Phosphatidylinositol 4,5-bisphosphate mediates Ca2+-induced platelet alpha-granule secretion - Evidence for type II phosphatidylinositol 5-phosphate 4-kinase function

To understand the molecular basis of granule release from platelets, we exa mined the role of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P-2) in alpha -granule secretion. Streptolysin O-permeabilized platelets synthesiz ed PtdIns(4,5)P-2 when incubated in the presence of ATP. Incubation of stre ptolysin O permeabilized platelets with phosphatidylinositol-specific phosp holipase C reduced PtdIns(4,5)P-2 levels and resulted in a dose and time-de pendent inhibition of Ca2+-induced alpha -granule secretion. Exogenously ad ded PtdIns(4,5)P-2 inhibited alpha -granule secretion, with 80% inhibition at 50 muM PtdIns(4,5)P-2. Nanomolar concentrations of wortmannin, 33.3 muM LY294002, and antibodies directed against PtdIns 3-kinase did not inhibit C a2+-induced alpha -granule secretion, suggesting that PtdIns 3-kinase is no t involved in alpha -granule secretion. However, micromolar concentrations of wortmannin inhibited both PtdIns(4,5)P-2 synthesis and alpha -granule se cretion by similar to 50%. Antibodies directed against type II phosphatidyl inositol-phosphate kinase (phosphatidylinositol 5-phosphate 4-kinase) also inhibited both PtdIns(4,5)P-2 synthesis and Ca2+-induced alpha -granule sec retion by similar to 50%. These antibodies inhibited alpha -granule secreti on only when added prior to ATP exposure and not when added following ATP e xposure, prior to Ca2+-mediated triggering. The inhibitory effects of micro molar wortmannin and anti-type II phosphatidylinositol-phosphate kinase ant ibodies were additive, These results show that PtdIns(4,5)P-2 mediates plat elet alpha -granule secretion and that PtdIns(4,5)P-2 synthesis required fo r Ca2+-induced alpha -granule secretion involves the type II phosphatidylin ositol 5-phosphate 4-kinase-dependent pathway.