BIPHASIC ACTIVATION OF PKB-ALPHA AKT IN PLATELETS - EVIDENCE FOR STIMULATION BOTH BY PHOSPHATIDYLINOSITOL 3,4-BISPHOSPHATE, PRODUCED VIA A NOVEL PATHWAY, AND BY PHOSPHATIDYLINOSITOL 3,4,5-TRISPHOSPHATE/

Stimulation of platelet thrombin receptors or protein kinase C causes fibrinogen-dependent aggregation that is a function of integrin alpha( IIb)beta(3) activation. Such platelets rapidly and transiently form ph osphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P-3) and a small amount of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P-2). Afte r aggregation, a larger amount of PtdIns(3,4)P-2 is generated. We repo rt that this latter PtdIns(3,4)P-2 arises largely through wortmannin-i nhibitable generation of PtdIns3P and then phosphorylation by PtdIns3P 4-kinase (PtdIns3P 4-K), a novel pathway apparently contingent upon t he activation of the Ca2+-dependent protease calpain. Elevation of cyt osolic Ca2+ by ionophore, without integrin/ligand binding, is insuffic ient to activate the pathway. PtdIns3P 4-K is not the recently describ ed ''PIP5KII alpha.'' Cytoskeletal activities of phosphatidylinositol 3-kinase and PtdIns3P 4-K increase after aggregation. Prior to aggrega tion, PtdIns3P 4-K can be regulated negatively by the beta gamma subun it of heterotrimeric GTP-binding protein. After aggregation, PtdIns3P 4-K calpain-dependently loses its susceptibility to G beta gamma and i s, in addition, activated. Both PtdIns(3,4,5)P-3 and PtdIns(3, 4)P-2 h ave been shown to stimulate PKB alpha/Akt phosphorylation and activati on by phosphoinositide-dependent kinase 1. We find that activation of PKB alpha Akt in platelets is phosphorylation-dependent and biphasic; the initial phase is PtdIns(3,4,5)P-3-dependent and more efficient, wh ereas the second phase depends upon PtdIns(3,4)P-2 generated after agg regation. There is thus potential for both pre-and post aggregation-de pendent signaling by PKB alpha Akt.