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的糖蛋白von Willebrand因子(VWF)之间的相互作用。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。这种相互作用需要将vWF固定在内皮下层或存在极高的流体剪应力，例如在动脉狭窄的区域。一旦血小板通过任何一种方式与vWF结合，信号就会通过质膜传递，产生一系列后果，其中最重要的是整合素AlphaIIbbeta3的激活和血小板的聚集，以及促进血栓巩固的促凝活性的产生。我们有三个具体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，以及剪切力诱导它并导致血小板激活的机制。在具体目标1中，我们将重点关注GP Ibalpha的富含亮氨酸的重复区域-我们假设它是GP Ibalpha最重要的vWF结合区，以精确定义与vWF结合的序列。我们将使用几种方法，包括对犬-人GP Ibalpha嵌合体的研究，以定义参与相互作用的特定物种序列，对三个已知GP Ibalpha序列的哺乳动物物种中保守的带电残基的突变，以及识别潜在相互作用残基的分子建模。在第二个具体目标中，我们将研究GP Ibalpha富含亮氨酸重复序列和C-末端的二硫键环，以确定它们在直接促进vWF结合或调节结合位点暴露方面的作用。在第三个特定目标中，我们将检验这样的假设，即剪切力不仅诱导vWF与GP Ibalpha结合，而且可能还会施加牵引力来启动信号转导。我们预计，这些目标的成功实现将有助于深入了解动脉血栓形成的第一步，并将推动这项SCOR应用的总体目标-开发改进的和特定的动脉血栓治疗方法。

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.