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

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Investigating the Role of Von Willebrand Factor in Fibrin Formation and Fibrinolysis INVESTIGATING THE ROLE OF VON WILLEBRAND FACTOR IN FIBRIN FORMATION AND FIBRINOLYSIS by Max A Mendez Lopez A thesis submitted in partial fulfilment of the requirements for the degree of Master of Science in Molecular Medicine Supervisor Thomas McKinnon. BSc, PhD. Imperial College of Science, Technology and Medicine September 2014 1 ACKNOWLEDGEMENTS I want to thank Adriana and both of our families for their patience, advice and unconditional support. I am especially grateful with Dr. McKinnon for his guidance and recommendations, these months have been really enjoyable. To Dr. Nowak, Prof. Laffan and the rest of the members of the lab thank you for let me be part of your group. I would also like to acknowledge the Ministry of Science and Technology of Costa Rica, whose financial support has allowed me to undertake this degree. Pura Vida! 2 ABBREVIATIONS Abs Absorbance ADAMTS13 A disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) aPTT Activated partial thromboplastin time GP Glycoprotein HK High molecular weight kininogen kDa KiloDaltons kDapp Apparent dissociation constant MW Molecular weight PBS Phosphate buffer saline PK Prekallikrein PT Prothrombin time TF Tissue Factor TAFI Thrombin-Activated Fibrinolysis Inhibitor t-PA Tissue-type plasminogen activator u-PA urokinase-type plasminogen activator VWF Von Willebrand factor 3 TABLE OF CONTENTS Title……………………………………..…………………………………………….…… 1 Acknowledgements……………………………………………………………………..… 2 Abbreviations………………………………………………………………………………3 Table of Contents…………………………………………………………………….…… 4 Abstract…………………………………………………………………………………… 6 List of Figures…………………………………………………………………..………… 7 List of Tables……………………………………………………………………………… 7 1. INTRODUCTION………………………..………………………………………..… 8 1.1 Haemostasis Overview……………………………………………………...…… 8 1.2 The Cell-Based Model of Coagulation…………………………………...……... 8 1.3 The Contact System and The Intrinsic Pathway…………………………..…… 10 1.4 The Extrinsic and Common Pathways……………………………………….… 12 1.5 Fibrinolysis…………………………………………………………………….. 16 1.6 The Von Willebrand Factor………………………………………………….… 17 1.6.1 Biology and Biosynthesis………………………………………….… 17 1.6.2 Structure and Functional Domains………………………..……….… 18 1.6.3 Von Willebrand Dynamics Under Flow…………………..……….… 20 1.7 The Role of Platelets…………………………………………………………… 23 2. AIMS………………………………………………………………………………... 25 3. MATERIALS AND METHODS………………………………………..…………. 26 3.1 Binding Assays……………………………………………………………….... 26 3.1.1 FXIIa-VWF Binding Assay………………………………………..… 26 3.1.2 FXIIIa-VWF Binding Assay………………………………………… 26 3.2 Turbidity Assays…………………………………………………………..…… 27 3.2.1 Fibrin Formation………………………………………………...…… 27 3.2.2 Fibrinolysis………………………………………………………..…. 27 3.3 Confocal Microscopy……………………………………………………..……. 28 4 4. RESULTS…………………………………………………………………………… 29 4.1 Turbidity Assays for Fibrin Generation………………………………………..... 29 4.2 The effects of VWF on Turbidity……………………………………………...… 31 4.3 The Role of Thrombin on Von Willebrand Factor-mediated Effects ………..…. 33 4.4 Von Willebrand Factor Enhances Fibrin Polymerisation……………………..… 34 4.5 Role of Von Willebrand Factor-FXIIa Complex and FXIIIa on Turbidity Assays…………. ...………………………………………………………..……35 4.5.1 Von Willebrand Factor Binds to FXIIa………………………………...…. 35 4.5.2 Von Willebrand Factor binds to FXIIIa………………………………...… 36 4.5.3 Effects of Von Willebrand Factor-FXIIa Complex and FXIIIa on Lag Times………………………………………………………….………….. 37 4.6 Turbidity Assays for Fibrinolysis……………………………………………….. 38 4.6.1 Effects of VWF on Fibrinolysis……………………………………….…… 38 5. DISCUSSION……………………………………….………………………..…….. 39 6. CONCLUSION AND FUTURE PERSPECTIVES…………………………….... 43 7. REFERENCES…………………………………………………………………...… 44 5 ABSTRACT Von Willebrand Factor (VWF) is a large multimeric glycoprotein produced by endothelial cells and megakaryocytes that mediates platelet adhesion to damaged vessel walls under conditions of high shear. In humans, its absence produces Von Willebrand Disease, a potentially life threatening condition characterised by a varied degree of bleeding phenotypes. Conversely, high levels of VWF increase the risk of ischemic heart disease and stroke. The majority of thrombotic events share a common clot phenotype characterised by the formation of a compact fibrin mesh-works with impaired lysability and permeability. Whilst the platelet capture activity of VWF and its influence in thrombus growth is well defined, the effects of VWF in fibrin formation have not been described; therefore, the interaction of VWF with fibrin, its influence in fibres formation and role in fibrinolysis must be investigated. For these purposes, optical end-point measurements of turbidity are used in this project to address the effects of VWF by the analysis of the shape of the turbidity curve and comparisons of lag times. In addition, the impact of VWF on fibrin structure is further examined by confocal microscopy imaging of clots formed in the presence and absence of VWF. Data arising from this project provide new information on the effects of VWF acting as a fibrin polymerisation enhancer and fibrinolysis protector, expanding current knowledge in the area of thrombus formation and presenting a novel mechanism by which VWF regulates clot stabilization. The results presented here are particularly important because they explain at least partly the increased risk of thrombosis in patients with high levels of VWF and serve as the basis for further investigations in translational haematology regarding the possible development of drugs that modulated this activity. 6 LIST OF FIGURES 1.1 The Coagulation Cascade……………………………………….…….…………….. 9 1.2 Activation of The Contact System………………………………….…….….…….. 11 1.3 Formation of The Tissue Factor:FVIIa Complex………………………………...... 13 1.4 Fibrinogen Structure and Conversion to Fibrin……………………………………. 15 1.5 Fibrinolysis……………………………………………………………………….... 17 1.6 The Von Willebrand Factor Domains……………………………………...……… 19 1.7 Representation of Blood Flow in a Vessel………………………………………… 22 4.1 Thrombin Determines the Turbidity Curve in a Concentration-Dependent Manner………………………………………………………………………………30 4.2.1 Von Willebrand Factor Effects on Turbidity Curves………………………………. 31 4.2.2 The Von Willebrand Factor Delays Fibres Aggregation and Increases Lag Times…32 4.3. The Effects of Von Willebrand Factor on Turbidity Assays are Independent of Thrombin Concentration………………………………………………………...…. 33 4.4 Von Willebrand Factor Enhances Fibrin Polymerisation………………………….. 34 4.5.1 Factor XIIa Binds to Von Willebrand Factor…………………………………….… 35 4.5.2 Von Willebrand Factor Binds FXIIIa…………………………………………….… 36 4.5.3 VWF-FXIIa Interaction Enhances the Effect on Lag Times……………………….. 37 4.6 Von Willebrand Factor Delays Fibrinolysis……………………………………..… 38 LIST OF TABLES 1.1. Laboratory Diagnosis of Von Willebrand Disease……………………………..……. 21 7 1. INTRODUCTION 1.1 Haemostasis Overview In 1905, Paul Morawitz postulated a theory for the blood coagulation with the four factors so far discovered: fibrinogen, protrombin, thrombokinase and calcium (I-IV). In his model, prothrombin was converted to thrombin in presence of calcium and thrombokinase and fibrinogen was transformed into fibrin by thrombin. His model persisted for almost 40 years until Paul Owren found a patient in Oslo who suffered nosebleeds and menorrhagia with a prolonged thrombin time. He showed that the abnormality was not due to a deficiency in the coagulation factors so far discovered. Following Morawitz’ nomenclature, Owren named the “missing factor” as Factor V and called the disease “parahaemophilia” (Stormorken H, 2003). Owren published a paper with the results of his doctoral thesis in 1947 and a new series of proteins emerged and were named sequentially in the following years. In 1964, two groups independently but simultaneously presented a new theory of blood coagulation called the “Cascade” or “Waterfall” in which a protein (factor) was enzymatically cleaved, sequentially activating the proteins downstream (Macfarlane RG, 1964; Davie EW & Ratnoff OD, 1964). The models suggested the presence of an extrinsic and an intrinsic pathway that converged into a common pathway forming a “Y” with the final goal of cleaving prothrombin to form thrombin (Figure 1.1). 1.2 The Cell-based Model of Coagulation Despite being clinically useful and easy to understand, the Cascade was not initially described as an in vivo model of physiology. The differences between the phenotypes of FXII and FVIII deficiencies and the finding that the Tissue Factor:Factor VIIa complex (TF:FVIIa) was able to activate FIX raised the possibility that the two pathways were acting together and not separately (Osterud B, 1977). In addition, the Cascade was taken for many years as a predictive model of bleeding in patients with prolonged TP or aPTT, which erroneously caused many surgical procedures to be cancelled or suspended in patients with no risks (Hoffman M and Monroe D, 2007). Considering emerging information on the interaction of the two pathways and lack of clarity in some aspects (e.g. the role of FXI), a new model emerged in 2001 that integrated aspects such as the role of activated platelets and the tissue factor bearing cells in the Cell Based Model of Blood Coagulation (Hoffman M, Monroe D, 2001). This model consists of three overlapping 8 steps; the first one or initiation is where the TF-bearing cells interact with injured endothelium; it resembles the extrinsic pathway of the Cascade and is characterised by TF binding to and activating FVII, which subsequently activates FX and
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