Rozenvayn N, Flaumenhaft R
Division of Hemostasis and Thrombosis Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.
J Biol Chem. 2001 Jun 22;276(25):22410-9. doi: 10.1074/jbc.M008184200. Epub 2001 Apr 13.
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.
为了解血小板颗粒释放的分子基础，我们研究了磷脂酰肌醇4,5 - 二磷酸（PtdIns(4,5)P(2)）在α颗粒分泌中的作用。经链球菌溶血素O通透处理的血小板在ATP存在下孵育时会合成PtdIns(4,5)P(2)。用磷脂酰肌醇特异性磷脂酶C孵育经链球菌溶血素O通透处理的血小板会降低PtdIns(4,5)P(2)水平，并导致对Ca(2+)诱导的α颗粒分泌产生剂量和时间依赖性抑制。外源添加的PtdIns(4,5)P(2)抑制α颗粒分泌，在50微摩尔PtdIns(4,5)P(2)时抑制率达80%。纳摩尔浓度的渥曼青霉素、33.3微摩尔LY294002以及针对磷脂酰肌醇3 - 激酶的抗体均不抑制Ca(2+)诱导的α颗粒分泌，这表明磷脂酰肌醇3 - 激酶不参与α颗粒分泌。然而，微摩尔浓度的渥曼青霉素对PtdIns(4,5)P(2)合成和α颗粒分泌的抑制率均约为50%。针对II型磷脂酰肌醇 - 磷酸激酶（磷脂酰肌醇5 - 磷酸4 - 激酶）的抗体也对PtdIns(4,5)P(2)合成和Ca(2+)诱导的α颗粒分泌产生约50%的抑制作用。这些抗体仅在ATP暴露之前添加时才抑制α颗粒分泌，而在ATP暴露之后、Ca(2+)介导的触发之前添加时则无此作用。微摩尔渥曼青霉素和抗II型磷脂酰肌醇 - 磷酸激酶抗体的抑制作用具有加和性。这些结果表明，PtdIns(4,5)P(2)介导血小板α颗粒分泌，且Ca(2+)诱导的α颗粒分泌所需的PtdIns(4,5)P(2)合成涉及II型磷脂酰肌醇5 - 磷酸4 - 激酶依赖性途径。
