Protein Kinase C (PKC) are family of serine threonine kinases, known to regulate various platelet functional responses. Among them novel class of PKC isoforms (nPKC) including delta(δ), theta(𝛉), eta(η), and epsilon(ε) are expressed in platelets. Although, the role of nPKC ε and η in platelets is fairly understood, not much is known about nPKC ε and η in platelets. In this study, we investigated the role of nPKC ε in platelet functional responses using ADP-induced signaling as our stereotype. ADP causes platelet activation via Gq-coupled P2Y1 receptor and Gi-coupled P2Y12 receptor. Thus, we primarily studied the role of P2Y1 receptor in nPKC ε activation. ADP activated nPKC ε in time- and concentration- dependent manner. In the presence of P2Y1 receptor antagonist MRS-2179 and in P2Y1 knockout (KO) murine platelets ADP failed to activate nPKC ε, suggesting that ADP activates nPKC ε via P2Y1 receptor. We further investigated the functional role of nPKC ε using specific nPKC ε inhibitory RACK peptide (ε V1-2). ε V1-2 is a peptide designed to compete with native nPKC ε to bind ε-Receptors for activated C Kinase (ε-RACK) and thereby inhibits nPKC ε catalytic activity due to decreased substrate accessibility. ADP-induced thromboxane generation in human platelets pretreated with ε V1-2 peptide was more compared to the platelets pretreated with control peptide. Similarly, ADP-induced thromboxane generation in platelets derived from nPKC ε KO mouse was more compared to the wild type (WT) littermates. However, ADP- induced alpha granule secretion and aggregation in aspirin treated platelets derived from PKC ε KO mice was not significantly different from platelets derived from wild type littermates. These data suggest that nPKC e regulates an unknown pathway, which primarily regulates thromboxane generation with minimal effects on aggregation and alpha granule secretion. Furthermore, we also investigated the role of nPKC ε in PAR- and GPVI- mediated platelet aggregation and dense granule secretion. Interestingly, in both aspirin-treated and non-aspirin-treated platelets PAR- and GPVI- mediated platelet aggregation and dense granule secretion were potentiated. Consistent with ex vivo studies, FeCl3-induced arterial thrombosis was enhanced in nPKC ε KO mice compared to WT littermates.

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