Role of von Willebrand Factor in Platelet Thrombus Formation

描述（由申请人提供）：本申请中提出的研究继续努力阐明VWFA1结构域（VWFA1）中的von Willebrand因子（VWF）可以差异调节特定的粘附功能。实验证据表明，糖蛋白（GP） Ib1与固定在表面上的VWFA1相互作用，导致血小板粘附，或在溶液中介导血小板聚集，可能通过不同的机制发生。我们的目标是确定这些不同VWF活动启动和调节的独特结构决定因素，并建立它们在血管损伤模型中的功能相关性。在目的2中，我们打算确定1-凝血酶是否是VWF粘附特性的生理调节剂。我们假设VWFA1和1-凝血酶在结合到相同的GPIb1受体时建立了分子间接触，这独立于剪切应力影响了VWFA1-GPIb1键的稳定性。我们建议鉴定支持与gpib1结合的1-凝血酶相互作用的VWFA1残基，从而确定在血栓形成过程中调节VWF功能的新机制。在目标3中，我们建议评估在人群中发现的特定IgG是否是VWF活性的调节剂。我们已经在人和小鼠血液中发现了一种特异性的IgG，可以选择性地与VWFA1结合，并获得了初步证据，表明该IgG可能在血栓形成中起作用。我们的目标是表征特异性IgG的结构，确定与VWFA1相互作用的模式，并获得其生理病理意义的确切体内证据。在目的4中，我们打算确定胶原结合VWF导致血小板活化的信号功能。我们描述了血小板在流动条件下粘附到固定的VWFA1或胶原蛋白后独特的胞浆内Ca++信号，并发现当血小板与胶原结合的VWF相互作用时，它们被增强。我们建议详细分析胶原- vwf复合物支持的血小板活化机制以及水动力对该过程的影响。这项研究的结果将提高我们影响动脉粥样硬化血栓中血小板的疾病过程的能力。

DESCRIPTION (provided by applicant): The studies proposed in this application continue efforts to elucidate von Willebrand factor (VWF) in the VWF A1 domain (VWFA1) can differentially regulate specific adhesive functions. Experimental evidence indicates that the interaction of glycoprotein (GP) Ib1 with VWFA1 immobilized onto a surface, leading to platelet adhesion, or in solution, mediating platelet aggregation, may occur through different mechanisms. Our goal is to define the distinctive structural determinants underlying the initiation and regulation of these diverse VWF activities and establish their functional relevance in models of vascular injury. In aim 2 we intend to ascertain whether 1-thrombin is a physiologic modulator of VWF adhesive properties. We hypothesize that VWFA1 and 1-thrombin establish inter- molecular contacts when bound to the same GPIb1 receptor, and this influences the stability of the VWFA1-GPIb1 bond independently of shear stress. We propose to identify the VWFA1 residues that support the interaction with GPIb1-bound 1-thrombin, thus defining a novel mechanism for the regulation of VWF function during thrombogenesis. In aim 3 we propose to evaluate whether a specific IgG found in the human population is a modulator of VWF activity. We have identified in human and mouse blood a specific IgG that binds selectively to VWFA1, and obtained preliminary evidence that this IgG may play a role in thrombus formation. Our goal is to characterize the structure of the specific IgG, define the mode of interaction with VWFA1 and obtain definitive in vivo evidence for its physiopathological significance. In aim 4 we intend to define the signaling function of collagen- bound VWF leading to platelet activation. We have characterized the distinctive intracytoplasmic Ca++ signals that follow platelet adhesion to immobilized VWFA1 or collagen under flow conditions, and found that they are enhanced when platelets interact with collagen-bound VWF. We propose to dissect the mechanisms of platelet activation supported by the collagen-VWF complex and the effects of hydrodynamic force on the process. The results of this research will improve our ability to influence disease processes that involve platelets in atherothrombosis. PUBLIC HEALTH RELEVANCE: Experimental and clinical evidence points to a key role of VWF in thrombus formation, orchestrated by interactions with other proteins and modulated through forces generated by flowing blood. Understanding these different functions requires addressing structural details as well as verification of concepts in intravidal models. This will lead to a better diagnosis and treatment for cardio- and cerebro-vascular diseases.