ROLE OF PROTEOLYSIS BY CANP IN PLATELET ACTIVATION

在刺激血小板时，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）。 这种酶催化几种蛋白质的有限蛋白水解 其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. 为了增加我们对参与的机制的了解， 血小板活化，这是至关重要的，我们了解的结构， 这些蛋白质的功能以及它们如何响应刺激而被修饰。 本提案的目的是描述CANP的特点， CANP可能参与血小板活化的机制。 CANP有 从人血小板中纯化并通过其他方法部分表征， investigators. 本研究将对CANP进行纯化， 其特征在于底物特异性和 两种形式的CANP。 一种形式的CANP（需要高 钙离子浓度）可以通过自身蛋白水解转化为钙离子浓度。 其他形式（需要更低的，更生理的，浓度的 钙离子）。 本报告所用方法的一个重要组成部分是： 该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 通过免疫电镜定位CANP。 该提案将涉及若干具体机制， 参与血小板活化。 是CANP敏感的高分子 糖蛋白Ib和肌动蛋白结合蛋白的重量复合物， 激活血小板？ 是CANP激活蛋白激酶C 参与血小板活化的各种机制， 不同的刺激？ CANP是否在激活 磷脂酶C 磷脂酶A2是否存在于血小板膜中， 能被CANP激活的非活性酶原？ 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。

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.