REGULATION OF VON WILLEBRAND MULTIMER SIZE

VON WILLEBRAND 多路复用器尺寸的调节

• 批准号: 2569056
• 负责人: T J LYNCH
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2569056
• 关键词: chemical kinetics; endopeptidases; enzyme activity; gene mutation; human tissue; immunocytochemistry; intermolecular interaction; platelet aggregation; posttranslational modifications; protein structure function; von Willebrand factor; von Willebrand's disease

Von Willebrand factor (vWF) is a large glycoprotein secreted by endothelial cells and platelets into the plasma and vascular connective tissue. In addition to binding to and stabilizing Factor VIII, vWF provides a bridge through which platelets adhere to the subendothelial connective tissue in the event that the endothelium is wounded. This interaction permits platelet aggregation and is one of the earliest events in hemostasis. The normal interaction between platelets and vWF depends in part on the size and structure of vWF, which is a multimeric protein composed of 2 to >20 disulfide-linked protomers. The protomers comprise two identical polypeptide chains of about 200 kDa each. In normal individuals, the majority of circulating vWF is in the form of intermediate to very large multimers, which have been shown to be more effective in mediating platelet adhesion and aggregation than the smaller forms of vWF. A number of mutations of the vWF gene cause a selective loss of the high molecular weight forms of circulating vWF, leading to a bleeding disorder termed Type IIA von Willebrand's disease. There is evidence that these variants of vWF are more susceptible to proteolysis, most likely by a calpain-like enzyme, but the protease has not been characterized. In order to identify the responsible protease, we have modified the standard method of analyzing vWF monomers to increase speed and sensitivity. With this technique, we will use purified, high molecular weight multimers of vWF to assay for protease activity in normal and Type IIA vWD plasma. The protease will then be purified and analyzed structurally (partial sequencing, peptide mapping and immunochemical) and kinetically. Kinetic analyses may be extended to synthetic peptides containing the cleavage site and/or recombinant domains of normal and variant vWF. The effect of shear stress on the cleavage of normal and variant vWF by the protease will be of particular interest.

血管性血友病因子 (vWF) 是一种大糖蛋白，由 内皮细胞和血小板进入血浆和血管结缔组织 组织。 除了结合和稳定因子 VIII 外，vWF 提供了血小板粘附到内皮下细胞的桥梁 在内皮受伤时形成结缔组织。 这 相互作用允许血小板聚集，并且是最早的相互作用之一 止血事件。 血小板和 vWF 之间的正常相互作用 部分取决于 vWF 的大小和结构，vWF 是一种多聚体 由 2 至 >20 个二硫键连接的原聚体组成的蛋白质。 原体 包含两条相同的多肽链，每条约200kDa。 在 正常个体，大多数循环 vWF 的形式为 中等至非常大的多聚体，已被证明更 更有效地介导血小板粘附和聚集 vWF 的形式。 vWF 基因的许多突变导致选择性 循环 vWF 高分子量形式的损失，导致 一种称为 IIA 型冯维勒布兰德病的出血性疾病。 有 有证据表明 vWF 的这些变体更容易被蛋白水解， 最有可能是由钙蛋白酶样酶引起，但该蛋白酶尚未被 特点。 为了确定负责的蛋白酶，我们有 修改了分析 vWF 单体的标准方法以提高速度 和敏感性。 通过这种技术，我们将使用纯化的、高 vWF 的分子量多聚体用于测定蛋白酶活性 正常和 IIA 型 vWD 血浆。 然后将蛋白酶纯化并 结构分析（部分测序、肽图谱和 免疫化学）和动力学。 动力学分析可以扩展到 含有切割位点的合成肽和/或重组肽 正常和变异 vWF 的结构域。 剪应力对的影响 蛋白酶对正常和变异 vWF 的切割具有特殊意义 兴趣。