Alpha-dystroglycan plays functional roles in platelet aggregation and thrombus growth by Reid Gallant A thesis submitted in conformity with the requirements For the degree of Master of Science Graduate Department of Laboratory Medicine and Pathobiology University of Toronto © Copyright by Reid Gallant 2017 i Alpha-dystroglycan Plays Functional Roles in Platelet Aggregation and Thrombus Growth Reid Gallant Master of Science Department of Laboratory Medicine and Pathobiology University of Toronto 2017 ABSTRACT Fibrinogen (Fg) and von Willebrand factor (VWF) have been considered essential for platelet adhesion and aggregation. However, platelet aggregation still occurs in mice lacking Fg and/or VWF but not β3 integrin, suggesting other, unidentified αIIbβ3 integrin ligand(s) mediate platelet aggregation. Through screening published platelet proteomics data, we identified a candidate, alpha-dystroglycan (α-DG). Using Western blot and flow cytometry, I found α-DG is expressed on platelets. Using aggregometry, I observed that antibodies against α-DG or its N- terminal Laminin-binding site, decreased platelet aggregation induced by various platelet agonists in both platelet-rich plasma and gel-filtered platelets. These antibodies also decreased platelet adhesion/aggregation in perfusion chambers independent of α-DG-Laminin interaction. Using laser injury intravital microscopy and carotid artery thrombosis models, we further found that these anti-α-DG antibodies decreased thrombus growth in vivo. Our results showed that α- DG may form an α-DG-fibronectin complex that binds to αIIbβ3 integrin, contributing to platelet adhesion/aggregation, and thrombosis growth. ii Acknowledgements ―It helps a man immensely to be a bit of a hero-worshipper, and the stories of the lives of the masters of medicine do much to stimulate our ambition and rouse our sympathies‖ – Sir William Osler I will always be grateful to my MSc supervisor, Dr. Heyu Ni, for providing me with an exciting two years of challenge and opportunity. When I look back at my time in this lab it has far exceeded my expectations. You encouraged me not only to think critically, but to always ask thoughtful questions about the work of myself and others. As a mentor you have truly set an example of hard work and dedication to every student that becomes a part of your lab. The way in which I approach challenges and goals in my own life has been changed by this example alone. To my thesis committee members, Dr. Margaret Rand and Dr. Walter Kahr, thank you for your insightful guidance, this has helped to shape my work as a graduate student. I genuinely appreciate the time you have taken to discuss and revise my work. I would especially like to thank all of the members of the Ni Lab who I have had the great good fortune of working with. Dr. Yiming Wang, you have been an incredible friend and mentor since the day I started, your contributions to this project are so great I don’t even know where to begin to thank you. Dr. Guangheng Zhu and Dr. Pingguo Chen, with the numerous questions I have asked you both over the past two years I will always appreciate your incredible patience and knowledge. To Dr. Miguel Neves, I would like to thank you for your very kind encouragement to persevere in science. Your deep understanding of chemistry, among many other things, was always very helpful. Xiaohong Ruby Xu, thank you for the endless help in reviewing my work iii your support has humbled me. To my colleagues that I have the privilege of calling friends Elaine Oswald, June Li, Tyler Stratton, Jade Sullivan, Miao Xu, Rebekah Yu, and Si-Yang Yu thank you for the great memories, even outside the lab. Mark Twain once said that, ―…the really great people make you believe that you too can become great.‖ I have always felt this way about working with such an intelligent group of people. To my family, I certainly would not be where I am today without your love and support. In particularly difficult times you have helped me to move forward. Lastly, I would like to thank Andrea, who has been a great friend to me for many years I will always look up to you. iv Table of Contents Section Title Page Acknowledgements iii Table of Contents v List of Figures and Tables vii Abbreviations ix Chapter 1 – Introduction 1 1.1 Introduction to Hemostasis and Thrombosis 1 1.1.1 Hemostasis 1 1.1.2 Thrombosis 3 1.1.3 The Vessel Wall 5 1.1.4 Platelets 7 1.1.5 Coagulation Cascade and Fibrinolysis 12 1.2 Platelets in Hemostasis and Thrombosis 16 1.2.1 Platelet Versatility 16 1.2.2 Platelets Linking the Protein Wave, First Wave, and Second Wave of Hemostasis 18 1.2.3 Disorders of Platelet Number and Function 19 1.2.4 Platelet Integrin Receptors 24 1.2.5 Agonist-induced Signaling 29 1.2.6 Targeting Platelets in Thrombosis 29 1.2.7 Fibrinogen and Von Willebrand Factor Independent Platelet Aggregation 30 1.3 The Dystroglycan Complex 31 1.4 Rationale and Hypothesis 34 Chapter 2 – Methods 37 2.1 Reagents and Animals 37 2.2 Preparation of Mouse Platelets for In Vitro Models 37 2.2.1 Western Blotting 38 2.2.2 Flow Cytometry 39 2.2.3 Light Transmission Aggregometry 39 2.2.4 Protein Co-Immunoprecipitation 40 2.3 Ex Vivo Perfusion Chamber 40 2.4 In vivo Thrombosis Models 41 2.4.1 Carotid Artery Thrombosis Model 41 2.4.2 Cremaster Arterial Thrombosis Model 42 2.5 Statistical Analysis 42 Chapter 3 – Results 43 43 3.1 α-DG was expressed on the platelet surface 3.2 Anti- α-DG antibodies decreased platelet aggregation 45 3.3 Anti- α-DG antibodies decreased thrombus formation in ex vivo perfusion chambers 48 3.4 Anti- α-DG antibodies decreased thrombus formation in small but not large vessels 51 3.5 Platelet α-DG interacted with integrin αIIbβ3, likely through fibronectin 54 Chapter 4 – Discussion 58 v Chapter 5 – Future Directions 64 References 67 vi List of Figures and Tables List of Figures Figure 1 Platelets play important roles in thrombosis and hemostasis Figure 2 The Coagulation Cascade Figure 3 Structure of the platelet integrin αIIbβ3. Schematic representation of the dystroglycan Figure 4 complex. α-DG is expressed in human and mouse Figure 5 platelets Figure 6 α-DG is present on the surface of mouse and human platelets Figure 7 Anti-α-DG antibody decreases mouse platelet aggregation in PRP and gel-filtered platelets Figure 8 Anti-α-DG antibody decreases human platelet aggregation in PRP and gel-filtered platelets Figure 9 Anti-α-DG antibody decreases thrombus formation in mouse whole blood ex vivo Anti-α-DG antibody did not alter adhesion to Figure 10 laminin ex vivo Figure 11 Anti-α-DG antibody decreases laser-induced thrombus formation in small vessels in vivo Figure 12 Anti-α-DG antibody does not inhibit thrombus formation in large vessels in vivo Figure 14 The Dystroglycan Complex interacts with integrin αIIbβ3 in the absence of VWF and Fg vii List of Tables Table 1 Platelet Integrin-Ligand Interactions Table 2 Antiplatelet Drugs viii List of Abbreviations α-DG Alpha-dystroglycan µL Microliter µM Micrometer Ab Antibody ADAMTS13 A disintegrin and metalloproteinase with a thrombospondin type 1 motif 13 BSA Bovine serum albumin CD40L Cluster of differentiation 40 ligand Co-IP Co-immunoprecipitation DAG Diacylglycerol DGC Dystroglycan complex DIT Drug-induced thrombocytopenia ECM Extracellular matrix Fg Fibrinogen FITC Fluorescein isothiocyanate FNAIT Fetal and neonatal alloimmune thrombocytopenia GP Glycoprotein GPCR G protein-coupled receptor HPA Human platelet antigens IP3 Inositol-1,4,5-triphosphate ITP Immune thrombocytopenia Min Minute mL Milliliter mM Millimolar NO Nitric oxide ix PAR Protease-activated receptor PBS Phosphate-buffered saline pFn Plasma fibronectin PDGF Platelet derived growth factor PRP Platelet-rich plasma PSI Plexin semaphorin integrin RGD Arginine-glycine-aspartic acid SD Standard deviation TGF-β Transforming growth factor beta t-PA Tissue plasminogen activator TTP Thrombotic thrombocytopenia purpura VWF Von Willebrand factor W/V Concentration percent weight by volume x 1. Introduction Blood is essential for the metabolic function of humans and other complex multicellular organisms. It is a medium of transport for a variety of cells and biomolecules that are required for the maintenance of homeostasis. It is composed of both a plasma and cellular component. On average, plasma accounts for 55% of blood volume and contains dissolved gases, ions, proteins and nutrients (glucose, amino acids, and lipids)1. On the other hand the cellular fraction of blood is composed of erythrocytes, leukocytes, and platelets accounts for roughly 45% of the blood volume2. Oxygen, hormones, and biological macromolecules are delivered to tissues through the blood, while carbon dioxide, urea, and other metabolic by-products are simultaneously cleared3, 4. In concert with the respiratory, renal and cardiovascular systems, the blood helps maintain complex concentration gradients of gasses and metabolites between the internal and external environment as well as the extracellular and intracellular space5, 6. Effective gas exchange in the blood requires proper maintenance of both intravascular volume and oxygen carrying capacity. Therefore, significant blood loss can lead to insufficient tissue perfusion, ischemia, tissue damage, and possibly death7, 8. 1.1 Hemostasis and Thrombosis 1.1.1 Hemostasis In vertebrates, the process of hemostasis minimizes blood loss after an injury and initiates wound repair. In 1905 the classic theory of coagulation was introduced and laid the foundation for further hemostasis research. This theory proposed that a ―prothrombin 1 activator‖ performed the conversion of prothrombin to thrombin in the presence of calcium; thrombin subsequently catalyzes the formation of fibrin from fibrinogen9.