ROLE AND REGULATION OF PLATELET PROTEIN KINASE C

蛋白激酶C（PKC）是一种重要的信号转导酶， 转导、细胞调节、细胞分化和肿瘤促进。 现在已知这种酶是作为一个密切相关的同种型家族存在的 其单独的机制和功能尚未确定。人 在血小板中，PKC与许多血小板功能有关 包括分泌和聚集以及负反馈机制 (on血小板活化）。我们已经确定了四种PKC同工酶， 血小板长期目标是明确PKC在血小板活化中的作用， 功能该建议旨在研究不同的PKC 同工酶被不同地调节以实现选择性功能。 我们的实验室在生物化学方面有着丰富的经验， PKC的特性和血小板生物学的研究，是唯一的 适合评估这些问题。这项建议的具体目标是， 因此，旨在明确PKC的作用机制和意义， 血小板的同工酶。这些将通过以下方法解决：1）纯化PKC 血小板同工酶（FPLC）及其体外研究 调节（使用我们开发的混合胶束方法）; 2） 确定静息和静息状态下PKC同工酶的细胞内定位， 活化血小板，分别（通过监测酶活性，佛波醇 结合和蛋白质印迹）和; 3）研究原位（生理） 血小板PKC同工酶的调节及其选择性 底物磷酸化（使用我们已经建立的体外模型， 发达）。这些研究将提供必要的生物化学背景 用于确定PKC同工酶的机制和生理调节。 这些知识对于提高我们对 血小板在止血中的作用以及在 动脉血管疾病这种生物化学知识的提高可能会导致 更有效、更合理的抗血小板药物开发。知识 从血小板模型中获得的信息应该被证明是非常有用的 研究PKC同工酶在各种疾病中的调节和作用的研究人员， 其他细胞系统。

Protein kinase C (PKC) has emerged as a critical enzyme in signal transduction, cell regulation, cell differentiation, and tumor promotion. The enzyme is now known to exist as a family of closely-related isoforms whose individual mechanism and function has not been defined. In human platelets, PKC has been implicated in a number of platelet functions including secretion and aggregation as well as negative feedback mechanisms (on platelet activation). We have identified four isoenzymes of PKC in platelets. The long-term goals are to define the role of PKC in platelet function. This proposal aims at studying the hypothesis that different PKC isoenzymes are differentially regulated to transduce selective functions. Our laboratory, which has extensive experience in the biochemical characterization of PKC and in the study of platelet biology, is uniquely suited to evaluate these questions. The specific aims of this proposal are, therefore, directed at determining the mechanism and significance of PKC isoenzymes from platelets. These will be addressed by: 1) purifying PKC isoenzymes from platelets (using FPLC) and studying their in vitro regulation (using mixed micellar methodologies that we have developed); 2) determining the intracellular localization of PKC isoenzymes in resting and activated platelets, respectively, (by monitoring enzyme activity, phorbol binding and by Western blots) and; 3) studying the in situ (physiologic) regulation of platelet PKC isoenzymes and determining their selective substrate phosphorylations (using an in vitro model that we have developed). These studies will provide the necessary biochemical background for determining the mechanism and physiologic regulation of PKC isoenzymes. Such knowledge is of great significance for improving our understanding of the role of platelets in hemostasis and in the pathophysiology of arteriovascular disease. This improved biochemical knowledge may lead to more efficacious and rational antiplatelet drug development. Knowledge gained from the platelet model should prove to be of great usefulness for investigators studying the regulation and role of PKC isoenzymes in various other cell systems.