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 examined the role of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) in alpha-granule secretion. Streptolysin O-permeabilized platelets synthesized PtdIns(4,5)P(2) when incubated in the presence of ATP. Incubation of streptolysin O-permeabilized platelets with phosphatidylinositol-specific phospholipase C reduced PtdIns(4,5)P(2) levels and resulted in a dose- and time-dependent inhibition of Ca(2+)-induced alpha-granule secretion. Exogenously added PtdIns(4,5)P(2) inhibited alpha-granule secretion, with 80% inhibition at 50 microm PtdIns(4,5)P(2). Nanomolar concentrations of wortmannin, 33.3 microm LY294002, and antibodies directed against PtdIns 3-kinase did not inhibit Ca(2+)-induced alpha-granule secretion, suggesting that PtdIns 3-kinase is not involved in alpha-granule secretion. However, micromolar concentrations of wortmannin inhibited both PtdIns(4,5)P(2) synthesis and alpha-granule secretion by approximately 50%. Antibodies directed against type II phosphatidylinositol-phosphate kinase (phosphatidylinositol 5-phosphate 4-kinase) also inhibited both PtdIns(4,5)P(2) synthesis and Ca(2+)-induced alpha-granule secretion by approximately 50%. These antibodies inhibited alpha-granule secretion only when added prior to ATP exposure and not when added following ATP exposure, prior to Ca(2+)-mediated triggering. The inhibitory effects of micromolar wortmannin and anti-type II phosphatidylinositol-phosphate kinase antibodies were additive. These results show that PtdIns(4,5)P(2) mediates platelet alpha-granule secretion and that PtdIns(4,5)P(2) synthesis required for Ca(2+)-induced alpha-granule secretion involves the type II phosphatidylinositol 5-phosphate 4-kinase-dependent pathway.