Mechanism of Activation and Functional Role of Protein Kinase Cη in Human Platelets

The novel class of protein kinase C (nPKC) isoform eta is expressed in platelets, but not much is known about its activation and function. In this study, we investigated the mechanism of activation and functional implications of nPKC eta using pharmacological and gene knock-out approaches. nPKC eta was phosphorylated (at Thr-512) in a time-and concentration-dependent manner by 2MeSADP. Pretreatment of platelets with MRS-2179, a P2Y(1) receptor antagonist, or YM-254890, a G(q) blocker, abolished 2MeSADP-induced phosphorylation of nPKC eta. Similarly, ADP failed to activate nPKC eta in platelets isolated from P2Y(1) and G(q) knock-out mice. However, pretreatment of platelets with P2Y(12) receptor antagonist, AR-C69331MX did not interfere with ADP-induced nPKC eta phosphorylation. In addition, when platelets were activated with 2MeSADP under stirring conditions, although nPKC eta was phosphorylated within 30 s by ADP receptors, it was also dephosphorylated by activated integrin alpha(II)b beta(3) mediated outside-in signaling. Moreover, in the presence of SC-57101, alpha(II)b beta(3) receptor antagonist, nPKC eta dephosphorylation was inhibited. Furthermore, in murine platelets lacking PP1C gamma, a catalytic subunit of serine/threonine phosphatase, alpha(II)b beta(3) failed to dephosphorylate n PKC eta. Thus, we conclude that ADP activates nPKC eta via P2Y(1) receptor and is subsequently dephosphorylated by PP1 gamma phosphatase activated by alpha(II)b beta(3) integrin. In addition, pretreatment of platelets with eta-RACK antagonistic peptides, a specific inhibitor of nPKC eta, inhibited ADP-induced thromboxane generation. However, these peptides had no affect on ADP-induced aggregation when thromboxane generation was blocked. In summary, nPKC eta positively regulates agonist-induced thromboxane generation with no effects on platelet aggregation.