A novel integrin-activated pathway forms PKB/Akt-stimulatory phosphatidylinositol 3,4-bisphosphate via phosphatidylinositol 3-phosphate in platelets

The aggregation of human platelets is an important physiological hemostatic event contingent upon receptor-dependent activation of the surface integrin alpha(IIb)beta(3) and subsequent binding of fabrinogen. Aggregating platelets form phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P-2), which has been reported to stimulate in vitro the activity of the proto-oncogenic protein kinase PKB/Akt, as has phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P-3. It has been assumed that PtdIns(3,4)P-2 is synthesized by either 5-phosphatase-catalyzed hydrolysis of PtdIns(3,4,5)P-3 produced by phosphoinositide 3-kinase (PI3K) or phosphorylation by PI3K. of PtdIns4P. We investigated the route(s) by which PtdIns(3,4)P, is formed after directly activating alpha(IIb)beta(3) with anti-ligand-induced binding site Fab fragment and report that aggregation does not lead to the generation of PtdIns(3,4,5)P-3, but to transient formation of PtdIns3P and generation of PMIns(3,4)P-2, the latter primarily by PMIns3P 4-kinase. Both this novel pathway and the activation of PKB/Akt are inhibited by the PI3K inhibitor, wort-mannin, and the calpain inhibitor, calpeptin, constituting the first evidence that PMIns(3,4)P-2 can stimulate PKB/Akt in vivo in the absence of PtdIns(3,4,5)P-3, Integrin-activated generation of the second messenger PMIns(3,4)P-2 thus depends upon a route distinct from that known to be utilized initially by growth factors. This pathway is of potential general relevance to the function of integrins.