Mechanism of Activation and Functional Role of Protein Kinase Cη in Human Platelets (original) (raw)

2009, Journal of Biological Chemistry

The novel class of protein kinase C (nPKC) isoform 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 using pharmacological and gene knockout approaches. nPKC 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. Similarly, ADP failed to activate nPKC in platelets isolated from P2Y 1 and G q knockout mice. However, pretreatment of platelets with P2Y 12 receptor antagonist, AR-C69331MX did not interfere with ADP-induced nPKC phosphorylation. In addition, when platelets were activated with 2MeSADP under stirring conditions, although nPKC was phosphorylated within 30 s by ADP receptors, it was also dephosphorylated by activated integrin ␣ IIb ␤ 3 mediated outside-in signaling. Moreover, in the presence of SC-57101, a ␣ IIb ␤ 3 receptor antagonist, nPKC dephosphorylation was inhibited. Furthermore, in murine platelets lacking PP1c␥, a catalytic subunit of serine/threonine phosphatase, ␣ IIb ␤ 3 failed to dephosphorylate nPKC. Thus, we conclude that ADP activates nPKC via P2Y 1 receptor and is subsequently dephosphorylated by PP1␥ phosphatase activated by ␣ IIb ␤ 3 integrin. In addition, pretreatment of platelets with-RACK antagonistic peptides, a specific inhibitor of nPKC, inhibited ADP-induced thromboxane generation. However, these peptides had no affect on ADP-induced aggregation when thromboxane generation was blocked. In summary, nPKC positively regulates agonist-induced thromboxane generation with no effects on platelet aggregation.