Alpha-Dystroglycan Plays Functional Roles in Platelet Aggregation and Thrombus Growth

Alpha-dystroglycan plays functional roles in platelet aggregation and thrombus growth

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

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.

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.

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

Chapter 5 – Future Directions 64 References 67

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

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

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

activator‖ performed the conversion of prothrombin to thrombin in the presence of calcium; thrombin subsequently catalyzes the formation of fibrin from fibrinogen9.

Surprisingly, only five years later Dr. William Duke began to make observations on the relationship between platelet count and bleeding time in patients with thrombocytopenia10. It is now well understood that following a vascular injury, platelets, the coagulation cascade, as well as the vessel wall itself contribute to the hemostatic process. Endothelial damage and exposure of platelets to subendothelial matrix proteins leads to platelet accumulation, known as the primary wave of hemostasis11. Meanwhile, the coagulation cascade results in the formation of a fibrin matrix, or the secondary wave of hemostasis12. Both primary and secondary hemostasis work together to halt bleeding and naturally, a defect in either primary or secondary hemostasis can result in abnormal bleeding13. More recent work has shown that a ―protein wave‖ of hemostasis occurs prior to the classical ―first wave‖ (primary hemostasis)14. During this protein wave phase, plasma fibronectin (pFn), plasma proteins, and possibly even platelet granule proteins deposit onto the injured vessel wall15. In the presence of fibrin, plasma fibronectin supports thrombus formation and platelet adhesion and aggregation16. In addition, spatial and temporal regulation of the hemostatic process is necessary to prevent thrombosis and maintain circulation. For example, it appears pFn has a dual role in thrombosis and hemostasis as pFn has also been shown to reduce aggregation and thrombus formation in the absence of fibrin thereby limiting excessive thrombosis17, 18. Given the consequences of imbalanced hemostasis and wound repair, any factor which has the potential to disrupt any wave of hemostasis—either the protein wave, or classical primary or secondary hemostasis—should be studied in depth.

1.1.2 Thrombosis

While platelets and the coagulation cascade are necessary to maintain a closed circulatory system, platelets can contribute to the pathological occlusion of a vessel. Thrombosis occurs under circumstances that initiate the hemostatic process even when it is not necessarily required, such as the rupture or erosion of an atherosclerotic plaque which, exposes the circulation to a prothrombotic plaque interior19. Vessel occlusion and ischemia result in major diseases such as myocardial infarction, stroke, and deep vein thrombosis. In 2015, the World Health Organization estimated that 17.7 million deaths

(31% of global deaths) were caused by cardiovascular disease, of which 14.1 million deaths were accounted for by coronary artery disease and stroke.

The pathology of atherothrombosis involves the formation and rupture of atherosclerotic lesions in the arterial circulation. Atherothrombosis—occlusive thrombus formation superimposed on pre-existing atherosclerotic plaques within vessels—is the most common cause of myocardial infarction and cardiovascular death, although other causes do exist (e.g. vasospasm, excessive metabolic demand, hypotension, and complications from revascularization procedures)20. Atherosclerotic plaques develop over time as lipids and white blood cells accumulate within the vessel wall21. These lesions consist of an atheromatus ―core‖ consisting of macrophages, recruited smooth muscle cells, lipids, cellular fibronectin, collagen, and other cells/cellular debris that is surrounded by a fibrous cap which separates the inner portion of the lesions from the vessel lumen21, 22.

Over time, these plaques become vulnerable, especially at the peripheral areas of the

fibrous cap and as the fibrous cap erodes it may eventually rupture19. Plaque rupture exposes prothrombotic core proteins like collagen and tissue factor to the circulation, which initiates platelet adhesion, activation, and aggregation as well as the coagulation cascade. A platelet-rich thrombus quickly forms at the site of injury and, depending on the extent of stenosis, can either occlude the vessel or embolize further downstream.

Platelets have been recognized for several decades to play a central role in the final stages of vessel occlusion23. More recent work has shown that platelets also play an interesting role in the initiation and progression of atherosclerotic plaques long before rupture occurs24. Thrombotic diseases should be treated acutely with the intention of re- establishing blood flow by reopening the occluded vessel. This can be achieved either through the use of (i) medical thrombolytic therapy and/or (ii) surgical revascularization procedures. Methods of surgical revascularization include either the use of stents which open up occluded vessels (coronary angioplasty with stenting) or artery grafts to bypass the occluded vessel (such as coronary artery bypass grafting)20. Medical thrombolytic therapy utilizes drugs which cause thrombolysis or clot breakdown. For example, patients can be given recombinant forms of the tissue plasminogen activator (t-PA) which activate plasmin, resulting in the degradation of fibrin clots (see Section 1.1.5). In addition to medical therapies which target fibrin clot degradation, patients with myocardial infarction are also treated with the anti-platelet agents such as P2Y12 antagonists, inhibitors of thromboxane A2 synthesis, phosphodiesterase inhibitors, and αIIbβ3 inhibitors (See section 1.1.4)25. As fibrinolytic therapies will not greatly reduce the platelet recruitment to an arterial thrombus, certain anti-platelet agents have been shown to have additional

benefits (reduce recurrence and improve outcomes) compared to aspirin and fibrinolytic therapy alone 26, 27.

1.1.3 The Vessel Wall

The three major components of hemostasis and thrombosis are the vessel wall, platelets, and coagulation cascade. When an injury to the vessel wall occurs, it must be intricately involved in hemostatic process in order to promote thrombus formation and prevent bleeding. The vessel wall has three layers in large veins and arteries: the tunica intima, tunica media, and tunica adventitia28.

The tunica intima is the closest layer to the circulation and is where the endothelial cells are located. This innermost layer of the vessel wall plays an extremely important role in regulating the balance between pro- and anticoagulant processes. Various stimuli including inflammation, trauma, and pathogens can alter this balance through interactions with platelets or the coagulation cascade29. In addition to acting as a physical barrier between thrombogenic extracellular matrix (ECM) proteins and platelets, the endothelium also produces proteins and molecules which have antiplatelet activity: CD39

(an ADPase), nitric oxide (through platelet cGMP production), and prostacyclin (through platelet cAMP production)30. This natural suppression of platelets by the intact endothelium can also quickly change following injury or activation of endothelial cells, and—in contrast to its antiplatelet activity—the endothelium itself is also capable of producing and exposing factors that contribute to the activation, adhesion and aggregation of platelets as well as the initiation of the coagulation cascade. Among the

prothrombotic factors secreted from activated endothelial cells is von Willebrand factor

(VWF), which is released from storage granules called Weibel-Palade bodies. VWF is predominantly produced by the endothelium, it binds platelet glycoproteins (GPIbα,

αIIbβ3, and αvβ3) and collagen contributing to both platelet adhesion/aggregation31.

VWF also contributes to coagulation by binding coagulation factor VIII and increasing its biological half-life31, 32. VWF is longest immediately following secretion and its length is regulated post secretion by A disintegrin and metalloproteinase with a thrombospondin type 1 motif 13 (ADAMTS13) cleaving the unfolded VWF A2 domain33. Desmopressin or DDVAP—an antidiuretic hormone analogue—is used to pharmacologically increase blood levels of VWF and factor VIII in some bleeding disorders by acting on endothelial

V2R receptors34, 35. Following the activation of the coagulation cascade, the endothelium also contributes to the cessation of coagulation as well as fibrinolysis by expressing thrombomodulin and t-PA36. Therefore, an intact endothelium protects against platelet aggregation, adhesion, and thrombus formation, while a damaged endothelium exposes platelets to prothrombotic factors, initiating clot formation.

Following vessel injury, the first phase of hemostasis is reflex vasoconstriction or

―vascular spasm‖. This primary response to injury is mediated by the middle layer of the vessel wall, the tunica media, between the tunica intima and adventitia. This layer contains smooth muscle cells that help to regulate vascular tone and varies in thickness depending on the vessel type and also produce cellular fibronectin, a protein that supports platelet adhesion22, 37. Reflex vasoconstriction in tunica media vascular smooth muscle

cells is mediated by factors released from the vascular endothelium within the tunica intima.

Lastly, the tunica adventitia is the outermost layer of blood vessels; it contains ECM proteins like collagen which are exposed to platelets after an injury. Collagen plays a major role in the supporting the deposition of pFn, VWF, and other plasma proteins in the early stages following an injury as well as the adhesion and activation of platelets15. The platelet glycoproteins α2β1 and GPVI mediate platelet adhesion and activation following an interaction with collagen38, 39.

1.1.4 Platelets

Platelets, the last major component of the hemostatic system, are small discoid shaped anucleate cell fragments that are approximately 2-4µm in size and decrease in size as they age40. They are derived from the myeloid lineage and shed from the surface of megakaryocytes in hematopoietic tissues like the bone marrow and lungs41.

Megakaryocyte development and proplatelet release are predominantly regulated by the binding of a cytokine produced in the liver and kidneys called thrombopoietin to its receptor c-Mpl42. Other cytokines like interleukin-6 and interleukin-11 also contribute to megakaryocyte maturation and platelet production43. Under normal conditions, platelets circulate in human blood at a concentration of approximately 150-450 x 103/µL44.

Platelets, because of their small size, are radially dispersed towards the vessel wall by larger circulating cells, allowing them to quickly react to vessel wall injuries. If human platelets are not exposed to an activating stimulus like vascular injury, they spend

approximately 7 to 10 days in circulation before being cleared by the reticuloendothelial system and hepatic Ashwell-Morell receptors45.

In the circulation, resting platelet have a discoid shape that is maintained by a spectrin- based cytoskeleton, marginal microtubule coil, and an actin cytoskeleton46. The typical platelet contains three types of granules important to their function: α-granules, δ- granules, and lysosomes47. α-granules are the most abundant secretory granule in platelets, accounting for up to 10% of the platelet volume (50-80 per platelet)48. These granules contain proteins that contribute to a variety of processes (additional integrin

αIIbβ3, cell growth, coagulation, adhesion, angiogenesis, and immune response)49-51.

While the smaller and less abundant δ-granules (3-8 per platelet) contain small molecules like: calcium, polyphosphate, ADP, and serotonin that contribute to platelet activation and hemostasis52, 53. Finally, lysosomes containing digestive glycosidase and protease enzymes are of intermediate size (175-250nm)54. Disorders in which the maturation of α- granules or δ-granules is absent result in coagulopathies referred to as a platelet storage pool defects, highlighting their importance in platelet function47, 55, 56.

Platelets adhere to sites of subendothelial matrix exposure following vascular injury, initiating rapid activation and further platelet aggregation. Platelet adhesion is a key initial step in hemostasis and thrombosis, which is supported by proteins like laminin, fibronectin, fibrinogen/fibrin, and collagen within the subendothelial matrix15, 57, 58.

Adhesion receptors on platelets allow them to overcome the shearing or drag force they experience upon immobilization. The shearing force occurs because of a gradient in fluid

velocity that increases from the wall to center of a vessel and is commonly assessed using shear rate (s-1)59. Under high shear rates, 1000 s-1 and above, platelet tethering in human blood becomes dependent on immobilized VWF multimers60. VWF binds subendothelial matrix collagen via the A3 domain and uncoil, exposing the A1 domain61. The binding between GPIbα and VWF A1 domain has a rapid on-off rate that independently supports platelet translocation but not stable adhesion62. VWF can then mediate platelet rolling through binding the platelet glycoprotein (GP) Ib-IX-V complex initiating inside-out signaling, integrin activation, and eventually stable adhesion60. Stable platelet adhesion to

VWF requires αIIbβ3 binding with arginine-glycine-aspartic acid residues in the C1 domain of VWF62, 63. These interactions are critical in platelet binding and aggregation at the site of injury. Bernard Soulier Syndrome is a rare autosomal recessive disease where patients are deficient in GP Ib-IX-V complex, and present with bleeding disorders which must be controlled with platelet transfusions of functional platelets expressing the GP Ib-

IX-V complex64. In addition to supporting platelet aggregation, VWF also binds factor

VIII prolonging its half-life in circulation65. While under low shear rates (20-200 s-1), many interactions between platelet integrins and their ligands contribute to adhesion66.

Examples of these integrin-ligand interactions are included in Table 1.

Table 1: Platelet Integrin-Ligand Interactions

Platelet Integrin Ligand αIIbβ3 fibrinogen (Fg)/fibrin, VWF, fibronectin αvβ3 vitronectin, osteopontin αLβ2 ICAM-1, ICAM-2, ICAM-3, JAM-A α2β1 collagen α5β1 fibronectin, collagen α6β1 laminin

Platelets in close proximity to an injured vessel wall may become activated by

subendothelial matrix protein interactions such as the binding of GP VI to collagen and

GPIbα with VWF, followed by further activation by soluble agonists (e.g. ADP, thromboxane A2) resulting in changes to both platelet shape and the conformation of integrin αIIbβ366. Several soluble agonists (serotonin, ADP, and epinephrine) and adhesive proteins (Fg, VWF, fibronectin, CD62P, etc.) are contained within platelet dense-granules and α-granules, respectively, and exocytosed during activation. Following inside-out signaling, conformational changes to integrin αIIbβ3 increase the ligand binding affinity for target proteins like Fg, VWF, and other unidentified ligands permitting further activation, adhesion, and aggregation.

Figure 1. Platelets play important roles in hemostasis and thrombosis. Following vessel injury, deposition of plasma and possibly platelet proteins onto the exposed extracellular matrix ―protein wave of hemostasis‖ occurs before the ―first wave of hemostasis‖. Platelet adhesion and aggregation on the damaged vessel wall is considered the ―first wave of hemostasis‖. The GPIb-IX-V complex and its ligand adherent VWF mediate platelet rolling along the vessel wall. Following GPVI-collagen stimulation, stable adhesion requires several integrin-ligand interactions. During platelet adhesion and subsequent activation the αIIbβ3 integrin changes conformation from a low to high affinity ligand-binding state for fibrinogen, VWF, and unidentified ligands. This leads to platelet aggregation and thrombus formation. Platelet activation and phosphatidylserine exposure contribute to cell based thrombin generation, enhancing platelet aggregation and blood coagulation the ―the first and second waves of hemostasis‖11.

1.1.5 Coagulation Cascade and Fibrinolysis

In addition to the vessel wall, the second important component of hemostasis is the coagulation cascade (―secondary hemostasis‖). The coagulation cascade is a series of enzymatic reactions which occur sequentially, resulting in the formation of thrombin, fibrin, and then cross-linked fibrin which stabilizes a platelet plug (Figure 2). One of two initial pathways can initiate the coagulation cascade: the extrinsic or intrinsic pathway. In the event of endothelial injury, the extrinsic pathway is believed to predominantly contribute to the quick, initial generation of thrombin, which is then re-enforced by activation of the intrinsic pathway. Extrinsic pathway activation begins when tissue factor exposed at the site of injury binds to and activates factor VII forming the extrinsic tenase67. This tissue factor-factor VIIa complex activates factor X in the presence of calcium and factor Xa (activated factor X) can then generate enough thrombin to initiate coagulation68. Alternatively, the intrinsic or contact activation pathway requires a negatively charged surface which activates factor XII. Factor XIIa then activates XI which can then activate factor IX67, 69. Factor IX in combination with its cofactor, factor

VIII is referred to as the extrinsic tenase.

Although the intrinsic and extrinsic pathways are activated by different initial triggers, both lead to the conversion of factor X to factor Xa and have a shared common pathway where factor Xa in the presence of calcium and factor Va forms the prothrombinase complex70. The prothrombinase complex converts prothrombin to thrombin, which causes positive feedback to further activate coagulation factors and thrombin itself. In addition to positive feedback, thrombin also cleaves the N-terminus of fibrinogen α and β chains

which forms fibrin monomers by exposing two knob regions (―A‖ and ―B‖) that interact with corresponding pockets or holes (―a‖ and ―b‖) on adjacent fibrin molecules and drives polymerization71. Fibrinogen, the precursor of fibrin, is a 340kDa glycoprotein that contains 6 peptides two copies of proteins: Aα, Bβ, and γ connected by 29 disulfide bonds72. Factor XIIIa then cross-links the fibrin polymer matrix increasing its strength by introducing covalent bonds between certain glutamine and lysine residues71, 73. Recent studies have also shown that pFn may represent another important hemostatic factor because it is covalently incorporated into the fibrin matrix by factor XIIIa, enhancing the mechanical strength and diameter of fibrin fibers15, 73.

Figure 2. The coagulation cascade.

Regulation of fibrin formation is controlled by fibrinolysis as well as several endogenous inhibitors of the coagulation cascade which aid in the degradation of a fibrin matrix and prevent extensive clotting, respectively. In the presence of thrombomodulin—a thrombin cofactor expressed on endothelial surfaces—thrombin is able to activate protein C, an endogenous anticoagulant protein. Activated protein C in the presence of protein S proteolytically inactivates factor VIIIa and factor Va. Therefore, because factors VIIIa and Va are important amplifiers in the coagulation cascade, by activating protein C, thrombin regulates its own negative feedback. Another polypeptide which contributes to coagulation inhibition is the tissue factor pathway inhibitor, which reversibly inhibits factor Xa as well as the factor VIIa-tissue factor complex shortly after it is formed.

Antithrombin is a small protein produced in the liver which inhibits the function of factor

Xa, IXa, and thrombin68. Antithrombin binds thrombin and Xa like a substrate in a 1:1 ratio however it does not easily dissociate, effectively blocking proteolytic activity until it is cleared from the blood74. The anticoagulant drug heparin binds antithrombin, causing a conformational change that significantly increases the binding affinity of antithrombin with thrombin and factor Xa75.

Deficiencies in certain coagulation factors, such as factors VIII and IX, lead to an increased risk of bleeding in the inherited diseases hemophilia A and B, respectively76.

While hemophilia represents an example of a relatively common genetic coagulation factor deficiency, patients may develop deficiencies in other factors. For example, factors

X, IX, VII, II, and proteins C and S are produced in the liver and their production relies on the presence of Vitamin K. Therefore, a patient deficient in vitamin K or with early

liver disease can develop deficiencies in these factors and have clinically prolonged bleeding times. The dependency on vitamin K is also the basis of the anticoagulant drug

Warfarin. Warfarin inhibits the synthesis (carboxylation) of vitamin K-dependent clotting factors by blocking Vitamin K epoxide reductase, making it a useful drug in the treatment of blood clots or stroke prevention in at-risk patients. Naturally, the main risk of Warfarin use—like acquired factor deficiency—is prolonged bleeding. While patients who have congenital or acquired deficiencies in clotting factors are at risk of bleeding, increased factor activity or prolonged half-life can increase the risk of thrombosis. This is exemplified in carriers of factor V Leiden, a genetic variant in factor V that is resistant to degradation by activated protein C.

1.2 Platelet Functions

1.2.1 Platelet Versatility

For many years scientists have wondered whether platelets are required for more than just hemostasis. This idea stems from the fact that most people appear to have many more platelets in circulation that are required for hemostasis. A normal platelet count is considered anywhere from 150-450 x 103/µL, but bleeding risk tends to increase when this number drops below 50 x 103/µL44. In addition to their role in thrombosis and hemostasis, studies are emerging that demonstrate platelets play a role in a wide range of biological processes including: inflammation, immunity, angiogenesis, lymphatic development, and cancer. Platelets contribute to an inflammatory response during activation and adhesion by releasing inflammatory cytokines (CD40L and interleukin 1β) and chemokines (CCL5 and CXCL4)77, 78,79. Following platelet adhesion, the

inflammatory response propagates as nuclear factor κB becomes active in endothelial cells as well80. Platelets have also been recognized to participate in the adaptive immune response through CD40L. This molecule has been shown to enhance CD8+ T Cell response and induce isotype switching in B cells from IgM to IgG production in germinal centers81. Additionally, platelet CD40L can bind endothelial CD40, leading to the increased expression and release of adhesion molecules and cytokines82.

Logically, platelets also contribute to angiogenesis or new vessel growth, which follows soon after vascular injury and hemostasis. Platelets have been shown to contain both pro- and anti-angiogenic factors which, depending on the context, may be may be differentially released83. These platelet proteins that regulate angiogenesis include: vascular endothelial growth factor, platelet derived growth factor, endostatin, platelet factor 4, thrombospondin 1, and basic fibroblast growth factor84. In experimental models, the presence of platelets has been shown to support angiogenesis and to some extent requires adhesion85. As well as blood vessels, lymphatic vessel development also seems to require platelet C lectin-like receptor 2 (CLEC2) to respond to podoplanin on lymphatic endothelial cells thereby activating platelets and maintaining a separation between the blood and lymph86.

In oncology, reports of an association between hemostasis and malignancy date back as far as 1865 when Professor Armand Trousseau reported several cases of thrombophlebitis, a form of vessel inflammation due to a blood clot, as the presenting feature of malignancy83. It has been well documented that for some malignancies,

thrombocytosis—a platelet count above 450 x 103/µL—is a useful prognostic marker because it is associated with poorer outcomes and shortened survival87,88. There seems to be a lot of interaction between platelets and tumors cells that supports growth, angiogenesis, and metastasis. Some studies have shown that platelets stimulate tumor angiogenesis and possibly even protect tumor cells from immune surveillance89. While tumor cells secrete cytokines like IL-6 and thrombopoetin induce thrombopoiesis and enhance platelet-tumor interactions87. As platelets become recognized in more and more biological processes, a greater understanding of platelet physiology may have implications beyond the field of thrombosis and hemostasis.

1.2.2 Platelets Linking the Protein Wave, First Wave, and Second Wave of Hemostasis

Hemostasis is a protective process that prevents hemorrhage following vascular injury.

Maintenance of a closed circulatory system depends on the precise and appropriate regulation of both platelets and the coagulation cascade following vessel injury. Under normal hemostatic conditions platelets adhere, activate, and aggregate at the site of injury

(primary hemostasis), while the coagulation cascade results in the generation of thrombin and conversion of fibrinogen into a cross-linked fibrin network (secondary hemostasis).

There is however, considerable overlap between primary and secondary hemostasis.

Platelets contain the coagulation factors V, VIII, and Fg in α-granules, while factors XIII and XI are located in the cytosol and membrane fraction, respectively90. Furthermore, anionic phospholipids exposed on a population of activated platelets enhance coagulation factor activity and thrombin generation. This is achieved by coagulation factors (intrinsic tenase and prothrombinase complex) binding exposed phosphatidylserine on the external

leaflet of the plasma membrane in a calcium-dependent manner91, 92. The thrombin generated by the coagulation cascade (including cell based thrombin generation) not only feeds back to increase coagulation cascade (See Section 1.1.4), but also feeds back to further activate platelets through protease-activated receptors. Interestingly, the platelet

αIIbβ3 integrin is also required for proper cell-based thrombin generation and the retraction of a clot once it is formed93, 94. Platelets can also promote later wound healing through releasing platelet derived growth factor (PDGF) and transforming growth factor beta (TGF-β) during activation95.

1.2.3 Disorders of Platelet Number and Function

Conditions and diseases that compromise either the function or number of circulating platelets often confer a risk of bleeding, highlighting their importance in hemostasis. For instance, mutations in the kindlin 3 gene have been proposed to cause Leukocyte

Adhesion Deficiency Type III, a disorder that is characterized by poor integrin activation in leukocytes and platelets because of a deficiency of the kindlin 3 protein96. This protein works with talin—a cytoskeletal protein—to bind β integrin subunit cytoplasmic tails and promote their separation from the α integrin subunit as integrins are activated from a low- to high-affinity ligand binding state. As a result of a kindlin 3 deficiency bleeding symptoms are observed due to this qualitative defect in the platelet integrin activation.

Another qualitative platelet defect which may occur is Glanzmann’s Thrombasthenia, which occurs due to a decrease in the number or function of αIIbβ3 which mediates platelet-platelet and platelet-vessel wall interactions. Without the ability for platelet- platelet interaction, Glanzmann’s patients experience bleeding symptoms similar to

Leukocyte Adhesion Deficiency Type III, and patients may require platelet transfusions to

stop bleeds.

In contrast to the above qualitative platelet defects, platelet number is another critical parameter for hemostasis. Thrombocytopenia, is defined as a platelet count below 100

000/μL, while the risk of spontaneous bleeding is not completely predicted by the platelet count it is much greater when it falls below 20 000/ μL97. Thrombocytopenia can be caused by: an increase in platelet consumption, decreased production, certain medications or increased sequestration. Thrombotic thrombocytopenia purpura (TTP), for example, is a thrombotic microangiopathy of low platelet number which can lead to purpura and bleeding (among many other symptoms) caused by ultra large VWF multimers binding platelets and increasing consumption. In TTP, either a genetic deficiency of or autoantibodies against the VWF-cleaving protease ADAMTS13 decreasing its activity. As a result, the size and prothrombotic activity of VWF multimers increases98. These ultra large VWF multimers cause systemic formation of platelet thrombi within the microvasculature, exhausting the available supply of circulating platelets, making patients prone to large bleeds. Treatment of TTP involves plasmapheresis which removes circulating autoantibodies against ADAMTS13 (if this is the cause) and replaces functional ADAMTS13.

Another important bleeding disorder that results from increased platelet clearance is immune thrombocytopenia (ITP). Low platelet counts (<100 000/μL) and mucocutaneous bleeding are characteristic of ITP99, 100. Since 1951 when Harrington showed that plasma from ITP patients was frequently able to cause transient thrombocytopenia in healthy

recipients, autoantibodies have been suspected to mediate platelet clearance and possibly platelet production99. More recently, autoantibodies targeting one or multiple platelet glycoproteins have been recognized to bind platelets leading to clearance via Fcγ receptors on macrophages in the reticuloendothelial system100. Immune thrombocytopenia is treated with corticosteroids, IVIG, thrombopoietin mimetics, and in some cases splenectomy101, 102. The effectiveness of intravenous immunoglobulin in treating ITP is hypothesized to decrease platelet clearance, to some extent, through the activation of Fcγ receptors on phagocytic cells, blocking interactions with opsonized platelets103. Interestingly, Fc-independent clearance has recently been observed in anti-

GPIbα mediated ITP, which could explain why IVIG is not always efficacious45. Anti-

GPIbα antibodies were shown to cause platelet desialylation and subsequent clearance in the liver through Ashwell-Morell receptors45. In addition to antibody mediated platelet clearance mechanisms, others have shown that CD8+ T-cells could mediate platelet desialylation and hepatic clearance as well as direct cytotoxicity to platelets in ITP104,105.

Although, murine models suggest CD8+ T-cells are required for effective corticosteroid therapy and a subset of CD8+ T-regulatory may supress the function of cytotoxic CD8+ T- cells, suggesting their role in ITP is somewhat more complex106.

Abnormal clearance of platelets by the immune system also contributes to the pathology fetal and neonatal alloimmune thrombocytopenia (FNAIT), the most common cause of neonatal thrombocytopenia. In FNAIT, a maternal alloimmune response is mounted against polymorphic paternal platelet antigens expressed on fetal platelets and possibly other cells107. The platelet integrin αIIbβ3 is most frequently associated with FNAIT; it

contains 20 (6 on αIIb and 14 on the β3 subunit) of the 33 recognized Human Platelet

Antigens (HPAs)108. Less frequently FNAIT is also cause by alloantibodies targeting the fetal human leukocyte antigen, which is also expressed on platelets109. Pathogenic maternal antibodies cross the placenta via the neonatal Fc receptor and opsonize fetal platelets, leading to their clearance110, 111. The maternal immune response and decreased platelet count increases the risk of intrauterine growth restriction, bleeding, and miscarriage112. Intracranial hemorrhage is a life-threatening bleeding complication that occurs in approximately 10% fetuses with FNAIT113. Recent laboratory studies have suggested a fundamental difference in the pathology of FNAIT mediated by an anti-β3 versus an anti-GPIbα immune response. Intracranial hemorrhage was observed in anti-β3 but not in an anti-GPIbα murine model of FNAIT although neonates both groups had comparable thrombocytopenia114. Interestingly, this study also showed that miscarriage was significantly more common in an anti-GPIbα murine model of FNAIT. Treatments that prevent the pathogenic maternal antibodies from crossing the placenta, like IVIG and anti-FcRn antibodies, appear to be effective treatments of FNAIT110, 115. It is currently unclear whether there is a difference between the immune response and severity of anti-

αIIb compared to anti- β3 mediated FNAIT. There is a possibility that pathogenic anti-

αIIb antibodies may target fetal hematopoietic stem cells in the fetal liver (and other tissues) as hematopoietic stem cells have been shown to express αIIb116.

Drug-induced thrombocytopenia (DIT) is caused by a therapeutic substance that increases platelet destruction or clearance. The platelet decrease in DIT is mechanistically different than drugs like DNA cross-linking agents that suppress hematopoiesis and platelet

production117. The association of quinine and thrombocytopenia was documented over

150 years ago, since that time many drugs have been found to cause thrombocytopenia independent of bone marrow suppresion117, 118. Usually a drug that causes DIT either increases the affinity of antibodies that are naturally present prior to treatment or induces the production of anti-platelet antibodies that mediate platelet clearance117,119. In most cases, the platelet count will return to normal several days after discontinuation of the suspected medication. An important medication that is well known to induce thrombocytopenia is heparin which was discovered over 100 years ago and is still used today as an anticoagulant for a variety of indications120. Heparin-induced thrombocytopenia has been described in the literature since 1942 and is caused by antibodies targeting the protein complex of heparin and α-granule protein platelet factor

4121. The thrombocytopenia is defined as a 50% or greater decrease in platelet count approximately one week following the initiation of heparin therapy and is more frequently observed in patients treated with unfractioned heparin122. However, only a subset of patients that generate antibodies will develop thrombocytopenia and in some cases thrombosis, which is generally the main concern and the most detrimental complication of heparin-induced thrombocytopenia123. Platelet activation and cell based- thrombin generation is mediated through the activation of the FcγRIIA receptor on platelets and monocytes by pathogenic antibodies124. The resulting thrombosis commonly results in deep vein thrombosis or pulmonary embolism123. The above examples of qualitative and quantitative platelet disorders demonstrate that adequate function and number of circulating platelets is critical in the maintenance of hemostasis.

1.2.4 Platelet Integrin Receptors

Platelet function requires the expression of several integrin receptors. Integrins are heterodimeric type 1 transmembrane proteins that are widely expressed cell adhesion and signal transduction molecules. In total, 24 integrins have been identified which are composed from several different α (18) and β (8) subunits. On average, the extracellular region of α- and β-subunits is about 1000 and 760 amino acids in length, respectively.

These receptors have a ligand binding area within the interface between the α- and β- subunit N-terminals that also depends on the presence of divalent cations. The prototypical integrin binding motif within ligands consists of an arginine-glycine-aspartic acid (RGD) sequence125. Frequently, integrins exist in a low-affinity ligand binding state, however, following agonist stimulation and inside-out signaling they can undergo a conformational change that increases ligand affinity21. The mobility of αIIbβ3 within the plane of the plasma membrane also increases after activation contributing to higher avidity and receptor clustering126, 127. In the case of platelet integrin αIIbβ3, the transition to a high-affinity state permits Fg binding and aggregation. Platelets express six integrins; one β2 (αLβ2), two β3 (αIIbβ3 and αVβ3) and three β1 integrins (α2β1, α5β1, α6β1)128.

The β1 integrins are important in adhesion to ECM proteins, but are unable to support platelet aggregation independently of β3 expression. β3 integrins also contribute to adhesion under low shear and αIIbβ3, even in the resting state, can support adhesion to fibrinogen and fibrin62. Furthermore, qualitative and quantitative defects in β3 integrin expression cause Glanzmann’s Thrombasthenia, a bleeding disorder associated with defective platelet aggregation and clot retraction (See Section 1.2.2). The expression of

the β3 subunit and αIIbβ3 on platelets is therefore essential for proper platelet aggregation.

Integrin αIIbβ3 mediates platelet aggregation through binding fibrinogen and other ligands129-132. It is the most highly expressed transmembrane protein on the surface with approximately 80 000 copies per resting platelet and accounts for approximately 17% of the total membrane protein133, 134. In fact, during activation, as α-granules containing membrane-bound αIIbβ3 fuse with the plasma membrane and release their contents, significantly more αIIbβ3 becomes expressed on the platelet surface135. Both subunits

αIIb and β3 contain large extracellular domains, a single α-helical transmembrane domain, and a short intracellular domain. The αIIb and β3 subunits are encoded by two separate genes on chromosome 17 that noncovalently associate in the endoplasmic reticulum of megakaryocytes and are eventually expressed on the surface of platelets136,

137. Known ligands of activated αIIbβ3 include: Fg, VWF, fibronectin, vitronectin, plasminogen, thrombospondin, prothrombin,and CD40L. Many of these ligands contain the classical RGD sequence, although some ligands, like the fibrinogen gamma chain dodecapeptide (CHHLGGAKQAGDV), only contain a conserved aspartic acid (D) residue138. In addition to playing a role in hemostasis and thrombosis, αIIbβ3 is a common target in many immune platelet disorders. Single nucleotide polymorphisms in the integrin αIIbβ3 account for 20 (6 on αIIb and 14 on the β3 subunit) of the 33 recognized Human Platelet Antigens (HPAs)108. Mismatch of HPAs is the cause of several immune platelet disorders including multitransfusion platelet refractoriness, fetal and neonatal immune thrombocytopenia, and posttransfusion purpura.

The αIIb subunit is approximately 1008 amino acids in length139. This subunit is transcribed as one protein and then proteolytically cleaved in the golgi complex of megakaryocytes into a heavy and a light chain125, 137. The light chain is 22 kDa and contains a 20 amino acid intracellular tail, α-helical transmembrane domain, and a short extracellular segment that is linked to the heavy chain through a disulfide bond. While the heavy chain of αIIb has a molecular weight of 114 kDa, it contains two calf domains, a thigh domain, and a β-propeller domain (Figure 2). An important feature of the αIIb subunit is a bend between the thigh region and first calf domain; this provides flexibility during extension125. The αIIb β-propeller domain is involved in both ligand binding and association to the β3 subunit. This domain contains 7 repeated regions of about sixty amino acids that form the propeller ―blades‖ as well as four divalent cation binding sites that are important for structural stability140.

The other half of the heterodimer, the β3 subunit, is 762 amino acids long and approximately 90 kDa125. There is a highly conserved 48 amino acid cytoplasmic sequence, and an important feature is the ionic clasp or salt bridge that is formed between the cytoplasmic tail of αIIb (R995) and β3 (D732) that help to regulate the conformation of the resting state141. This idea has been further demonstrated in experiments that showed when the cytoplasmic domain was truncated in either the αIIb or the β3 subunit the active conformation is stabilized, suggesting this region has a regulatory role142. The extracellular portion of the β3 subunit consists of the β tail domain, EGF (Epidermal

Growth Factor) domains 1-4, PSI (Plexin Semaphorin Integrin) domain, hybrid domain,

127 and βA domain . The head region of the β3 subunit (the hybrid and βA domains) allows heterodimerization with the αIIb subunit by making contact with the β-propeller domain.

The βA domain contains three divalent cation binding sites, which are required to maintain the correct structure for ligand binding143. The PSI domain, which is located near the N-terminus of β3 is a cysteine rich region that has been shown to have thiol isomerase activity (2 CXXC thioredoxin motifs) which is believed to play a major role in disulfide rearrangement and conformational changes during activation144. Similar to αIIb, a genu also exists in the β3 subunit between EGF 1 and 2 which, in the resting state, creates a bend in the crystal structure145. This region acts as a hinge that allows extension of the globular head of the protein as it transitions to a high affinity conformation.

Figure 3. Structure of the platelet integrin αIIbβ3. The equilibrium between the resting (left) and active (right) conformation is shown. Extracellular domains of the αIIb subunit consist of a β-propeller region, a thigh region and calf domains 1 and 2. The β3 subunit extracellular region consists of the βA domain, hybrid domain, PSI domain, EGF domains 1-4, and the β tail domain146.

1.2.5 Agonist-induced Signaling

Although many agonists lead to platelet activation (collagen, thrombin, TxA2, ADP), they all lead to an increase in calcium concentration within platelets. After G protein-coupled receptor (GPCR) activation a hetertrimeric G-protein complex exchanges GDP for GTP

147 in its Gα subunit resulting in dissociation from the βγ subunits . Then, Gαq or Gαi subunit

(depending on the GPCR) of the heterotrimeric protein complex activates various isoforms of phospholipase C148. Phospholipase C hydrolyzes phosphatidylinositol-4,5- bisphosphate in the plasma membrane forming inositol-1,4,5triphosphate (IP3) and diacylglycerol (DAG) both of which lead to an increase in intracellular calcium. IP3 binds to a receptor on the dense tubular system leading to a release of stored calcium and opening of the Orai1 channel on the membrane. Whereas DAG binds to the plasma membrane receptor transient receptor potential channel 6 permitting the influx of extracellular calcium149. During activation the calcium concentration in platelets increases from 100-500nM into the micromolar range150. This spike in intracellular calcium concentration as well as DAG activate many proteins including protein kinase C isoforms which are necessary for platelet activation as it precedes shape change, integrin activation, and granule secretion.

1.2.6 Targeting Platelets in Thrombosis

Although platelets are very important in hemostasis, platelets can also respond to abnormal lesions on the vessel wall. Thrombosis is often preceded by one or a combination of three changes in the circulation called Virchow’s Triad: endothelial injury, abnormal blood flow (stasis or turbulence), and hypercoagulability151. Typically stasis

precedes venous thrombosis, while endothelial injuries contribute to arterial thrombosis.

Arterial thrombosis or atherothrombosis involves the formation of a platelet-rich thrombus following atherosclerotic plaque rupture (endothelial injury) and exposure of an underlying thrombogenic surface. Following these initial changes, inappropriate platelet activation and aggregation contribute to the formation of a partially or completely occlusive blood clot. The importance of platelets in thrombosis, particularly arterial thrombosis, is further highlighted by the efficacy of anti-platelet agents in the treatment of thrombosis recurrence and progression. Currently several FDA approved antiplatelet agents are used to antagonize PAR-1, P2Y12, and αIIbβ3 in the prevention of morbidity and mortality from cardiovascular disease25 (Table 2).

Table 2: Antiplatelet Drugs

Drug Class Examples Drug Target/Mechanism PAR-1 Antagonists Vorapaxar Inhibit the PAR-1 receptor, blocking thrombin- mediated platelet aggregation P2Y12 Antagonists Clopidogrel Inhibit the P2Y12, the ADP receptor on platelets Prasugrel preventing their activation Ticagrelor αIIbβ3 Inhibitors Abciximab Inhibit αIIbβ3 ligand binding to prevent platelet Eptifibatide aggregation and adhesion Tirofiban COX Inhibitors Asprin Inhibit COX to decrease thromboxane A2 NSAIDs production, thereby decreasing platelet activation

1.2.7 Fibrinogen and Von Willebrand Factor Independent Platelet Aggregation

While Fg and VWF were believed to be required for platelet adhesion and aggregation, it has been well reported that in fact adhesion and aggregation occur in vivo in mice lacking

130 both of these proteins in a ferric chloride (FeCl3) mesenteric artery injury model . While

Fg and VWF may not be absolute requirements for the formation of thrombi, the expression of integrin αIIbβ3 on platelets is essential for platelet aggregation. As a result of this requirement, platelets lacking the β3 integrin subunit are unable to incorporate into wild-type platelet thrombi130. More recent work has shown that the incorrect view that platelet aggregation depends on Fg and VWF may have come from the use of anticoagulants while performing in vitro platelet aggregation assays. Unexpectedly, platelet aggregation has been observed in non-anticoagulated platelet rich plasma (PRP) from mice lacking both Fg and VWF129. Therefore certain anticoagulants appear to make ex vivo platelet aggregation depend on Fg and VWF. In addition to mouse models, residual platelet aggregation has also been reported in the platelet-rich plasma from a patient with severe hypofibronoginemia152. These observations suggest that novel ligands of αIIbβ3 contribute to the formation of a platelet plug and may play a role in thrombotic disease. Identifying novel proteins will lead to a deeper understanding of platelet physiology and possibly the pathophysiology of thrombotic disease.

1.3 Dystroglycan complex

The dystroglyan complex is a membrane-spanning complex that is critical in connecting the ECM to the cytoskeleton in the heart, brain, and nerves in a calcium-dependent manner153. The dystroglycan complex is a heterodimeric transmembrane protein encoded by a single gene DAG1 on chromosome 3154. This protein is transcribed and translated into a single 895 amino acid precursor protein and then cleaved into two non-covalently associated subunits alpha-dystroglycan (α-DG) and beta-dystroglycan (β-DG) which are

expressed on cell membranes (Figure 3)155. The noncovalent interaction occurs between the N-terminal region of β-DG and the C-terminal region of α-DG153.

Figure 4. Schematic representation of the dystroglycan complex. The dystroglycan complex in myocytes interacts with both the basement membrane and the actin cytoskeleton155.

The dystroglycan complex is a key link between the intracellular cytoskeleton and proteins in the extracellular matrix156. β-DG is a 431 amino acid, 43 kDa single pass transmembrane protein, in platelets the cytoplasmic tail of β-DG binds actin filaments, utrophin, as well as the carboxyl terminal of a truncated isoform of dystrophin Dp71Δ110, which itself associates with the actin cytoskeleton (Figure 4)157, 158. The α-DG subunit is a peripheral membrane protein which associates non-covalently with β-DG. Functionally, the Ig-like domain within the N-terminal of alpha subunit acts as a receptor for a number of extracellular matrix proteins including laminin, agrin, neurexin, perlecan and possibly fibronectin156, 159, 160. α-DG is 156 kDa dumbbell shaped protein with globular regions located in both the N- and C-terminal connected by a long central mucin region that undergoes extensive glycosylation161. This post-translational glycosylation is an important modification for dystroglycans in order for them to interact with their binding partners. Post-translational glycosylation of a phosphorylated O-mannosylated threonine in the mucin-like domain of α-DG by like-acetylglucosaminotransferase (LARGE), a golgi protein, provides the binding site on α-DG for ECM proteins [-3-xylose-α1,3- glucuronic acid-β1-]n (LARGE-glycan)162-164. This unique LARGE-glycan on α-DG allows it to bind laminin globular domains ~180 amino acid globular domains LG4 and

LG5 within the alpha chain of laminin.

Clinically, the dystroglycan complex was first studied in a subset of muscular dystrophies related to a hypoglycosylation—and therefore reduced LARGE-glycan—on α-DG, which abolishes this interaction with laminin, agrin, and perlecan165. As a result of a deficiency in α-DG binding with ECM proteins, myocytes and many other cells are unable to form a

strong connection with the ECM. Therefore, the muscle becomes unstable during contraction and over time this leads to a variety of problems including oxidative stress, fibrosis, inflammation, atrophy of myocytes, and muscular weakness166. Aberrant glycosylation or expression of α-DG is associated with a variety of muscular dystrophies including: muscle eye brain disease, Walker Warburg syndrome, Fukuyama type congenital muscular dystrophy, as well as limb girdle muscular dystrophy167. In contrast,

Duchenne muscular dystrophy is an X-linked disease caused by mutations in the dystrophin gene not α-DG that acts as a scaffold between the entire dystroglycan complex and actin cytoskeleton (Figure 4). Although an absence of dystrophin may also lead to a decreased expression of components of the dystroglycan complex like α-DG.

1.4 Rationale and Hypothesis

Although it is widely expressed and involved in cell adhesion, α-DG and the dystroglycan complex have never been studied in the context of platelet function. Interestingly, a recent quantitative proteomic study found the copy number of the dystroglycan complex in platelets was approximately 1900, suggesting it is at least as abundant as the integrin subunit alpha V, which had an estimated copy number of 1400 per platelet168.

Furthermore, the integrin αvβ3 has been shown to play a role in platelet adhesion to osteopontin and vitronectin despite significantly lower expression than αIIbβ3169. It is therefore possible that α-DG plays a role in platelet function. Other research into dystroglycans has revealed that germline knockout of the dystroglycan gene DAG1 in mice results in embryonic lethality at E7.0 days170. Alternatively mice lacking the expression of the glucuronyltransferase LARGE—which adds the laminin binding site on

α-DG without affecting β-DG—are viable and may be a good model for studying the function of platelet α-DG171. However, little is known about the function of the dystroglycan complex outside of myocytes, especially with regards to the hemostatic and thrombotic function of this complex within platelets.

We reasoned that for a protein to support platelet aggregation as a receptor or ligand it needed to either have a sufficient plasma concentration, expression on the platelet surface, or be shed/secreted from the surface of platelets during activation. Through screening published data on the platelet sheddome, we identified a potential candidate, α-

DG172. α-DG is a glycoprotein found in the dystroglycan complex, which bridges the intracellular actin cytoskeleton with extracellular matrix by binding proteins like laminin.

Based on the function of α-DG in myocyte ECM adhesion, we believe that α-DG may serve a similar function during platelet-platelet or platelet-ECM interaction during adhesion and aggregation. The aim of this study is to identify if and how α-DG contributes to platelet adhesion, aggregation, and thrombus formation.

One possibility that α-DG contributes to platelets function is through an interaction with a known ligand. Laminin is the major non-collagenous component of the subendothelial matrix and therefore in an ideal position to interact with platelets following vascular

173 injury. Blood vessels contain 4 laminin isoforms: laminin-411, -421, -511, and -521 .

Platelets have also been found to contain and secrete laminin isoforms 411 and 511, which contribute to adhesion174. The major laminin receptors on platelets include: αvβ3,

58 α2β1, ribosomal protein SA (67kDa laminin receptor), integrin α6β1, GPVI and α-DG .

Developmental studies using embryos lacking the β1 integrin subunit have shown that a compensatory overexpression of the dystroglycan complex in epiblasts, not integrins occurs suggesting a partial overlap in function175.

A recent study has also suggested α-DG facilitates adhesion of extracellular matrix fibronectin following a cleavage of the α-DG N-terminus by proprotein convertase 5/6160.

This observation suggests that there is a possibility for α-DG on platelets to interact with pFn or cellular fibronectin in the extracellular matrix following post translational cleavage of the α-DG N-terminus. This potential interaction could contribute not only to adhesion but also platelet aggregation. pFn functions as a ligand of platelet αIIbβ3 but also has the ability to become incorporated into a fibrin clot and deposit on exposed collagen fibronectin15. Therefore, fibronectin could act as a bridge between α-DG and collagen or αIIbβ3.

Hypothesis

α-DG is expressed on the platelet surface and affects platelet adhesion, activation, aggregation, and thrombus formation through an interaction with αIIbβ3.

2. Materials and Methods

2.1 Reagents and Animals

Fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG Fab, thrombin, and ADP were purchased from Sigma-Aldrich (Oakville, ON). Collagen fibrils for platelet aggregation were purchased from Chrono-Log (Havertown, PA, USA). Thrombin receptor activating peptide 4 (AYPGKF-NH2) was ordered from Tocris Bioscience

(Bristol, UK). Anti-α-DG monoclonal antibody IIH6C4 Millipore (Etobicoke, ON), Anti-

α-DG monoclonal antibody VIA4-1 Millipore (Etobicoke, ON), and Anti-α-DG polyclonal antibody H-300 SantaCruz Biotech (Mississauga, ON) antibodies were used.

DiOC6 fluorescent dye for mitochondrial membranes was purchased from Invitrogen

(Burlington, ON). FITC-conjugated human fibrinogen was also purchased from Thermo-

Fisher Scientific (Burlington, ON). Sepharose protein G and sepharose 2B beads were purchased from Sigma-Aldrich (Oakville, ON). Type I collagen fibrils (equine collagen

Horm; Nycomed, Roskilde, Denmark) and human laminin 111 or laminin 521

(BioLamina; Sundbyberg, Sweden) were also purchased for perfusion experiments. Six to eight week old C57BL/6 mice were purchased from Charles River Laboratories

(Montreal, QC). All mice were housed in the St. Michael’s Hospital Research Vivarium and the experimental procedures were approved by the Animal Care Committee.

2.2 Preparation of Mouse Platelets for In Vitro Models

C57BL/6 Mice (Charles River Laboratories, Montreal, QC) were anesthetized with an intraperitoneal injection (10µl/g body weight) of 10% 2,2,2-tribromoethanol (Sigma-

Aldrich, Oakville, ON) and bled from the retro-orbital plexus using heparin-coated glass capillary tubes. Blood was collected in a tube containing either 3.8% ACD (38mM citric acid, 75 mM trisodium citrate, 100mM detrose) or sodium citrate ratio of 9:1 blood:anticoagulant (BD Biosciences, San Jose, CA). PRP and platelet-poor plasma was obtained by centrifugation of anticoagulated whole blood (10 minutes at 300g and 1050g respectively). Gel-filtered platelets were acquired by gel filtration of PRP with PIPES buffer (PIPES 5mM, NaCl 1.37mM, KCl 4mM, glucose 0.1% W/V, pH 7.0) in a sepharose-2B column (Sigma-Aldrich, Oakville, ON).

2.2.1 Western Blotting

In order to test for the expression of α-DG on platelets, we gel-filtered human and mouse platelet-rich plasma (PRP) with PIPES buffer. Platelet lysates (human and murine) were then prepared by boiling the samples for five minutes in loading dye. Protein concentrations were quantified and 30μg of platelet lysate per well was run on a 10% polyacrylamide gel, separated and transferred to a PVDF membrane. Membranes were blocked in 5% BSA (bovine serum albumin) (Sigma-Aldrich, Oakville, ON) and incubated with a mouse monoclonal antibody against α-DG IIH6C4 (Millipore,

Etobicoke, ON) at a concentration of 1:1000 in PBS with 5% BSA. A goat anti-mouse

IgG secondary antibody conjugated to horseradish peroxidase (SantaCruz Biotech,

Mississauga, ON) and PierceTM ECL chemiluminescent substrate (Thermo Fischer,

Mississauga, ON) were used to develop the membrane and visualize protein.

2.2.2 Flow Cytometry

PRP and gel-filtered platelets from human and mouse blood were collected and diluted in

PIPES with 4% BSA to a concentration of 3 x105 platelets/mL. Then, samples of resting or activated platelets were incubated with a mouse monoclonal anti-α-DG antibody

IIH6C4 (Millipore, Etobicoke, ON) 1:100 dilution for 45 minutes at 4°C to detect surface expression of α-DG. Following this, samples were incubated with a donkey-anti mouse

IgG Fab FITC-labeled secondary antibody (Sigma-Aldrich, Oakville, ON) for 30 minutes in the dark and fixed with 4% paraformaldehyde. Finally the fluorescence activated cell sorted samples were tested and the number of FITC positive cells compared between samples treated with primary and secondary antibody or secondary alone (n=10000 cells per sample) using the LSRFortessa X-20 flow cytometer (BD Biosciences, San Jose, CA) and FlowJo LLC data analysis software (Ashland, OR).

2.2.3 Light Transmission Aggregometry

Platelet aggregometry was performed as previously described176. Platelet concentration in

PRP or gel-filtered platelets was determined with a hemocytometer and adjusted to 3x108 platelets/mL. Platelets were incubated with antibodies against α-DG or control and aggregation was initiated with 5-20 µM ADP (Sigma-Aldrich, Oakville, ON), 0.2 U/mL thrombin (Sigma-Aldrich, Oakville, ON), 250 µM TRAP4 (Tocris Bioscience, Bristol,

UK) or 5-10 µg/mL collagen (Chrono-Log, Havertown, PA). The change in light transmission was recorded for a minimum of 10 minutes using the Chrono-log aggregometer (Chrono-Log, Havertown, PA) with the stir bar rate and temperature set to

1000 rpm at 37℃.

2.2.4 Protein G Agarose Co-Immunoprecipitation

Resting/activated gel-filtered platelet lysates (human and mouse) were incubated with protein G sepharose beads (Sigma-Aldrich, Oakville, ON) for 1.5 hours at 4°C to pre- clear any non-specific IgG present in the lysates. Lysates were then incubated with 6 µg of an antibody against either the β3 integrin subunit or α-DG at 4°C overnight. The following day, beads were pelleted and washed three times to remove unbound proteins.

Protein G agarose beads were then boiled in 30 µl SDS loading dye and dithiothreitol. 30

μg of platelet lysate was added to each well of a 10% polyacrylamide gel, separated and transferred to a PVDF membrane. The membrane was blocked and incubated with either a mouse monoclonal antibody against α-DG IIH6C4 (Millipore, Etobicoke, ON) for the

β3 integrin co-immunoprecipitation (co-IP) or a mouse monoclonal antibody against β3 integrin PSI E1 for the α-DG co-IP. The α-DG co-IP was also stripped and incubated with a monoclonal antibody against fibronectin to test for an interaction between these two proteins160.

2.3 Ex vivo Perfusion Chamber

Rectangular slides with microcapillary channels (height 0.1 x width 1.0 x length 17 mm;

Ibidi North America, Madison, Wisconsin) were coated with 100 µg/mL type I collagen fibrils (equine collagen Horm; Nycomed, Roskilde, Denmark), laminin 111, or laminin

521 (human laminin BioLamina; Sundbyberg, Sweden) for 48 hours at 4°C. Human and murine whole blood was labeled with DiOC6 (1 µM; Sigma-Aldrich) for 20 minutes at

37°C. DiOC6-labeled blood was perfused through the coated microcapillary channels

using an automatic syringe pump (Harvard Apparatus, Holliston, MA, USA) for 3 minutes at various shear rates between 100 seconds–1 and 3000 seconds–1. Platelet adhesion was then recorded over the course of perfusion under a Zeiss Axiovert 135 inverted fluorescent microscope by a computer (IBM IntelliStation Z Pro) using the

Slidebook software (Intelligent Imaging Innovations). After 3 minutes of perfusion, 3- dimensional images of thrombi were captured with a confocal cell imaging system

(CARV; Atto Bioscience, Rockville, MD) attached to the microscope. Thrombi volume was analyzed as well using the Slidebook program (Intelligent Imaging Innovations).

2.4 In vivo Thrombosis Models

In both in vivo models, 6- to 8- week old male C57BL/6 mice were anesthetized and a tracheal tube was inserted to facilitate breathing.

2.4.1 Carotid Artery Thrombosis Model

Anaesthetized mice were administered IV PBS or anti-α-DG (10 µg in 200 µL) in the tail vein. The carotid artery was dissected and attached to a Doppler sensor (TS420 transit- time perivascular flowmeter, Transonic System Inc., Ithaca, New York) as previously decribed177. Then, a strip of Whatman No.1 filter paper immersed in 10% ferric chloride

(Sigma-Aldrich, Oakville, ON) was used to induce injury to the carotid artery at a point upstream of the ultrasound probe. Using the probe, blood flow was monitored and occlusion time measured for both groups (injected with anti-α-DG antibody or PBS control). Occlusion was defined as a decrease of blood flow of 75% or more for at least 3 minutes.

2.4.2 Cremaster Arterial Thrombosis Model

Laser injury of an arteriole in the cremaster muscle was used as previously described178.

The cremaster muscle was prepared under a dissecting microscope and superfused throughout the experiment with preheated bicarbonate-buffered saline. Platelets were labeled by injecting a rat anti–mouse CD41 antibody (Leo.A1; EMFRET Analytics; 0.1

µg/g) into a jugular vein canula and detected with Alexa-660–conjugated goat anti–rat

IgG (Invitrogen, Carlsbad, CA; 2 mg/mL). Multiple independent upstream injuries were performed on the cremaster arteriole of each mouse with the use of an Olympus BX51WI microscope with a pulsed nitrogen dye laser. Mice were infused with an anti-α-DG or

PBS and following injury, the accumulation of fluorescently labeled platelets at the thrombus was captured during a five minute period and analyzed using Slidebook software (Intelligent Imaging Innovations). Stability and size of thrombi was compared between control and treatment groups.

2.5 Statistical Analysis

In vitro, in vivo, and ex vivo data from various experiments between anti-α-DG antibody treated and control was compared and analyzed using GraphPad 6 (GraphPad Software,

Inc. La Jolla, CA). Statistical significance was assessed by Student’s unpaired t-test and chi-square test and represented as mean ± SD. P<0.05 was considered statistically significant.

3. Results

3.1 α-DG is expressed on platelet surfaces

Our first aim was to confirm whether α-DG is expressed in human and murine platelets.

To determine α-DG protein expression, we performed western blots with a monoclonal antibody specific for the binding site of laminin on α-DG. Immunoblots confirmed α-DG is expressed in both human and mouse platelets (Figure 5), and the detection of α-DG on gel-filtered platelets by flow cytometry (Figure 6) confirmed that α-DG is a cell surface protein, and therefore may be capable of functioning as a platelet receptor.

Human platelets Mouse platelets

250 kDa α-DG 170 kDa 150 kDa 156kDa α-DG 130 kDa 156 kDa

Figure 5. α-DG is expressed in human and mouse platelets. Representative western blots of α-DG expression in protein extracts from washed human and mouse platelet lysates.

Secondary A Primary and Secondary

Secondary B

Primary and Secondary Ab

Figure 6. α-DG is present on the surface of mouse and human platelets. Flow cytometry of gel-filtered (A) wild-type mouse and (B) human platelets labeled with either a primary monoclonal antibody against α-DG (IIH6C4) and a FITC labeled secondary (blue curve), or secondary alone (red curve).

3.2 Anti-α-DG antibodies decreased platelet aggregation

To assess whether or not α-DG plays a role in in platelet function, we first tested the role of α-DG in platelet aggregation in vitro. Gel-filtered platelets and PRP were isolated from

C57BL/6 mice and incubated with an anti-α-DG or vehicle control (PBS) for 15 minutes.

In experiments, four different antibodies were used: polyclonal (H-300) and monoclonal

(VIA4-1 and IIH6C4) antibodies against α-DG, and one polyclonal against the N- terminal region of α-DG, specifically (anti-α-DG N-term). Using light transmission aggregometry to assess platelet aggregation revealed that in PRP, ADP-induced platelet aggregation was decreased in platelets treated with the anti-α-DG antibody H-300 (Figure

7A). Similarly, in gel-filtered mouse platelets, thrombin-induced platelet aggregation was decreased in platelets incubated with both anti-α-DG antibodies (H-300 and anti-α-DG

N-term) compared to control (Figure 7B,C). We next treated human platelets and PRP with anti-α-DG antibodies to determine whether α-DG had the same effect in human platelets. ADP-induced PRP aggregation was significantly decreased in platelets incubated with both polyclonal and monoclonal anti-α-DG antibodies, H-300 and VIA4-1

(Figure 8A,C). Additionally, platelet aggregation initiated by collagen was also significantly decreased in human gel-filtered platelets treated with anti-α-DG antibody

(Figure 8B). Together, these interesting observations suggest that the interaction between

α-DG and its ligand(s) plays a role in platelet function by supporting platelet aggregation through either a physical and/or signaling mechanism in platelets.

PRP A ADP

B Gel-filtered Thrombin

C Gel-filtered

Thrombin

Figure 7. Anti-α-DG polyclonal antibody decreases mouse platelet aggregation in PRP and gel-filtered platelets. Platelet aggregation (% light transmission) of (A) Wild-type mouse PRP induced by 5μM ADP with or without anti-α-DG polyclonal antibody (H-300, 2μg/mL, n=3). (B) Gel- filtered mouse platelet aggregation induced by 0.2U/mL thrombin with or without anti-α-DG polyclonal antibody (H-300, 2μg/mL, n=4). (C) Wild-type mouse platelet aggregation induced by 0.2U/mL thrombin with or without anti-α-DG polyclonal antibody against N-terminus (anti-α-DG N term, 2μg/mL, n=3). Data presented as % light transmission ± SEM (*P<0.05). PRP: platelet-rich plasma.

PRP

ADP

B Gel-filtered

Collagen

PRP C

ADP

Figure 8. Anti-α-DG polyclonal and monoclonal antibodies decrease human platelet aggregation in PRP and gel-filtered platelets. Human platelet aggregation (% light transmission) in (A) PRP induced by 5μM ADP with or without anti-α-DG polyclonal antibody (H-300, 1μg/mL, n=3). (B) Human platelet aggregation in gel-filtered platelets induced by 5μg/mL collagen with or without anti-α-DG polyclonal antibody (H-300, 1μg/mL, n=3). (C) Human platelet aggregation in PRP induced by 10μM ADP with or without anti-α-DG monoclonal antibody (VIA4-1, 10μg/mL, n=3). Data presented as % light transmission ± SEM (*P<0.05). PRP: platelet-rich plasma.

3.3 Anti-α-DG antibodies decreased thrombus formation in ex vivo perfusion chambers

We next used an ex vivo model of thrombus formation under high shear rate (1800/s) to evaluate the role of α-DG in murine thrombus growth. DiOC6-labelled whole blood was first incubated with anti-α-DG antibody (H-300), and then perfused through collagen coated microcapillary channels—mimicking in vivo capillaries—and thrombus formation was measured by mean flouresence intensity (MFI). Thrombus formation was significantly decreased in anti-α-DG antibody (H-300) treated platelets, suggesting that under flow, the platelet glycoprotein α-DG may contribute to stable adhesion or the subsequent recruitment of platelets following exposure to collagen (Figure 9). We also investigated the effect of anti-α-DG antibodies on adhesion to laminin, as α-DG is a well- known high affinity receptor for laminin. Compared to control, the polyclonal antibody against α-DG (H-300) did not have an effect on adhesion in laminin-coated chambers at a low or high shear rate (Figure 10).

Control α-dystroglycan

IgG Ab

Figure 9. Anti-α-DG polyclonal antibody decreases thrombus formation ex vivo in mouse whole blood perfusion chambers. (A) Representative images and (B) quantification of DiOC6-labelled platelets treated with or without anti-α-DG polyclonal antibody (H-300, 10µg/mL) perfused through a collagen-coated chamber (shear rate 1800/s). Data presented as MFI of labeled platelets ±SD. MFI: Mean Flourescence Intensity, n=3.

Control α-dystroglycan

Figure 10. Anti- α-DG polyclonal antibody did not alter adhesion to laminin under low shear. (A) Representative images and (B) quantification of DiOC6-labelled platelets treated with or without anti-α-DG polyclonal antibody (H-300, 8µg/mL) perfused through a laminin-coated chamber (shear rate 100/s). Data presented as MFI of labeled platelets ±SD. MFI: Mean Flourescence Intensity.

3.4 Anti- α-DG antibodies decreased thrombus formation in small but not large vessels

Our next aim was to determine whether α-DG may play a role on platelet function in vivo. We first used an in vivo model of injury in a small vessel, the cremaster arterial thrombosis model. Intravital microscopy was performed in the arterioles of the cremaster muscle of mice before and after infusion with either 4 or 8 µg of polyclonal anti-α-DG

(H-300). Injection of polyclonal antibody against α-DG significantly reduced laser- induced thrombus formation in vivo (Figure 11). In antibody-infused mice, thrombi were unstable and more likely to embolize, therefore as well as decreasing thrombus formation this antibody may also alter thrombus stability in vivo. We next used a model of large vessel injury (the carotid artery thrombosis model) to assess the anti-thrombotic effect of anti-α-DG antibodies in large arteries. Intravenous anti-α-DG antibody treatment prolonged the occlusion time in the carotid artery, although statistically significance was not reached (Figure 12).

Figure 11. Anti α-DG polyclonal antibody decreased laser-induced thrombus formation in small vessels in vivo. (A) Representative images one minute after laser injury in mice treated with anti-α-DG polyclonal antibody (H-300, 4μg/mouse or 8μg/mouse), and (B) MFI of labeled platelets for four minutes following injury. (C) Quantification of the area under the MFI curve. Data presented as MFI ± SD and AUC ± SEM (**P< 0.01). MFI: Mean Fluorescence Intensity, n=3 mice.

PBS control A 6 min

10µg anti- α-DG IIH6

9 min

Figure 12. Anti-α-DG antibody did not significantly inhibit thrombus formation in large vessels in vivo. (A) Representative tracing of vessel occlusion time of carotid artery flow after FeCl3 injury with or without anti-α-DG antibody. (B) Quantification occlusion times from several mice (H-300, 10μg/mouse, n=3). Data presented as mean ± SD.

3.5 Platelet α-DG interacts with integrin αIIbβ3, likely through fibronectin

In order to determine if α-DG interacts with integrin αIIbβ3 directly or indirectly, reciprocal co-IP experiments were performed in human gel-filtered platelet lysates. When

α-DG and interacting proteins were pulled down from resting and activated platelet lysates, a band was detected at the approximate weight of the β3 integrin subunit with a monoclonal antibody (PSI E1) (Figure 13A). Conversely, when the β3 integrin subunit was pulled down from resting and activated platelet lysates, a band corresponding to the approximate molecular weight of α-DG was detected with a monoclonal antibody (VIA4)

(Figure 13B). Interestingly, after stripping and re-probing the human α-DG co-IP for fibronectin with a monoclonal antibody, fibronectin was also detected (Figure 13C).

A α-DG pull down B β3 pull down R A R A

~170kDa ~130kDa α-DG

~93kDa β3 subunit

~70kDa

β3 primary (PSI β3 primary (PSI

E1) E1), re-probed for α-dystroglycan

C α-DG pull down

R A

~170kDa

~130kDa

~93kDa

~70kDa

re-probed for human Fn (~235kDa)

Figure 13. The Dystroglycan Complex interacts with integrin αIIbβ3. Representative blots of Co-IP pull down of (A) α-DG and (B) β3 in human platelet lysates Resting or Activated with collagen (20µg/mL collagen). (C) Co-IP pull down of α-DG re-probed for fibronectin. R: Resting, A: Activated n=2.

VWF and Fg are known ligands for β3 integrin. In order to determine whether α-DG could be more efficiently bound to β3 in the absence of competition from VWF and Fg, co-IP experiments were repeated using platelet lysates from Fg and VWF double knockout mice. Although the signal was weaker than the co-IP from human platelet lysates, the result appears to be consistent. When α-DG and interacting proteins were pulled down from resting and activated platelet lysates, β3 was detected (Figure 14A).

Alternatively, when the β3 integrin subunit was pulled down from resting and activated platelet lysates, α-DG was detected (Figure 14B).

α-DG pull down β3 pull down

R A R A

~170kDa ~170kDa

α-DG ~130kDa ~130kDa

~93kDa β3 ~93kDa

~70kDa ~70kDa

β3 primary (PSI α-dystroglycan

primary E1)

Figure 14. The Dystroglycan Complex interacts with integrin αIIbβ3 in the absence of VWF and Fg. Representative blots of co-IP pull down of (A) α-DG and (B) β3 in Fg/VWF double knockout mouse platelet lysates Resting or Activated with collagen (20µg/mL collagen). R: Resting, A: Activated.

4. Discussion

A delicate balance in hemostasis is essential for human physiology: a tip in this balance could lead to life-threatening bleeds or, on the opposite end of the spectrum, hypercoagulable pro-thombotic states may lead to the formation of dangerous clots. At the center of clot formation (platelet aggregation and coagulation) is αIIbβ3, a heterodimeric integrin complex found on platelets, responsible for platelet-platelet interaction, adherence of platelets to damaged vessel walls, and clot retraction93.

Research within the past two decades has revealed that αIIbβ3-mediated platelet aggregation is more complex than previous theories. Notably, loss of both VWF and Fg

(in double VWF and Fg mice) does not prevent platelet aggregation, suggesting that other ligands for the αIIbβ3 integrin exist and may independently mediate platelet adhesion and aggregation. Here, we have shown that α-DG is expressed on the surface of platelets and contributes to platelet adhesion, aggregation, and thrombus formation. This contribution to platelet interaction appears to depend on αIIbβ3 and may be a novel protein that contributes Fg/VWF-independent platelet aggregation. Identification of novel proteins that contribute to platelet aggregation could help to explain some of the variability in platelet function. As well, these novel proteins may provide insight into why current antithrombotic therapies can fail in some patients as well as facilitate the development of new therapies. In contrast to antithrombotic therapeutics, the commonly used anticoagulant heparin has been shown to inhibit α-DG ligand binding representing another possible target of the drug161.

We found that first found that a polyclonal antibody (H-300) against α-DG decreased human and mouse platelet aggregation using light transmission aggregometry. To better determine the region on α-DG mediating this effect we repeated the aggregation experiments with an α-DG N-terminus antibody and observed a similar decrease in platelet aggregation. This suggested that the N-terminus may act as a platelet receptor and we further confirmed this finding with two monoclonal antibodies (IIH6C4 and VIA4-1) against the specific laminin binding epitope in α-DG. Perfusion chambers were used with both laminin and collagen to assess the effect of antibodies blocking α-DG, a well characterized laminin receptor, on platelet adhesion and aggregation in whole blood under flow. Consistent with previous reports, our anti- α-DG antibodies were unable to decrease adhesion to laminin, likely due to the fact that platelet adhesion to laminin is predominantly mediated by integrin α6β158. Interestingly, in collagen-coated perfusion chambers we found that an antibody against α-DG decreased ex vivo thrombus formation at high shear rates (1800s-1). We also found that an anti- α-DG antibody significantly decreased thrombus formation following laser injury in the arterioles of the mouse cremaster muscle using intravital microscopy. These ex vivo and in vivo findings were consistent with our aggregometry data and suggested that anti- α-DG antibodies may in fact decrease platelet-platelet interactions.

There are several possible mechanisms which could explain the observation that antibodies against α-DG decrease platelet adhesion, aggregation, and thrombus formation. One possibility is that α-DG is a direct ligand of a platelet receptor involved in aggregation. The two most likely receptors which α-DG may interact with are the

abundant glycoproteins GPIbα and αIIbβ3. Because antibodies targeting α-DG had no significant effect on ristocetin-induced platelet aggregation, GPIbα is an unlikely candidate. Utilizing co-IP, however, we found a possible binding interaction between

αIIbβ3 and α-DG.

This finding, although interesting, does not confirm a direct interaction between the two proteins and leads to the next possible mechanism by which α-DG alters platelet physiology: α-DG may not interact with platelet GP receptors directly, but bind a ligand which bridges two platelets. α-DG may indirectly interact with a platelet receptor like

GPIbα or αIIbβ3 via a shared ligand that is bi- or multivalent. We decided to further test this with co-IP experiments as it has previously been shown that α-DG is capable of binding fibronectin160. We found that anti-α-DG antibodies were able to pull-down both the β3 integrin subunit and fibronectin. Therefore, fibronectin may represent a possible mechanistic link between platelet α-DG and integrin αIIbβ3 during aggregation. In addition to this, we are also interested in using pFn knockout mice or a sepharose 4B column coupled to gelatin to remove pFn from PRP and examine whether the presence of fibronectin is required for α-DG to contribute to platelet aggregation and adhesion179. If

α-DG does in fact bind pFn, it may play an important role in adhesion, as pFn has been shown to deposit at sites of vascular injury shortly after vascular injury15. This will be further tested by coating perfusion chambers and 96-well plates with pFn and determining whether anti- α-DG antibodies are able to specifically decrease adhesion to pFn.

α-DG antibodies may also alter platelet function through an interaction where a peptide closely associated with α-DG in the dystroglycan complex mediates the binding to an adjacent platelet. In the future, the possibility that our anti- α-DG antibodies are sterically hindering an adjacent protein in the dystroglycan complex could be ruled out by confirming a direct interaction between α-DG and GPIbα or αIIbβ3 in a more specific system than co-IPs, like surface plasmon resonance. Lastly, we cannot yet rule out that a signaling mechanism that contributes to inside-out signaling and integrin activation could also be inhibited by anti-α-DG antibodies, leading to the observed decrease in platelet aggregation. It is however more likely that the effects of anti-α-DG antibodies occur due to a ligand receptor binding event because platelet aggregation was decreased by several agonists.

One limitation of this study is the use of murine in vivo models. Although the in vivo thrombosis models we used provide a good representation of thrombosis in the vessels of mice, mouse models are not directly comparable to human thrombus formation. There are some important differences between murine and human platelets that may affect their function in response to agonists and under shear. Notably, murine platelets circulate at a much higher concentration and are significantly smaller than human platelets. The average platelet count and volume of murine platelets is approximately 1000 x 109/L

(450-1690 x 109/L) and 4.7±0.3 fL respectively180, 181. In contrast to this, the normal platelet concentration in humans is 150-450 x 109/L and the mean platelet volume is 7.2 to 11.7 fL for ninety-five percent of people182. Human platelets are also well known to only express protease activated receptors (PAR) PAR1 and PAR4 while murine platelets

PAR3 and PAR4183. While both PAR1 and PAR4 mediate thrombin signaling in human platelets, mouse platelet PAR3 has only been shown to act as a cofactor for PAR4 signaling184, 185. Lastly, although they are highly homologous the human and mouse α-DG sequences are not completely identical, only 93% of amino acids are shared153. In order to address this inherent challenge of animal models, we performed as many in vitro models as possible to confirm our findings in both mouse and human blood. Another important limitation of this work is the possibility of the antibodies we used having off-target effects. We tried to address this concern in two different ways. First we performed western blots of platelet lysates in order to visualize the specificity of antibodies. Blots had very few additional bands, suggesting the antibodies were relatively specific.

Secondly, we used several antibodies (both monoclonal and polyclonal) to increase the generalizability of our findings. We obtained similar findings with several antibodies; therefore the possibility that our results are due to off-target effects on proteins other than

α-DG is unlikely. It is difficult to determine to what extent α-DG plays in normal hemostasis from mouse models and in vitro experiments, but some human diseases that affect the expression of α-DG and related proteins in the DGC may have shed some light on this.

Here, we study blockade of α-DG in the context of platelet physiology, but another disease process where it is known to be deficient is muscular dystrophy. Interestingly, it has been reported the observation that compared to a control group, Duchenne muscular dystrophy patients typically experience significantly more bleeding during corrective surgical procedures for scoliosis186, 187. Some authors attribute this abnormal bleeding to a

platelet abnormality because there was no difference in coagulation while GPIV (CD36 a collagen receptor) expression and ristocetin-induced platelet aggregation were decreased187. While another group has proposed that Duchenne muscular dystrophy patients have impaired collagen signaling due to increased Gs signaling which generates cAMP, an inhibitory molecule to platelet function188. In contrast to the idea that

Duchenne muscular dystrophy may cause a minor platelet abnormality, it has also been proposed that decreased vessel reactivity (i.e. reflexive vasoconstriction) is the main hemostatic deficiency189. Our findings may provide another possible explanation as we have shown that α-DG, a protein that is deficient in all the cells of patients with muscular dystrophy, may contribute to platelet aggregation and possibly hemostasis. One interesting study has shown that platelets from Dmdmdx-3Cv mice, a line used to model muscular dystrophy have decreased adhesion to collagen157. Future research could certainly try to further elucidate whether anti- α-DG antibodies have any effect on platelets from patients that have a deficiency of α-DG or lack this protein entirely.

5. Future Directions

In the near future we would like to confirm the co-IP result indicating a possible interaction between α-DG and αIIbβ3 with surface plasmon resonance, currently we are working on covalently attaching both extracellular subunits of integrin αIIbβ3 to gold surfaces using carbodiimide cross linking chemistry190. We could then use these surfaces to test whether α-DG directly interacts with αIIbβ3 or Fn. If we are able to successfully show that α-DG is a ligand of integrin αIIbβ3, we would also like to test whether the anti-

α-DG antibodies play a role in Fg/VWF independent platelet aggregation using double knockout mice. It is possible that in the absence of Fg and VWF α-DG contributes more significantly to platelet aggregation than in wild type mice. Following these animal studies, it would also be useful to establish the role of α-DG in human blood samples from patients with both afibrinogenemia and von Willebrand Disease, because their platelets might also depend on α-DG to a greater extent during adhesion, aggregation, and thrombus formation. Evaluating the effect of blocking or removing α-DG on lamellipodia and filopodia formation during platelet shape change and spreading as the discoid structure disappears in response to various agonists and ECM proteins would also be important. It is possible that α-DG may be involved in shape change because α-DG and the dystroglycan complex act as an important link between the actin cytoskeleton and

ECM. Following ligand binding the dystroglycan complex may contribute to signaling that contributes to the reorganization of cytoskeletal proteins in platelets. Especially since the number of actin filaments within platelets significantly increases during shape change191. α-DG has also been shown to play a role in the synaptic cytoskeleton and

clustering of acetylcholine receptors192. Based on this previous work in neurons, it would also be very interesting to investigate whether α-DG is involved in avidity modulation or integrin clustering in platelets following activation.

An evaluation of whether α-DG might be a useful protein target in thrombosis would require a good understanding of its role in hemostasis. This is important to assess because current antithrombotic drugs often have a risk of bleeding since they inhibit hemostasis as well. In order to determine this, I would like to perform in vitro and in vivo experiments to gain a better understanding of the role of α-DG and antibodies targeting this protein in hemostasis. Thromboelastography experiments would be performed in human and mouse blood to determine whether antibodies against α-DG effect the kinetics of clot formation as well as the clot strength. Tail vein bleeding times could be measured in mice following intraparitoneal or intravenous injection with monoclonal and polyclonal antibodies against α-DG. The ability of anti-α-DG antibodies to decrease thrombus formation and platelet aggregation would suggest they may also alter hemostasis and requires further investigation. Although it is possible that some proteins could be contribute predominantly to thrombosis.

Platelets are known to contribute to a variety of other biological processes including atherosclerotic development24. However αIIbβ3 may not be the only platelet receptor that contributes to this process, as deficiency does not confer protection193. It would be interesting to test whether murine platelet deficient in α-DG confer any protection from the development of atherosclerosis. This could be studied using mice specifically

deficient in platelet α-DG or transplantation of bone marrow from α-DG deficient mice into LDLR knockout mice which are used to study atherosclerosis194.

Studying the aggregation of platelets from patients with dystroglycanopathies or muscular dystrophy would also help to further elucidate the function of α-DG in human platelets. Several important experiments could be performed using these samples. First, it would be interesting to see whether platelet aggregation from humans which lack functional α-DG could also be decreased by monoclonal and polyclonal antibodies against this protein. Studies have also reported collagen adhesion deficiencies in both platelets from human Duchenne muscular dystrophy patients as well as a mouse model of muscular dystrophy157, 186. Platelets from Duchenne muscular dystrophy patients may also decreased adhesion to other ECM proteins like fibronectin and laminin since both are reported ligands of α-DG160, 165.

References

1. Michelsen TM, Holme AM, Henriksen T. Transplacental nutrient transfer in the human in vivo determined by 4 vessel sampling. Placenta. 2017

2. Wu X, Zhao M, Pan B, Zhang J, Peng M, Wang L, et al. Complete blood count reference intervals for healthy han chinese adults. PLoS One. 2015;10:e0119669

3. Toffaletti JG, Rackley CR. Monitoring oxygen status. Adv Clin Chem. 2016;77:103-124

4. Withers PC. Urea: Diverse functions of a 'waste' product. Clin Exp Pharmacol Physiol. 1998;25:722-727

5. Marano M, D'Amato A, Guiotto G, Schiraldi F. [central venous blood gas analysis]. G Ital Nefrol. 2015;32

6. Lau WL, Vaziri ND. Urea, a true uremic toxin: The empire strikes back. Clin Sci (Lond). 2017;131:3-12

7. Pacagnella RC, Souza JP, Durocher J, Perel P, Blum J, Winikoff B, et al. A systematic review of the relationship between blood loss and clinical signs. PLoS One. 2013;8:e57594

8. Veith NT, Histing T, Menger MD, Pohlemann T, Tschernig T. Helping prometheus: Liver protection in acute hemorrhagic shock. Ann Transl Med. 2017;5:206

9. Czerchawski L. [on the 80th anniversary of the classical theory of blood coagulation]. Postepy Hig Med Dosw. 1986;40:515-554

10. Duke WW. The relation of blood platelets to hemorrhagic disease. By w.W. Duke. JAMA. 1983;250:1201-1209

11. Xu XR, Gallant RC, Ni H. Platelets, immune-mediated thrombocytopenias, and fetal hemorrhage. Thromb Res. 2016;141 Suppl 2:S76-79

12. Brandt JT. Current concepts of coagulation. Clin Obstet Gynecol. 1985;28:3-14

13. Lippi G, Franchini M, Favaloro EJ. Diagnostics of inherited bleeding disorders of secondary hemostasis: An easy guide for routine clinical laboratories. Semin Thromb Hemost. 2016;42:471- 477

14. Wang Y, Ni H. Fibronectin maintains the balance between hemostasis and thrombosis. Cell Mol Life Sci. 2016;73:3265-3277

15. Wang Y, Reheman A, Spring CM, Kalantari J, Marshall AH, Wolberg AS, et al. Plasma fibronectin supports hemostasis and regulates thrombosis. J Clin Invest. 2014;124:4281-4293

16. Wang Y, Ni H. Fibronectin: Extra domain brings extra risk? Blood. 2015;125:3043-3044

17. Huynh K, Gyenes M, Hollenberg CP, Nguyen TH, Van Vo T, Stoldt VR. Fibronectin unfolded by adherent but not suspended platelets: An in vitro explanation for its dual role in haemostasis. Thromb Res. 2015;136:803-812

18. Reheman A, Yang H, Zhu G, Jin W, He F, Spring CM, et al. Plasma fibronectin depletion enhances platelet aggregation and thrombus formation in mice lacking fibrinogen and von willebrand factor.

Blood. 2009;113:1809-1817

19. Falk E. Why do plaques rupture? Circulation. 1992;86:III30-42

20. Anderson JL, Morrow DA. Acute myocardial infarction. N Engl J Med. 2017;376:2053-2064

21. Abela GS. Cholesterol crystals piercing the arterial plaque and intima trigger local and systemic inflammation. J Clin Lipidol. 2010;4:156-164

22. Glukhova MA, Frid MG, Shekhonin BV, Vasilevskaya TD, Grunwald J, Saginati M, et al. Expression of extra domain a fibronectin sequence in vascular smooth muscle cells is phenotype dependent. J Cell Biol. 1989;109:357-366

23. Jorgensen L, Rowsell HC, Hovig T, Mustard JF. Resolution and organization of platelet-rich mural thrombi in carotid arteries of swine. Am J Pathol. 1967;51:681-719

24. Huo Y, Schober A, Forlow SB, Smith DF, Hyman MC, Jung S, et al. Circulating activated platelets exacerbate atherosclerosis in mice deficient in apolipoprotein e. Nat Med. 2003;9:61-67

25. Xu XR, Carrim N, Neves MA, McKeown T, Stratton TW, Coelho RM, et al. Platelets and platelet adhesion molecules: Novel mechanisms of thrombosis and anti-thrombotic therapies. Thromb J. 2016;14:29

26. Sabatine MS, Cannon CP, Gibson CM, Lopez-Sendon JL, Montalescot G, Theroux P, et al. Addition of clopidogrel to aspirin and fibrinolytic therapy for myocardial infarction with st- segment elevation. N Engl J Med. 2005;352:1179-1189

27. Investigators E. Use of a monoclonal antibody directed against the platelet glycoprotein iib/iiia receptor in high-risk coronary angioplasty. N Engl J Med. 1994;330:956-961

28. Seidel CL. Cellular heterogeneity of the vascular tunica media. Implications for vessel wall repair. Arterioscler Thromb Vasc Biol. 1997;17:1868-1871

29. Kazmi RS, Boyce S, Lwaleed BA. Homeostasis of hemostasis: The role of endothelium. Semin Thromb Hemost. 2015;41:549-555

30. Wang GR, Zhu Y, Halushka PV, Lincoln TM, Mendelsohn ME. Mechanism of platelet inhibition by nitric oxide: In vivo phosphorylation of thromboxane receptor by cyclic gmp-dependent protein kinase. Proc Natl Acad Sci U S A. 1998;95:4888-4893

31. Wagner DD. Cell biology of von willebrand factor. Annu Rev Cell Biol. 1990;6:217-246

32. Michiels JJ, Gadisseur A, Vangenegten I, Schroyens W, Berneman Z. Recessive von willebrand disease type 2 normandy: Variable expression of mild hemophilia and vwd type 1. Acta Haematol. 2009;121:119-127

33. Springer TA. Von willebrand factor, jedi knight of the bloodstream. Blood. 2014;124:1412-1425

34. Kaufmann JE, Vischer UM. Cellular mechanisms of the hemostatic effects of desmopressin (ddavp). J Thromb Haemost. 2003;1:682-689

35. Stoof SCM, Schutte LM, Leebeek FWG, Cnossen MH, Kruip M. Desmopressin in haemophilia: The need for a standardised clinical response and individualised test regimen. Haemophilia. 2017

36. Huber D, Cramer EM, Kaufmann JE, Meda P, Masse JM, Kruithof EK, et al. Tissue-type

plasminogen activator (t-pa) is stored in weibel-palade bodies in human endothelial cells both in vitro and in vivo. Blood. 2002;99:3637-3645

37. Pugsley MK, Tabrizchi R. The vascular system. An overview of structure and function. J Pharmacol Toxicol Methods. 2000;44:333-340

38. Moroi M, Jung SM, Okuma M, Shinmyozu K. A patient with platelets deficient in glycoprotein vi that lack both collagen-induced aggregation and adhesion. J Clin Invest. 1989;84:1440-1445

39. Holtkotter O, Nieswandt B, Smyth N, Muller W, Hafner M, Schulte V, et al. Integrin alpha 2- deficient mice develop normally, are fertile, but display partially defective platelet interaction with collagen. J Biol Chem. 2002;277:10789-10794

40. Holinstat M. Normal platelet function. Cancer Metastasis Rev. 2017

41. Lefrancais E, Ortiz-Munoz G, Caudrillier A, Mallavia B, Liu F, Sayah DM, et al. The lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors. Nature. 2017;544:105-109

42. Kaushansky K. Lineage-specific hematopoietic growth factors. N Engl J Med. 2006;354:2034- 2045

43. Senchenkova EY, Komoto S, Russell J, Almeida-Paula LD, Yan LS, Zhang S, et al. Interleukin-6 mediates the platelet abnormalities and thrombogenesis associated with experimental colitis. Am J Pathol. 2013;183:173-181

44. Thon JN, Macleod H, Begonja AJ, Zhu J, Lee KC, Mogilner A, et al. Microtubule and cortical forces determine platelet size during vascular platelet production. Nat Commun. 2012;3:852

45. Li J, van der Wal DE, Zhu G, Xu M, Yougbare I, Ma L, et al. Desialylation is a mechanism of fc- independent platelet clearance and a therapeutic target in immune thrombocytopenia. Nat Commun. 2015;6:7737

46. Thon JN, Italiano JE. Platelets: Production, morphology and ultrastructure. Handb Exp Pharmacol. 2012:3-22

47. Chen CH, Lo RW, Urban D, Pluthero FG, Kahr WH. Alpha-granule biogenesis: From disease to discovery. Platelets. 2017;28:147-154

48. Blair P, Flaumenhaft R. Platelet alpha-granules: Basic biology and clinical correlates. Blood Rev. 2009;23:177-189

49. Coppinger JA, Cagney G, Toomey S, Kislinger T, Belton O, McRedmond JP, et al. Characterization of the proteins released from activated platelets leads to localization of novel platelet proteins in human atherosclerotic lesions. Blood. 2004;103:2096-2104

50. Maynard DM, Heijnen HF, Horne MK, White JG, Gahl WA. Proteomic analysis of platelet alpha- granules using mass spectrometry. J Thromb Haemost. 2007;5:1945-1955

51. Suzuki H, Nakamura S, Itoh Y, Tanaka T, Yamazaki H, Tanoue K. Immunocytochemical evidence for the translocation of alpha-granule membrane glycoprotein iib/iiia (integrin alpha iib beta 3) of human platelets to the surface membrane during the release reaction. Histochemistry. 1992;97:381-388

52. Ruiz FA, Lea CR, Oldfield E, Docampo R. Human platelet dense granules contain polyphosphate and are similar to acidocalcisomes of bacteria and unicellular eukaryotes. J Biol Chem.

2004;279:44250-44257

53. King SM, Reed GL. Development of platelet secretory granules. Semin Cell Dev Biol. 2002;13:293-302

54. Rendu F, Brohard-Bohn B. The platelet release reaction: Granules' constituents, secretion and functions. Platelets. 2001;12:261-273

55. Raccuglia G. Gray platelet syndrome. A variety of qualitative platelet disorder. Am J Med. 1971;51:818-828

56. McNicol A, Israels SJ. Platelet dense granules: Structure, function and implications for haemostasis. Thromb Res. 1999;95:1-18

57. Sonnenberg A, Modderman PW, Hogervorst F. Laminin receptor on platelets is the integrin vla-6. Nature. 1988;336:487-489

58. Schaff M, Tang C, Maurer E, Bourdon C, Receveur N, Eckly A, et al. Integrin alpha6beta1 is the main receptor for vascular laminins and plays a role in platelet adhesion, activation, and arterial thrombosis. Circulation. 2013;128:541-552

59. Ruggeri ZM. Platelets in atherothrombosis. Nat Med. 2002;8:1227-1234

60. Ruggeri ZM. Old concepts and new developments in the study of platelet aggregation. J Clin Invest. 2000;105:699-701

61. Springer TA. Biology and physics of von willebrand factor concatamers. J Thromb Haemost. 2011;9 Suppl 1:130-143

62. Savage B, Saldivar E, Ruggeri ZM. Initiation of platelet adhesion by arrest onto fibrinogen or translocation on von willebrand factor. Cell. 1996;84:289-297

63. Ruggeri ZM, De Marco L, Gatti L, Bader R, Montgomery RR. Platelets have more than one binding site for von willebrand factor. J Clin Invest. 1983;72:1-12

64. Lopez JA, Andrews RK, Afshar-Kharghan V, Berndt MC. Bernard-soulier syndrome. Blood. 1998;91:4397-4418

65. Swystun LL, Georgescu I, Mewburn J, Deforest M, Nesbitt K, Hebert K, et al. Abnormal von willebrand factor secretion, factor viii stabilization and thrombus dynamics in type 2n von willebrand disease mice. J Thromb Haemost. 2017

66. Davi G, Patrono C. Platelet activation and atherothrombosis. N Engl J Med. 2007;357:2482-2494

67. Palta S, Saroa R, Palta A. Overview of the coagulation system. Indian J Anaesth. 2014;58:515-523

68. Triplett DA. Coagulation and bleeding disorders: Review and update. Clin Chem. 2000;46:1260- 1269

69. Gailani D, Renne T. Intrinsic pathway of coagulation and arterial thrombosis. Arterioscler Thromb Vasc Biol. 2007;27:2507-2513

70. Mueck W, Stampfuss J, Kubitza D, Becka M. Clinical pharmacokinetic and pharmacodynamic profile of rivaroxaban. Clin Pharmacokinet. 2014;53:1-16

71. Doolittle RF. The conversion of fibrinogen to fibrin: A brief history of some key events. Matrix Biol. 2017;60-61:5-7

72. Weisel JW, Litvinov RI. Mechanisms of fibrin polymerization and clinical implications. Blood. 2013;121:1712-1719

73. Matsuka YV, Migliorini MM, Ingham KC. Cross-linking of fibronectin to c-terminal fragments of the fibrinogen alpha-chain by factor xiiia. J Protein Chem. 1997;16:739-745

74. Rosenberg RD, Damus PS. The purification and mechanism of action of human antithrombin- heparin cofactor. J Biol Chem. 1973;248:6490-6505

75. Harter K, Levine M, Henderson SO. Anticoagulation drug therapy: A review. West J Emerg Med. 2015;16:11-17

76. Bertamino M, Riccardi F, Banov L, Svahn J, Molinari AC. Hemophilia care in the pediatric age. J Clin Med. 2017;6

77. Henn V, Slupsky JR, Grafe M, Anagnostopoulos I, Forster R, Muller-Berghaus G, et al. Cd40 ligand on activated platelets triggers an inflammatory reaction of endothelial cells. Nature. 1998;391:591-594

78. Lindemann S, Tolley ND, Dixon DA, McIntyre TM, Prescott SM, Zimmerman GA, et al. Activated platelets mediate inflammatory signaling by regulated interleukin 1beta synthesis. J Cell Biol. 2001;154:485-490

79. von Hundelshausen P, Weber KS, Huo Y, Proudfoot AE, Nelson PJ, Ley K, et al. Rantes deposition by platelets triggers monocyte arrest on inflamed and atherosclerotic endothelium. Circulation. 2001;103:1772-1777

80. Gawaz M, Brand K, Dickfeld T, Pogatsa-Murray G, Page S, Bogner C, et al. Platelets induce alterations of chemotactic and adhesive properties of endothelial cells mediated through an interleukin-1-dependent mechanism. Implications for atherogenesis. Atherosclerosis. 2000;148:75- 85

81. Elzey BD, Tian J, Jensen RJ, Swanson AK, Lees JR, Lentz SR, et al. Platelet-mediated modulation of adaptive immunity. A communication link between innate and adaptive immune compartments. Immunity. 2003;19:9-19

82. Michel NA, Zirlik A, Wolf D. Cd40l and its receptors in atherothrombosis-an update. Front Cardiovasc Med. 2017;4:40

83. Battinelli EM, Markens BA, Italiano JE, Jr. Release of angiogenesis regulatory proteins from platelet alpha granules: Modulation of physiologic and pathologic angiogenesis. Blood. 2011;118:1359-1369

84. Peterson JE, Zurakowski D, Italiano JE, Jr., Michel LV, Fox L, Klement GL, et al. Normal ranges of angiogenesis regulatory proteins in human platelets. Am J Hematol. 2010;85:487-493

85. Kisucka J, Butterfield CE, Duda DG, Eichenberger SC, Saffaripour S, Ware J, et al. Platelets and platelet adhesion support angiogenesis while preventing excessive hemorrhage. Proc Natl Acad Sci U S A. 2006;103:855-860

86. Uhrin P, Zaujec J, Breuss JM, Olcaydu D, Chrenek P, Stockinger H, et al. Novel function for blood platelets and podoplanin in developmental separation of blood and lymphatic circulation. Blood.

2010;115:3997-4005

87. Stone RL, Nick AM, McNeish IA, Balkwill F, Han HD, Bottsford-Miller J, et al. Paraneoplastic thrombocytosis in ovarian cancer. N Engl J Med. 2012;366:610-618

88. Koeppen KM, Schmidt S, Paulisch R, Schneider D, Gerhartz H. [thrombocytosis as a paraneoplastic syndrome]. Verh Dtsch Ges Inn Med. 1975;81:1161-1163

89. Stegner D, Dutting S, Nieswandt B. Mechanistic explanation for platelet contribution to cancer metastasis. Thromb Res. 2014;133 Suppl 2:S149-157

90. Walsh PN. Platelets and coagulation proteins. Fed Proc. 1981;40:2086-2091

91. Rand ML, Wang H, Bang KW, Packham MA, Freedman J. Rapid clearance of procoagulant platelet-derived microparticles from the circulation of rabbits. J Thromb Haemost. 2006;4:1621- 1623

92. Reddy EC, Wang H, Bang KWA, Packham MA, Rand ML. Young steady-state rabbit platelets do not have an enhanced capacity to expose procoagulant phosphatidylserine. Platelets. 2017:1-7

93. Hodivala-Dilke KM, McHugh KP, Tsakiris DA, Rayburn H, Crowley D, Ullman-Cullere M, et al. Beta3-integrin-deficient mice are a model for glanzmann thrombasthenia showing placental defects and reduced survival. J Clin Invest. 1999;103:229-238

94. Wang H, Bang KW, Blanchette VS, Nurden AT, Rand ML. Phosphatidylserine exposure, microparticle formation and mitochondrial depolarisation in glanzmann thrombasthenia platelets. Thromb Haemost. 2014;111:1184-1186

95. Nurden AT, Nurden P, Sanchez M, Andia I, Anitua E. Platelets and wound healing. Front Biosci. 2008;13:3532-3548

96. Moser M, Nieswandt B, Ussar S, Pozgajova M, Fassler R. Kindlin-3 is essential for integrin activation and platelet aggregation. Nat Med. 2008;14:325-330

97. Samuels P, Bussel JB, Braitman LE, Tomaski A, Druzin ML, Mennuti MT, et al. Estimation of the risk of thrombocytopenia in the offspring of pregnant women with presumed immune thrombocytopenic purpura. N Engl J Med. 1990;323:229-235

98. Tsai HM, Lian EC. Antibodies to von willebrand factor-cleaving protease in acute thrombotic thrombocytopenic purpura. N Engl J Med. 1998;339:1585-1594

99. Harrington WJ, Minnich V, Hollingsworth JW, Moore CV. Demonstration of a thrombocytopenic factor in the blood of patients with thrombocytopenic purpura. J Lab Clin Med. 1951;38:1-10

100. Cines DB, Blanchette VS. Immune thrombocytopenic purpura. N Engl J Med. 2002;346:995-1008

101. Bussel JB, Pham LC. Intravenous treatment with gammaglobulin in adults with immune thrombocytopenic purpura: Review of the literature. Vox Sang. 1987;52:206-211

102. Kuter DJ. Thrombopoietin and thrombopoietin mimetics in the treatment of thrombocytopenia. Annu Rev Med. 2009;60:193-206

103. Siragam V, Crow AR, Brinc D, Song S, Freedman J, Lazarus AH. Intravenous immunoglobulin ameliorates itp via activating fc gamma receptors on dendritic cells. Nat Med. 2006;12:688-692

104. Qiu J, Liu X, Li X, Zhang X, Han P, Zhou H, et al. Cd8(+) t cells induce platelet clearance in the liver via platelet desialylation in immune thrombocytopenia. Sci Rep. 2016;6:27445

105. Olsson B, Andersson PO, Jernas M, Jacobsson S, Carlsson B, Carlsson LM, et al. T-cell-mediated cytotoxicity toward platelets in chronic idiopathic thrombocytopenic purpura. Nat Med. 2003;9:1123-1124

106. Ma L, Simpson E, Li J, Xuan M, Xu M, Baker L, et al. Cd8+ t cells are predominantly protective and required for effective steroid therapy in murine models of immune thrombocytopenia. Blood. 2015;126:247-256

107. Zdravic D, Yougbare I, Vadasz B, Li C, Marshall AH, Chen P, et al. Fetal and neonatal alloimmune thrombocytopenia. Semin Fetal Neonatal Med. 2016;21:19-27

108. Curtis BR, McFarland JG. Human platelet antigens - 2013. Vox Sang. 2014;106:93-102

109. Porcelijn L, Van den Akker ES, Oepkes D. Fetal thrombocytopenia. Semin Fetal Neonatal Med. 2008;13:223-230

110. Chen P, Li C, Lang S, Zhu G, Reheman A, Spring CM, et al. Animal model of fetal and neonatal immune thrombocytopenia: Role of neonatal fc receptor in the pathogenesis and therapy. Blood. 2010;116:3660-3668

111. Jakoi ER, Cambier J, Saslow S. Transepithelial transport of maternal antibody: Purification of igg receptor from newborn rat intestine. J Immunol. 1985;135:3360-3364

112. Vadasz B, Chen P, Yougbare I, Zdravic D, Li J, Li C, et al. Platelets and platelet alloantigens: Lessons from human patients and animal models of fetal and neonatal alloimmune thrombocytopenia. Genes Dis. 2015;2:173-185

113. Bonstein L, Haddad N. Taking a wider view on fetal/neonatal alloimmune thrombocytopenia. Thromb Res. 2017;151 Suppl 1:S100-S102

114. Yougbare I, Lang S, Yang H, Chen P, Zhao X, Tai WS, et al. Maternal anti-platelet beta3 integrins impair angiogenesis and cause intracranial hemorrhage. J Clin Invest. 2015;125:1545-1556

115. Li C, Piran S, Chen P, Lang S, Zarpellon A, Jin JW, et al. The maternal immune response to fetal platelet gpibalpha causes frequent miscarriage in mice that can be prevented by intravenous igg and anti-fcrn therapies. J Clin Invest. 2011;121:4537-4547

116. Mitjavila-Garcia MT, Cailleret M, Godin I, Nogueira MM, Cohen-Solal K, Schiavon V, et al. Expression of cd41 on hematopoietic progenitors derived from embryonic hematopoietic cells. Development. 2002;129:2003-2013

117. Aster RH, Bougie DW. Drug-induced immune thrombocytopenia. N Engl J Med. 2007;357:580- 587

118. Weintraub RM, Pechet L, Alexander B. Rapid diagnosis of drug-induced thrombocytopenic purpura. Report of three cases due to quinine, quinidine, and dilantin. JAMA. 1962;180:528-532

119. Aster RH. Immune thrombocytopenia caused by glycoprotein iib/iiia inhibitors. Chest. 2005;127:53S-59S

120. Wardrop D, Keeling D. The story of the discovery of heparin and warfarin. Br J Haematol. 2008;141:757-763

121. Arepally GM. Heparin-induced thrombocytopenia. Blood. 2017;129:2864-2872

122. Greinacher A. Clinical practice. Heparin-induced thrombocytopenia. N Engl J Med. 2015;373:252- 261

123. Greinacher A, Farner B, Kroll H, Kohlmann T, Warkentin TE, Eichler P. Clinical features of heparin-induced thrombocytopenia including risk factors for thrombosis. A retrospective analysis of 408 patients. Thromb Haemost. 2005;94:132-135

124. Reilly MP, Taylor SM, Hartman NK, Arepally GM, Sachais BS, Cines DB, et al. Heparin-induced thrombocytopenia/thrombosis in a transgenic mouse model requires human platelet factor 4 and platelet activation through fcgammariia. Blood. 2001;98:2442-2447

125. Quinn MJ, Byzova TV, Qin J, Topol EJ, Plow EF. Integrin alphaiibbeta3 and its antagonism. Arterioscler Thromb Vasc Biol. 2003;23:945-952

126. Constantin G, Majeed M, Giagulli C, Piccio L, Kim JY, Butcher EC, et al. Chemokines trigger immediate beta2 integrin affinity and mobility changes: Differential regulation and roles in lymphocyte arrest under flow. Immunity. 2000;13:759-769

127. Xiao T, Takagi J, Coller BS, Wang JH, Springer TA. Structural basis for allostery in integrins and binding to fibrinogen-mimetic therapeutics. Nature. 2004;432:59-67

128. Nieswandt B, Varga-Szabo D, Elvers M. Integrins in platelet activation. J Thromb Haemost. 2009;7 Suppl 1:206-209

129. Yang H, Reheman A, Chen P, Zhu G, Hynes RO, Freedman J, et al. Fibrinogen and von willebrand factor-independent platelet aggregation in vitro and in vivo. J Thromb Haemost. 2006;4:2230- 2237

130. Ni H, Denis CV, Subbarao S, Degen JL, Sato TN, Hynes RO, et al. Persistence of platelet thrombus formation in arterioles of mice lacking both von willebrand factor and fibrinogen. J Clin Invest. 2000;106:385-392

131. Dunne E, Spring CM, Reheman A, Jin W, Berndt MC, Newman DK, et al. Cadherin 6 has a functional role in platelet aggregation and thrombus formation. Arterioscler Thromb Vasc Biol. 2012;32:1724-1731

132. Reheman A, Tasneem S, Ni H, Hayward CP. Mice with deleted multimerin 1 and alpha-synuclein genes have impaired platelet adhesion and impaired thrombus formation that is corrected by multimerin 1. Thromb Res. 2010;125:e177-183

133. Wagner CL, Mascelli MA, Neblock DS, Weisman HF, Coller BS, Jordan RE. Analysis of gpiib/iiia receptor number by quantification of 7e3 binding to human platelets. Blood. 1996;88:907-914

134. Ni H, Freedman J. Platelets in hemostasis and thrombosis: Role of integrins and their ligands. Transfus Apher Sci. 2003;28:257-264

135. Niiya K, Hodson E, Bader R, Byers-Ward V, Koziol JA, Plow EF, et al. Increased surface expression of the membrane glycoprotein iib/iiia complex induced by platelet activation. Relationship to the binding of fibrinogen and platelet aggregation. Blood. 1987;70:475-483

136. Sosnoski DM, Emanuel BS, Hawkins AL, van Tuinen P, Ledbetter DH, Nussbaum RL, et al. Chromosomal localization of the genes for the vitronectin and fibronectin receptors alpha subunits and for platelet glycoproteins iib and iiia. J Clin Invest. 1988;81:1993-1998

137. Duperray A, Berthier R, Chagnon E, Ryckewaert JJ, Ginsberg M, Plow E, et al. Biosynthesis and processing of platelet gpiib-iiia in human megakaryocytes. J Cell Biol. 1987;104:1665-1673

138. Takeoka S, Okamura Y, Teramura Y, Watanabe N, Suzuki H, Tsuchida E, et al. Function of fibrinogen gamma-chain dodecapeptide-conjugated latex beads under flow. Biochem Biophys Res Commun. 2003;312:773-779

139. Coller BS, Seligsohn U, Little PA. Type i glanzmann thrombasthenia patients from the iraqi-jewish and arab populations in israel can be differentiated by platelet glycoprotein iiia immunoblot analysis. Blood. 1987;69:1696-1703

140. Springer TA. Folding of the n-terminal, ligand-binding region of integrin alpha-subunits into a beta-propeller domain. Proc. Natl. Acad. Sci. U. S. A. 1997;94:65-72

141. Hughes PE, Diaz-Gonzalez F, Leong L, Wu C, McDonald JA, Shattil SJ, et al. Breaking the integrin hinge. A defined structural constraint regulates integrin signaling. J Biol Chem. 1996;271:6571-6574

142. O'Toole TE, Katagiri Y, Faull RJ, Peter K, Tamura R, Quaranta V, et al. Integrin cytoplasmic domains mediate inside-out signal transduction. J Cell Biol. 1994;124:1047-1059

143. Xiong JP, Stehle T, Goodman SL, Arnaout MA. Integrins, cations and ligands: Making the connection. J Thromb Haemost. 2003;1:1642-1654

144. Zhu G, Zhang Q, Reddy EC, Carrim N, Chen Y, Xu XR, et al. The integrin psi domain has an endogenous thiol isomerase function and is a novel target for antiplatelet therapy. Blood. 2017;129:1840-1854

145. Xiong JP, Stehle T, Zhang R, Joachimiak A, Frech M, Goodman SL, et al. Crystal structure of the extracellular segment of integrin alpha vbeta3 in complex with an arg-gly-asp ligand. Science. 2002;296:151-155

146. Coller BS, Shattil SJ. The gpiib/iiia (integrin alphaiibbeta3) odyssey: A technology-driven saga of a receptor with twists, turns, and even a bend. Blood. 2008;112:3011-3025

147. Wacker D, Stevens RC, Roth BL. How ligands illuminate gpcr molecular pharmacology. Cell. 2017;170:414-427

148. Stalker TJ, Newman DK, Ma P, Wannemacher KM, Brass LF. Platelet signaling. Handb Exp Pharmacol. 2012:59-85

149. Varga-Szabo D, Braun A, Nieswandt B. Calcium signaling in platelets. J Thromb Haemost. 2009;7:1057-1066

150. Assinger A, Volf I, Schmid D. A novel, rapid method to quantify intraplatelet calcium dynamics by ratiometric flow cytometry. PLoS One. 2015;10:e0122527

151. Bagot CN, Arya R. Virchow and his triad: A question of attribution. Br J Haematol. 2008;143:180- 190

152. Xu X, Wu J, Zhai Z, Zhou R, Wang X, Wang H, et al. A novel fibrinogen bbeta chain frameshift mutation in a patient with severe congenital hypofibrinogenaemia. Thromb Haemost. 2006;95:931- 935

153. Covaceuszach S, Bozzi M, Bigotti MG, Sciandra F, Konarev PV, Brancaccio A, et al. Structural

flexibility of human alpha-dystroglycan. FEBS Open Bio. 2017;7:1064-1077

154. Ibraghimov-Beskrovnaya O, Ervasti JM, Leveille CJ, Slaughter CA, Sernett SW, Campbell KP. Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix. Nature. 1992;355:696-702

155. Khurana TS, Davies KE. Pharmacological strategies for muscular dystrophy. Nat Rev Drug Discov. 2003;2:379-390

156. Chamberlain J. The dynamics of dystroglycan. Nat Genet. 1999;23:256-258

157. Austin RC, Fox JE, Werstuck GH, Stafford AR, Bulman DE, Dally GY, et al. Identification of dp71 isoforms in the platelet membrane cytoskeleton. Potential role in thrombin-mediated platelet adhesion. J Biol Chem. 2002;277:47106-47113

158. Cerecedo D, Cisneros B, Suarez-Sanchez R, Hernandez-Gonzalez E, Galvan I. Beta-dystroglycan modulates the interplay between actin and microtubules in human-adhered platelets. Br J Haematol. 2008;141:517-528

159. Reed UC. Congenital muscular dystrophy. Part ii: A review of pathogenesis and therapeutic perspectives. Arq Neuropsiquiatr. 2009;67:343-362

160. Heng S, Paule SG, Li Y, Rombauts LJ, Vollenhoven B, Salamonsen LA, et al. Posttranslational removal of alpha-dystroglycan n terminus by pc5/6 cleavage is important for uterine preparation for embryo implantation in women. FASEB J. 2015;29:4011-4022

161. Brancaccio A, Schulthess T, Gesemann M, Engel J. Electron microscopic evidence for a mucin- like region in chick muscle alpha-dystroglycan. FEBS Lett. 1995;368:139-142

162. Goddeeris MM, Wu B, Venzke D, Yoshida-Moriguchi T, Saito F, Matsumura K, et al. Large glycans on dystroglycan function as a tunable matrix scaffold to prevent dystrophy. Nature. 2013;503:136-140

163. Barresi R, Campbell KP. Dystroglycan: From biosynthesis to pathogenesis of human disease. J Cell Sci. 2006;119:199-207

164. Clarke NF, Maugenre S, Vandebrouck A, Urtizberea JA, Willer T, Peat RA, et al. Congenital muscular dystrophy type 1d (mdc1d) due to a large intragenic insertion/deletion, involving intron 10 of the large gene. Eur J Hum Genet. 2011;19:452-457

165. Inamori K, Yoshida-Moriguchi T, Hara Y, Anderson ME, Yu L, Campbell KP. Dystroglycan function requires xylosyl- and glucuronyltransferase activities of large. Science. 2012;335:93-96

166. Lawler JM. Exacerbation of pathology by oxidative stress in respiratory and locomotor muscles with duchenne muscular dystrophy. J Physiol. 2011;589:2161-2170

167. Taniguchi-Ikeda M, Morioka I, Iijima K, Toda T. Mechanistic aspects of the formation of alpha- dystroglycan and therapeutic research for the treatment of alpha-dystroglycanopathy: A review. Mol Aspects Med. 2016;51:115-124

168. Burkhart JM, Vaudel M, Gambaryan S, Radau S, Walter U, Martens L, et al. The first comprehensive and quantitative analysis of human platelet protein composition allows the comparative analysis of structural and functional pathways. Blood. 2012;120:e73-82

169. Bennett JS, Chan C, Vilaire G, Mousa SA, DeGrado WF. Agonist-activated alphavbeta3 on

platelets and lymphocytes binds to the matrix protein osteopontin. J Biol Chem. 1997;272:8137- 8140

170. Williamson RA, Henry MD, Daniels KJ, Hrstka RF, Lee JC, Sunada Y, et al. Dystroglycan is essential for early embryonic development: Disruption of reichert's membrane in dag1-null mice. Hum Mol Genet. 1997;6:831-841

171. Martins PC, Ayub-Guerrieri D, Martins-Bach AB, Onofre-Oliveira P, Malheiros JM, Tannus A, et al. Dmdmdx/largemyd: A new mouse model of neuromuscular diseases useful for studying physiopathological mechanisms and testing therapies. Dis Model Mech. 2013;6:1167-1174

172. Fong KP, Barry C, Tran AN, Traxler EA, Wannemacher KM, Tang HY, et al. Deciphering the human platelet sheddome. Blood. 2011;117:e15-26

173. Hallmann R, Horn N, Selg M, Wendler O, Pausch F, Sorokin LM. Expression and function of laminins in the embryonic and mature vasculature. Physiol Rev. 2005;85:979-1000

174. Geberhiwot T, Ingerpuu S, Pedraza C, Neira M, Lehto U, Virtanen I, et al. Blood platelets contain and secrete laminin-8 (alpha4beta1gamma1) and adhere to laminin-8 via alpha6beta1 integrin. Exp Cell Res. 1999;253:723-732

175. Li S, Harrison D, Carbonetto S, Fassler R, Smyth N, Edgar D, et al. Matrix assembly, regulation, and survival functions of laminin and its receptors in embryonic stem cell differentiation. J Cell Biol. 2002;157:1279-1290

176. P VK, Kumar CU, J CS, P U, E C. Effect of oral anti-platelet regimens on platelet aggregation using chronolog light transmittance aggregometry in coronary heart disease patients: An observational study. J Clin Diagn Res. 2013;7:2478-2482

177. Day SM, Reeve JL, Myers DD, Fay WP. Murine thrombosis models. Thromb Haemost. 2004;92:486-494

178. Celi A, Merrill-Skoloff G, Gross P, Falati S, Sim DS, Flaumenhaft R, et al. Thrombus formation: Direct real-time observation and digital analysis of thrombus assembly in a living mouse by confocal and widefield intravital microscopy. J Thromb Haemost. 2003;1:60-68

179. Isemura M, Sato N, Yosizawa Z. Isolation and characterization of glycine-rich gelatin-binding protein, a plasma glycoprotein which interacts with gelatin, arginine, heparin, and hyaluronic acid. J Biol Chem. 1982;257:14854-14857

180. Levin J, Ebbe S. Why are recently published platelet counts in normal mice so low? Blood. 1994;83:3829-3831

181. Corash L. The relationship between megakaryocyte ploidy and platelet volume. Blood Cells. 1989;15:81-107

182. Demirin H, Ozhan H, Ucgun T, Celer A, Bulur S, Cil H, et al. Normal range of mean platelet volume in healthy subjects: Insight from a large epidemiologic study. Thromb Res. 2011;128:358- 360

183. Arachiche A, de la Fuente M, Nieman MT. Platelet specific promoters are insufficient to express protease activated receptor 1 (par1) transgene in mouse platelets. PLoS One. 2014;9:e97724

184. Coughlin SR. Thrombin signalling and protease-activated receptors. Nature. 2000;407:258-264

185. Nakanishi-Matsui M, Zheng YW, Sulciner DJ, Weiss EJ, Ludeman MJ, Coughlin SR. Par3 is a cofactor for par4 activation by thrombin. Nature. 2000;404:609-613

186. Maurin N, Forst J, Leithe H, Forst R. Deficiency of platelet glass bead adhesion and platelet membrane glycoprotein iv (cd36) in duchenne muscular dystrophy. Thromb Haemost. 1998;79:1067

187. Forst J, Forst R, Leithe H, Maurin N. Platelet function deficiency in duchenne muscular dystrophy. Neuromuscul Disord. 1998;8:46-49

188. Labarque V, Freson K, Thys C, Wittevrongel C, Hoylaerts MF, De Vos R, et al. Increased gs signalling in platelets and impaired collagen activation, due to a defect in the dystrophin gene, result in increased blood loss during spinal surgery. Hum Mol Genet. 2008;17:357-366

189. Turturro F, Rocca B, Gumina S, De Cristofaro R, Mangiola F, Maggiano N, et al. Impaired primary hemostasis with normal platelet function in duchenne muscular dystrophy during highly- invasive spinal surgery. Neuromuscul Disord. 2005;15:532-540

190. Blaszykowski C, Sheikh S, Benvenuto P, Thompson M. New functionalizable alkyltrichlorosilane surface modifiers for biosensor and biomedical applications. Langmuir. 2012;28:2318-2322

191. Hartwig JH, Barkalow K, Azim A, Italiano J. The elegant platelet: Signals controlling actin assembly. Thromb Haemost. 1999;82:392-398

192. Campanelli JT, Roberds SL, Campbell KP, Scheller RH. A role for dystrophin-associated glycoproteins and utrophin in agrin-induced achr clustering. Cell. 1994;77:663-674

193. Shpilberg O, Rabi I, Schiller K, Walden R, Harats D, Tyrrell KS, et al. Patients with glanzmann thrombasthenia lacking platelet glycoprotein alpha(iib)beta(3) (gpiib/iiia) and alpha(v)beta(3) receptors are not protected from atherosclerosis. Circulation. 2002;105:1044-1048

194. Getz GS, Reardon CA. Animal models of atherosclerosis. Arterioscler Thromb Vasc Biol. 2012;32:1104-1115