A TYPE-II PHOSPHOINOSITIDE 3-KINASE IS STIMULATED VIA ACTIVATED INTEGRIN IN PLATELETS - A SOURCE OF PHOSPHATIDYLINOSITOL 3-PHOSPHATE

We have observed that aggregation of human platelets, caused by activa tion of integrin alpha(IIb)beta(3) and its consequent binding of fibri nogen, stimulates a novel pathway for synthesis of phosphatidylinosito l 3,4-bisphosphate, thereby activating protein kinase B/Akt. Such synt hesis depends upon both the generation of phosphatidylinositol 3-phosp hate (PtdIns3P), which is sensitive to wortmannin (IC50 7 nM) and calp ain inhibitors, and the phosphorylation of PtdIns3P by PtdIns3P 4-kina se. We now report that a recently characterized C2 domain-containing p hosphoinositide 3-kinase iso form (HsC2-P13K) is present in platelets and a leukemic cell line (CHRF-288) derived from megakaryoblasts, and is likely to be responsible for the stimulated synthesis of PtdIns3P o bserved in platelets. HsC2-P13K, identifiable by Western blotting and immunoprecipitatable activity, is sensitive to wortmannin (IC50 6-10 n m), requires Mg2+, and shows strong preference for PtdIns over PtdIns4 P or phosphatidylinositol 4,5-bisphosphate as substrate. HsC2-P13K is activated severalfold when platelets aggregate in an alpha(IIb)beta(3) -dependent manner or when platelet or CHRF-288 lysates are incubated w ith Ca2+. Activation is prevented by calpain inhibitors. CHRF-288, whi ch cannot undergo activation of alpha(IIb)beta(3) and thereby aggregat e in response to platelet agonists, do not generate PtdIns3P or activa te HsC2-P13K under conditions that stimulate other phosphoinositide 3- kinases. HsC2-P13K may thus be an important effector for integrin-depe ndent signaling.