Protein kinase C (PKC) isoforms have been implicated in several platelet functional responses, but the contribution of individual isoforms has not been thoroughly evaluated. Novel PKC isoform PKC-is activated by glycoprotein VI (GPVI) and protease-activated receptor (PAR) agonists, but not by adenosine diphosphate. In human platelets, PKC--selective antagonistic (RACK; receptor for activated C kinase) peptide significantly inhibited GPVI and PARinduced aggregation, dense and ␣-granule secretion at low agonist concentrations. Consistently, in murine platelets lacking PKC-, platelet aggregation and secretion were also impaired. PKCmediated phosphorylation of tSNARE protein syntaxin-4 was strongly reduced in human platelets pretreated with PKC-RACK peptide, which may contribute to the lower levels of granule secretion when PKC-function is lost. IntroductionPlatelet activation plays an important role in hemostasis, and the abnormal activation of platelets leads to thrombosis. 1 After circulating platelets are exposed to collagen-rich subendothelium at the site of vascular injury, platelets become activated, release granule contents, and generate thrombin and the lipid mediator thromboxane A 2 (TXA 2 ). 2,3 Secreted adenosine diphosphate (ADP), serotonin, and TXA 2 amplify the initial stimulus in a positive feedback activation of platelets. 2,3 In addition, ␣-granule proteins, such as P-selectin, mediating adhesive interactions between platelets, leukocytes, and endothelial cells, play a pivotal role in the pathogenesis of thrombosis and inflammation. 4 Glycoprotein VI (GPVI) and G-protein-coupled protease-activated receptors (PARs) are 2 dominant signaling receptors that mediate many of the important functional responses in platelets. [1][2][3] There are significant similarities in GPVI and PAR signaling, as phospholipase C (PLC) is activated by both pathways, which results in the generation of inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 mediates the release of Ca 2ϩ from intracellular stores, whereas DAG causes direct protein kinase C (PKC) activation. 3,5 Platelet aggregation requires the ␣ IIb ␤ 3 receptor to undergo a conformational change from a low-to a high-affinity state to bind ligands, such as fibrinogen, which is considered inside-out signaling. On the other hand, the pathway of outside-in signaling is induced by ligand binding to ␣ IIb ␤ 3 . 6,7 Human platelets express several PKC isoforms: ␣, ␤, , ⑀, ␦, , and . 8,9 Many functional responses, including platelet secretion, aggregation, and actin reorganization, have been shown to be positively regulated by PKC isoforms. 10 PKC-, as a member of PKC novel subfamily, is Ca 2ϩ -insensitive but This isoform contains a carboxyl-terminal catalytic domain with 2 conserved regions, C3 and C4, which are essential for catalytic activity and substrate binding, but lacks the calcium-binding C2 region. 12,13 After activation, PKC-is phosphorylated at threonine, serine (autophosphorylation site), and tyrosine residues. Among these, phosphorylation ...

Objective-Protein Kinase C delta (PKC␦) is expressed in platelets and activated downstream of protease-activated receptors (PAR)s and glycoprotein VI (GPVI) receptors. The purpose of this study was to investigate the role of PKC␦ in platelets. Methods and Results-We evaluated the role of PKC␦ in platelets using two approaches-pharmacological and molecular genetic approach. In human platelets pretreated with isoform selective antagonistic RACK peptide (␦ V1-1)TAT, and in the murine platelets lacking PKC␦, PAR4-mediated dense granule secretion was inhibited, whereas GPVI-mediated dense granule secretion was potentiated. These effects were statistically significant in the absence and presence of thromboxane A 2 (TXA 2 ). Furthermore, TXA 2 generation was differentially regulated by PKC␦. However, PKC␦ had a small effect on platelet P-selectin expression. Calcium-and PKC-dependent pathways independently activate fibrinogen receptor in platelets. When calcium pathways are blocked by dimethyl-BAPTA, AYPGKF-induced aggregation in PKC␦ null mouse platelets and in human platelets pretreated with (␦ V1-1)TAT, was inhibited.

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 cPLA 2 phosphorylation and thromboxane generation as well as ERK activation and intracellular calcium (Ca 2+ i ) mobilization, 2 signaling molecules, upstream of cPLA 2 activation. Thus, PKCs inhibit cPLA 2 activation by inhibiting ERK and Ca 2+ 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 PKCε null murine platelets compared with platelets from wild-type littermates. Interestingly, when thromboxane release is blocked, ADP-induced aggregation in PKCε knockout and wild-type was similar, suggesting that PKCε does not affect ADP-induced aggregation directly. PKCε knockout mice exhibited shorter times to occlusion in an FeCl 3 -induced arterial injury model and shorter bleeding times in tail-bleeding experiments. Conclusion— We conclude that PKCε negatively regulates ADP-induced thromboxane generation in platelets and offers protection against thrombosis.

PI3Ks (phosphoinositide 3-kinases) play a critical role in platelet functional responses. PI3Ks are activated upon P2Y12 receptor stimulation and generate pro-aggregatory signals. P2Y12 receptor has been shown to play a key role in the platelet aggregation and thromboxane A2 generation caused by co-stimulation with Gq or Gz, or super-stimulation of Gi pathways. In the present study, we evaluated the role of specific PI3K isoforms alpha, beta, gamma and delta in platelet aggregation, thromboxane A2 generation and ERK (extracellular-signal-regulated kinase) activation. Our results show that loss of the PI3K signal impaired the ability of ADP to induce platelet aggregation, ERK phosphorylation and thromboxane A2 generation. We also show that Gq plus Gi- or Gi plus Gz-mediated platelet aggregation, ERK phosphorylation and thromboxane A2 generation in human platelets was inhibited by TGX-221, a PI3Kbeta-selective inhibitor, but not by PIK75 (a PI3Kalpha inhibitor), AS252424 (a PI3Kgamma inhibitor) or IC87114 (a PI3Kdelta inhibitor). TGX-221 also showed a similar inhibitory effect on the Gi plus Gz-mediated platelet responses in platelets from P2Y1-/- mice. Finally, 2MeSADP (2-methyl-thio-ADP)-induced Akt phosphorylation was significantly inhibited in the presence of TGX-221, suggesting a critical role for PI3Kbeta in Gi-mediated signalling. Taken together, our results demonstrate that PI3Kbeta plays an important role in ADP-induced platelet aggregation. Moreover, PI3Kbeta mediates ADP-induced thromboxane A2 generation by regulating ERK phosphorylation.