Riddell D R, Graham A, Owen J S
University Department of Medicine, Royal Free Hospital School of Medicine, London, United Kingdom.
J Biol Chem. 1997 Jan 3;272(1):89-95. doi: 10.1074/jbc.272.1.89.
We have previously reported that plasma apolipoprotein (apo) E-containing high density lipoprotein particles have a potent anti-platelet action, apparently by occupying saturable binding sites in the cell surface. Here we show that purified apoE (10-50 microg/ml), complexed with phospholipid vesicles (dimyristoylphosphatidylcholine, DMPC), suppresses platelet aggregation induced by ADP, epinephrine, or collagen. This effect was not due to sequestration of cholesterol from platelet membranes; apoE x DMPC chemically modified with cyclohexanedione (cyclohexanedione-apoE x DMPC) did not inhibit aggregation but nevertheless removed similar amounts of cholesterol as untreated complexes, about 2% during the aggregation period. Rather we found that apoE influenced intracellular platelet signaling. Thus, apoE x DMPC markedly increased cGMP in ADP-stimulated platelets which correlated with the resulting inhibition of aggregation (r = 0.85; p < 0.01, n = 10), whereas cyclohexanedione-apoE x DMPC vesicles had no effect. One important cellular mechanism for up-regulation of cGMP is through stimulation of nitric oxide (NO) synthase, the NO generated by conversion of L-arginine to L-citrulline, binds to and activates guanylate cyclase. This signal transduction pathway was implicated by the finding that NO synthase inhibitors of distinct structural and functional types all reversed the anti-platelet action of apoE, whereas a selective inhibitor of soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (100 nM), had a similar reversing action. Direct confirmation that apoE stimulates NO synthase was obtained by use of L-[3H]arginine; platelets pretreated with apoE x DMPC produced markedly more L-[3H]citrulline (0.71 +/- 0.1 pmol/h/10(9) platelets) than controls (0.18 +/- 0.03; p < 0.05). In addition, hemoglobin which avidly binds NO also suppressed the anti-aggregatory effect, indicating that apoE stimulated sufficient production of NO by platelets for extracellular release to occur. We conclude that apoE inhibits platelet aggregation through the L-arginine:NO signal transduction pathway.
我们之前曾报道，血浆中含有载脂蛋白（apo）E的高密度脂蛋白颗粒具有强大的抗血小板作用，显然是通过占据细胞表面的饱和结合位点来实现的。在此我们表明，与磷脂囊泡（二肉豆蔻酰磷脂酰胆碱，DMPC）复合的纯化apoE（10 - 50微克/毫升）可抑制由ADP、肾上腺素或胶原诱导的血小板聚集。这种作用并非由于从血小板膜中隔离胆固醇所致；用环己二酮化学修饰的apoE x DMPC（环己二酮 - apoE x DMPC）并不抑制聚集，但在聚集期间仍能去除与未处理复合物相似量的胆固醇，约为2%。相反，我们发现apoE影响细胞内血小板信号传导。因此，apoE x DMPC在ADP刺激的血小板中显著增加cGMP，这与随后的聚集抑制相关（r = 0.85；p < 0.01，n = 10），而环己二酮 - apoE x DMPC囊泡则无此作用。cGMP上调的一个重要细胞机制是通过刺激一氧化氮（NO）合酶，由L - 精氨酸转化为L - 瓜氨酸所产生的NO，与鸟苷酸环化酶结合并激活它。这一信号转导途径可由以下发现得到证实：不同结构和功能类型的NO合酶抑制剂均能逆转apoE的抗血小板作用，而可溶性鸟苷酸环化酶的选择性抑制剂1H - [1,2,4]恶二唑并[4,3 - a]喹喔啉 - 1 - 酮（100 nM）具有类似的逆转作用。通过使用L - [3H]精氨酸直接证实了apoE刺激NO合酶；用apoE x DMPC预处理的血小板产生的L - [3H]瓜氨酸（0.71 ± 0.1皮摩尔/小时/10⁹个血小板）明显多于对照组（0.18 ± 0.03；p < 0.05）。此外，能 avidly结合NO的血红蛋白也抑制了抗聚集作用，表明apoE刺激血小板产生了足够的NO以进行细胞外释放。我们得出结论，apoE通过L - 精氨酸：NO信号转导途径抑制血小板聚集。
