Role of Von Willebrand Factor in Platelet Thrombosis Formation

在本申请中，我们建议继续努力，旨在阐明血管性血友病因子（VWF）的结构和功能，重点是四个目标。在目标1中，我们将测试是否在VWF A1结构域（VWFA 1）的结构元件可以差异调节特定的粘附性能。初步数据表明，糖蛋白（GP）Iba与固定在表面上的VWFA 1的相互作用，导致血小板粘附，可能通过不同的机制发生相比，可溶性VWF介导的血小板聚集的结合。我们的目标是确定启动和调节特定VWF活性的独特结构元件，并建立其在血管损伤模型中的功能相关性。在目标2中，我们打算确定α-凝血酶是否可以作为VWF粘附特性的生理调节剂。我们假设VWFA 1和α-凝血酶在与相同的GPIba受体结合时建立分子间接触，并且这可能独立于剪切应力影响VWFA 1-GPIba键的稳定性。我们建议鉴定支持与GPIba结合的α-凝血酶相互作用的VWFA 1残基，从而定义血栓形成期间调节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异性IgG是否是VWF活性的调节剂。我们已经确定了在人类和小鼠血液中的特异性IgG，选择性结合VWFA 1，并获得了初步证据，这种IgG可能在血栓形成中发挥作用。我们的目标是表征特异性IgG的结构，定义与VWFA 1的相互作用模式，并获得其生理病理学意义的体内证据。在目标4中，我们将定义胶原结合的VWF导致血小板活化的信号传导功能。我们的特点是不同的胞浆内Ca++信号，血小板粘附到固定的VWFA 1或胶原蛋白在流动条件下，并发现它们增强血小板与胶原蛋白结合的VWF相互作用时。我们建议解剖的胶原蛋白-VWF复合物支持的血小板活化的机制和流体动力学的影响的过程中。这项研究的结果将提高我们的能力，影响疾病的进程，涉及血小板在动脉粥样硬化血栓形成。

In this application we propose to continue efforts aimed at elucidating von Willebrand factor (VWF) structure and function focusing on four aims. In aim 1 we will test whether structural elements in the VWF A1 domain (VWFA1) can differentially regulate specific adhesive properties. Preliminary data indicate that the interaction of glycoprotein (GP) Iba with VWFA1 immobilized onto a surface, leading to platelet adhesion, may occur through different mechanisms as compared to binding of soluble VWF mediating platelet aggregation. Our goal is to define the distinctive structural elements underlying the initiation and regulation of specific VWF activities and establish their functional relevance in models of vascular injury. In aim 2 we intend to ascertain whether a-thrombin can act as a physiologic modulator of VWF adhesive properties. We hypothesize that VWFA1 and a-thrombin establish inter-molecular contacts when bound to the same GPIba receptor, and this may influence the stability of the VWFA1-GPIba bond independently of shear stress. We propose to identify the VWFA1 residues that support the interaction with a-thrombin bound to GPIba, 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 will define the signaling function of collagen-bound VWF leading to platelet activation. We have characterized distinct 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.