• unlimited access with print and download
    $ 37 00
  • read full document, no print or download, expires after 72 hours
    $ 4 99
More info
Unlimited access including download and printing, plus availability for reading and annotating in your in your Udini library.
  • Access to this article in your Udini library for 72 hours from purchase.
  • The article will not be available for download or print.
  • Upgrade to the full version of this document at a reduced price.
  • Your trial access payment is credited when purchasing the full version.
Buy
Continue searching

Organization and function of platelet glycoprotein Ib-IX complex

ProQuest Dissertations and Theses, 2009
Dissertation
Author: Xi Mo
Abstract:
Glycoprotein (GP) Ib-IX complex, the second most abundant receptor expressed on the platelet surface, plays critical roles in haemostasis and thrombosis by binding to its ligand, von Willebrand factor (vWF). Defect or malfunction of the complex leads to severe bleeding disorders, heart attack or stroke. Comprised of three type I transmembrane subunits--GPIbα, GPIbβ and GPIX, efficient expression of the GPIb-IX complex requires all three subunits, as evident from genetic mutations identified in the patients and reproduced in transfected Chinese hamster ovary (CHO) cells. However, how the subunits are assembled together and how the complex function is regulated is not fully clear. By probing the interactions among the three subunits in transfected cells, we have demonstrated that the transmembrane domains of the three subunits interact with one another, facilitating formation of the two membrane-proximal disulfide bonds between GPIbα and GPIbβ. We have also identified the interface between extracellular domains of GPIbβ and GPIX, and provided evidence suggesting a direct interaction between extracellular domains of GPIbα and GPIX. All of these interactions are not only critical for correct assembly and consequently efficient expression of the GPIb-IX complex on the cell surface, but also for its function, such as the proper ligand binding, since removing the two inter-subunit disulfide bonds significantly hampers vWF binding to the complex under both static and physiological flow conditions. The two inter-subunit disulfide bonds are also critical for regulating the ectodomain shedding of GPIbα by the GPIbβ cytoplasmic domain. Mutations in the juxtamembrane region of the GPIbβ cytoplasmic domain deregulate GPIbα shedding, and such deregulation is further enhanced when the two inter-subunit disulfide bonds are removed. In summary, we have established the overall organization of the GPIb-IX complex, and the importance of proper organization on its function.

Table of Contents List of Illustrations xiv List of Tables xxiii Abbreviations and Terms Used xxiv Chapter 1 General Introduction 1 1.1 Overview 2 1.2 Platelet and Haemostasis 2 1.2.1 Platelet 2 1.2.2 Role of platelets in haemostasis 3 1.2.3 Role of platelets beyond haemostasis 13 1.3 Glycoprotein Ib-IX-V 13 1.3.1 Milestones in studying glycoprotein Ib-IX-V 14 1.3.2 Structure and function of the GPIb-IX-V complex 20 1.4 CHO Kl cell line 31 Chapter 2 Characterization of the Inter-Subunit Disulfide Bonds in the Glycoprotein Ib-IX Complex 34 2.1 Overview 35 2.2 Introduction 35 2.2.1 Stoichiometry of the glycoprotein Ib-IX-V complex 35 2.2.2 Cysteine residues in glycoprotein Iba 36 vi

2.3 General Methodology 37 2.3.1 Site-direct mutagenesis and C-terminal epitope tagging of glycoprotein Ib-IX 37 2.3.2 Culture of CHO Kl cells 38 2.3.3 Transient transfection of CHO Kl cells 39 2.3.4 Detection of the surface expression level of the glycoprotein Ib-IX complex by flow cytometry 40 2.3.5 Preparation of cell lysate, immunoprecipitation and Western blot 41 2.3.6 Preparation of human platelets and glycoprotein Iba VNTR genotyping 42 2.3.7 Deglycosylation of glycoprotein Iba from platelet and CHO cells 43 2.4 Results 44 2.4.1 Effects of cysteine mutations on the glycoprotein Iboc/Ibp disulfide bond 44 2.4.2 The stoichiometry of the subunits in glycoprotein Ib-IX 51 2.4.3 Glycoprotein lb from human platelets and transfected CHO cells are equivalent 57 2.5 Discussion 62 2.5.1 Potential disulfide re-arrangement in glycoprotein Ib-IX 62 2.5.2 Impact of the revised stoichiometry on studying downstream-signaling going through glycoprotein Ib-IX 68 Chapter 3 Role of the Transmembrane Domains of the Glycoprotein Ib-IX Complex on Its Expression and Assembly 70 3.1 Overview 71 vii

3.2 Introduction 71 3.2.1 Expression and assembly of glycoprotein Ib-IX 71 3.2.2 Importance of the transmembrane domains of the glycoprotein Ib-IX complex 73 3.3 General Methodology 74 3.3.1 Mutagenesis in the transmembrane domains of the glycoprotein Ib-IX complex 74 3.3.2 Transient transfection of CHO Kl cells 75 3.3.3 Detection of the glycoprotein Ib-IX complex by flow cytometry and Western blot 75 3.4 Results 78 3.4.1 Assays for detecting the mutational effects 78 3.4.2 Role of the transmembrane domain of glycoprotein Ib-IX in the complex expression and assembly 80 3.4.3 Site scanning in the glycoprotein Ib(3 transmembrane domain 89 3.4.4 Site scanning in the glycoprotein Iba transmembrane domain 98 3.4.5 Interaction of the transmembrane domains of glycoprotein Ib-IX 106 3.5 Discussion 114 Chapter 4 Interaction Between the Extracellular Domains of the Glycoprotein Ib-IX Complex 119 4.1 Overview 120 4.2 Introduction 121 viii

4.2.1 Leucine-rich repeats proteins 121 4.2.2 Structure modeling of the extracellular domains of glycoprotein Ib(3 and glycoprotein IX 125 4.3 General Methodology 130 4.3.1 Mutagenesis of the glycoprotein Ib|3 and glycoprotein IX mutant constructs 130 4.3.2 Transient transfection of CHO Kl cells 131 4.3.3 Flow cytometry 132 4.3.4 Immnuoprecipitation, SDS-Polycrylamide Gel Electrophoresis and Western blot 132 4.4 Results 133 4.4.1 Both the transmembrane and extracellular domains of glycoprotein Ib(3 are required for efficient expression of glycoprotein IX in transfected CHO cells 133 4.4.2 Identification of a stable IbpVIX chimeric extracellular domain 138 4.4.3 The Ib^Eabc chimera preserves the interaction of glycoprotein IX with glycoprotein lbp 143 4.4.4 The three convex loops on glycoprotein Ib(3 are required for interacting with glycoprotein IX 153 4.4.5 Evidence suggesting a direct link between the glycoprotein Iba and glycoprotein IX extracellular domains 158 4.5 Discussion 162 ix

4.5.1 Interactions between the extracellular domains of glycoprotein Ib|3 and glycoprotein IX 162 4.5.2 Importance of the potential interaction between the extracellular domains of glycoprotein Iba and glycoprotein IX 165 Chapter 5 Role of the Glycoprotein Ib|3 Cytoplasmic Domain in Trafficking of the Glycoprotein Ib-IX Complex to the Plasma Membrane 168 5.1 Overview 169 5.2 Introduction 169 5.2.1 General secretion pathway in mammalian cells 169 5.2.2 Trafficking of the glycoprotein Ib-IX complex to the plasma membrane 173 5.3 General Methodology 174 5.3.1 Mutagenesis in the glycoprotein Ib(3 cytoplasmic domain 174 5.3.2 Expression of the glycoprotein Ib-IX complex in transiently transfected CHO cells detected by flow cytometry and Western blot 175 5.3.3 Localization of the glycoprotein Ib-IX complex in transiently transfected CHO cells detected by fluorescence microscopy 176 5.4 Results 176 5.4.1 The N-terminal HA-tag does not affect the expression level and assembly of the glycoprotein Ib-IX complex 177 5.4.2 Role of the cytoplasmic domain of glycoprotein Ib(3 on expression and assembly of the glycoprotein Ib-IX complex 177 x

5.4.3 The juxtamembrane residues of the glycoprotein Ib(3 cytoplasmic domain is critical for the complex trafficking to the plasma membrane assembly 192 5.4.4 The glycoprotein Ib|3 cytoplasmic domain is sufficient to mediate protein trafficking to the plasma membrane 200 5.5 Discussion 203 Chapter 6 Effects of Inter-molecular Disulfide Bonds on Ligand Binding to the Glycoprotein Ib-IX Complex 211 6.1 Overview 212 6.2 Introduction 213 6.2.1 von Willebrand factor 213 6.2.2 Interaction of von Willebrand factor and glycoprotein Iba 215 6.2.3 Regulation of von Willebrand factor binding to glycoprotein Iba 220 6.3 General Methodology 221 6.3.1 Generation of stable cell lines 221 6.3.2 Flow cytometry 224 6.3.3 Co-immunoprecipitation and Western blot 224 6.3.4 von Willebrand factor binding under static conditions 226 6.3.5 von Willebrand factor binding under flow conditions 226 6.4 Results 227 6.4.1 Generation of stable cell lines 227 6.4.2 Antibody binding to the double cysteine mutant glycoprotein Ib-IX complex 229 xi

6.4.3 Characterization of the structural changes in the double cysteine mutant glycoprotein Ib-IX complex 234 6.4.4 von Willebrand factor binding to the double cysteine mutant glycoprotein Ib-IX complex under static and flow conditions 242 6.4.5 Antibody binding to the single cysteine mutant glycoprotein Ib-IX complex 245 6.4.6 Characterization of the structural changes in the single cysteine mutant glycoprotein Ib-IX complex 250 6.4.7 von Willebrand factor binding to the single cysteine mutant glycoprotein Ib-IX complex under static and flow conditions 256 6.5 Discussion 259 6.5.1 Impact of this study on the putative allosteric regulation of the glycoprotein Ib-IX complex 259 6.5.2 Questions remained for future study 264 Chapter 7 Regulation of Glycoprotein Iba Shedding 265 7.1 Overview 266 7.2 Introduction 267 7.2.1 Protein ectodomain shedding and a metalloprotease and disintegrin family....267 7.2.2 Regulation of protein shedding 269 7.2.3 CHO Ml and CHO M2 cell lines 272 7.2.4 Shedding of glycoprotein Iba and its relationship with platelet clearance 273 7.3 General Methodology 275 xii

7.3.1 Generation of stable cell lines 275 7.3.2 Drug-induced or inhibited shedding in transiently or stably transfected Cells 275 7.3.3 Detection of glycoprotein Iba shedding by flow cytometry, immunoprecipitation, TCA precipitation and Western blot 276 7.4 Results 278 7.4.1 Ectodomain shedding of glycoprotein Iba in CHO Kl cells stably or transiently expressing the glycoprotein Ib-IX complex 278 7.4.2 Glycoprotein Iba is shed by ADAM17 in CHO Kl cells 284 7.4.3 Role of the cytoplasmic domains of glycoprotein Ib-IX on regulation of glycoprotein Iba shedding 288 7.4.4 Trans-subunit regulation of glycoprotein Iba shedding by the juxtamembrane region of the glycoprotein Ibp cytoplasmic domain 291 7.4.5 Regulation of glycoprotein Iba shedding by the glycoprotein lb(3 cytoplasmic domain is through the interaction between the two subunits 302 7.5 Discussion 319 7.5.1 Significance of studying glycoprotein Iba shedding 319 7.5.2 Potential application of inhibition of glycoprotein Iba shedding in platelet storage and transfusion 321 References 324 Vita 369 xiii

List of Illustrations Figure 1-1. Illustration of platelet adhesion, activation and aggregation upon vessel damage 5 Figure 1 -2. Illustration of the platelets under resting and activated states 9 Figure 1-3. Illustration of the pathways for coagulation 12 Figure 1-4. A contemporary version of a model of the GPIb-V-IX complex 21 Figure 1-5. GPIb-IX-V signaling pathways 24 Figure 1-6. A "toggle switch" model for the regulation of vWF binding to the GPIb-IX-V complex 29 Figure 1-7. Morphology of CHO Kl cells under contract phase microscope 32 Figure 2-1. The cysteine residues in the GPIba and GPIbp subunits 45 Figure 2-2. Effects of the cysteine mutations on surface expression of the GPIb-IX complex 46 Figure 2-3. Effects of the cysteine mutations on formation of the inter-subunit disulfide bonds between GPIba and GPIb|3 49 Figure 2-4. Effects of the C-terminal tags on the surface expression level of the GPIb-IX complex 52 Figure 2-5. Co-immunoprecipitation of HA- and c-myc-tagged GPIba, GPIb(3 and GPIX in the GPIb-IX complex 55 Figure 2-6. Comparison of GPIb, GPIba and GPIbp from human platelets and transiently transfected CHO Kl cells 59 Figure 2-7. Contribution of the VNTR polymorphism to the molecular weight xiv

of GPIba 60 Figure 2-8. Contribution of different glycosylation to the molecular weight of GPIba 63 Figure 2-9. Schematic drawing of the GPIb-IX-V complex on resting platelet surface with the revised stoichiometry 65 Figure 2-10. Proposed disulfide rearrangement of the GPIb-IX complex 66 Figure 3-1. Sequences of the transmembrane domains of the GPIb-IX complex 82 Figure 3-2. Effects of the transmembrane replacement on the surface expression levels of the GPIb-IX complex 83 Figure 3-3. Effects of the transmembrane replacements on the cellular expression levels of the GPIb-IX complex 86 Figure 3-4. Effects of the transmembrane replacements on the assembly of the GPIb-IX complex 88 Figure 3-5. Side-scanning mutagenesis of the GPIbp transmembrane domain 90 Figure 3-6. Effects of the side-scanning mutagenesis in the GPIb(3 transmembrane domain on the expression levels of the GPIb-IX complex 93 Figure 3-7. Effects of the single site mutations in the GPIb|3 transmembrane domain on the expression levels of the GPIb-IX complex 96 Figure 3-8. Side-scanning mutagenesis of the GPIba transmembrane domain 99 Figure 3-9. Effects of the side-scanning mutagenesis in the GPIba transmembrane domain on the expression levels of the GPIb-IX complex 101 xv

Figure 3-10. Effects of P488A in the GPIba transmembrane domain 104 Figure 3-11. Potential interactions between the transmembrane domains of GPIba and GPIbp 107 Figure 3-12. Mutation S503L in GPIba only reverses the decreasing effect on the complex expression of HI 39L inGPIbp 109 Figure 3-13. Mutation S503V in GPIba only reverses the decreasing effect on the complex expression of HI 39L inGPIb|3 112 Figure 3-14. Proposed model of a four-helical bundle for the transmembrane domains of the GPIb-IX complex 116 Figure 4-1. Tertiary and quaternary structures of the example leucine-rich repeat domains viewed from the sides of the respective solenoids 123 Figure 4-2. Sequence analysis and structural models of the GPIb(3 and GPIX extracellular domains 127 Figure 4-3. Illustration of the GPIb|3 and GPIX constructs containing the full length or extracellular domain used in the study 135 Figure 4-4. Both the transmembrane and extracellular domains of GPIb|3 are required for expression of GPIX in CHO cells 136 Figure 4-5. Illustration of various chimeric HA-Ib|3E/IXE constructs 140 Figure 4-6. Identification of a stable HA-Ib(3E/IXE chimera 141 Figure 4-7. Illustration of various IbpVIX chimeric constructs used in the study 144 Figure 4-8. Expression of HA-Ib|3Eat>c chimera, in the context of full-length GPIX subunit, could be enhanced with coexpression of GPIb(3, the xvi

extent of which mimics that of the GPIX extracellular domain 146 Figure 4-9. Expression of HA-IbpEabc chimera, in the context of full-length GPIX subunit, could be enhanced with coexpression of GPIb(3, the extent of which mimics that of the GPIX extracellular domain 148 Figure 4-10. The convex loops of GPIX preserve the interaction of GPIX with GPIb(3, in the context of full-length subunits 151 Figure 4-11. The convex loops on GPIb(3 are required for the interaction with GPIX 154 Figure 4-12. The convex loops of GPIbp are required for the interaction of GPIbp with GPIX, in the context of full-length subunits 156 Figure 4-13. The convex loops of IXE sufficiently preserve the reliance of GPIX expression on GPIb(3 159 Figure 4-14. Inclusion of HA-IbpEabc-IXic in the GPIb-IX complex uncovers a direct link between the GPIba and GPIX extracellular domains 160 Figure 4-15. A potential function of the hypothesized weak link between the GPIba and GPIX extracellular domains 167 Figure 5-1. Secretory pathway of protein sorting 171 Figure 5-2. The N-terminal HA-tag on GPIb(3 does not affect the expression or assembly of the GPIb-IX complex 178 Figure 5-3. The GPIbp cytoplasmic domain is critical for efficient expression and correct assembly of the GPIb-IX complex 181 Figure 5-4. Series of truncating mutations in the GPIb(3 cytoplasmic domain and their effects on the surface expression level of the GPIb-IX xvii

complex, 183 Figure 5-5. Series of truncating mutations in the GPIb|3 cytoplasmic domain and their effects on the cellular expression level and assembly oftheGPIb-IX complex 185 Figure 5-6. The juxtamembrane basic residues in the GPIbp cytoplasmic domain are critical for the surface expression of the GPIb-IX complex 188 Figure 5-7. The juxtamembrane basic residues in the GPIb(3 cytoplasmic domain are critical for the cellular expression and assembly of the GPIb-IX complex 190 Figure 5-8. The juxtamembrane basic residues in the GPIb(3 cytoplasmic domain are critical for GPIbp trafficking to the plasma membrane 193 Figure 5-9. The juxtamembrane residues in the GPIbcx cytoplasmic domain are not required for the proper assembly of the GPIb-IX complex 195 Figure 5-10. Cellular localization of GPIba in the transfected CHO cells 198 Figure 5-11. Cellular localization of GPIb(3 in the transfected CHO cells 199 Figure 5-12. Expression of the single chain GPIba in CHO Kl cells 201 Figure 5-13. Surface expression level of the single chain GPIba in CHO M2 cells 204 Figure 5-14. C148G in the GPIb|3 cytoplasmic domain did not affect surface expression of the GPIb-IX complex 206 Figure 5-15. C148G in the GPIb(3 cytoplasmic domain did not affect cellular expression or the assembly of the GPIb-IX complex 208 xvm

Figure 6-1. Domain structure of von Willebrand factor (pre-pro vWF and mature vWF) 216 Figure 6-2. Crystal structures of the isolated GPIba ligand binding domain and the complex of GPIba andthevWF Al domain 218 Figure 6-3. Epitopes of the antibodies against GPIba and GPIX 230 Figure 6-4. Surface expression of the GPIb-IX complex in stable CHOaccPIX and CHOaSspIX cells 232 Figure 6-5. The double cysteine mutation C484S/C485S in GPIba abolished the formation of the GPIba/GPIbp inter-subunit disulfide bonds while maintained its cellular expression level 233 Figure 6-6. The double cysteine mutation C484S/C485S in GPIba largely maintained the structural integrity of the mutant GPIb-IX complex in CHOasspIX cells 237 Figure 6-7. The double cysteine mutation C484S/C485S in GPIba led to formation of a disulfide-linked GPIb|3 homodimer 240 Figure 6-8. Ristocetin-induced vWF binding to various stable CHO cells lines in solution 244 Figure 6-9. Schematic diagram of the parallel plate flow chamber 246 Figure 6-10. Rolling of CHOa

CH0

Figure 7-8. The stable CHO cell lines expressing the cytoplasmic domain- mutant GPIba constructs 292 Figure 7-9. The GPIba cytoplasmic domain is not required for regulating GPIba shedding in stable CHO cell lines 294 Figure 7-10. Effects of the single E mutations on the assembly of the GPIb-IX complex and the cellular expression levels of GPIbp and GPIX 296 Figure 7-11. Effects of the single E mutagenesis in the GPIb(3 cytoplasmic domain on GPIba shedding 298 Figure 7-12. The juxtamembrane region of the GPIbp cytoplasmic domain was critical fro regulating GPIba shedding 300 Figure 7-13. GPIba in the E-mutant complexes was shed by ADAM17 303 Figure 7-14. Effects of the double E-mutations on the assembly of the GPIb-IX complex and the cellular expression levels of GPIbp and GPIX 305 Figure 7-15. Comparison of the surface expression levels of GPIba in various complexes in CHO Kl (red bar) and CHO M2 (yellow bar) cells 307 Figure 7-16. Incorrect assembly of the GPIb-IX complex did not induce GPIba Shedding 309 Figure 7-17. The juxtamembrane region of the GPIb(3 cytoplasmic domain regulated GPIba shedding through the interaction between the two subunits 311 Figure 7-18. The juxtamembrane region of the GPIbp cytoplasmic domain regulated GPIba shedding through the interaction between the two subunits 313 xxi

Figure 7-19. GPIba in the double mutant complexes (Ibctss + E-mutant GPIb(3) was shed by AD AMI 7 315 Figure 7-20. Comparison of the surface expression levels of GPIba in various complexes in CHO Kl and CHO M2 cells 317 Figure 7-21. The double-E mutations in the juxtamembrane region of the GPIb|3 cytoplasmic domain significantly reduced calmodulin binding 320 xxii

List of Tables Table 1 -1. Typical ranges of wall shear rates and wall shear stresses 7 Table 1 -2. Ligands for the platelet GPIb-IX-V complex and their functions 19 Table 1-3. Primers used for removing or creating the restriction cleavage sites around the transmembrane domains of the GPIb-IX complex 76 xxm

Chapter 1 General Introduction CHAPTER 1 GENERAL INTRODUCTION 1

Chapter 1 General Introduction 1.1 Overview Platelets, the small anuclear cells circulating in the blood stream, play a vital role in haemostasis. This is achieved by the activation of platelets, recruiting of the clotting factors, aggregation of platelets, recruiting of the red blood cells, and finally formation of thrombi. Platelet carries out its function mostly by utilizing the receptors on its surface, and glycoprotein (GP) Ib-IX-V complex, the second most abundant receptor on platelets, is one of the most important. The GPIb-IX-V complex, by binding to the ligand von Willebrand factor (vWF), mediates the rolling and tethering of platelets to the damaged blood vessel wall under high shear stress, and subsequently leads to the activation and aggregation of platelets. Lacking of efficient expression of the GPIb-IX- V complex on the cell surface, which requires three of its four subunits, leads to severe bleeding disorders. On the other hand, deregulated binding of the complex to its ligands will cause pathological thrombi formation in the blood vessels, leading to heart attack or stroke. Therefore, it is critical to understand the organization and function of the complex. In this study, transfected Chinese hamster ovary (CHO) cell line is used as a model, due to the lack of nucleus in platelets, to study the organization of the GPIb-IX complex and the roles of the correct organization on the proper function of the complex. 1.2 Platelet and Haemostasis 1.2.1 Platelets Platelet, also called thrombocyte, is a minute (2-4 um in diameter), non-nucleated, disc-like cytoplasmic body found in the blood plasma of mammals. The physiological range for platelets is 150-400 x 109/L, and on average, about 1 x 1011 platelets are produced each day by a healthy adult. Platelets are produced in blood cell formation 2

Chapter 1 General Introduction (thrombopoiesis) in bone marrow, derived from fragmentation of precursor megakaryocyte. Each megakaryocyte can produce 5,000 ~ 10,000 platelets, and the process is regulated by thrombopoietin, a glycoprotein hormone usually produced by the liver and kidney. The platelets circulating in the blood stream have the average lifespan between 7 and 10 days, and the old platelets are destroyed by phagocytosis in the spleen and by Kupffer cells in the liver. The first description of the platelet could be dated back to the middle of 1800s, when the German anatomist Max Schultze (1825-1874) first described '"spherules' much smaller than red blood cells that are occasionally clumped and may participate in collections of fibrous material" in his newly founded journal Archivfur mikroscopische Anatomie (Schultze, 1865). Then later on, based on Schultze's findings, Giulio Bizzozero (1846-1901) utilized "living circulation" to study blood cells of amphibians microscopically in vivo. He found that platelets clumped at the injury site of the blood vessel, initiating the formation of a blood clot (Bizzozero, 1882). This observation confirmed the role of platelets in coagulation. 1.2.2 Role of platelets in haemostasis Over the past twenty years, the functions of platelets and how they carry out their functions is a lot more understood, especially regarding the specific functions of those adhesion proteins and receptors on the platelet surface. The major function of the platelets is to maintain haemostasis, and this is primarily achieved by formation of thrombi at the injured endothelium of the blood vessels. However, thrombus formation must be inhibited when the endothelium of the blood vessels is not damaged; otherwise, it will induce various cardiovascular diseases, such as heart attack or stroke. Platelets 3

Chapter 1 General Introduction carry out all these functions undergoing a regular process, including adhesion, activation, degranulation, and aggregation (Figure 1-1). Adhesion Under normal conditions, a thin layer of endothelial cells lined on the inner surface of the blood vessels produce endothelial ADPase and prostacyclin (PGI2) to inhibit platelet activation. Endothelial ADPase clears away the platelet activator ADP from platelet surface receptors, while PGI2 acts as a vasodilator and inhibits platelet aggregation. Under the layer of endothelial cells is a layer of collagen. Under physiological conditions, a glycoprotein called von Willebrand factor (vWF), a cell adhesion ligand produced by the endothelial cells, helps endothelial cells adhere to collagen in the basement membrane so that collagen does not pass into the blood stream. When endothelial damage occurs, immediate reflex that promotes vasoconstriction takes place to diminish blood loss. In addition, upon endothelial damage, collagen is exposed, and vWF is immobilized on the exposed collagen. The immobilized vWF as well as other proteins on the endothelial cells will recruit the platelets so that the platelets come into contact with exposed collagen and immobilized vWF, and subsequently adhere to the damaged site, leading to the decrease in secretion of those endothelium platelet inhibitors. Platelet adhesion to the exposed subendothelium, the initial step for platelet activation and aggregation, is a complex process including multiple steps. It involves various adhesive ligands, such as collagen, vWF, fibronectin, thrombospondin and potentially laminin, and receptors on the platelet surface, such as GPVI, GPIb-IX-V, 4

Chapter 1 General Introduction Subendothelial Matrix Proteins Figure 1-1. Illustration of platelet adhesion, activation and aggregation upon vessel damage. Following damage of the blood vessels, exposed collagen from the damaged site and vWF immobilized on collagen will promote the platelets to adhere. After a complex signaling network inside the platelets, the platelets are activated, undergoing cytoskeleton rearrangement, morphology change and releasing the granules stored inside the platelets. The released ADP, Thromboxane A2 and serotonin attracts more platelets to the area, promotes platelet aggregation, and vasoconstriction, respectively. ADP and thromboxane A2 promote more platelet adhesion and therefore more ADP and thromboxane. The positive feedback promotes the formation of a platelet plug. 5

Chapter 1 General Introduction integrin cxnbfc, 012P1, cxsPi and a$\. The adhesive interactions during platelet adhesion is largely determined by the prevailing rheological conditions—the flow rate or the wall shear stress (Agbanyo, et ah, 1993; Beumer, et ah, 1995; Beumer, et ah, 1994; Bonnefoy, etah, 2001; Denis, et ah, 1998; Hindriks, et ah, 1992; Houdijk, etah, 1985; Kroll, et ah, 1996; Moroi, et ah, 1996; Nievelstein, et ah, 1988; Savage, et ah, 1998; Savage, et ah, 1996; Turitto, et ah, 1985). The blood flow can be described as an "infinite number of infinitesimal laminae sliding across one another, each lamina suffering some frictional interaction with its neighbors", while the shear stress is defined as "the force per unit area between laminae" (Kroll, et ah, 1996). Thus the wall shear stress of Newtonian fluids for flow in tubular vessels can be calculated as a function of volumetric flow rate: xw = ^Q/jtr3, where fi is viscosity, Q is the volumetric flow rate, and r = radial distance of the tubular chamber, and shear forces in vivo have been calculated by using this formula (Table 1-1) (Kroll, et ah, 1996). Under low shear conditions, such as those experienced in large arteries and veins, adhesion of platelets to the damaged subendothelium is primarily mediated by one or more fibrillar collagens, fibronectin and laminin (Agbanyo, et ah, 1993; Beumer, et ah, 1995; Beumer, et ah, 1994; Hindriks, et ah, 1992; Kroll, et ah, 1996; Nievelstein, et ah, 1988). In contrast, under high shear conditions, such as those experienced in arterioles and stenotic vessels, the initial tethering and adhesion of platelets to the subendothelial vessel wall is critically dependent on the binding of the platelet GPIb-IX-V complex to the immobilized vWF (Alevriadou, et ah, 1993; Bolhuis, et ah, 1981; Fressinaud, et ah, 1988; Meyer, et ah, 1987; Ruggeri, 1997; Sakariassen, et ah, 1987; Sakariassen, et ah, 1986; Weiss, etah, 1978). 6

Full document contains 395 pages
Abstract: Glycoprotein (GP) Ib-IX complex, the second most abundant receptor expressed on the platelet surface, plays critical roles in haemostasis and thrombosis by binding to its ligand, von Willebrand factor (vWF). Defect or malfunction of the complex leads to severe bleeding disorders, heart attack or stroke. Comprised of three type I transmembrane subunits--GPIbα, GPIbβ and GPIX, efficient expression of the GPIb-IX complex requires all three subunits, as evident from genetic mutations identified in the patients and reproduced in transfected Chinese hamster ovary (CHO) cells. However, how the subunits are assembled together and how the complex function is regulated is not fully clear. By probing the interactions among the three subunits in transfected cells, we have demonstrated that the transmembrane domains of the three subunits interact with one another, facilitating formation of the two membrane-proximal disulfide bonds between GPIbα and GPIbβ. We have also identified the interface between extracellular domains of GPIbβ and GPIX, and provided evidence suggesting a direct interaction between extracellular domains of GPIbα and GPIX. All of these interactions are not only critical for correct assembly and consequently efficient expression of the GPIb-IX complex on the cell surface, but also for its function, such as the proper ligand binding, since removing the two inter-subunit disulfide bonds significantly hampers vWF binding to the complex under both static and physiological flow conditions. The two inter-subunit disulfide bonds are also critical for regulating the ectodomain shedding of GPIbα by the GPIbβ cytoplasmic domain. Mutations in the juxtamembrane region of the GPIbβ cytoplasmic domain deregulate GPIbα shedding, and such deregulation is further enhanced when the two inter-subunit disulfide bonds are removed. In summary, we have established the overall organization of the GPIb-IX complex, and the importance of proper organization on its function.