Role of Von Willebrand Factor in Platelet Thrombosis Formation

在本申请中，我们建议继续致力于阐明von Willebrand因子(VWF)的结构和功能，重点关注四个目标。在目标1中，我们将测试VWF A1结构域(VWFA1)中的结构元件是否可以不同地调节特定的粘合剂属性。初步数据表明，糖蛋白(GP)Iba与固定在表面的VWFA1相互作用，导致血小板黏附，与结合可溶性VWF介导的血小板聚集相比，可能通过不同的机制发生。我们的目标是确定特定VWF活动启动和调节的独特结构元素，并建立它们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a-凝血酶是否可以作为VWF粘附性的生理调节器。我们假设，当VWFA1和α-凝血酶结合到同一个GPIba受体上时，会建立分子间的联系，这可能会影响VWFA1-GPIba键的稳定性，而不受剪应力的影响。我们建议确定支持与GPIba结合的α-凝血酶相互作用的VWFA1残基，从而定义一种新的机制来调节VWF在血栓形成过程中的功能。在目标3中，我们建议评估在人类群体中发现的特定免疫球蛋白是否是VWF活性的调节器。我们已经在人和小鼠的血液中发现了一种选择性地与VWFA1结合的特异性免疫球蛋白，并获得了初步证据，表明该免疫球蛋白可能在血栓形成中发挥作用。我们的目标是确定特定免疫球蛋白的结构，确定与VWFA1的相互作用模式，并获得明确的体内证据，证明其生理病理意义。在目标4中，我们将定义胶原结合的VWF导致血小板激活的信号功能。在流动条件下，我们研究了血小板与固定的VWFA1或胶原蛋白黏附后的胞浆内钙信号，发现当血小板与胶原结合的VWF相互作用时，细胞内钙信号增强。我们建议剖析胶原-VWF复合体支持的血小板激活的机制以及流体动力对这一过程的影响。这项研究的结果将提高我们影响涉及血小板参与动脉粥样硬化血栓形成的疾病过程的能力。

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.