The PKB inhibitor Akti-1/2 potentiates PAR-1-mediated platelet function independently of its ability to block PKB.

Abstract

The role of PKB in platelet function is poorly defined due to the lack of genuinely selective small-molecule inhibitors and limiting genetic models. Recently, a selective, non-ATP-competitive PKB inhibitor, Akti-1/2 has been reported, but the efficacy and specificity against PKB activation in platelet function is unknown. To determine the effect of the PKB inhibitor Akti-1/2 on PKB activation and platelet function by Western blotting and aggregometry/flow cytometry, respectively. Akti-1/2 potently inhibited thrombin-mediated PKB phosphorylation on Thr(308) and Ser(473) and phosphorylation of its downstream substrate GSK3beta, with a negligible effect on the phosphorylation of pleckstrin, p38, ERK and JNK. Surprisingly, Akti-1/2 strongly potentiated PAR-1-mediated platelet aggregation. This effect persisted in the presence of PI3 kinase inhibitors, indicating a mechanism of action that is independent of PKB. Potentiation of aggregation by Akti-1/2 was associated with increased [Ca(++)](i), PKC activation and degranulation and was ablated by agents that antagonized this pathway. The PKB inhibitor Akti-1/2 increases PAR-1-mediated platelet responses in a PKB-independent, Ca(++)/PKC-dependent manner. This effect is strong and rapid and may impact on the therapeutic application of Akti-1/2 and structurally related compounds.