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.
Rozenvayn
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N
Division of Hemostasis and Thrombosis Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.
Flaumenhaft
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R
D000730:Androstadienes / Q000494:pharmacology; D000906:Antibodies / Q000276:immunology; D001792:Blood Platelets / Q000201:enzymology / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002867:Chromones / Q000494:pharmacology; D003594:Cytoplasmic Granules / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000276:immunology / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000502:physiology*; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D010738:Type C Phospholipases / Q000378:metabolism; D000077191:Wortmannin
D000730:Androstadienes; D000906:Antibodies; D002118:Calcium; D002867:Chromones; D004791:Enzyme Inhibitors; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D019269:Phosphatidylinositol 4,5-Diphosphate; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017853:Phosphotransferases (Alcohol Group Acceptor); D010738:Type C Phospholipases; D000077191:Wortmannin; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C020077:1-phosphatidylinositol-4-phosphate 5-kinase