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.

von Willebrand因子（VWF）是一种大的糖蛋白，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，VWF 提供了一个桥梁，血小板粘附在下皮上 结缔组织，如果内皮受伤。 这 相互作用允许血小板聚集，并且是最早的 止血事件。 血小板和VWF之间的正常相互作用 一部分取决于VWF的大小和结构，这是一个多聚体 蛋白质由2至> 20个二硫键的原型组成。 原始人 包括两个相同的多肽链每个约200 kDa。 在 普通人，大多数循环vwf的形式是 中间到非常大的多聚体，已显示出更多 比较小的有效介导血小板粘附和聚集 vwf的形式。 VWF基因的许多突变引起选择性 循环vWF的高分子重量形式的损失，导致 一种流血疾病，称为IIA型von Willebrand病。 有 证据表明这些VWF的变体更容易受到蛋白水解的影响， 最有可能是类似钙蛋白酶的酶，但蛋白酶尚未 特征。 为了识别负责任的蛋白酶，我们有 修改了分析VWF单体以提高速度的标准方法 和灵敏度。 使用这种技术，我们将使用纯化的高 VWF的分子量多聚体以分析蛋白酶活性 正常和类型IIA VWD等离子体。 然后将蛋白酶纯化，并 在结构上进行分析（部分测序，肽映射和 免疫化学）和动力学。 动力学分析可能扩展到 包含切割位点和/或重组的合成肽 正常和变体VWF的域。 剪切应力对 蛋白酶对正常和变体VWF的裂解将特别 兴趣。