Structure/Functional Characterization of vWF-A1 Domain

描述(申请人提供)：血小板快速附着到固定在血管损伤部位的vWF的能力，以及对维持血管完整性至关重要的事件，依赖于GPIBα和vWF的A1结构域(vWF-A1)之间的相互作用。到目前为止，对这种受体-配体之间的结构关系的了解已经取得了相当大的进展，但还需要更多的努力来阐明这些黏附分子的物理化学编码的血小板黏附的动力学，以及血小板形状在促进止血过程中所起的作用。前者是通过与任一分子相关的功能增强突变而变得明显的，分别称为血板型和2B型vWD，这些突变通过分析复合体的晶体结构确定的不同机制改变这对黏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，在开启速度和延长这种相互作用的寿命方面也有类似的增强。根据我们的研究，我们假设这种相互作用的内在开关比率是决定血小板-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GPIBα-vWF-A1键的动力学属性以及确定血小板形状对粘附性的影响来建立这些概念。在目标1中，我们将使用微球、重组小鼠和人vWF-A1蛋白以及缺乏β-微管蛋白的动物的血小板来评估细胞大小和形状对该键的力驱动动力学的影响。结果将被用来开发一个计算机模型，旨在复制血小板-vWF的相互作用。在目标2中，我们将确定小鼠vWF-A1中促进与GPIBα相互作用的关键结构元件。将对候选残基进行定点突变，并通过流动研究和动态力光谱分析确定重组蛋白的动力学。在目标3中，我们将在目标2的结果的基础上，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的生物物理性质在体内调节血小板黏附中的贡献。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，使其能够与天然配体对竞争。

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.