Protein Kinase C Isoform ε Negatively Regulates ADP-Induced Calcium Mobilization and Thromboxane Generation in Platelets

Objective— Members of the protein kinase C (PKC) family are shown to positively and negatively regulate platelet activation. Although positive regulatory roles are extensively studied, negative regulatory roles of PKCs are poorly understood. We investigated the mechanism and specific isoforms involved in PKC-mediated negative regulation of ADP-induced functional responses. Methods and Results— A pan-PKC inhibitor, GF109203X, potentiated ADP-induced cPLA2 phosphorylation and thromboxane generation as well as ERK activation and intracellular calcium (Ca2+i) mobilization, 2 signaling molecules, upstream of cPLA2 activation. Thus, PKCs inhibit cPLA2 activation by inhibiting ERK and Ca2+i mobilization. Because the inhibitor of classic PKC isoforms, GO-6976, did not affect ADP-mediated thromboxane generation, we investigated the role of novel class of PKC isoforms. ADP-induced thromboxane generation, calcium mobilization, and ERK phosphorylation were potentiated in PKCe null murine platelets compared with platelets from wild-type littermates. Interestingly, when thromboxane release is blocked, ADP-induced aggregation in PKCe knockout and wild-type was similar, suggesting that PKCe does not affect ADP-induced aggregation directly. PKCe knockout mice exhibited shorter times to occlusion in an FeCl3-induced arterial injury model and shorter bleeding times in tail-bleeding experiments. Conclusion— We conclude that PKCe negatively regulates ADP-induced thromboxane generation in platelets and offers protection against thrombosis.