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 hemos tatic event contingent upon receptor-dependent activation of the surfa ce integrin alpha(IIb)beta(3) and subsequent binding of fabrinogen. Ag gregating 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 phosphatidylinos itol 3,4,5-trisphosphate (PtdIns(3,4,5)P-3. It has been assumed that P tdIns(3,4)P-2 is synthesized by either 5-phosphatase-catalyzed hydroly sis of PtdIns(3,4,5)P-3 produced by phosphoinositide 3-kinase (PI3K) o r phosphorylation by PI3K. of PtdIns4P. We investigated the route(s) b y which PtdIns(3,4)P, is formed after directly activating alpha(IIb)be ta(3) with anti-ligand-induced binding site Fab fragment and report th at aggregation does not lead to the generation of PtdIns(3,4,5)P-3, bu t 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-ma nnin, and the calpain inhibitor, calpeptin, constituting the first evi dence 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 messe nger 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 potenti al general relevance to the function of integrins.