ROLE OF PROTEOLYSIS BY CANP IN PLATELET ACTIVATION

CANP 蛋白水解在血小板激活中的作用

• 批准号: 3448822
• 负责人: JANICE R OKITA
• 金额: $ 5.37万
• 依托单位: VERSITI WISCONSIN, INC.
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-04-01 至 1988-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3448822
• 关键词: calcium; chemical structure function; chromatography; coagulation factor VIII; fibrinogen; histochemistry /cytochemistry; human tissue; immunochemistry; immunoelectron microscopy; immunohematology; membrane structure; molecular weight; monoclonal antibody; peptidases; phospholipase A2; phospholipase C; platelet activating factor; platelet aggregation; protein kinase C; proteolysis

Upon stimulation of platelets, a number of different events occur. One of these events is the activation of a calcium-activated neutral protease (CANP). This enzyme catalyzes the limited proteolysis of several proteins which are intimately involved in platelet function: glycoprotein Ib, actin-binding protein, von Willebrand factor, figrinogen and protein kinase C. In order to increase our knowledge of the mechanisms involved in platelet activation, it is essential that we understand the structure and function of these proteins and how they are modified in response to stimuli. The objectives of this proposal are to characterize CANP and to investigate mechanisms by which CANP may be involved in platelet activation. CANP has been purified from human platelets and partially characterized by other investigators. In this investigation, CANP will be purified and further characterized with respect to substrate specificity and the relationship between the two forms of CANP. One form of CANP (which requires a high concentration of calcium ions) may be converted by autoproteolysis to the other form (which requires a lower, more physiological, concentration of calcium ions). An important component of the methodology used in this project will be the development of a murine monoclonal antibody against CANP. The monoclonal antibody will be used to determine the subcellular localization of CANP by immunoelectron microscopy. This proposal will address several specific mechanisms by which CANP may be involved in platelet activation. Are the CANP-sensitive high molecular weight complexes of glycoprotein Ib and actin-binding protein degraded upon activation of platelets? Is the activation of protein kinase C by CANP involved in any of the various mechanisms of platelet activation by different stimuli? Does CANP play a role in the activation of phospholipase C? Is phospholipase A2 present in platelet membranes as an inactive proenzyme which can be activated by CANP? This investigation will lead to a better understanding of the events that take place upon platelet stimulation and aggregation and thus provide new insight into the etiology of thrombotic and hemostatic disorders.

当血小板受到刺激时，会发生许多不同的事件。 之一 这些事件是钙激活中性蛋白酶的激活 （CANP）。 该酶催化多种蛋白质的有限蛋白水解 与血小板功能密切相关：糖蛋白 Ib， 肌动蛋白结合蛋白、血管性血友病因子、figrinogen 和蛋白激酶 C. 为了增加我们对所涉及机制的了解 血小板活化，我们必须了解其结构和 这些蛋白质的功能以及它们如何响应刺激而被修改。 本提案的目标是描述 CANP 的特征并调查 CANP可能参与血小板活化的机制。 CANP 有 从人类血小板中纯化出来，并通过其他方法进行部分表征 调查人员。 在本次调查中，CANP将被纯化并进一步 就底物特异性和关系进行表征 CANP 的两种形式之间。 CANP 的一种形式（需要高 钙离子浓度）可以通过自蛋白水解转化为 其他形式（需要更低、更生理性的浓度） 钙离子）。 本研究中使用的方法的一个重要组成部分 项目将开发一种鼠单克隆抗体 CANP。 单克隆抗体将用于确定亚细胞 通过免疫电子显微镜定位 CANP。 该提案将解决 CANP 可能通过的几个具体机制 参与血小板活化。 CANP 敏感的高分子吗 糖蛋白 Ib 和肌动蛋白结合蛋白的重量复合物降解 血小板活化？ CANP激活蛋白激酶C吗 参与血小板活化的任何各种机制 不同的刺激？ CANP 是否在激活中发挥作用？ 磷脂酶C? 血小板膜中是否存在磷脂酶 A2 CANP可以激活无活性的酶原吗？ 这项调查将有助于更好地了解事件 在血小板刺激和聚集时发生，从而提供新的 深入了解血栓和止血疾病的病因。