The Plasma Contact System

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LIST OF PAPERS

This thesis is based on the following four papers, which will be referred to in the text by their Roman numerals:

I Bäck J, Sanchez J, Elgue G, Nilsson Ekdahl K, Nilsson B. Activated human platelets induce factor XIIa-mediated contact activation. Biochem Biophys Res Commun 2010; 391(1): 11-7.

II Bäck J, Lang MH, Elgue G, Kalbitz M, Sanchez J, Nilsson Ekdahl K, Nilsson B. Distinctive regulation of contact activation by antithrombin and C1-inhibitor on activated platelets and material surfaces. Biomaterials 2009; 30(34): 6573-80.

III Bäck J, Faxälv L, Nilsson Ekdahl K, Nilsson B. Clot formation triggers factor XII activation by the fibrin network and subsequent inhibition by antithrombin. Manuscript.

IV Bäck J, Lood C, Bengtsson AA, Nilsson Ekdahl K, Nilsson B. Distinc- tive contact activation in systemic lupus erythematosus: The basis for new potential biomarkers to evaluate the risk of thrombotic events? Manuscript.

Reprints were made with permission from the respective publishers.

CONTENTS

INTRODUCTION ...... 11 THE PLASMA CONTACT SYSTEM ...... 13 Proteins of the contact system ...... 13 High molecular weight kininogen ...... 13 Coagulation factor XII ...... 14 Coagulation factor XI ...... 14 Prekallikrein ...... 15 Mechanisms of contact activation ...... 15 Material-induced activation ...... 15 Cell-induced activation ...... 16 In vivo-activating compounds ...... 17 Regulation of contact activation ...... 18 Effects of contact activation ...... 18 Coagulation ...... 18 The kallikrein-kinin system ...... 19 Activation of the innate immune response via the generation of antibacterial peptides ...... 19 Thrombin-inhibitory and profibrinolytic activities ...... 20 The complement system ...... 20 HEMOSTASIS ...... 21 Platelets and platelet plug formation – primary hemostasis ...... 22 Platelets ...... 22 Platelet adhesion ...... 23 Platelet activation ...... 24 Platelet aggregation ...... 24 Coagulation – secondary hemostasis ...... 25 In vivo model of coagulation – tissue factor activation ...... 26 Fibrin network formation ...... 26 Procoagulant properties of platelets ...... 27 Open questions and possible roles of FXII ...... 28 AIMS OF THE STUDY ...... 29 General aim ...... 29

Specific aims ...... 29 Paper I ...... 29 Paper II ...... 29 Paper III ...... 30 Paper IV ...... 30 MATERIALS AND METHODS ...... 31 Heparin coating ...... 31 Platelet activation ...... 31 Enzyme-linked immunosorbent assays ...... 32 Flow cytometry ...... 32 Chromogenic substrates ...... 33 Clotting time ...... 33 Platelet aggregation ...... 34 Tubing loop models ...... 34 Phospholipid vesicles ...... 35 Fibrin clots ...... 35 RESULTS AND DISCUSSION ...... 36 Paper I ...... 36 Paper II ...... 38 Paper III ...... 40 Paper IV ...... 42 CONCLUSIONS ...... 44 Paper I ...... 44 Paper II ...... 44 Paper III ...... 44 Paper IV ...... 45 CONCLUDING REMARKS AND FUTURE PERSPECTIVES ...... 46 SUMMARY IN SWEDISH ...... 51 ACKNOWLEDGMENTS ...... 55 REFERENCES ...... 57

ABBREVIATIONS

ADP Adenosine diphosphate AT Antithrombin ATP Adenosine triphosphate AU Arbitrary unit C1INH C1 inhibitor CK1 Cytokeratin 1 CTI Corn trypsin inhibitor ELISA Enzyme-linked immunosorbent assay F Factor FP Fibrinopeptide gC1qR Receptor for the globular heads of C1q Gla Gamma-carboxyglutamic acid GP Glycoprotein HAE Hereditary angioedema HK High molecular weight kininogen HRP Horseradish peroxidase HUVECs Human umbilical vein endothelial cells LK Low molecular weight kininogen MFI Mean fluorescence intensity OR Odds ratio PAR Protease-activated receptor PC Phosphatidylcholine PE Phosphatidylethanolamine PPP Platelet-poor plasma PRP Platelet-rich plasma PS Phosphatidylserine PVC Polyvinyl chloride RNA Ribonucleic acid SBTI Soybean trypsin inhibitor Serpin Serine protease inhibitor SLE Systemic lupus erythematosus SM Sphingomyelin TAT Thrombin-antithrombin TF Tissue factor TFPI Tissue factor pathway inhibitor

tPA Tissue plasminogen activator TRAP Thrombin receptor agonist peptide TSP-1 Thrombospondin-1 TxA2 Thromboxane A2 uPA Urokinase plasminogen activator u-PAR Urokinase plasminogen activator receptor vWf von Willebrand factor

INTRODUCTION

Blood is one of the body tissues that is a vital part of the human physiology. It constantly circulates in our vascular system, providing cells with nutrients and oxygen and removing carbon dioxide and other waste products. The blood cells, together with protein systems, are also involved in the body’s defense, keeping foreign substances and organisms outside the vascular system and preventing them from causing damage. To sustain these functions, it is extremely important that the blood be kept flowing and contained within the vascular system. The acute response mechanisms that are initiated to stop bleeding when a blood vessel is injured are all encompassed by the term hemostasis. These vessel repair mechanisms must act very rapidly and efficiently to prevent excessive bleeding; at the same time, the hemostatic system involves strong regulatory mechanisms that keep it in perfect balance to prevent excessive hemostatic responses. An imbalance between these procoagulant and regulatory mechanisms can lead to thrombosis, with subsequent vessel occlusion or embolism, or it can cause bleeding problems; in the end, both conditions can result in substantial tissue damage and a lethal outcome. Platelets are blood cells that are crucial for hemostasis. Together with coagulation factors, they cooperate to prevent the bleeding that would other- wise occur as a result of damage to the blood vessels. These cooperative interactions are very complex, and the mechanisms that lead to coagulation and clot formation are not fully understood. The plasma contact system is a cascade system that can initiate coagulation when the blood comes into contact with foreign surfaces, but its role in the physiological coagulation initiated by vessel damage is not yet clear. The absence of an increased bleeding risk in individuals deficient in the first factor (F) in this pathway, FXII (1, 2), has contributed to the assumption that the contact system has minor importance for normal hemostasis. Recent findings from in vivo studies have generated renewed interest in the contact system. Mice deficient in FXII have been shown to exhibit defective thrombus formation that produces unstable and non-occlusive, platelet- rich thrombi (3). The investigators who conducted this research stated that these mice are protected from thrombosis while still retaining an apparently normal hemostasis. In addition, selective depletion and inhibitors of FXII have been shown to exert antithrombotic effects in animal models, seeming- ly without producing any related side effects such as bleeding (4, 5).

11 Thus, FXII has been suggested to have importance for pathological thrombus formation but to be dispensable for hemostasis. Whether humans deficient in FXII are less prone to suffer from thrombosis is currently debat- ed although it is obvious that deficiencies of factors of the contact system do not provide protection from thrombotic events (6, 7), and indeed, John Hageman, who was the first described patient deficient in FXII, which resulted in the discovery of this protein in 1955, himself died from pulmonary embolism (8). Several studies have found that low FXII levels are associated with an increased risk of venous (9-12) as well as arterial thromboembolism (13-17), but these results have not been confirmed in other studies (18-23). It is important to recognize that a total deficiency in FXII is an extremely rare condition and that all these studies have necessarily been based on studies of individuals with low FXII levels or genetic polymorphisms leading to lower FXII plasma activity (24, 25). However, if the role proposed for FXII in thrombosis cannot yet be defini- tively confirmed or refuted, the papers in this thesis nevertheless provide a deeper understanding of the physiological activation and function of the contact system. In line with the finding that FXII is essential for thrombus formation, it provides evidence for contact activation initiated by activated platelets, which is further enhanced by fibrin formation, contributing to clot formation. Moreover, the regulation of the platelet- and fibrin-induced contact activation occurring during the clotting process differs from material- induced contact activation, suggesting a new approach to measuring the activation of this system. With regard to the ongoing discussion about the possible association with thrombosis, we found that altered levels of contact activation products were correlated with previous thrombotic events in patients with systemic lupus erythematosus (SLE). This result is very interesting from a clinical perspective and suggests that, instead of being limited to looking at plasma levels of FXII, we may be able to use these contact activation products, i.e., enzyme-serpin complexes, as new biomarkers for platelet activation and thrombotic risk that could conceivably be utilized in the future to assess the risk of thrombotic events.

12 THE PLASMA CONTACT SYSTEM

Blood clots immediately when exposed to various foreign surfaces. In the 1950s it was observed that blood clotted when added to glass tubes, and thus the concept of contact activation was born (26, 27). Four plasma proteins ap- peared to be involved in this contact-mediated coagulation and were termed the “plasma contact system.” Since then, contact activation has been the subject of detailed in vitro studies involving both purified proteins and plasma, with several different artificial surfaces used to trigger the activation. Anionic compounds have been shown to be the most efficient activators of the contact system and initiators of blood coagulation. However, direct confirmation of such in vivo activation is not yet available, although activating compounds are present in the platelets and presumably also in the extravascular tissue.

Proteins of the contact system The plasma contact system consists of three zymogens, coagulation factors FXII and FXI and prekallikrein, and one non-enzymatic cofactor, high molecular weight kininogen (HK). Like all coagulation enzymes, the zymogens are serine proteases, with a catalytic triad of serine, histidine, and aspartic acid in the active site of each enzyme. All four contact proteins are mainly synthesized in the liver and released into the bloodstream. If not referred to any other reference, the following protein information comes from the book Hemostasis and Thrombosis (28).

High molecular weight kininogen Human blood plasma contains two kininogens, HK and low molecular weight kininogen (LK), and the same gene located in the third chromosome encodes the synthesis of both proteins. LK has no evident function in blood coagulation, but HK participates in the contact activation process. HK is a ∼120-kDa α-globulin with a plasma concentration of 65-130 mg/L (29). It circulates as a non-covalent complex with prekallikrein or FXI (30, 31). HK is composed of a heavy chain region, the bradykinin moiety, and a light chain region. The heavy chain is similar to that of LK; the light chain is associated with the cofactor properties of HK, and both prekallikrein and FXI bind to this chain (32, 33).

13 HK is cleaved by plasma kallikrein in three sequential steps (34): The first cleavage yields a nicked kininogen composed of two disulfide-linked chains. The second cleavage releases the vasoactive nonapeptide bradykinin and an intermediate kinin-free protein. The third cleavage results in a stable, kinin-free protein composed of two disulfide-linked chains.

Coagulation factor XII FXII, encoded by a gene located on human chromosome 5, is a glycoprotein consisting of a single polypeptide chain of ∼80-kDa. Its concentration in blood is 20-30 mg/L. FXII can be divided into two regions, a heavy chain and a light chain. The heavy chain contains artificial surface-binding domains, whereas the light chain contains the active site and is the binding domain for the serpins C1- inhibitor (CINH) and antithrombin (AT). FXII has multiple domains with high sequence homology to regions of tissue-type plasminogen activator, epidermal growth factor, and fibronectin (35, 36). Upon contact with negatively charged surfaces and macromolecules, FXII binds to these initiators and becomes autoactivated (37). FXII can also be activated by the action of kallikrein, which cleaves FXII between Arg353- Val354 to generate α-FXIIa (38). Surface-bound α-FXIIa can be cleaved by kallikrein, releasing β-FXIIa into the fluid phase. β-FXIIa can only activate prekallikrein whereas α-FXIIa cleaves and activates both FXI and prekallikrein (38).

Coagulation factor XI A gene located on chromosome 4 encodes human FXI. This ∼160-kDa protein is composed of two identical polypeptide chains linked by disulfide bonds. FXI circulates in the blood in a non-covalent complex with HK at a concentration of 4-6 mg/L (31). The two FXI monomers are cleaved into an N-terminal heavy chain and a C-terminal light chain containing the serine protease catalytic triad. Each heavy chain contains four tandem repeat sequences, designated apple domains, that contain binding sites for platelets, heparin, and many other proteins such as HK, thrombin, and FXIIa (39). In complex with HK, FXI is adsorbed to negatively charged surfaces and macromolecules. There it is activated by α-FXIIa, and the active enzyme either remains bound to the surface or is released into the fluid phase. FXI can also be activated by thrombin (40). FXIa activates FIX if calcium ions are available. This activation is the only enzyme-substrate reaction in the coagulation cascade that does not require any specific cofactor other than calcium ions.

14 Prekallikrein Prekallikrein is a single-chain serine protease encoded by a gene that maps to chromosome 4. It circulates in the blood in two forms with molecular sizes of 85 and 88 kDa, respectively. The difference in the two forms is probably related to their relative degree of glycosylation. Prekallikrein is a γ- globulin with a plasma concentration of 35-50 mg/L. At least 75% of plasma prekallikrein circulates in an equimolar complex with HK, and the rest as free prekallikrein (30). Prekallikrein and FXI are 58% homologous with regard to their amino acid sequences, and therefore they have a similar domain structure. The high homology between the heavy chain of FXI and prekallikrein suggests a common origin for these two zymogens (39, 41). Conversion of prekallikrein to kallikrein is catalyzed by surface-bound α- FXIIa, augmented by HK or by β-FXIIa in the fluid phase.

Mechanisms of contact activation The mechanism of contact activation has almost exclusively been studied by using purified proteins and various negatively charged substances such as glass, kaolin, dextran sulfate, ellagic acid, endotoxins, and glycosaminoglycans to initiate the activation (42-46). Recently, studies of the interaction and activation of the contact components on endothelial cell membranes have been published, suggesting an alternative to the mechanism operating when negatively charged surfaces and macromolecules are involved.

Material-induced activation In the presence of a negatively charged surface, FXII becomes bound, and then autoactivated as a result of conformational changes that expose the catalytic serine protease region (37). The activated surface-bound form of FXII, α-FXIIa, cleaves and activates plasma prekallikrein and FXI, generating active plasma kallikrein and FXIa (38). FXIa then activates FIX, initiating coagulation and thus thrombin generation. Plasma kallikrein amplifies contact activation by cleaving FXII into FXIIa and surface-bound α-FXIIa, liberating soluble β-FXIIa, which produces more kallikrein. Thus, the contact activation cascade is an autocatalytic process. Kallikrein also cleaves HK, liberating the vasoactive and pro-inflammatory nonapeptide bradykinin (34). HK is a required cofactor for the contact activation process since both prekallikrein and FXI bind to negatively charged surfaces via HK. Contact activation induced by a material surface is illustrated in Figure 1.

Figure 1. Schematic illustration of contact activation induced by a negatively charged material surface.

Cell-induced activation The concept of contact activation in the plasmatic environment of platelets was raised in the 1960s, when data indicating the presence of activated FXII and FXI on the platelet surface were presented (47, 48). In the 1980s, it was shown that isolated platelets in buffer systems containing purified proteins could promote the activation of FXII (49, 50). Washed platelets were shown to bind HK and FXI in a zinc-dependent manner (51-53). Apart from the observation that FXIIa binds to washed platelets via glycoprotein (GP) Ib, which competes with HK (53-55), little has been published regarding the direct binding of FXII to platelets or platelet-triggered contact activation. Recent research concerning blood plasma has indicated that platelets are able to amplify the contact activation induced by a negatively charged substance or material such as high molecular-weight dextran sulfate (56). As opposed to the relatively few studies of the interactions with platelets, the interaction between endothelial cells and proteins of the contact system has been studied in detail. FXII and HK have been found to bind to human umbilical vein endothelial cells (HUVECs) via the C1q receptor for globular heads (gC1qR), the urokinase plasminogen activator receptor (uPAR), and cytokeratin 1 (CK1) (57-59). Prekallikrein that binds to HK assembled on endothelial cells can be converted to plasma kallikrein in a manner independent of FXIIa, and prolylcarboxypeptidase has been identified as the main endothelial prekallikrein activator in this system (60-63). This activation does not initiate coagulation but acts as a regulator of vascular function,

16 in particular via the generation of bradykinin (64-66). Endothelial cell bound FXII can be autoactivated, but it is believed to be activated mainly by kallikrein (61, 67). The interaction of both HK and FXII with endothelial cells is restricted by the availability of free Zn2+, but FXII requires a 30-fold higher Zn2+ concentration, indicating that FXII binding and activation occur in vivo when there is an increased concentration of Zn2+: for instance, when platelets, leukocytes, or endothelial cells are activated or injured (59, 63).

In vivo-activating compounds Several negatively charged surfaces bind to and activate FXII in vitro. Many biological substances also promote FXII autoactivation, but it has been difficult to identify these activating compounds in the intravascular compartment in non-disease states. Negatively charged phospholipids and sulfatides expressed in platelets would be expected to activate FXII, but such activation has never been convincingly shown in vivo (68). Extracellular nucleic acids, and particularly ribonucleic acid (RNA), enhance coagulation and have been found to be associated with fibrin-rich thrombi (and attendant thrombus formation) in mice experiencing arterial thrombosis (69). FXII binds to and is autoactivated by extracellular RNA. Therefore, contact activation is thought to be responsible for the procoagulant properties of RNAs (69) and extracellular RNA derived from damaged or necrotic cells, particularly under pathological conditions or in severe tissue damage, may represent an in vivo trigger for FXII-mediated contact activation. Another recently described mechanism to account for the possible in vivo activation of FXII involves inorganic polyphosphate, which is stored in the dense granules of platelets and is released when the platelets are activated (70). In in vitro model systems, inorganic polyphosphate has been shown to accelerate blood clotting by triggering FXII activation and promoting FV activation (71). In mouse models, polyphosphate triggers contact activation- mediated bradykinin-induced edema, and mice deficient in FXII, FXI, or both proteins survive a little longer than wild-type mice in a model of lethal pulmonary thromboembolism produced by intravenous infusion of polyphosphate (72). Collagen (type I) can bind to and activate FXII, thereby enhancing clotting and thrombin generation in vitro and theoretically also in vivo when collagen is exposed as a result of vessel damage (73, 74). FXII is also activated by misfolded-protein aggregates, and increased levels of FXIIa and kallikrein have been found in the blood of patients with amyloidosis (75). Heparin derived from human mast cells activates FXII, inducing contact activation, and is thought to be responsible for the generation of kinins in allergic reactions (76, 77).

17 Regulation of contact activation Inhibition of the enzymes involved in contact activation has been studied almost exclusively in solutions of purified proteins or in plasma with artificial negatively charged surfaces as the initiators of contact activation. Sever- al protease inhibitors, including C1INH, AT, α1-antitrypsin, α2-antiplasmin, protein C inhibitor, and α2-macroglobulin, are able to inhibit the enzymes of the contact system (78-85). Except for α2-macroglobulin, all these proteins belong to the superfamily of serine protease inhibitors known as serpins. Several studies have strongly indicated that C1INH is the predominant inhibitor of all the enzymes of the contact system, followed by α-2- macroglobulin, α-2-antiplasmin, and AT (82, 85-87). Glycosaminoglycans such as heparin and heparan sulfate enhance the inhibitory effect of AT on all contact enzymes (88-91) and have also been reported to enhance, to a lesser degree, the inhibitory effect of C1INH on kallikrein and FXIa, but not FXIIa (87, 92).

Effects of contact activation In addition to the ability of FXII to initiate coagulation, the proteins of the contact system also participate in the initiation of the inflammatory response via kinin formation and possibly also via complement activation. Further- more, it has been shown that the contact system contributes to the innate immune response against invasive bacteria by generating antibacterial peptides. The proteins of the contact system have been reported to influence fibrinolysis as well.

Coagulation If there is an FXII-activating surface present, the autoactivation of FXII easily initiates coagulation, and if no anticoagulants are present, it leads to clot formation even if there are no cells present. FXIIa activates FXI, which in turn activates FIX and thereby launches the plasma cascade reactions that lead to thrombin formation. That fact that contact activation initiates coagulation can certainly present a clinical challenge and is an important issue today, since blood-contacting biomaterials are commonly used for both diagnostic and treatment purposes. However, the contact system is believed to be of only minor importance in terms of stopping the bleeding that occurs after vessel damage, and it is not included in the current model of in vivo coagulation. As previously mentioned, an FXII deficiency is not associated with any bleeding tendency, and alternative activation mechanisms involving FXI, which in contrast to FXII has a hemostatic role, have replaced FXII in the in vivo model. This current model of coagulation and possible influence of the contact system is discussed in the hemostasis section under “Coagulation” below.

18 The kallikrein-kinin system FXII, prekallikrein, and HK are also components of the kallikrein-kinin system, which is responsible for the release of the multipotent proinflammatory mediator bradykinin. Bradykinin is a nonapeptide released from HK by kallikrein-mediated cleavage. Bradykinin is an exceedingly potent vasoactive peptide that can cause venular dilatation, activation of arterial endothelial cells, increased vascular permeability, hypotension, constriction of uterine and gastrointestinal smooth muscle and the coronary and pulmonary vascula- ture, bronchoconstriction, and activation of phospholipase A2 to augment arachidonic acid metabolism (93). Bradykinin is thought to be a major contributor to the innate inflammatory response; however, it also has thrombin- inhibitory and profibrinolytic activities (see below). Contact activation is also considered a potent local regulator of blood pressure through its deliv- ery of bradykinin. In hereditary angioedema (HAE) types I and II, which are the result of inactive C1INH or low levels of this inhibitor, bradykinin mediates swelling that most commonly occurs in the face, gastrointestinal tract, extremities, penis, and scrotum (94). A deficiency of C1INH leads to an uncontrolled production of bradykinin via the kallikrein-kinin system, whose proteins are mainly supposed to interact in the fluid phase but have recently also been suggested to initiate bradykinin production on endothelial cells. An additional type of HAE has been described, HAE type III, in which patients, most commonly women, have normal, functional levels of C1INH (95, 96). This type includes both HAE caused by known mutations in the FXII gene and HAE with an unknown genetic cause. One subgroup with mutations in the FXII gene expresses a mutant FXII which, when activated to FXIIa, has a specific activity much higher than that of the normal, unmutated protein, with a resulting augmentation of bradykinin formation (97). Estrogens can regulate the expression and plasma levels of FXII (98), and high estrogen levels (e.g., during pregnancy or treatment with oral contraceptives) are very often implicated in the initiation angioedema attacks in type III patients.

Activation of the innate immune response via the generation of antibacterial peptides The proteins of the contact system can bind to the surface of several important bacterial pathogens, resulting in the assembly and activation of the system (99-101). One of the components that triggers this contact activation is lipopolysaccharides (45, 46). In human plasma, activation of the contact system at the surface of several bacterial pathogens has been shown to result in the generation of an antibacterial peptide (102). This peptide is a fragment derived from HK and includes a 26-amino acid sequence that is mainly responsible for the peptide’s antibacterial activity. Thus, contact activation

19 appears to contribute to the innate immune response and to play a role in the defense against invasive bacterial infection.

Thrombin-inhibitory and profibrinolytic activities Several thrombin-inhibitory and profibrinolytic activities of the contact system have been described. HK has been shown to inhibit thrombin-induced platelet activation by means of several different mechanisms (103, 104). FXIIa, kallikrein, and FXIa can cleave plasminogen directly but much less efficiently than do tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) (105-107). The FXII-dependent fibrinolytic activity is relatively weak and also difficult to demonstrate in plasma when inhibitors are present. On the other hand, bradykinin has been characterized as a potent in vivo inducer of tPA release in humans (108).

The complement system FXIIa can activate the complement C1-complex, resulting in the activation of the complement system via the classical pathway (109, 110). Kallikrein can also directly activate complement components C3 and C5 (111). Since this activation only has been demonstrated with purified proteins, its importance in vivo is still uncertain.

20 HEMOSTASIS

Hemostasis is the complex physiological process that controls blood fluidity and maintains the balance between bleeding and thrombosis within the blood. It is always available to rapidly form a hemostatic plug to prevent unrestricted bleeding after vessel injury and also to resolve the plug after the vessel has been repaired, in order to restore unobstructed blood circulation. The normal hemostatic response occurs after a vessel has been damaged and consists of four major steps: 1) vasoconstriction, 2) platelet plug formation, 3) coagulation, and 4) fibrinolysis. Vasoconstriction is the immediate narrowing of a blood vessel as the result of a contraction of the muscular wall of the vessels. The response is mediated by vasoconstrictors released by the damaged endothelium. The vasoconstriction temporarily decreases the local blood flow, thereby reducing blood loss. Platelets then bind to the damaged endothelium and form a platelet plug that stops the bleeding. The formation of the primary platelet plug involves the adhesion of platelets followed by their activation and aggregation; this step is designated primary hemostasis. To stabilize the platelet plug, a fibrin network is formed via the coagulation process; this is secondary hemostasis. The primary and secondary hemostasis following a vessel injury is summarized in Figure 2. After the vessel has been repaired, the clot must be removed; this goal is accomplished by the fibrinolytic system. The fibrinolysis pathway has important similarities to the coagulation cascades. The key event in fibrinolysis is the conversion of plasminogen to plasmin, a powerful enzyme that dissolves the fibrin network into small soluble fragments that can be transported away by the blood and thereby cleared from the circulation.

Figure 2. The sequential events following a vessel injury. A) Schematic illustration of a vessel. B) Platelet adhesion to collagen in the subendothelial matrix. C) Platelet aggregation and activation leading to formation of a platelet plug that initially stops bleeding. D) Fibrin network formation, the result of coagulation, stabilizes the platelet plug.

Platelets and platelet plug formation – primary hemostasis

Platelets Platelets are small non-nucleated cells circulating in the vascular system; their main role is to prevent bleeding after vessel damage through the formation of a platelet plug. They also play a critical role in the retraction of clots, wound healing, and inflammation. Platelets are produced in the bone marrow, where they are derived from large megakaryocytes by exocytotic budding (112). After leaving the marrow space, approximately one-third of the platelets are sequestered in the spleen, while the other two-thirds circulate in the vascular system for 7 to 10 days, at concentrations ranging from 150 to 450 x 109 cells/L (113). Platelets are the second-most common cellular component in healthy blood; normally, only a small number of these cells are consumed during hemostatic processes, and the remainder circulate in resting form through the vascular system for the course of their entire life- time.

22 To avoid extensive hemorrhage, a rapid and effective response by the platelets is vital. Therefore, platelets are very sensitive to external stimuli and can be quickly activated. The high density of receptors on the platelet cell membrane facilitates the platelet’s activation and its interaction with the subendothelial matrix and other blood cells, including other platelets. Alt- hough platelets have developed extranuclear mechanisms to allow them to process and efficiently translate mRNAs (retained from their megakaryocyte precursors) into proteins, their protein synthesis is limited and not sufficient to allow the cell to adapt to different situations (114). Instead, the platelet contains several granules that, upon activation, rapidly release their contents into the surrounding medium. Platelets have three different types of granules. Two are platelet-specific: the largest and most abundant, the alpha granules, and the smaller dense granules. The third type of granules is the lysosomes, which are also present in most other cells. Alpha granules contain a large variety of secretory proteins involved in hemostasis, such as platelet activation and adhesion molecules, plasma coagulation factors, and fibrinolytic proteins (115). The dense granules mainly contain cations and smaller signal molecules such as adenosine diphosphate (ADP), adenosine triphosphate (ATP), and serotonin (115). Lysosomes contain acid hydrolas- es, cathepsin and lysosomal membrane proteins (115).

Platelet adhesion Platelets are sentinels that continuously scan the vascular bed for defects. As soon as the endothelial cell layer of a vessel wall is damaged, platelets rapidly adhere to exposed components of the subendothelial matrix. Several adhesive macromolecules serve as ligands for different platelet receptors, but adhesion is primarily mediated via fibrillar collagen type I and III (116) and von Willebrand factor (vWf), a multimeric plasma protein that rapidly binds to exposed collagen and acts as a link between collagen and platelets. The initial contact adhesion between platelets from the flowing blood and collagen molecules is facilitated by the GP Ib-IX-V receptor, a heterotrimer composed of two disulfide-linked GPIbα-GPIbβ in complex with two GPIX and one GPV (117, 118). The binding is mediated by vWf, which contains several binding sites for the GPIb-IX-V receptor. The binding of vWf to this receptor mediates outside-in signaling leading to platelet activation (119, 120). The importance of this interaction is supported by the observation that individuals with deficiencies in GPIb-IX-V (Bernard-Souiler syndrome) or vWf (von Willebrand disease) have severe bleeding diatheses (121). Plate- lets then become more firmly adherent to the subendothelium through the mediation of two receptors, membrane-bound integrin α2β1 and GP VI, which both directly bind to collagen (122).

23 Platelet activation The adhesion of platelets initiates the release of granule contents and a change in the shape of the cells, with the platelets forming pseudopods that extend to cover the injured surface and bridge exposed fibers. The platelets secrete several substances that promote the activation, aggregation, and degranulation of additional platelets. Important for this process are ADP and ATP, which activate neighboring platelets via ADP- and ATP-sensitive receptors (123), and thromboxane A2 (TxA2), which is rapidly synthesized and acts as a vasoconstrictor while also promoting platelet aggregation and degranulation (124). The activated platelets also release and express P- selectin on their surface; this adhesion molecule plays an essential role in the initial recruitment of leukocytes to the site of injury during inflammation (125). During clot formation, the activation of the coagulation results in the generation of the key enzyme thrombin, which in addition to facilitating the fibrin network formation, is also the most potent physiologic agonist of platelets, activating the two distinct protease-activated receptors (PARs)-1 and -4. Activation of these receptors leads to an increase in cytosolic Ca2+ as well as protein kinase C activation. These events precede all the major events associated with platelet activation, for instance the release of granule contents and their expression on platelets, the shape change, and integrin activation. Therefore, thrombin essentially drives platelet activation (126). It is believed that PAR-1 activation initiates the signal transduction in platelets that is necessary for their activation, whereas subsequent activation of PAR- 4 may be necessary to sustain the signaling events that optimize the propagation of platelet activation.

Platelet aggregation Platelet adhesion and activation are followed by platelet aggregation, during which platelets from the freely flowing blood bind to adherent platelets at the wound site. The activated platelets expose several receptors, but the integrin αIIbβ3 (GP IIb-IIIa), the most abundant platelet surface membrane GP, is also the most important for mediating platelet aggregation (127). The αIIbβ3 receptor binds to fibrinogen and serves to crosslink adjacent platelets because two of the receptors on different platelets can bind to the same fibrinogen molecule (128). The αIIbβ3 receptor has both a low- and a high- affinity state for binding fibrinogen; the high-affinity state is achieved when platelets are activated by agonists such as thrombin, ADP, collagen, or immobilized vWf (129). The modulation of the affinity of αIIbβ3 for fibrinogen is a result of conformational changes and a consequence of inside-out signaling, and this change in affinity is the primary driving force for the formation of initial platelet-platelet contacts and the initiation of the aggregation. The αIIbβ3 receptor can also mediate outside-in signaling by binding fibrinogen,

24 which drives the expression of various secondary effects of platelets, for instance the secretion of TxA2 and serotonin (130). One of the principal processes by which αIIbβ3 appears to mediate the transfer of information from the extracellular to the intracellular milieu is by tyrosine phosphorylation of cytoplasmic proteins. In addition to fibrinogen, the receptor also has an affinity for vWF, vitronectin, fibronectin, and thrombospondin (128). The platelet aggregation leads to the formation of a soft fibrinogen-rich platelet plug that arrests bleeding at the site of injury.

Coagulation – secondary hemostasis Coagulation is the general term for the complex enzymatic mechanisms leading to the formation of a fibrin network that stabilizes the primary platelet plug. It consists of a series of complex, and not yet fully understood, interactions between coagulation proteins and cells. In a way, coagulation can been viewed as a plasma cascade system in which one enzyme activates the next proenzyme by proteolytic cleavage, with every step causing the activation of more and more molecules that results in an overall amplification of the entire process. It is important to note, however, that the coagulation reactions occur as overlapping steps and take place to a great extent on the surface of cells, where tissue factor (TF)-bearing cells initiate and activated platelets amplify the coagulation. Thus, these cells also direct and control the coagulation process. The ultimate goal of the process is the formation of thrombin, which transforms the soluble fibrinogen molecules into insoluble fibrin fibers. The growing fibrin fibers are built up into a strong, intercon- nected network in which blood cells become entangled and the resulting blood clot is formed. Several coagulatory and also some regulatory proteins of the blood contain a characteristic domain rich in gamma-carboxyglutamic acid (Gla), which is synthesized from glutamic acid in a reaction that requires vitamin K as a cofactor (131). These vitamin K-dependent proteins interact with cell membranes through the Gla domain, which in the presence of Ca2+ can bind to phospholipids (132). Inhibition of the gamma-carboxylation of these proteins results in a drastic attenuation of coagulation and has for a long time been used in anticoagulation therapy in the form of warfarin administration (133). As described in the previous section, coagulation can be initiated via contact activation, but initiation via TF is the main contributor to the activation of physiologic coagulation in vivo.

25 In vivo model of coagulation – tissue factor activation The process of blood coagulation can be divided into an initiation and a propagation phase. The initiation phase is activated by the exposure of flowing blood to TF, an integral membrane glycoprotein found in extravascular tissue (134, 135). The zymogen FVII that circulates in blood binds to the membrane-bound TF, and the TF-FVIIa complex (known as a tenase complex) is rapidly formed. TF itself has no enzymatic function but instead serves as a cofactor to facilitate the rapid activation of FVII and significantly enhance the enzymatic activity of FVIIa (136). TF also binds to the activated form of FVII, FVIIa; although FVII is mainly found in the circulation as the inactive zymogen, some reports have stated that 1-2% of the total amount of FVII is in the activated form (137). The TF-FVIIa complex can activate both FX and FIX to produce their active forms, FXa and FIXa (137). Tissue factor pathway inhibitor (TFPI), which circulates in the plasma and is released by activated platelets, rapidly inactivates TF-FVIIa. TFPI requires the formation of an active TF-FVIIa complex and FXa generation before it inhibits the complex by forming a stable quaternary complex, TF-FVIIa-FXa-TFPI (138). In the absence of its cofactor, activated FV (FVa), the small amounts of FXa that are initially formed by TF-FVIIa can only generate trace amounts of thrombin. The minor amount of thrombin produced is insufficient for complete fibrin formation but nevertheless activates platelets through PAR receptors and converts cofactors FV and FVIII to their active forms. It is this feedback activation that accelerates further thrombin generation and leads to the explosive generation of thrombin during the propagation phase. The activated FVIIIa binds to FIXa, and the FIXa-FVIIIa (a tenase complex) prefer- entially assembles on the phospholipid membrane of activated platelets (139, 140), where it very effectively activates FX. The FIXa-FVIIIa complex activates FX with much higher conversion rate than that of the TF-FVIIa complex and is required to sustain the coagulation (141). The fact that a deficiency of either FVIII or FIX causes a severe bleeding disorder, hemophilia A or B, respectively (142), demonstrates that this is the main mechanism of FX activation in vivo. The rapidly forming FXa thereafter forms a complex with its cofactor FVa. This complex, FXa-FVa (prothrombinase complex), is also located on the phospholipid membrane (143), where it converts prothrombin to thrombin. The FIXa-VIIIa and the FXa-FVa complexes are both highly efficient and can in a short time raise thrombin concentration levels sufficiently to facilitate fibrin network formation.

Fibrin network formation The end goal of the coagulation process is to create an insoluble fibrin network that stabilizes the platelet plug. Fibrinogen is converted to fibrin via

26 proteolytic cleavage by thrombin, which removes two distinctive peptides, fibrinopeptide A and B (FPA and FPB), from the fibrinogen molecule. The fibrin monomers polymerize into protofibrils that subsequently assemble to form a three-dimensional network. After being activated by thrombin, the transglutaminase FXIII then crosslinks adjacent fibrin chains, enhancing the stiffness of the clot and its resistance to fibrinolysis (144). FXIIIa also incor- porates fibronectin into the fibrin network, increasing both size and density of the fibrin fibers (145). The fibrin formation process is sensitive to environmental factors, and the conditions present during the conversion will affect the final network structure. There are several known factors that influence the fibrin quality, with thrombin being one of the most thoroughly studied. Low thrombin concentrations produce coarse, unbranched networks of thick fibrin fibers, whereas high thrombin concentrations produce dense, highly branched networks of thin fibers (146). In general, coarse networks have lower porosity and elastic modulus and increased susceptibility to fibrinolysis, whereas dense networks have higher porosity and elastic modulus and are relatively resistant to fibrinolysis.

Procoagulant properties of platelets Coagulation and platelet activation are closely connected and integrated processes. Thrombin, the key enzyme in the coagulation cascade, is a very strong platelet agonist, acting on the PAR-1 and -4. At the same time, activated platelets enhance and contribute to the coagulation process via several independent mechanisms. When activated, one of their most important contributions is to provide a procoagulant phospholipid membrane that catalyzes several enzymatic steps in the coagulation cascade (147, 148). The major constituents of the platelet membrane are phosphatidycholine (PC), 38%; phosphatidylethanolamine (PE), 27%; sphingomyelin (SM), 17%; and phosphatidylserine (PS), 10%. In the resting platelet membrane there is a significant asymmetry, with almost no detectable PS, and only 20% of the total PE present on the outer leaflet of the platelet surface (149). Activation of the platelets leads to a rapid loss of asymmetry, exposing PS and more PE, and thus making the outer surface of the platelet slightly anionic. In concert with Ca2+, the platelet surface efficiently binds the coagulation factor complexes through the proteins Gla domain, thereby significantly increasing their activity (150). Another procoagulant mechanism is the release of α-granule contents during platelet activation; this process results in a drastic increase in the local concentration of coagulation factors and of fibrinolysis inhibitors at the site of the growing clot (115).

27 Open questions and possible roles of FXII Even though the TF-FVIIa complex initially activates FIX to FIXa, TFPI rapidly inactivates this complex, and further activation of FIX occurs via FXIa. The importance of this amplification is certainly variable from one individual to another, because a deficiency in FXI (hemophilia C) is associated with a mild-to-moderate bleeding tendency that most often is injury-related but can also be asymptomatic. FXI can be activated by FXII through contact activation, but this pathway is difficult to reconcile with the observation that patients affected by a deficiency of FXII do not have a bleeding tendency (1, 2). Instead thrombin has been suggested as the in vivo activator of FXI (40, 151). However, whether thrombin activation of FXI can occur in blood is questionable. Thrombin proteolytically cleaves fibrinogen and FXI with similar Km values, but the molar concentration of fibrinogen in the blood is about 300 times higher than that of FXI. In addition, phosphorylation of FXI by a casein kinase released by activated platelets increases its susceptibility to activation of FXIIa, but only to a lesser extent to that by thrombin (152). Although several questions have been raised concerning these reactions (153-155), the hemostasis research community has accepted that the activation of FXI occurs by thrombin instead of FXIIa as a way to explain why FXI has hemostatic activity and FXII does not. One other thing that is not fully explained by the model of in vivo coagulation is the activation of FVII to FVIIa during the initiation phase. Several proteases can activate FVII, including thrombin, TF-FVIIa, FXa, and FIXa, as well as FXII (156-158), but if FVIIa comes first, what other protease can then first activate FVII? Although some degree of autoactivation could occur, and activated FVIIa may exist in the circulation, it is likely that something else contributes to the initial activation. FXII is the only protease that does not need proteolytic cleavage to become activated, and both FXIIa and its product FIXa directly activate FVII in plasma (158, 159). Thus, even though it is not essential for life, it is possible that contact activation could be involved in the initial activation of FVII that occurs when coagulation is initiated by vessel damage.

28 AIMS OF THE STUDY

General aim The physiological role of the plasma contact system still remains a partial enigma. The general aim of the work described in this thesis was to expand our understanding of the plasma contact system, focusing on its physiological activation and function, principally from a hemostatic perspective, and to determine whether analysis of its activation products might be useful in clinical settings.

Specific aims Paper I Studies in the 1970s and 1980s indicated that isolated platelets in buffer systems containing purified proteins can promote the activation of FXII (49), and more recent studies have demonstrated that platelets can amplify the contact activation induced by negatively charged macromolecules or materials (56).

The aims of paper I were to:

1) Determine whether activated human platelets alone could induce FXI- Ia-mediated contact activation under physiological conditions. 2) Determine whether this activation had any influence on clot formation.

Paper II In paper I, we observed that the regulation of contact activation elicited by activated platelets differed from that previously described when purified proteins and negatively charged material surfaces had been used to initiate the activation. We found that platelet-induced contact activation mainly led to the formation of complexes between FXIIa or FXIa and AT, but not C1INH, which had been postulated to be the main inhibitor of these enzymes (82, 85-87). Inorganic polyphosphate, released from activated platelets, had been suggested to be an in vivo contact-activating compound (70, 71).

29 The aims of paper II were to:

1) Compare the regulation of contact activation triggered by materials and activated platelets. 2) Examine the contact-activating properties of inorganic polyphosphate in blood. 3) Determine whether FXIIa-AT could be used as a biomarker for platelet activation.

Paper III In papers I and II, we found that platelet activation induces contact activation, and high amounts of FXIIa-AT, FXIa-AT, and kallikrein-AT complexes are generated in clotted blood. The identity of the compound that triggers the activation of FXII is not known, but immobilized fibrin has recently been shown to possibly activate the contact system (160).

The aims of paper III were to:

1) Determine whether platelets are necessary for the formation of the AT complexes. 2) Determine whether fibrin could be responsible for activating factor XII in clotting blood.

Paper IV In paper I, we found that activated platelets trigger contact system activation, and paper III identified a possible mechanism to account for the propagation of this activation that occurs during clot formation. Recent in vivo studies have also demonstrated that the contact system is engaged in thrombus formation (3). Patients with systemic lupus erythematosus (SLE) have persistent platelet activation and an increased risk of thrombotic events (161-163), which cannot be explained by traditional cardiovascular risk factors (164). We wanted to test the clinical significance of our experimental results and determine whether the contact system could be involved in the inflammation and vascular disease associated with SLE, leading us to collect samples from SLE patients.

The aims of paper IV were to:

1) Determine whether the contact system is activated in patients with SLE. 2) Compare SLE patients with and without previous vascular disease with regard to their contact system activation. 3) Determine whether FXIIa-AT complexes might serve as a new biomarker for platelet activation and vascular disease in SLE.

30 MATERIALS AND METHODS

Protocols for all the experimental procedures employed are described in the four papers that are the basis of this thesis. Here in the thesis itself, some additional information is provided, and some of the advantages and disad- vantages of the chosen methods are discussed in greater detail.

Heparin coating Since FXII can be activated on material surfaces, we used tubes made of polypropylene, which is a poor activator of FXII. In some experiments, we wanted to further minimize the surface activation of FXII. Therefore, the materials in contact with blood were furnished with the Corline heparin surface. This immobilized heparin surface has previously been shown not to promote any contact activation (165, 166). The inhibition of contact activation is the result of an AT-mediated mechanism, in which AT binds to the heparin surface and inhibits FXIIa. This mechanism does not produce significant increases in AT complexes because FXIIa-AT remains bound to the surface. The immobilized heparin surface is very stable, with no leakage of heparin (167), and it also prevents the binding of leukocytes and platelets, further demonstrating its non-reactive character (168).

Platelet activation Thrombin mediates the activation of platelets through PAR-1 and -4. In our platelet function studies, we used a thrombin receptor-activating peptide (TRAP, SFLLRN-amide), which selectively binds to PAR-1, in the presence of a thrombin inhibitor, lepirudin. This combination allowed platelet activation to occur without clot formation or any amplification mechanisms mediated by thrombin. In addition, the presence of lepirudin guaranteed that FXI was activated exclusively by FXIIa and not by thrombin, which has been suggested as an in vivo activator of FXI. In the regulation studies, platelets were also activated with ADP and collagen, which are two physiological and well-characterized activators of platelets. Since platelets are easily activated, the handling of the blood during the experimental procedures can potentially induce the activation of the plate-

31 lets. Therefore, in the functional studies we included control platelet-rich plasma (PRP) to which no platelet activators were added (i.e., non-activated platelets were used); these control samples underwent the same treatment as the activated platelets. To avoid any possible influence of non-specific activation, all the results for the activated platelets were compared to those for the non-activated controls. The experiments in PRP were sometimes also compared to those in plasma without any cells present (i.e., platelet-poor plasma [PPP]), to allow us to really see the influences of the platelets.

Enzyme-linked immunosorbent assays The measurements of the enzyme-serpin complexes in plasma were performed using sandwich enzyme-linked immunosorbent assays (ELISAs). A capture antibody specific for the enzyme and a detecting antibody specific for the inhibitor in the complexes were used. This approach made the ELISAs very specific and kept the risk of false-positive results to a mini- mum. In addition, despite the fact that complexes formed are irreversible, they tend to become non-detectable if the samples are kept at ambient tem- perature for too long and are not immediately frozen. One disadvantage is that since polyclonal antibodies were used for these experiments, there could have been some minor variations in the specificity of different batches of antibodies. Figure 4 of this thesis shows the quantification of plasma FVIIa-AT complexes. The complexes were measured using sheep anti-human FVII (Affini- ty Biological, Ancaster, ON, Canada) as capture antibody and a biotinylated rabbit anti-human antithrombin as detecting antibody (Dako, Glostrup, Denmark), followed by horseradish peroxidase (HRP)-conjugated streptavi- din (Pharmacia Amersham, Uppsala, Sweden). A standard was prepared by mixing FVIIa (NovoSeven®; Novo Nordisk, Bagsvaerd, Denmark) and antithrombin (Pharmacia, Uppsala, Sweden) in the presence of heparin (3 IU/mL). Values were expressed as AU/mL.

Flow cytometry We wanted to determine whether platelets activated in PRP exposed contact- activation proteins. To remove non-specifically bound proteins on the surface of activated platelets, we washed the platelets before staining them. The wash procedure has the disadvantage that specifically but weakly bound proteins can be washed away; in addition, the procedure can to some extent activate the platelets. Since we compared the results to those of non- activated platelets that had undergone the same treatment, the potential platelet activation did not pose a major problem. However, we were only able to

32 detect exposed proteins on a small proportion of the platelets, and it is possible that some specifically bound proteins became detached. The advantage of our approach is that the risk of false positive results is negligible and that the proteins detected are clearly exposed on the surface. Since only some of the platelets exposed the proteins and no total shift in the platelet populations occurred, the results are presented as mean fluorescence intensities (MFI).

Chromogenic substrates In paper I, we investigated the enzymatic activity of the proteins detected on the surface of activated platelets. Since the enzymes of the contact system and other enzymes that can cleave the substrates are all present in plasma, the platelets were washed in order to remove all plasma proteins. The chromogenic substrate used for detection of both kallikrein and FXI- Ia is more liable to be cleaved by kallikrein than by FXIIa, and the FXIIa- assay was therefore amplified with prekallikrein. The FXIIa assay was also performed on platelets from a FXII-deficient patient without showing any FXIIa activitity. The FXIa and kallikrein assays were not amplified, and only low-level increases in the enzymatic cleavage were detected. Such results were expected, since flow cytometry experiments with washed platelets alone showed that a few percent of the cells exposed the proteins. We used corn trypsin inhibitor (CTI), a specific inhibitor of FXIIa, and soybean trypsin inhibitor (SBTI), with a broad inhibitory capacity to block both kallikrein and FXIa, to attenuate the enzymatic activity. In paper III, we tested the ability of purified FXII to become activated by negatively charged phospholipids and fibrin clots. The fibrin clots were prepared by incubating fibrinogen with thrombin, and thrombin can cleave the chromogenic substrate used for detection of FXIIa. To mimic the conditions in the blood experiment and to reduce the thrombin activity, the thrombin inhibitor lepirudin was added to the samples. The enzymatic activities of FXIIa and thrombin were then measured using two different chromogenic substrates. A high level of enzymatic activity was detected for FXIIa, but only a minor increase in the absorbance of the substrate used for thrombin detection, which is a much more sensitive and selective chromogenic substrate than the one used for the detection of FXIIa. Thus, we could ensure that the enzymatic activity measured using the chromogenic substrate for FXIIa was mainly derived from FXIIa, and not from thrombin.

Clotting time To investigate the influence of platelet-induced contact activation on clot formation, clotting times in PRP, where the platelets were activated with

33 TRAP specific for PAR-1, were measured using a coagulometer. These assays were performed in the presence and absence of the FXIIa inhibitor CTI. All materials in contact with blood and PRP were coated with heparin in order to minimize material-induced contact activation. The heparin surface causes relatively long clotting times, since it reduces platelet adhesion as well as enhances the inhibitory capacity of AT, and consequently the clot formation induced by platelets has to take place in the fluid phase.

Platelet aggregation Whole blood impedance aggregometry, in the absence and presence of CTI, was used to investigate the influence of FXIIa on platelet aggregation. This method measures the increase in impedance between two electrodes caused by platelet aggregates. The use of whole blood eliminates the need for cen- trifugation and thus allows the examination of platelet aggregation under more physiological conditions than does optical aggregometry, which de- mands PRP. The electrodes used for the measurements are made of silver and therefore also initiate contact activation. In addition, they allow the platelets to aggregate on a surface, reflecting the events that may occur on a biomaterial surface or a biological surface such as a damaged vessel wall. In every measurement two pair of electrodes are used, which reduces the variability of the results. As many as four samples can be analyzed at the same time, allowing the simultaneous analysis of control samples and samples with CTI (to inhibit FXIIa). A potential concern is the fact that although impedance aggregometry was introduced as early as the 1980s, optical aggregometry has been and is still the most frequently used test for studying platelet aggregation. Nevertheless, when comparisons between the two methods have been performed, they have yielded identical results with regard to platelet functions (169).

Tubing loop models The main advantage of the tubing loop model is that it permits the study of circulating blood without any soluble anticoagulant being present. To study contact activation under these conditions, we used polyvinyl chloride (PVC) tubing, with its inner surface coated with heparin, to resemble a blood vessel. The tubing was filled with fresh non-anticoagulated blood, and the tubing loops were closed to form circuits by using surface-heparinized connectors. The tubing loops were placed on a rotator device in a 37°C incubator and rotated vertically at 33 rpm for 30 min. After incubation, blood samples were collected and analyzed for platelet count and enzyme-serpin complexes.

34 Phospholipid vesicles In order to study clotting reactions that are triggered without any cells being present, and to assess the influence on contact activation of the negatively charged phospholipids phosphatidylserine (PS) and phosphatidylethanolamine (PE) exposed on the surface of activated platelets, we used vesicles composed of phospholipids. Since we do not know exactly how the phospholipids are distributed on the surface of activated platelets, and their distribution has been shown to change with the platelets’ stage of activation, we made eight different vesicle preparations containing various weight ratios of phosphatidylcholine (PC, an uncharged phospholipid), PS, and PE, based on platelets’ total percentage content of the various phospholipids. The vesicles were carefully filtered several times to obtain vesicles of uniform size that had a diameter less than 1 µm.

Fibrin clots In order to study the contact activation induced by fibrin, we made fibrin clots. Since the structure seemed to be of the greatest importance for our studies, we wanted to have as little modification of the proteins as possible; therefore, the clots were prepared by incubating thrombin with various amounts of fibrinogen. Three different amounts of fibrinogen were incubated with the same amount of thrombin, not only to produce different sizes of clots but, more importantly, to obtain fibrin networks with different struc- tures.

35 RESULTS AND DISCUSSION

Paper I In paper I we wanted to investigate whether activated platelets could themselves induce contact activation in blood. Platelets were activated with TRAP specific for PAR-1 in lepirudin-anticoagulated PRP, resulting in the formation of FXIIa-C1INH, FXIIa-AT, FXIa-C1INH, and FXIa-AT complexes in plasma. The presence of these complexes demonstrated that the contact system had been activated. To ensure that the complex generation was derived from activated platelets, activation was also triggered in the presence of a TF-blocking monoclonal antibody and using heparin-coated tubes. The generation of the contact enzyme-serpin complexes was not affected by these two conditions, confirming that the activation of the contact proteins had occurred as a consequence of specific platelet activation, independent of TF or any possible contact activation on the tube surface. It should also be pointed out that FXIIa mediated the activation of FXI, since thrombin was inhibited by the presence of lepirudin and could not contribute to this activation. Since we found evidence of contact activation in plasma, we also wanted to study the events occurring on the platelet surface. Using flow cytometry we were able to demonstrate that all the proteins of the plasma contact system were exposed on the surface of activated platelets. The exposure was not pronounced, with only a small proportion of the platelet population exposing the contact activation proteins, but it was consistently observed. It is possible that this proportion is an underestimate because the washing procedure performed in order to remove all plasma proteins may have also resulted in the detachment of some specifically bound proteins. Experiments with chromogenic substrates demonstrated that the FXIIa, FXIa, and kallikrein from the activated platelets were enzymatically active, thus verifying the flow cytometric results and corroborating the presence of these proteins on the platelet surface. The enzymatic activity of all the proteases was reduced or completely abolished by the addition of CTI or SBTI, providing further evidence that the enzymatic activity was derived from the contact system proteases. To study contact activation during clot formation, clotting was triggered by the PVC surface in the loop model or by thromboplastin. PVC is a rather weak contact activation surface and only induced clotting in blood from half

36 of the donors, presumably as a result of the activation of platelets; in contrast, the physiological activator thromboplastin induced clotting in all the blood samples tested. In both models, substantial amounts of FXIIa-AT and FXIa-AT were generated when clotting was achieved, demonstrating that contact activation occurred in the clotting blood. The amount of FXIIa-AT was correlated with both thrombin activity and the level of thrombin-AT (TAT) complex formation, indicating its contribution to thrombin formation. Neither FXIIa-C1INH nor FXIa-C1INH complexes were detected, suggesting that C1INH does not primarily regulate these enzymes in clotting blood. The hypothesis that activated platelets are responsible for the initiation of the contact activation occurring during clot formation was corroborated by the use of abciximab, an inhibitor of the fibrinogen receptor GP IIb/IIIa. In the presence of abciximab, which inhibits platelet aggregation, no complexes were formed, and clotting induced by PVC loops was prevented in all the blood donors tested. To further assess the influence of platelet-triggered contact activation during clot formation, a coagulometer (with all its blood-contacting surfaces coated with heparin, to minimize material-induced activation) was used to measure clotting times. In PRP, TRAP-activated platelets produced shorter clotting times than did non-activated control platelets. CTI-mediated inhibition of FXIIa clearly prolonged the clotting time, whereas blocking TF had no effect. The clots formed when FXIIa was inhibited were also clearly less stable than those formed when TF was inhibited and also less stable than control clots, corroborating reports from several previous studies that FXII activation contributes to clot stability (3, 170, 171). In conclusion, the study performed in paper I demonstrated that FXII- mediated contact activation occurs on the surface and in the vicinity of activated platelets and contributes to clot formation. Activated platelets thereby constitute a nidus for contact activation inside the blood vessels and are able to recruit proteins of the contact system into the clot formation process.

37 Paper II In paper II, we explored contact activation induced by either activated platelets or negatively charged materials or macromolecules. Platelets activated in lepirudin-anticoagulated whole blood and PRP induced the activation of FXII, regardless of whether TRAP (specific for PAR-1), ADP, or collagen was used for platelet stimulation. FXIIa-AT and FXIa-AT, but almost no C1INH complexes, were detected in the resulting plasma. After platelet activation with TRAP and collagen, small amounts of kallikrein-AT were also found. Thus, the platelet-induced activation of FXII resulted in activation of both FXI and prekallikrein. The regulation of the contact activation enzymes was also studied in tubing loop models with circulating non-anticoagulated blood. In the loops, clotting was triggered via platelet stimulation with TRAP or by shear force. In all the clotted samples, high levels of FXIIa-AT, FXIa-AT, and kallikrein- AT were found, but hardly any C1INH complexes, directly paralleling the results of the clotting induced by the two other methods analyzed in paper I. To study the contact activation induced by negatively charged surfaces, we used two well-known contact-activating substances, glass and kaolin. We also tested inorganic polyphosphate, since this compound is released by activated platelets and has been thought to activate FXII in vivo. (70, 71). Con- tact activation triggered by these three substances in lepirudin-anticoagulated PPP, PRP, and whole blood generated high amounts of FXIIa-C1INH. Re- gardless of the activating compound used, low or negligible amounts of FXI- Ia-AT were generated. Kaolin caused the formation of both FXIa-C1INH and FXIa-AT, but higher amounts of C1INH complexes were generated. In the case of glass and polyphosphate, negligible amounts of FXIa complexes were formed, despite the strong activation of FXII. Kaolin and (to a lesser extent) glass, but not polyphosphate induced formation of kallikrein-C1INH. Polyphosphate was further analyzed in the loop model with non- anticoagulated blood. As we had observed for lepirudin-anticoagulated blood, polyphosphate gave a strong activation of FXII, as indicated by high amounts of FXIIa-C1INH, but no further activation of FXI or prekallikrein occurred. Thus, inorganic polyphosphate most likely has little importance for contact activation occurring during clot formation, even though it activates FXII. All the activation experiments were also performed in the presence of CTI, a specific inhibitor of FXIIa; the results obtained revealed a remarkable difference between platelet-induced and material-induced FXII activation. CTI totally abolished the generation of FXIIa-C1INH after activation with glass, kaolin, and polyphosphate, whereas the generation of FXIIa-AT after platelet activation was only decreased to some extent. During platelet activation, FXI and (to a lesser extent) prekallikrein were activated despite the fact that thrombin was inhibited and that CTI was assumed to inhibit FXIIa.

38 Thus, CTI was not able to inhibit platelet-induced contact activation, and it presumably had a lower affinity for FXIIa than that of AT, and it was therefore unable to compete with AT for binding to platelet-activated FXIIa. To investigate the influence of contact activation on platelet function, we measured platelet aggregation in the presence and absence of CTI by using whole-blood impedance aggregometry. In this method, the detection and measurement of aggregation are based on changes in impedance between two silver electrodes, which indicate that contact activation is triggered by both activated platelets and the electrode surface. The aggregation of platelets stimulated by ADP, collagen, and TRAP was reduced in the presence of CTI, confirming the involvement of FXIIa in this process. Contact activation occurring on a surface thus influences platelet functions such as aggregation and reflects the events that may occur on biomaterial surfaces and biological surfaces such as damaged vessel walls. To analyze the contact activation enzyme-serpin complexes and charac- terize the relationship between AT complexes and platelet activation in vivo, we analyzed plasma samples from patients treated for severe trauma, e.g., after traffic accidents. Blood was drawn at admission to the hospital and at repeated time points for up to 10 days. At the time points closest to the trauma, the highest amounts of FXIIa-AT were detected, and the levels subsequently decreased. In addition, low levels of FXIa-AT were detected and followed the same pattern as FXII-AT. These results were seen in several patients. The amount of FXIIa-AT was correlated with the amount of thrombospondin-1 (TSP-1), which is released by activated platelets. In contrast, the generation of FXIIa-C1INH complexes was only detected several hours after admission and on days 1-10, when FXIIa-AT levels were low. No association was found between FXIIa-C1INH and TSP-1. FXIIa-C1INH seemed to appear during treatments involving biomaterials and drugs in which surfaces were exposed, e.g., catheters, plasma expanders (dextran), and intrave- nously lipids, suggesting that these complexes were generated on an artificial surface. No FXI-C1INH or kallikrein-C1INH and only minute amounts of kallikrein-AT complexes were detected in the patients’ samples. In conclusion, the results reported in paper II demonstrate that contact activation triggered by activated platelets and occurring during the entire clotting process is regulated by AT, whereas C1INH is the main inhibitor when artificial material surfaces initiate the activation of FXII. In addition, the inhibition of FXIIa by CTI seems to differ for the different activation sites, and CTI had only a minor effect on platelet-mediated contact activation. Since enzyme-AT complexes are formed during platelet-mediated contact activation and FXIIa-AT is correlated with TSP-1 released by activated platelets in vivo, we suggest that FXIIa-AT is an interesting new biomarker candidate for platelet activation.

39 Paper III From the studies in papers I and II, we knew that activated platelets can induce contact activation, which is regulated by AT, and that high amounts of contact enzyme-AT complexes are formed in clotted blood. What compound triggered the activation of FXII, however, was not known. In paper III, we focused on how FXII is activated and how this activation leads to the generation of AT complexes during clot formation. To determine whether the presence of platelets is necessary for the formation of the AT complexes during clot formation, we triggered clotting in two different ways: First, clotting was triggered by thromboplastin in non- anticoagulated plasma, which resulted in a clear generation of FXIIa-AT and FXIa-AT, although no cells were present. The longer the clotting was allowed to continue, the higher amount of FXIIa-AT was formed; in contrast, FXIa-AT levels reached a plateau. Second, clotting was triggered in re- calcified citrated plasma by vesicles consisting of different phospholipid mixtures that mimicked the surface of activated platelets. In this experiment, platelet microparticles were removed, although FXIIa-AT and FXIa-AT were consistently formed in the samples in which clotting was initiated. Both these plasma experiments showed that platelets, or platelet-released substances, are not necessary for the generation of FXIIa-AT and FXIa-AT complexes during clot formation. To determine whether the negatively charged phospholipids PS and PE could be responsible for the initiation of the contact activation, phospholipid vesicles were also incubated in lepirudin-anticoagulated plasma and with purified FXII, in addition to re-calcified citrated plasma. No FXIIa-complexes could be found in lepirudin-anticoagulated plasma, and we detected no enzymatic activity from FXII in the purified system. Thus, the negatively charged phospholipids PS and PE exposed on the surface of activated platelets do not seem to initiate the activation of FXII. The clotting induced by the phospholipids mimicking activated platelets in re-calcified citrated plasma is then likely to be triggered only because the coagulation factors accumulate on the vesicles or because of an amplification of material-induced contact activation from the test tubes. In contrast to the results obtained for phospholipid vesicles, activated platelets triggered FXIIa-AT and FXIa-AT generation in lepirudin- anticoagulated PRP, and the generation of these complexes was not affected if the test tubes were coated with immobilized heparin, which prevents material- induced contact activation (165). These results support the view that activated platelets, in addition to amplifying material-induced contact activation, are themselves able to induce the activation of FXII. The thrombin-mediated conversion of fibrinogen to fibrin is the key event in clot formation, and it has previously been shown that surface-adsorbed fibrin may activate the contact system (160). This makes fibrin a very interesting candidate for FXII activation during clot formation. We therefore

40 prepared fibrin clots by incubating various amounts of fibrinogen with thrombin. By the use of chromogenic substrates, we were able to show that these clots were able to activate purified FXII. When the fibrin clots were incubated in lepirudin-anticoagulated PPP, PRP, or whole blood, there was a significant generation of FXIIa-AT and FXIa-AT complexes, with the highest response in whole blood. This response was comparable to what we had previously seen in clotted blood. The highest levels of AT complexes were generated by the fibrin clots prepared with the highest amount of thrombin in relation to the amount of fibrinogen, which would likely yield the clots with most dense fibrin network (146). These clots also had some remaining thrombin activity. Even though thrombin cannot activate FXII, and thrombin alone could not induce the same events in PPP, PRP or whole blood, we wanted to ensure that the generation of AT complexes was not mediated by thrombin. Therefore, in addition to lepirudin, we also added melagatran, a low molecular weight thrombin inhibitor that is likely to inhibit thrombin within clots more efficiently than does lepirudin. There was no difference in the generation of AT complexes if both lepirudin and melagatran were present, demonstrating that AT complexes were formed independently of any possible free thrombin. Thus, it is likely that differences in the fibrin structure provide the explanation for the variations in the complex generation, and presumably a dense fibrin network with higher porosity is a more- activating structure than a coarse network with lower porosity. How activated platelets induce contact activation is still unclear, but it seems, at least, that the mechanism does not involve negatively charged phospholipids; it is possible, however, that fibrin is involved in this activation. Although the studies with platelets in paper I and II were performed in lepirudin-anticoagulated blood, the activated platelets might have induced minor thrombin formation that, in the close vicinity of the platelets, could cleave fibrinogen. It is also possible that fibrinogen that binds to platelet receptors undergoes conformational changes, making it capable of activating FXII. The conclusion from paper III is that platelets are not mandatory for the generation of contact enzyme-AT complexes and that, in parallel with platelets, fibrin activates FXII, and fibrin formation helps amplify and propagate the contact activation that occurs during clot formation. Another key finding is that fibrin seems to increase the affinity of AT for the contact enzymes, competing out C1INH and leading to the generation of AT complexes. Combined with our previous results, the results described in paper III have led us to suggest that FXII is initially activated and forms FXIIa-AT complexes when platelets become activated, but further contact activation and formation of AT complexes during clot formation are mainly achieved by fibrin network formation. Thus, in vivo, activated platelets regulate the assembly of these complexes via the initiation and amplification of the coagulation process, leading to the generation of thrombin and subsequently fibrin formation. These complexes are therefore interesting candidates as bi-

41 omarkers for monitoring the activation of platelets and the coagulation system.

Paper IV In paper IV, we wanted to test the clinical significance of our experimental results. Since SLE patients have persistent platelet activation and an increased risk of thrombotic events (161-163), which cannot be accounted for by traditional cardiovascular risk factors (164), we deemed them appropriate subjects for the study in paper IV. We found that SLE patients have altered levels of complexes between serpins and activated proteases of the contact system, supporting the concept that the contact system is involved in the pathology of the disease. We expected to find elevated levels of FXIIa-AT but saw no statistically significant difference from the levels in the healthy controls, although there was a larger variation in levels among the SLE patients. When we examined only those patients who had a history of thrombotic events, we found a difference between their levels and those of patients without a history of thrombosis, and the patients who had experienced thrombotic events had higher levels of FXIIa-AT than did those who had not. That the levels of FXIIa-AT were not even higher is probably a result of FXII consumption and the fact that SLE patients are treated with a wide range of drugs, including some that inhibit platelets and coagulation. The levels of the other AT complexes, i.e. FXIa- AT and kallikrein-AT, were strongly correlated with those of FXIIa-AT but were rather similar between patients and controls, although we did see statistically lower levels of FXIa-AT in the patients. Despite the fact that both FXIa-AT and kallikrein-AT complexes followed the same pattern as FXIIa- AT, the differences in their levels did not allow us to statistically distinguish between those patients with previous thrombosis and those without. Since the levels of the three different types of AT complexes were correlated with each other, they likely were generated together and were derived from the same activating source. The levels of all the AT complexes with the strongest rs values for FXIIa-AT were correlated with those of several markers of platelet activation: P-selectin exposure, TSP-1, and platelet-monocyte complexes. These results are consistent with our previous finding that activated platelets are clearly involved in the contact activation leading to the formation of complexes between activated proteases of the contact system and AT. In contrast to the levels of FXIIa-AT, the levels of FXIIa-C1INH in SLE patients were lower than those of controls and were lowest (often below the detection limit of the assay) in the SLE patients with previous thrombotic events. The levels of FXIa-C1INH were similar in the patients and controls, but the levels of kallikrein-C1INH were clearly elevated in the SLE patients.

42 As we saw for the corresponding AT complexes, neither the levels of FXIa- C1INH nor kallikrein-C1INH were sufficiently different between those with and without previous thrombotic events to allow us to distinguish between them. Both of them were correlated with FXIIa-C1INH, but FXIa-C1INH showed a much higher rs value than did kallikrein-C1INH. We do not know the activation source for the C1INH complexes, but based on what is known about HAE, a lack of functional regulation by C1INH leads to an uncontrolled interaction with and activation of the contact proteases in the fluid phase, and it has been suggested that this activation is initiated on endothelial cells. In addition, under experimental conditions, prekallikrein can be activated on endothelial cells independently of FXIIa, with prolylcarboxylase identified as the main endothelial prekallikrein activator instead of FXIIa. There seem to be two subgroups of kallikrein-C1INH in SLE patients that may be generated by different mechanisms, one correlated with the levels of FXIIa-C1INH, and one occurring in the absence of any detectable FXIIa-C1INH complexes. Thus, it is possible that in the second group, endothelial prolylcarboxylase is the activating source for the prekallikrein. The endothelial lining of the blood vessels of SLE patients is damaged to a greater or lesser extent (172, 173), and we hypothesize that endothelial dysfunction could be the reason for their altered levels of both kallikrein-C1INH and FXIIa-C1INH. The fact that altered levels of FXIIa-AT and FXIIa-C1INH were correlated with previous thrombosis and the lack of reliable laboratory parameters for evaluating the risk of thrombotic events in SLE patients prompted us to calcu- late the odds ratio (OR) for thrombosis with regard to the altered levels of the FXIIa-serpin complexes. The OR for vascular disease among the SLE patients was 8.9 if they had low levels of FXIIa-C1INH, 6.1 if they had high levels of FXIIa-AT, and 23.4 if they had both decreased levels of FXIIa- C1INH and increased levels of FXIIa-AT. These ORs could be compared to that calculated from the existence of anti-cardiolipin antibodies (which, taken together with lupus anticoagulant, are the state-of-the-art criteria for evaluating the risk for thrombotic events); this OR was as low as 2.0 in our study. Most patients had thrombosis several years ago, and the median time since the last thrombotic event was as long as 8 years in our sample. Yet the patients with a history of vascular disease could still be distinguished from the others, suggesting that they have more pronounced pathological changes affecting the contact system. We also believe that it is likely that the contact activation enzyme-serpin complexes show significantly greater change in relation to and prior to the occurrence of any thrombosis, making it very interesting to evaluate these potential relationships in prospective studies. Neverthless, the evidence presented in paper IV strongly suggests that the FXIIa-serpin complexes are potential biomarkers for evaluating the risk of thrombosis and may in the future serve to complement other analyses in producing an accurate assessment of the risk of thrombotic events in SLE.

43 CONCLUSIONS

Paper I

• Activated human platelets initiate FXIIa-mediated contact activation in blood.

• In a model with minimized material-induced activation, platelet- triggered contact activation contributes to clot formation by shortening the clotting time and enhancing of clot stability.

Paper II

• Platelet-triggered contact activation and the activations taking place in clotting blood are regulated by AT, whereas material-induced activation is regulated by C1INH.

• Inorganic polyphosphate does not seem be the FXII-activating compound in clotting blood.

• In trauma patients, the levels of complexes formed between activated proteases of the contact system and AT are heightened, and the levels of FXIIa-AT complexes are strongly correlated with those of TSP-1 released by activated platelets.

Paper III

• Platelets, or platelet-released substances, are not mandatory for the activation of FXII and formation of FXIIa-AT complexes during clot formation.

• Fibrin formation is one underlying mechanism for the amplification and propagation of the contact activation that occurs when blood clots. Fi-

44 brin both activates FXII and seems to increase the affinity of AT for the proteases of the contact system, leading to the generation of contact enzyme-AT complexes.

Paper IV

• SLE patients have altered levels of complexes between serpins and activated proteases of the contact system, supporting the concept that the contact system is involved in the pathology of this disease.

• The contact enzyme-AT complexes are clearly linked to platelet activation in vivo.

• Altered levels of both FXIIa-AT and FXIIa-C1INH are correlated with previous vascular disease, and may be potential biomarkers for accurate assessment of the risk of future thrombotic events in SLE patients.

45 CONCLUDING REMARKS AND FUTURE PERSPECTIVES

When platelets become activated, they bind to proteins of the contact system and initiate contact activation. This activation triggers coagulation, and if the coagulation process proceeds, it is further enhanced by fibrin formation that occurs as a result of the generation of thrombin. The contact activation induced by platelets and fibrin is subsequently regulated by AT, leading to formation of complexes between activated proteases of the contact system and AT in plasma. These events occur when blood clots, and they influence the clot formation by amplifying thrombin generation (Figure 3), leading to enhanced clot stability. It is also possible that the platelet-induced contact activation is involved in the initiation phase of coagulation and contributes to the initial activation of FVII. This contention is supported not only by previous studies but also by the fact that platelet activation in our experimental systems, resulted in the activation of FVII, measured as an increase in FVIIa-AT complexes, as well as the induction of contact activation (Figure 4). It is also possible that platelet-exposed TF is a strong contributor to the activation of FVII. Although the significance of blood-borne TF is somewhat controver- sial, TF is present in platelets and platelet microparticles and is believed to be exposed on platelets after their activation, either as a result of de novo synthesis or derived from stores of preformed TF (174). The possible involvement of platelet-triggered contact activation in the initiation phase of coagulation is illustrated in Figure 5. Obviously, the amplification mediated by the contact system can be compensated by other coagulation factors, and initiation via TF is sufficient to stop the bleeding caused by a vascular injury, since humans deficient in FXII do not have any bleeding problems. In contrast, a deficiency of FXI causes a mild-to-moderate bleeding disorder with great interpersonal variability in severity. Although thrombin has been suggested to be the in vivo activator of FXI, this notion has also been questioned (153-155), and it is possible that the contribution to thrombin generation via contact activation is of different levels of importance in different individuals.

Figure 3. Amplification by platelets and fibrin-triggered contact activation in the in vivo model of coagulation. Tissue factor-bearing cells in the damaged vessel wall initiate the coagulation. Trace amounts of thrombin are produced by factor Xa, which in turn activates the cofactors factor VIII and factor V. The resulting FIXa- FVIIIa and FXa-FVa complexes assemble on the surface of activated platelets and very efficiently activate FX and prothrombin, respectively. In addition, platelet- triggered contact activation amplifies the coagulation process by activating FIX. Massive amounts of thrombin are generated, which cleave the fibrinopeptides from fibrinogen molecules. The fibrin that is formed then polymerizes into a fibrin network, which also triggers contact activation and further enhances the generation of thrombin. Finally, the fibrin network is stabilized by the cross-linking activity of factor XIIIa.

Figure 4. Platelet activation results in activation of FVII. Complexes between activated FVII (FVIIa) and antithrombin (AT) were measured in plasma obtained from lepirudin-anticoagulated PRP with non-activated and activated platelets. The platelets were activated with a thrombin receptor agonist peptide (TRAP) specific for the protease-activated receptor 1 (PAR-1). Data presented are means ± SEM, n = 12. Significantly higher levels (p < 0.0001, Mann-Whitney rank-sum test) of FVIIa- AT were generated when the platelets were activated.

Figure 5. Possible involvement of platelet-triggered contact activation in the initiation phase of coagulation. Following vessel damage, tissue factor (TF) is exposed to factor VII in the flowing blood. Factor VII binds to TF, which facilitates the rapid activation of FVII and significantly enhances the enzymatic activity of FVIIa. It is possible that FIXa, as well as FXIIa itself (both in fluid phase and on the platelet surface), derived from platelet-induced contact activation, contribute to the activation of FVII to FVIIa in this initiation phase of coagulation.

48 However, since the existence of the contact protease-AT complexes in blood is linked to activated platelets and thrombin generation in vivo, and since increased levels of FXIIa-AT in SLE patients are correlated with previous thrombotic events, these complexes are very interesting from a clinical perspective. In SLE, the increased levels of FXIIa-AT probably re- flect pathological activity that leads to an undesirable activation of platelets and coagulation. Decreased levels of FXIIa-C1INH are also correlated with previous thrombotic events, and even though the basis for this relationship is unknown, we hypothesize that it is associated with endothelial dysfunction, because C1INH regulates contact activation on endothelial cells, and damage to the endothelium is a progressive pathological process in SLE. There is currently a lack of reliable laboratory parameters for evaluating the risk of thrombotic events in SLE, and both FXIIa-serpin complexes are promising biomarkers that in the future might be used for this type of assessment. To continue our investigation of the contact system in SLE patients, we have initiated a new study in collaboration with Department of Clinical Sci- ences Lund, Section of Rheumatology, Skåne University Hospital. In this study, we will consider endothelial functions and use a non-invasive diagnostic cardiac tool, EndoPat, to measure the current endothelial health of SLE patients as well as several plasma parameters reflecting endothelial activation and dysfunction. This analysis will give us more information about the possible relationship between endothelial dysfunction and the contact system, including the altered levels of contact enzyme-C1INH complexes found in SLE patients. We will also consider traditional cardiovascular risk factors, so that we can compare and make a more accurate assessment of the possible use of the FXIIa-serpin complexes as biomarkers of thrombotic risk. Of course, we would also like to perform prospective studies in which we can follow the levels of the complexes over time and see if altered levels are associated with thrombotic outcome. Alterations in the levels of the complexes between activated proteases of the contact system and serpins are likely to occur, not only in SLE patients but also in other individuals with vascular disease and those more prone to thrombotic events. Measuring these complexes in other patient groups, for instance those with different cardiovascular diseases such as myocardial infarctions and atherosclerosis, would be very interesting and is likely to be the subject of future studies. Since platelets are very sensitive cells and one of the first to be affected in several various diseases and pathological conditions, the AT complexes may be altered in other types of patients as well. Systemic sclerosis is a clinically heterogeneous autoimmune disease, which affects the connective tissue of the skin, internal organs, and walls of blood vessels (hence the impact on both endothelium and platelets), which makes it a very interesting disease that we would like to explore with regard to the contact system. The pursuit of clinical applications of contact protease-serpin complexes has just begun, and there are certainly a number

49 of diseases in which they can help to explain the pathological mechanisms involved and hopefully be used as biomarkers to measure disease activity.

50 SUMMARY IN SWEDISH

Populärvetenskaplig sammanfattning

Blod är en livsnödvändig vätska som cirkulerar i vårt kärlsystem och förser kroppens vävnader och organ med syre och näring samtidigt som det även transporterar bort koldioxid och andra avfallsprodukter. Cellerna som finns i blodet tillsammans med blodproteiner är också involverade i kroppens för- svar mot bakterier och andra främmande organismer. För att upprätthålla dessa funktioner är det extremt viktigt att blodet hålls flödande och bevaras inuti våra blodkärl. Hemostas är benämningen på kroppens system för att snabbt täta uppkomna skador på blodkärl och därmed hindra skadlig blodför- lust. Hemostasen omfattas också av dämpande mekanismer som ser till att de snabba reparationssystem som startas när ett blodkärl går sönder inte blir för kraftiga. Blodplättar (trombocyter) är de celler i blodet som är nödvändiga för hemostasen och tillsammans med speciella blodproteiner, så kallade koagulationsfaktorer, samverkar de till att stoppa blödningar från skadade blodkärl. Denna samverkan är mycket komplicerad och man vet inte idag helt säkert hur mekanismerna som leder till koagulation, det vill säga att blodet levrar sig, fungerar. Kontaktaktiveringssystemet består av fyra blodproteiner och är ett kas- kadsystem som kan starta koagulation om blodet kommer i kontakt med en främmande yta, som t.ex. glas i ett provrör, titan i ett implantat eller cellytan på en bakterie. Kontaktaktiveringssystemets roll vid fysiologisk koagulation, som startar när ett blodkärl går sönder, är dock oklar. Det har varit svårt att hos friska individer hitta naturligt förekommande ytor eller komponenter i blodet som kan aktivera detta system. Personer som saknar den första kom- ponenten av detta system, faktor XII, vilket är mycket ovanligt, har inte heller någon ökad blödningsrisk. Detta har bidragit till att man har antagit att kontaktaktiveringssystemet har mindre betydelse för normal hemostas. Nyligen visades det att möss som saknar faktor XII har en felaktig blodproppsbildning. De blodproppar som bildas efter det att man på konstgjord väg orsakat en kärlskada är inte täta eller stabila utan det lossnar hela tiden små delar från blodproppen samtidigt som den byggs upp. I olika djurmo- deller har man också testat att blockera faktor XII och sett att blockeringen kan hindra bildandet av konstgjort framkallade blodproppar utan att ge nå- gon risk för blödning. Faktor XII har därför förmodats ha betydelse för sjuk- domsframkallande blodproppsbildning men inte för normal hemostas. Det

51 finns också motsatta studier som visar att brist eller låga nivåer av faktor XII hos människa ger en ökad risk för olika typer av blodproppar. Dessa resultat förtydligar hur komplicerat kontaktaktiveringssystemet är och man vet egentligen inte riktigt vad det har för betydelse för hemostasen eller för ris- ken att utveckla en blodpropp, och forskarna är oeniga och har presenterat olika motstridiga teorier. I denna avhandling har kontaktaktiveringssystemet studerats för att få bredare kunskap. Det generella målet var att från ett hemostas-perspektiv fokusera på fysiologisk aktivering och funktion samt att undersöka om de produkter som bildas i blodet när systemet aktiveras kan användas i någon klinisk tillämpning. I den första artikeln så frågar vi oss om trombocyter som är centrala i hemostasen kan starta kontaktaktiveringssystemet. Mycket riktigt kan vi visa att när trombocyterna aktiveras så binder de in proteinerna i detta system och gör så att de aktiveras, vi får en så kallad kontaktaktivering. Den här aktiveringen bidrar till bildning av trombin, som är ett nyckelprotein i koagulat- ionen. Vi visar även att om man blockerar faktor XII, som är det centrala proteinet i kontaktaktiveringssystemet, så tar det längre tid för blodet att koagulera och det gör även att det blodkoagel som bildas inte är lika stabilt jämfört med om faktor XII inte har blockerats. I den andra artikeln så tittar vi på skillnader mellan kontaktaktivering som startas av antingen aktiverade trombocyter eller av materialytor. Vi kan visa att regleringen av dessa aktiveringar är olika och det är två olika blodproteiner, så kallade inhibitorer, som reglerar respektive aktivering. När aktiveringen startas utav främmande materialytor så reglerar C1 inhibitor, vilket man har vetat sen tidigare. När trombocyter aktiverar systemet och när kontaktaktivering sker i blod som koagulerar, regleras aktiveringen istället av en annan inhibitor som kallas för antitrombin. Dessa båda inhibitorer binder till de aktiverade proteinerna i kontaktaktiveringssystemet och bildar så kallade komplex, som man kan mäta i blodet och använda som ett mått på hur omfattande aktiveringen varit. Från de två första artiklarna så visste vi att aktiverade trombocyter kan starta kontaktaktiveringssystemet och att det i blod som koagulerar sker en omfattande kontaktaktivering. Vi vet dock inte exakt vilken eller vilka komponenter som gör att aktiveringen startas och varför antitrombin reglerar denna aktivering. I den tredje artikeln försöker vi hitta sådana komponenter och att förstå varför det bildas komplex med antitrombin. Vi kan visa att trombocyter inte är nödvändiga för att man ska få en kontaktaktivering utan det sker även om blod utan dessa celler tillåts koagulera. När blodet koagulerar så bildas som tidigare nämnt trombin och detta protein omvandlar ett annat protein i blodet, fibrinogen, till en ny form som kallas fibrin. Flera sådana fibrinenheter bygger sedan upp ett nätverk som hjälper till att stabili- sera ett blodkoagel. Vi kan visa att detta fibrinnät aktiverar faktor XII och startar en kontaktaktivering. Fibrinet verkar även förstärka antitrombinets

52 reglerande effekt och bidra till att det bildas komplex mellan aktiverade kon- taktaktiveringsproteiner och antitrombin. I den fjärde artikeln undersöker vi kontaktaktiveringssystemet hos patienter med den reumatiska sjukdomen SLE (systemisk lupus erythematosus). Denna sjukdom kan drabba flera av kroppens organ. För vårt arbete är denna sjukdom intressant därför att trombocyterna hos dessa patienter är ofta aktiverade även fast de inte borde vara det. Patienter med SLE har också en mycket större risk att drabbas av olika typer av blodproppar än friska personer. I artikeln jämför vi SLE patienter som inte har haft någon blodpropp med dem som tidigare har drabbats av olika typer av blodproppar som t.ex. hjärtinfarkt, stroke och ventrombos. Vi kan visa att dessa två grupper skiljer sig åt och att de patienter som tidigare har drabbats av någon typ av blodpropp har förändrade nivåer av kontaktaktiveringsprodukter i blodet. Bland annat så har de högre nivåer av komplex mellan aktiverat faktor XII och antitrombin, och dessa sammanfaller även med att det finns aktiverade trombocyter. Detta visar att kontaktaktiveringssystemet har aktiverats och det tyder på att det är en del av SLE sjukdomen. Idag är det svårt att bedöma när en SLE patient riskerar att få en blodpropp och man har inte några riktigt bra markörer i blodet som man kan mäta och använda för att förutspå när det finns en ökad risk för att drabbas. Detta har gjort att många utav de personer som har SLE dör i förtid till följd av att de drabbas av just hjärt- kärlsjukdomar. Vi jämförde förändringen av två olika kontaktaktiveringsprodukter med den markör som idag används för att bedöma när det finns en ökad risk för att bilda blodproppar. Det visade sig att kontaktaktiveringspro- dukterna var mycket bättre än den befintliga markören. Detta gör att vi tror att dessa kontaktaktiveringsprodukter är möjliga markörer som i framtiden kanske kan användas för att kunna göra en bättre bedömning om när det finns en ökad risk för en SLE patient att drabbas av en blodpropp. Vet man detta så kan man behandla patienten med förebyggande mediciner och där- med förhoppningsvis undvika att de drabbas. Sammanfattningsvis så har resultaten från de artiklar som denna avhandling bygger på gett oss en mycket bredare kunskap om hur kontaktaktiveringssystemet fungerar. Vi vet nu att proteinerna i detta system aktiveras och att det startas en kontaktaktivering när trombocyterna av någon anledning blir aktiverade. Det fibrinnät som bildas när blodet koagulerar förstärker sedan denna kontaktaktivering. Effekten av kontaktaktiveringen är att koagel byggs upp fortare och blir mer stabila. Kontaktaktivering utgör därmed en förstärkningsmekanism för koagulationssystemet. Detta är med stor sanno- likhet bra om man får en skada på ett blodkärl eftersom det då hjälper till att snabbare täta skadan så att skadlig blodförlust hindras. Om man däremot har någon sjukdom eller om trombocyterna av någon annan anledning felaktigt aktiveras så kan kontaktaktiveringssystemet troligen bidra till att en blodpropp bildas. Mycket intressant ur ett kliniskt perspektiv är det faktum att det bildas mätbara produkter i blodet när systemet aktiveras och att dessa har ett

53 samband med blodproppar hos SLE-patienter. Kanske kan dessa kontaktaktiveringsprodukter i framtiden användas som markörer för att bedöma när en SLE-patient, eller en patient med någon annan typ av sjukdom, har en ökad risk för att drabbas av en blodpropp. En möjlig förklaring till att man i vissa studier har sett att personer med låga nivåer av faktor XII i blodet har en ökad risk att drabbas av blodproppar är att de koagel som bildas när blodkärl spontant går sönder inte är stabila. De kan stoppa en blödning men samtidigt kanske det lossnar små fragment som följer med blodet och sedan fastnar i mindre blodkärl där det bidrar till att en blodpropp bildas. Slutsatsen är att det är mycket viktigt med en perfekt balans av kontaktaktiveringssystemet och när denna råder så bidrar systemet till att stoppa blödningar men om denna balans störs och man får för mycket eller för lite kontaktaktivering kan det troligen istället bidra till att blodproppar bildas.

54 ACKNOWLEDGMENTS

The work presented in this thesis was carried out at the division of Clinical Immunology, Department of Immunology, Genetics, and Pathology, Faculty of Medicine, Uppsala University. Financial support was granted from the Swedish Research Council, the Swedish Research Council/SSF/Vinnova contract grant number 60761701, the National Institute of Health Grant, the Swedish Rheumatism Association, the Gustav V foundation, and faculty grants from the Linnéaus University, Sweden.

I would like to express my appreciation and thanks to all those who in various ways have helped me with the work that resulted in this thesis. I would especially like to thank:

My supervisors, Bo Nilsson and Kristina Nilsson Ekdahl, for their guidance and support during these years: for trusting in me and for always listen- ing to my ideas and allowing me to test them, and for being inspiring and always enthusiastically discussing results and thoughts.

My co-authors and former colleagues, Graciela Elgue and Javier Sanchez. You are both people with very lovely and colorful personalities, and thanks to you, I was introduced to the field of coagulation and the contact system.

Other co-authors, especially Christian Lood and Anders Bengtsson, for good advice, suggestions, and discussions about the SLE paper.

Lars Faxälv, for pleasant collaboration and laboratory guidance during my visits to Clinical Chemistry in Linköping, and Tomas Lindahl, for allowing me to come and work in your lab. Thanks to you both, and to all other nice people from Linköping, for always very friendly welcomed me to join your group during congresses.

Many thanks to our excellent lab technicians, Lillemor Funke and Susanne Lindblom, for all the laboratory help, support, and for making the lab such a nice place to work in.

55 Jaan Hong and Osama Hamad, and all other present and past collegues of our group, and our collegues in Kalmar, Kerstin Sandholm and Per Nils- son, for many valuable contributions.

Debbie McClellan, for your editorial help, carried out in a professional, rapid, and very friendly way.

The administrative personnel, for all your help with practical issues.

All PhD students, technicians, researchers, students, staff, and others at C5, C11, the satellite lab and GIN lab, for contributing to a dynamic research environment and for making Clinical Immunology to a very rewarding workplace.

Thank you also, all blood donors and all patients who have provided samples. Without you there would certainly not have been any thesis!

My beloved family: my parents Eva and Kenneth and my sisters Jessica and Camilla. Thanks for all your love and encouragement during the years!

Most important of all, my Ole, thanks for always being there and helping me with everything, and for all support and love, not least during the hectic time of writing this thesis, and my son Teodor – despite your young age you are such a great joy-spreader who makes me smile every day. You are the best, and I look forward to our life together. Love you both!

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