ROLE AND REGULATION OF PLATELET PROTEIN KINASE C

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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.