Structure/Functional Characterization of vWF-A1 Domain

描述（由申请人提供）：血小板快速附着于固定在血管损伤部位的vWF的能力，以及维持血管完整性的重要事件，依赖于GPIb α和vWF的A1结构域（vWF-A1）之间的相互作用。迄今为止，在理解这种受体-配体对之间的结构关系方面已经取得了相当大的进展，但需要更多的努力来阐明这些粘附分子的物理化学编码的血小板粘附动力学以及血小板形状在促进止血过程中的作用。前者通过与任一分子（分别称为血小板型和2B型vWD）相关的功能增强突变得以证明，通过对复合物晶体结构的分析，它们通过不同的机制改变了这对粘附分子之间的相互作用。然而，只有对这些突变对键形成的影响进行详细的动力学评估，才能证明它们具有共同的生物物理属性，类似的on-rate增强和这种相互作用的寿命延长。根据我们的研究，我们假设这种相互作用的内在开启和关闭率是决定血小板- vwf相互作用发生的时间和地点的关键。我们目前的目标是通过进一步定义GPIb α - vwf - a1键的动力学特性以及确定血小板形状对粘附的影响来建立这些概念。在Aim 1中，我们将使用微球、重组小鼠和人vWF-A1蛋白以及缺乏β -微管蛋白的动物血小板，评估细胞大小和形状对这种键的力驱动动力学的影响。结果将用于开发一个计算机模型，旨在复制血小板- vwf相互作用。在Aim 2中，我们将确定小鼠vWF-A1中促进与GPIb α相互作用的关键结构元件。候选残基的定点诱变将被执行，重组蛋白的动力学将通过流动研究和动态力谱确定。在Aim 3中，基于Aim 2的结果，我们将产生具有动态改变的A1结构域的小鼠，以确定这种相互作用在体内调节血小板粘附中的生物物理特性。来自这些研究的信息可能有助于设计具有特定动力学性质的抗血栓药物，可以与天然配体对竞争。

DESCRIPTION (provided by applicant): The ability of platelets to rapidly attach to vWF immobilized at sites of vascular injury, and event paramount to maintaining vascular integrity, is reliant upon the interactions between GPIb alpha and the A1 domain of vWF (vWF-A1). To date, considerable progress has been made in understanding the structural relationship between this receptor-ligand pair, but more effort is required to elucidate the dynamics of platelet adhesion encoded by the physical chemistry of these adhesion molecules and the role that platelet shape plays in facilitating the process of hemostasis. The former is made evident by function-enhancing mutations associated with either molecule, termed platelet type and type 2B vWD, respectively, which modify interactions between this pair of adhesion molecules through distinct mechanisms as determined by analysis of the crystal structure of the complex. Only a detailed kinetic evaluation, however, of the impact of these mutations on bond formation was able to demonstrate that they share common biophysical attributes, a similar enhancement in on-rate and prolongation the lifetime of this interaction. Based on our studies, we hypothesize that the intrinsic on and off-rates of this interaction are key to determining the time and place where platelet-vWF interactions can occur. Our current goal is to build on these concepts by further defining the kinetic properties of the GPIb alpha-vWF-A1 bond as well as determining the impact of platelet shape on adhesion. In Aim 1, we will assess the impact of cell size and shape on the force-driven kinetics of this bond using microspheres, recombinant murine and human vWF-A1 proteins, and platelets from animals deficient in betal-tubulin. Results will be used to develop a computer model designed to replicate platelet-vWF interactions. In Aim 2, we will identify key structural elements within the murine vWF-A1 that promote interactions with GPIb alpha. Site directed mutagenesis of candidate residues will be performed and the kinetics of recombinant proteins ascertained in flow studies and by dynamic force spectroscopy. In Aim 3, we will generate mice with kinetically altered A1 domains, based on results in aim 2, to establish the contribution of the biophysical properties of this interaction in regulating platelet adhesion in vivo. Information from these studies may aid in the design of antithrombotics with specific kinetic properties that can compete with the native-ligand pair.