier patients, in which the lack of GPIb/IX avoids normal binding of platelets to damaged endothelial cells, causing a bleeding phenotype (Diz-Kücükkaya R, 2013). It has also been demonstrated that contact-dependent signalling by platelet-platelet interaction are mediated by JAM- and SLAM- family members, which establish the required interactions for the stabilization of the thrombus (Brass LF et al, 2005; Brass LF et al, 2011)

2. AIMS

Accumulating evidence suggests that most cases of arterial and venous thrombotic events share a prothrombotic clot phenotype characterised by the formation of a compact fibrin mesh-works with impaired lysability and permeability (Undas A, 2014). Von Willebrand Factor binds to collagen and mediates platelet capture at a shear rate of 1000s-1 (Sugimoto M et al, 1999), in addition, it acts as the carrier molecule for FVIII. I hypothesise that VWF is also involved in fibrin polymerisation and thrombus stability.

For this project specifically, I will investigate the effects of VWF on fibrin formation using optical end-point methods based on turbidity measurements. In addition, the structure of the fibrin clots will be assessed using Laser Scanning Confocal Microscopy and correlated with turbidity assays. Finally, a possible role of VWF in fibrinolysis will also be investigated.

3. MATERIALS AND METHODS

3.1 Binding Assays

For binding assays, VWF was provided by Dr. Thomas McKinnon from the Haemostasis Laboratory, Imperial College London. FXIIa and FXIIIa were from Enzyme Research Laboratories, U.K.

3.1.1 FXII-VWF Binding Assay

120nM of activated FXII (FXIIa) was diluted into 50 mM sodium bicarbonate buffer (3.7g Sodium Bicarbonate, 0.64g Sodium Carbonate, 1L distilled water) pH 9.6, added onto Maxisorp high protein-binding plates (Nunc, Denmark) and incubated overnight at 4 ºC. The plate was washed with buffer PBS-tween (Phosphate Buffer Saline, 0.1% tween), blocked with 2% BSA (Bovine Serum Albumin) and incubated at room temperature for one hour. Increasing concentrations of plasma derived, full length recombinant or deletion A1 VWF (0–50µg/ml) were diluted in Tris Buffer (20mM Tris, pH: 7.4), added to the wells and incubated at room temperature for 60 minutes. A negative control containing Tris Buffer was included in all experiments. The plate was washed with 0.1% PBS-Tween and bound VWF was detected with polyclonal rabbit anti-human VWF-HRP (Dako, U.K). A final washing step was performed with PBS-Tween and then the colour change was induced by adding 100 µl of peroxidase substrate (SigmaFast Tablets; Sigma, U.K), stopped with 50 µl of 3M H2SO4 and read at 492nm in a µQuant plate reader (Biotek Instruments). All experiments were performed in duplicate.

3.1.2 FXIII-VWF Binding Assay

Activated FXIII was diluted into bicarbonate buffer to a final concentration of 5µg/ml and added to Maxisorp high protein-binding plates (Nunc) to incubate overnight at 4 ºC. Subsequent steps followed the protocol described in section 3.1.1.

3.2 Turbidity Assays

Thrombin, fibrinogen and FXIIa used for fibrin formation assays were from Enzyme Research Laboratories, U.K. Plasma purified VWF, VWF fragments and VWF-∆A1 were provided by Dr. Thomas McKinnon and Dr. Agata Nowak from the Haemostasis Laboratory, Imperial College London. For fibrinolysis assays, plasminogen and t-PA were from Enzyme Research Laboratories. All experiments were performed with fibrinogen thawed in a water bath at 37°C. All assays were performed at room temperature and in a final volume of 200 µl.

3.2.1 Fibrin Formation

Working solutions of VWF [100µg/ml], FXIIa [400nM], FXIIIa [10 µg/ml], thrombin [10nM] and fibrinogen [4µg/ml] were prepared in HEPES/calcium buffer (25mM HEPES, 122mM NaCl, 5mM CaCl, pH 7.4). For sample preparation, VWF [1-10µg/ml], FXIIa [125nM] or FXIIIa [3µg/m] were mixed with thrombin [0.1nM] in 1.5ml tubes, and added immediately to low binding polystyrene 96-well plates (Sterilin, U.K). Fibrinogen [1mg/ml] diluted in HEPES/calcium buffer was added with a multichannel pipette to start the reaction. Turbidity was measured at 405nm every minute for a minimum of one hour using the µQuant plate reader (Biotek Instruments, U.K).

To analyse the effect of the VWF-FXIIa complex, both proteins were incubated at room temperature for 30 minutes before following the previously described method.

3.2.2 Fibrinolysis

Fibrin lysis experiments were performed in low binding polystyrene 96-well plates (Sterilin, U.K). Plasminogen [240nM] and t-PA [0.1-1nM] diluted in HEPES/calcium buffer were mixed with VWF [10µg/ml] and thrombin [01.nM]. The reaction was started by the addition of fibrinogen [1mg/ml] diluted in HEPES/calcium buffer. Turbidity was measured at 405nm every minute for two hours using the µQuant plate reader (Biotek Instruments, U.K). The baseline was the value at 0 minutes. Time to start of lysis was the

27 first decreasing value after the plateau of the curve. Time to half lysis was calculated by subtracting the time at the start of lysis from the time at the curve returned to the baseline value. A second experimental approach was design to analyse fibrin lysis. Clots were formed at room temperature by mixing thrombin [1nM], plasminogen [240nM], VWF [10µg/ml] and fibrinogen [1mg/ml]. After one hour of incubation, t-PA [1nM] was added to the surface of the clots and the time to start of lysis was calculated by measuring change in turbidity every minute for two hours.

3.3 Confocal Microscopy Fibrinogen [450µg/ml] diluted in HEPES/calcium buffer was added to a chambered coverslip (µ-slide 8 well; Ibidi 80826) with 50µg/mL of Alexa-488 labelled fibrinogen (Life Technologies, UK). After addition of thrombin [0.75nM], VWF [10 µg/ml] and FXIIa [125nM], clots were formed for at least 2 hours in a humid atmosphere at room temperature. Laser confocal microscopy was performed at the Imperial College Cardiovascular Sciences Microscopy Laboratory, Hammersmith Campus, using the Zeiss LSM-510 META Axioplan 2. Clots were visualized with a 40X oil objective, using LSM 510 Software Version 4.2. Labelled fibrinogen was excited with a 488 nm argon laser and images were averaged 4 times.

4 RESULTS

4.1 Turbidity Assays for Fibrin Generation Turbidity assays rely on measuring the change in optical density as a function of time. In the case of fibrin formation, absorbance values increase as fibres aggregate. The initial experiments were aimed at establishing the standard turbidity curve. As shown, at low thrombin concentration (0.1nM) fibres start to aggregate 15 minutes after the initiation of the reaction (lag time), followed by a continuous increase in turbidity values to reach a maximum just before the curve plateaus. Titration experiments were also performed to analyse the effects of thrombin on turbidity. As shown, thrombin determines the shape of the curve in a concentration-dependent manner, with higher concentrations increasing maximum turbidity values and decreasing lag times.

Figure 4.1. Thrombin Determines the Turbidity Curve in a Concentration-Dependent Manner

Thrombin [0.1-0.5nM] was mixed with fibrinogen [1mg/ml] in HEPES/calcium buffer and turbidity was monitored each minute for 2 hours at room temperature. (A) Time course of the clot formed with thrombin [0.1nM]. The lag time for the curve is 15 minutes and turbidity values increase to a maximal before reaching a plateau. (B) Turbidity as a function of thrombin concentration. Thrombin increases maximum turbidity and decreases lag time in a concentration-dependent manner.

4.2. The Effects of Von Willebrand Factor on Turbidity

To determine the effects of VWF on turbidity curves, VWF [2, 5 and 10µg/ml] diluted in HEPES/calcium buffer was mixed with thrombin [0.1nM] and fibrinogen [1mg/ml] as described. Interestingly, at all concentrations examined VWF decreased maximum turbidity and increased lag times, displaying a concentration-dependent effect that suggests it enhances clot stability by promoting polymerisation of fibrin fibres before aggregation occurs.

Figure 4.2.1 Von Villebrand Factor Effects on Turbidity Curves VWF [2,5,10µg/ml] was mixed with thrombin [0.1nM] and fibrinogen [1mg/ml]. Turbidity was measured each minute for 90 minutes. At all concentrations examined la presence of VWF decreased maximum turbidity values and prolonged lag times, suggesting it delays fibre aggregation..

FXIIa was previously shown to decrease turbidity, increase lag times and enhance clot formation (Konigs et al, 2011). To validate the previous results, FXIIa was used as an internal control in subsequent fibrin formation assays with VWF. Interestingly, turbidity values were consistently lower in the clots formed with VWF, reinforcing the novel finding that it enhances fibrin polymerisation. In addition to the concentration-dependent effects on turbidity, a prolonged lag time was also seen to be determined by VWF concentration.

Figure 4.2.2 Von Willebrand Factor Delays Fibres Aggregation and Increases Lag Times

VWF [0-10µg/ml] and FXIIa [125nM] were mixed with thrombin [0.1nM] and turbidity change was started by adding fibrinogen [1mg/ml] as described. (A) Clots formed in the presence of VWF displayed turbidity curves with lower maximum values compared to its absence. FXIIa was used as an internal control and the effects previously described were reproduced (Konigs et al, 2011) (B) VWF increases lag time in a concentration-dependent manner, suggesting it delays lateral aggregation of non-polymerised fibres and favour the stabilisation of the clot.

4.3 The Role of Thrombin on Von Willebrand Factor-Mediated Effects

VWF and thrombin produce opposite effects on turbidity curves. To determine the influence of thrombin on the VWF-mediated effects, clots were formed with different thrombin concentrations in the presence or absence of VWF. As expected, there was a correlation between increasing thrombin concentrations and shorter lag times. In addition, VWF increased lag times at all concentrations tested, with stronger effects at the lower end.

Figure 4.3 The Effects of Von Willebrand Factor are Independent of Thrombin Concentration.

Clots were formed with increasing thrombin concentrations [0.1-2nM] in the presence and absence of VWF. Shorter lag times were obtained with higher thrombin concentrations. VWF increased lag times, with stronger effects at the lower end of the thrombin concentrations tested.

4.4 Von Willebrand Factor Enhances Fibrin Polymerisation

The structure of the clot has been intensively investigated. Most models of study have been implemented in two dimensions, but with the advent of confocal microscopy, the structure of the clot can be visualised in 3D by reconstruction of several thin layers of the specimen. With confocal microscopy, fibrin gels appear as a collection of fibres relatively monodisperse in diameter and connected together at nodal points with a branching order (Magatti D et al, 2011). To correlate the change in turbidity with the structure and polymerisation of fibrin fibres, clots formed in the presence and absence of VWF were imaged with confocal microscopy. As expected, clots formed with VWF had increased fibrin mesh-works compared to those formed with thrombin alone, confirming that VWF directly influences fibrin formation by enhancing fibres polymerisation.

A B

Figure 4.4 Von Willebrand Factor Enhances Fibrin Polymerisation

Confocal microscopy images of clots prepared by mixing thrombin [0.1nM], fibrinogen [1mg/ml] and VWF [0-10µg/ml] in HEPES/calcium buffer for two hours at room temperature. (A) Negative control represent clots formed without VWF. (B) Clots formed in the presence of VWF showed significantly more fibrin polymerisation.

4.5 Role of Von Willebrand Factor-FXIIa Complex and FXIIIa on Turbidity Assays

4.5.1 Von Willebrand Factor Binds to FXIIa

The next phase of the project was to investigate the possibility of an interaction between FXIIa and VWF. Microtitre plates were coated with 120nM of FXIIa and incubated with increasing concentrations of either pdVWF (plasma derived VWF), recombinant wtVWF or delta-A1 VWF (VWF-∆A1). Bound VWF was detected with polyclonal anti-VWF HRP conjugated antibodies. Significantly, VWF bound to FXIIa with high affinity; KD,app = ~4nM. No difference was observed between the ability of the recombinant wtVWF or pdVWF to bind to FXIIa. Interestingly, VWF-∆A1 had reduced affinity for FXIIa, suggesting that the major binding site on VWF for FXIIa is located within the VWF A1 domain.

Figure 4.5.1 Factor XIIa Binds to Von Willebrand Factor

FXIIa binds pdVWF and wtVWF with high affinity (KD,app= 4nM). VWF-∆A1 showed reduced affinity for FXIIa, suggesting this is the site of major binding.

4.5.2 Von Willebrand Factor binds to Factor XIIIa.

FXIIIa is the last protein in the coagulation system and its main function is to cross-link fibrin to form a stable insoluble clot. Because VWF enhances fibrin formation, a possible interaction between VWF and FXIIIa was examined. FXIIIa was immobilised onto microtiter plates and incubated with increasing concentrations of wtVWF. VWF bound to

FXIIIa with high affinity (KD,app = ~15nM), and interestingly, binding was not mediated by the A1 domain, suggesting that both FXIIIa and FXIIa form a complex with VWF interacting at different domains.

A B

Figure 4.5.2 Von Willebrand Factor Binds FXIIIa

FXIIIa was immobilised onto microtiter plates and incubated with increasing concentrations of wtVWF [0-60nM]. (A) Binding occurs with high affinity

KD,app = ~15nM. (B) Affinity is not affected for VWF-∆A1, suggesting FXIIIa-VWF interaction is not mediated by A1 domain.

4.5.3 Effects of the Von Willebrand Factor-FXIIa Complex and FXIIIa on Lag Times

To further investigate the effects of the VWF-FXIIa complex and FXIIIa, lag times were analysed for all turbidity assays performed in the presence of FXIIa, VWF and the VWF- FXIIa complex. Interestingly, the effect of the VWF-FXIIa complex on lag times was greater than the effect of the proteins separately, suggesting that FXIIa enhances VWF- mediated effects on clot formation or vice versa. In addition, the effect of the complex on turbidity is stronger than the FXIIIa effect, providing supporting evidence for an enhanced fibrin polymerisation in the presence of VWF.

Figure 4.5.3 VWF-FXIIa Complex Enhances the Effect on the Lag Time.

A complex of VWF-FXIIa was formed by incubated both proteins in HEPES/calcium buffer at room temperature for 30 minutes. (A) Lag times increased proportionally in the presence of VWF, FXIIa and VWF-FXII complex. (B) The effect of the VWF-FXIIa complex on turbidity is stronger than that of FXIIIa, providing supporting evidence for a role of VWF in fibrin polymerisation.

4.6 Turbidity Assays for Fibrinolysis

4.6.1 Effect of Von Willebrand Factor on Fibrinolysis

The effects of VWF on fibrinolysis have not been described. To investigate these, fibrin lysis assays were performed by adding 240nM of plasminogen and increasing concentrations of t-PA to the standard turbidity experiment. Time at the start of lysis was the first decreasing value after the plateau of the curve. Time to half lysis was calculated by subtracting the time at the start of the lysis from the time when the curve returned to baseline. The baseline was the value at 0 minutes. An alternative overlay lysis assay was performed with 0.2nM of thrombin in order to decrease the time needed to reach maximum turbidity; in these experiments, clots were formed at room temperature for one hour, after which t-PA was added to the surface of the forming clot. Because the first absorbance value was not available in this experiment, time at the start of lysis was calculated as the first decreasing value after the plateau. Significantly, time to half-lysis is prolonged in clots formed in the presence of VWF, with stronger effects at lower thrombin concentrations. In line with this, time to start of lysis is also longer with VWF, suggesting that VWF may be acting by protecting the clot form lysis or delaying it.

A B

Figure 4.6 Von Willebrand Factor Delays Fibrinolysis.

t-PA [0.2nM] and plasminogen [240nM] were added to turbidity experiments and clots were formed for one hour. (A) Fibrin lysis experiments show that VWF increases time to half-lysis. (B) VWF delays the time at the start of lysis.

5. DISCUSSION

VWF main activities in vivo are to mediate platelet adhesion to endothelial cells under shear and to act as the carrier molecule for FVIII. This project provides insight into a novel mechanism by which VWF regulates fibrin polymerisation and suggests a role of VWF in delaying fibrinolysis. These findings are particularly important because it has been shown that ex vivo clots of patients with heart disease form a denser fibrin network that are more difficult to lyse (Collet et al, 2006).

The formation of a clot at the site of injury is a complex process, but regardless of the mechanisms involved in thrombus formation the majority of events share a common clot phenotype characterised by the formation of a compact fibrin mesh-works with impaired lysability and permeability (Undas A, 2014). In vitro, clots form following three sequential steps: fibrinopeptides cleavage, association of fibrin monomers into protofibrils and aggregation of protofibrils into branching fibres (Weisel J and Nagaswami C, 1992). In addition, the presence of FXIIIa allows covalent cross-linking of α-α and γ-γ chains of the fibrin mesh, protecting it from fibrinolysis and favouring polymerisation (Bagoly et al, 2012).

Clot formation in vitro can be studied by mechanical and optical methods. The mechanical methods include conductivity measure, electromagnetic detection and viscometry. Viscometry is the basis for the development of tromboelastometry, a method that allows to study platelet function and coagulation at the same time. Optical end-point detection methods are the most commonly used for monitoring thrombus formation. Of particular importance for this project is turbidity, which was introduced in the 1970s (Davey et al, 1972; Gogstad et al, 1986) and determines fibrin formation by measuring a change in turbidity as a function of time (Carr and Hermanns, 1978). This technique provides a high degree of structural correlation with fibrin clots in purified systems (Weisel J and Nagaswami C, 1992; Carr M, 1988). In the classical turbidimetric assay, a detector is placed opposite a light source shining through the specimen, resulting in a change in the optical density as the sample transforms from a liquid to a solid state. The lag time or lag period is the time required for the protofibrils to grow to sufficient length before they aggregate and is represented in the curve as the time from the start of the reaction to the rise in turbidity values (Figure 5.1). An increase in lag times correlates with a delay in

39 lateral aggregation of fibrin monomers, which means that higher turbidity values translate into aggregation of thicker protofibrils as soon as they form; thus, lower maximal turbidity values represent fibres that have more time to polymerise before they aggregate, producing more stable clots.

A large volume of experimental work has been performed to investigate the effects of various modifications on fibrin polymerisation; nevertheless, under most conditions, turbidity is proportional to the square of the average radius of the fibres (Weisel J and Nagaswami C, 1992). Factors that impact turbidity are divided into four broad categories: hereditary and acquired variations in fibrin structure; environmental conditions such as pH or ionic conditions; cellular effects such as the secretion of platelet polyphosphates and finally flow conditions, because in static assays the clot stops forming when all fibrinogen is converted to fibrin, whereas under flow there is a continuous source of fibrinogen to keep to clot forming (Weisel J and Litvinov T, 2013). Thrombin also influences the shape of the turbidity curve, with higher concentrations producing shorter lag times (Figure 5.1).

Figure 5.1 Turbidity Curves

Turbidity is measured as a function of time. The lag time is the time required for the protofibrils to grow before they aggregate and is seen in the curve as a rise in turbidity values. Thrombin concentration determines the shape of the curve: higher concentrations produce shorter lag times. 1: 1U/mL, 2: 0.1U/mL, 3: 0.01U/mL, 4: 0.001U/mL. Reproduced from Weisel J and Nagaswami C, 1992.

The main objective of this project was to investigate the effects of VWF on fibrin formation. In the first stage of the project I optimised the assay and reproduced the effects of thrombin on turbidity previously described. Then, the role of VWF on fibrin formation was examined. VWF increased lag times and decreased turbidity at fixed thrombin concentrations. Interestingly, VWF-mediated effects were still significant when thrombin was titrated, suggesting a new role of VWF in regulating fibrin formation. This hypothesis is supported by the confocal microscopy images that demonstrate more fibrin fibres on the clots formed with VWF compared to those formed with thrombin alone. In humans, high levels of VWF have been associated with an increased risk of stroke (Wieberdink RG et al, 2010; Roldan V et al, 2006) and ischemic heart disease (Smith FB et al, 1997; Rumley A et al, 1999). It has also been suggested that VWF acts as an acute-phase reactant associated with thrombo-inflammation (Claus RA et al, 2010), a mechanism of occurrence and propagation of acute ischemic stroke (Nieswandt B, 2011). In addition, VWF-deficient mice are protected from cerebral ischemia (Kleinchnitz C et al, 2009) and the role of VWF as a mediator of platelet adhesion has been shown to be important for the development of deep vein thrombosis (Brill A et al, 2011). Data arising from this project show a novel effect of VWF in regulating fibrin polymerisation, a finding of particular importance considering that research on VWF has been mostly focused on its platelet-binding functions and there is no information describing the role of VWF in fibrin polymerisation.

FXIIa stabilises the structure of the fibrin clot, prolongs the lag time and decreases maximum turbidity values (Konings J et al, 2011). In addition, at a shear rate of 1000s-1 type I collagen activates FXIIa and enhances clotting and thrombin generation in a FXII- dependent manner (van der Meijden et al, 2009). Interestingly, VWF is also necessary to capture platelets at the same shear and binding assays showed high affinity binding between FXIIa and VWF-A1 domain, suggesting that both proteins bind and act together to enhance the effect on fibrin polymerisation. This is supported by the demonstration of increased lag times in the presence of the VWF-FXIIa complex compared to FXIIa alone, indicating that more fibrin is formed in the presence of both proteins.

To fully support the role of VWF in fibrin polymerisation, VWF effects were tested against FXIIIa. It has been shown that the Gln residues of the Pre-Pro-VWF react with FXIII as an amine receptor (Takagi J et al, 1995). Significantly, VWF-FXIIa complex displayed curves

41 with lower turbidity values than FXIIIa, suggesting that polymerisation of the fibres is higher with this complex compared to the achievable with FXIIIa alone. VWF also bound to FXIIIa with high affinity and surprisingly the interaction was not affected for ΔA1- VWF, indicating that FXIIa and FXIIIa bind to different domains on the VWF structure and act as a complex. It will be interesting to examine in the future the influence of VWF in polymerisation and lysis of FXIIIa-mediated fibrin cross-linking.

With the evidence that VWF influenced fibrin formation, plasmin and t-PA were added to the experiments to evaluate the possible role of VWF on fibrinolysis. VWF binds to fibrin and platelet GPIb receptor (Loscalzo J et al, 1986). Plasmin cleaves both molecules removing fibrin, VWF and platelets from the thrombus, for these reasons plasminogen activators (e.g. t-PA) have been used successfully for treatment of thrombotic disorders (Wardlaw JM et al, 2009). In these experiments, clots were formed in the presence and absence of VWF and were subsequently lysed. As expected, VWF increased time to half lysis and the time at start of lysis, suggesting that VWF delays fibrin lysis by its enhanced polymerising effect. It is also attractive to suggest that it protects from fibrinolysis by increasing cross-linking in a similar manner than FXIIIa; however, more experimental data is needed to confirm this and it is worth remembering that these effects were assessed in static conditions and the elongated structure of VWF could influence these results under flow conditions.

VWF-deficient mice have a phenotype of a defective thrombus formation (Denis C et al, 1998) and delayed platelet aggregation (Pendu R et al, 2009); however, reconstitution of VWF levels produce similar aggregation, rate of thrombus growth and thrombus size compared to wtVWF+/+ mice (De Meyer S et al, 2008). In addition, VWF-deficient mice form thrombi, but these are ineffective in promoting vessel occlusion possibly because of the absence of platelet-mediated clot retraction (Denis C et al, 2007). Considering data from mice models and results from this project, there is supporting evidence to suggest a role of VWF in fibrin polymerisation and fibrin lysis protection which may partly explain the higher risk of stroke and ischemic cardiac disease in patients with increased VWF levels.

6. CONCLUSIONS AND FUTURE PERSPECTIVES

This project provides supporting evidence for a novel function of VWF in regulating fibrin polymerisation and fibrinolysis, expanding current knowledge in the process of fibrin clot formation and stabilisation.

The role of VWF in enhancing polymerisation and delaying fibrin lysis may also explain at least partly the higher risk of ischemic heart disease and stroke observed in patients with increased VWF levels. The results obtained are encouraging, however, experiments need to be performed under flow to investigate the role and precise interactions of VWF with the previously studied proteins. In addition, the presence of platelets and inflammatory mediators may also influence VWF effects in fibrin polymerisation in vivo.

The information generated here may also be expanded in the future by several ways. The effects of VWF on turbidity compared to those of FXIIIa must to be expanded to determine the influence of VWF on FXIIIa-mediated fibrin cross-linking. It would be of particular interest to determine if the fibrinolysis protection effect provided by VWF can be inhibited in a way that clots become more susceptible to be lysed and cleared. The binding sites of VWF with FXIIa and FXIIIa must be investigated, and the use of site-directed mutagenesis could help in the understanding of the interactions between the proteins and their effects under flow. Finally, it would also be important to investigate the effects of plasmin on VWF-FXIIa complex, and to determine its implications in clot stability.

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