Molecular Basis of Platelet Adhesion

描述（由申请方提供）：血小板粘附于血管内皮下是正常止血和动脉血栓形成的初始步骤。血浆蛋白血管性血友病因子（vonWillebrand Factor，VWF）在血小板血栓形成中起重要作用，因为它与内皮下的胶原和血小板受体GPIb强烈结合。在动脉和微循环中存在的高剪切和高流量条件下，这种桥梁功能对于稳定血小板和内皮下层之间的相互作用至关重要。A1、A2和A3结构域介导VWF的关键相互作用。A1结构域结合GPIb、肝素、硫酸酯和VI型胶原。构象变化被认为是在VWF中的其他区域与胶原结合后暴露VWF-A1结构域。VWF-A2结构域含有与血浆中金属蛋白酶ADAMTS 13相关的生理切割位点。VWF-A3结构域包含纤维状胶原的主要胶原结合位点。VWF中A结构域的重要性被遗传性出血性疾病2型血管性血友病（VWD）强调，其对应于VWF中的功能异常。在VWF-A1和VWF-A2结构域中发现了引起2A、2B和2 M型VWD变体的突变残基。这项资助的目的是了解VWF-胶原相互作用诱导VWF-A1呈现导致与血小板GPIb结合的活性构象的分子机制。在第一个目标中，我们计划描述VWF-A3结构域中与胶原接触的残基。我们还将检验VWF-A1和VWF-A3结构域具有直接相互作用的假设。在第二个目标中，我们将制备重组VWF-A2、VWF-A1 A2和VWF-A2 A3蛋白，以分析VWF-A2结构域在VWF与GPIb和胶原结合中的作用。我们还将研究2A型突变增加VWF-A2结构域蛋白水解敏感性的分子机制。第三个目的是表征重组VWF-AIA 2A 3蛋白与GPIb和胶原的结合以及与血浆金属蛋白酶的相互作用。我们将研究在前两个目标中鉴定的感兴趣的突变体以及一些2B型突变对VWF-A1 A2 A3的亲和力和调节的影响。对于最后两个具体目标，我们计划分析糖基化对GPIb和胶原结合以及血浆金属蛋白酶活性的作用。从这项工作中获得的新信息将建立VWF多聚体被激活和调节的结构基础。这也可能有助于发展一种有效的测定方法来检测血浆中金属蛋白酶的活性。它将为治疗动脉血栓形成的试剂的产生提供分子指导。

DESCRIPTION (provided by applicant): Platelet adhesion to vascular subendothelium is the initial step in both normal hemostasis and arterial thrombosis. The plasma protein von Willebrand Factor (VWF) plays an important role in platelet thrombus formation, because it binds strongly to the collagen in the subendothelium and to the platelet receptor GPIb. This bridge function is critical to stabilize the interaction between platelets and subendothelium under high shear and high flow conditions present in the arterial and microcirculation. The A1, A2, and A3 domains mediate key interactions for VWF. The A1 domain binds to GPIb, heparin, sulphatides and collagen Type VI. Conformational changes are thought to expose the VWF-A1 domain after the binding of other regions in VWF to collagen. The VWF-A2 domain contains a physiological cleavage site that is associated with the metalloprotease ADAMTS13 in plasma. The VWF-A3 domain contains the major collagen-binding site for fibrillar collagens. The importance of the A domains in VWF is underscored by the inherited bleeding disorder Type 2 von Willebrand disease (VWD), which corresponds to functional abnormalities in VWF. The mutated residues that cause the variants of Type 2A, 2B, and 2M VWD have been found in the VWF-A1 and VWF-A2 domains. The goal of this grant is to understand the molecular mechanism by which the VWF-collagen interaction induces the VWF-A1 to assume the active conformation that results in binding to platelet GPIb. In the first aim, we plan to describe the residues that make contact with collagen within the VWF-A3 domain. We will also test the hypothesis that the VWF-A1 and VWF-A3 domains have a direct interaction. In the second aim we will make recombinant VWF-A2, VWF-AIA2, and VWF-A2A3 proteins to analyze the role of the VWF-A2 domain in the VWF-binding to both GPIb and collagen. We will also study the molecular mechanism by which Type 2A mutations increase the sensitivity of the VWF-A2 domain for proteolysis. The third aim will characterize the binding of the recombinant VWF-AIA2A3 protein to GPIb and collagen and the interaction with plasma metalloprotease. We will study the effects of interesting mutants identified in the two previous aims together with some Type 2B mutations on the affinity and regulation of the VWF-A1A2A3. For the last two specific aims, we plan to analyze the role of glycosylation on the GPIb and collagen binding and in plasma metalloprotease activity. The new information obtained from this work will establish the structural basis by which VWF multimers is activated and regulated. It may also contribute to the development of an effective assay to detect the activity of the metalloprotease in plasma. It will provide molecular guidance for the generation of reagents to treat arterial thrombosis.