Activation of phospholipase C and protein kinase C has little involvement in ADP-induced primary aggregation of human platelets: effects of diacylglycerols, the diacylglycerols, the diacylglycerol kinase inhibitor R59022, staurosporine and okadaic acid.
Activation of phospholipase C and protein kinase C has little involvement in ADP-induced primary aggregation of human platelets: effects of diacylglycerols, the diacylglycerols, the diacylglycerol kinase inhibitor R59022, staurosporine and okadaic acid.
复制标题
磷脂酶 C 和蛋白激酶 C 的激活与 ADP 诱导的人血小板初级聚集关系不大：二酰基甘油、二酰基甘油、二酰基甘油激酶抑制剂 R59022、星形孢菌素和冈田酸的作用。
作者：
M. Packham;A. Livne;D. H. Ruben;M. L. Rand
ADP诱导的人血小板聚集的第一阶段不涉及可察觉的血栓素A2的形成或颗粒内容物的释放；也注意到缺乏三磷酸肌醇的形成。由于血小板对ADP的这些反应与对其他聚集剂的反应明显不同，因此我们研究了ADP诱导的二酰甘油聚集、蛋白激酶C聚集、胞浆[Ca~(2+)]升高、Pleckstrin(47 KDa)磷酸化以及磷酸酶1和2a的作用。用[14C]5-羟色胺预先标记的洗涤过的人血小板悬浮在Tyrode溶液(2 mM Ca~(2+)，1 mM Mg~(2+))中，比较2-4微米ADP在纤维蛋白原存在下和0.05单位/毫升凝血酶诱导的聚集。二酰甘油激酶抑制剂6-(2-[(4-fluorophenyl)phenyl-methylene]-1-piperidinylethyl)-7-meth-YL-5H-噻唑并[3，2-a]-嘧啶-5-酮(R59022；25微米)对腺苷二磷酸诱导的聚集没有或仅有轻微的促进作用，但在更大程度上增强了凝血酶诱导的反应。1，2-二己酰基-n-甘油或1-油酰基-2-乙酰基-n-甘油(25微米)在ADP前加入或30-90 S可显著增强聚集而不形成血栓素；蛋白激酶C的抑制剂星形孢子素可减弱这种增强作用。星形孢子素(25 NM)虽然能强烈抑制凝血酶诱导的聚集和[14C]5-羟色胺的释放，但不能抑制ADP诱导的聚集。所有这些观察表明，ADP诱导的初级聚集很少或根本不依赖于二酰甘油的形成或蛋白激酶C的激活。在2-4微米的范围内，ADP并不显著增加Pleckstrin的磷酸化(用[~(32)P]正磷酸标记的血小板进行研究)，但1，2-二己酰-sn-甘油诱导的Pleckstrin的磷酸化却被ADP增加。令人惊讶的是，在增强ADP诱导的聚集的同时，二酰甘油强烈地抑制了ADP诱导的胞浆[Ca~(2+)]升高；因此，初级聚集的程度与胞浆[Ca~(2+)]升高的水平无关。1，2-二己酰-n-甘油或1-油酰基-2-乙酰-n-甘油与血小板孵育数分钟，可逆转其促进聚集的作用，并观察到抑制作用。用冈田酸(一种磷酸酶1和2a的抑制剂)孵育血小板可以抑制ADP和凝血酶诱导的聚集；尽管这种作用的原因尚不清楚，但它不太可能涉及对磷脂酶C的抑制，因为二酰甘油的形成似乎与ADP诱导聚集的初级阶段几乎没有关系。
The primary phase of ADP-induced aggregation of human platelets does not involve appreciable formation of thromboxane A2 or release of granule contents; lack of formation of inositol trisphosphate has also been noted. Because these responses of platelets to ADP differ so markedly from their responses to other aggregating agents, the roles in ADP-induced aggregation of diacylglycerol, protein kinase C, increases in cytosolic [Ca2+], phosphorylation of pleckstrin (47 kDa) and phosphatases 1 and 2a were investigated. Washed human platelets, prelabelled with [14C]5-hydroxytryptamine and suspended in Tyrode solution (2 mM Ca2+, 1 mM Mg2+), were used for comparisons between the aggregation induced by 2-4 microM ADP, in the presence of fibrinogen, and that induced by 0.05 units/ml thrombin. The diacylglycerol kinase inhibitor 6-(2-[(4-fluorophenyl)phenyl-methylene]-1-piperidinylethyl)-7-meth yl-5H-thiazolo[3,2-a]-pyrimidin-5-one (R59022; 25 microM) had no, or only a slight, enhancing effect on ADP-induced aggregation, but potentiated thrombin-induced responses to a much greater extent. 1,2-Dihexanoyl-sn-glycerol or 1-oleoyl-2-acetyl-sn-glycerol (25 microM) added with or 30-90 s before ADP greatly potentiated aggregation without formation of thromboxane; staurosporine, an inhibitor of protein kinase C, reduced this potentiation. Staurosporine (25 nM) did not inhibit ADP-induced aggregation, although it strongly inhibited thrombin-induced aggregation and release of [14C]5-hydroxytryptamine. All these observations indicate little or no dependence of primary ADP-induced aggregation on the formation of diacylglycerol or on the activation of protein kinase C. At 2-4 microM, ADP did not significantly increase the phosphorylation of pleckstrin (studied with platelets prelabelled with [32P]orthophosphate), but 1,2-dihexanoyl-sn-glycerol- induced phosphorylation of pleckstrin was increased by ADP. Surprisingly, the diacylglycerols strongly inhibited the ADP-induced rise in cytosolic [Ca2+] concurrently with potentiation of ADP-induced aggregation; thus the extent of primary aggregation is independent of the level to which cytosolic [Ca2+] rises. Incubation of platelets with 1,2-dihexanoyl-sn-glycerol or 1-oleoyl-2-acetyl-sn-glycerol for several minutes reversed their potentiating effects on aggregation, and inhibition was observed. Incubation of platelets with okadaic acid, an inhibitor of phosphatases 1 and 2a, inhibited ADP- and thrombin-induced aggregation; although the reason for this effect is unknown, it is unlikely to involve inhibition of phospholipase C, since formation of diacylglycerol appears to have little involvement in the primary phase of ADP-induced aggregation.