Molecular determinants of glycoprotein Ib/vonWillibrand factor interaction

止血系统识别动脉病变的第一步是血小板表面的大糖蛋白（GP）复合体GP Ib-IX-V复合体和附着在血管基质上的更大糖蛋白血管性血友病因子（vWF）的相互作用。在GP Ib-IX-V复合体中，GP Ibalpha包含所有已知的vwf相互作用序列。VWF也在血浆中循环，但在某些情况下不与血小板结合。这种相互作用需要vWF固定在内皮下层或存在极高的流体剪切应力，例如可能在动脉狭窄区域发现的剪切应力。一旦血小板以任何一种方式与vWF结合，信号就会通过质膜传递，这有许多后果，最重要的是整合素alphaIIbbeta3的激活和血小板聚集，以及促凝活性的产生，促进血栓的巩固。我们有三个特定的目标来继续我们的工作，以确定GP Ib-IX-V-vWF相互作用的分子性质以及剪切应力诱导它并导致血小板活化的机制。在Specific Aim 1中，我们将重点关注GP Ibalpha中富含亮氨酸的重复区域，我们假设这是GP Ibalpha中最重要的vWF结合区域，以精确定义与vWF结合相关的序列。我们将使用几种方法，包括研究犬-人GP Ibalpha嵌合体，以确定参与相互作用的特定物种序列，在已知GP Ibalpha序列的三种哺乳动物物种中保守的带电残基的诱变，以及分子建模以确定潜在的相互作用残基。在第二个特定目标中，我们将研究GP α α和c端富含亮氨酸的重复序列两侧的二硫环，以了解它们在直接促进vWF结合或调节结合位点暴露中的作用。在第三个特定目标中，我们将验证剪切应力不仅诱导vWF结合GP Ibalpha，而且还可能施加牵引力来启动信号转导的假设。我们期望这些目标的成功实现将使我们深入了解动脉血栓形成的第一步，并进一步实现SCOR应用的总体目标——开发出针对动脉血栓形成的改进和特异性治疗方法。

The first step in the recognition of arterial lesions by the hemostatic system is an interaction between a large glycoprotein (GP) complex on the platelet surface, the GP Ib-IX-V complex, and an even larger glycoprotein affixed to the vascular matrix, von Willebrand factor (vWF). Within the GP Ib-IX-V complex, GP Ibalpha contains all of the known vWF-interacting sequences. VWF also circulates in the plasma but under circumstances does not bind platelets. That interaction requires that vWF be immobilized onto the subendothelium or the presence of extremely high fluid shear stresses, such as may be found in regions of artery narrowing. Once the platelets bind vWF by either means, signals are transmitted across the plasma membrane that have a number of consequences, the most important being the activation of the integrin alphaIIbbeta3 and the platelet aggregation, and the generation of pro- coagulant activity that promotes consolidation of the thrombus. We have three Specific Aims to continue our work to define the molecular nature of the GP Ib-IX-V-vWF interaction and the mechanisms by which shear stress induces it and leads to platelet activation. In Specific Aim 1, we will focus on the leucine-rich repeats region of GP Ibalpha-which we hypothesize is the mot important vWF-binding region of GP Ibalpha, to precisely define the sequences involved in binding vWF. We will use several approaches, including studies of canine-human GP Ibalpha chimeras to define specific species sequences involved in the interaction, mutagenesis of charged residues conserved in three mammalian species for which the GP Ibalpha sequence is known, and molecular modeling to identify potentially interactive residues. In the second Specific Aim, we will examine the disulfide loops that flank the GP Ibalpha leucine-rich repeats on both the and C-termini for their role in either directly facilitating vWF binding or regulating exposure of binding sites. In the third Specific Aim, we will test the hypothesis that shear stress not only induces vWF to bind GP Ibalpha but not it may also exerts a traction force to initiate signal transduction. We expect that successful accomplishment of these aims will yield insight into the very first step in the formation of an arterial thrombus, and will further the overall goal of this SCOR applications-to develop improved and specific therapies for arterial thrombosis.