Abstract
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.
Highlights
To understand the molecular basis of granule release from platelets, we examined the role of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in ␣-granule secretion
A role for PtdIns(4,5)P2 synthesis in PC12 cell granule secretion was demonstrated when type I PIPK and PtdIns transfer protein were identified as proteins within cytosol responsible for reconstituting ATP-mediated priming of granule secretion (29, 30)
The fact that Ca2ϩinduced ␣-granule secretion from SL-O-permeabilized platelets is inhibited by PtdIns-specific PLC (which cleaves PtdIns(4,5)P2), by exogenously added PtdIns(4,5)P2 (which may act by displacing proteins bound to endogenous PtdIns(4,5)P2), and by two different anti-type II PIPK antibodies (which inhibit PtdIns(4,5)P2 synthesis) provides strong evidence that PtdIns(4,5)P2 mediates ␣-granule secretion in this system
Summary
Chemicals and Reagents—All buffer constituents, solvents, ATP, CaCl2, PtdIns, PtdIns 4-phosphate, PtdIns(4,5)P2, and PtdIns-specific PLC (from Bacillus cereus) were purchased from Sigma. The anti-carboxyl-terminal end antibody was raised against a peptide consisting of the 18 carboxyl-terminal amino acids of type II␣ PIPK. Gel-filtered platelets (20 l) were incubated with 25 M fluorescein isothiocyanate-dextran sulfate of various molecular masses in the presence or absence of SL-O for 15 min. Phosphate-buffered saline (500 l) was added to the sample after a 20-min incubation, and the platelets were analyzed immediately by flow cytometry as described below. Immunoblot Analysis—Gel-filtered platelets (1–2 ϫ 107/ml) or GSTtagged PIPK was diluted in sample buffer (62.5 mM Tris-HCl, 2% SDS, 0.5% -mercaptoethanol, 10% glycerol, and 0.01% bromphenol blue) at 95 °C for 5 min. Data were analyzed using CellQuest software on a Macintosh Power PC
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