Platelet shape change is mediated by both calcium-dependent and -independent signaling pathways - Role of p160 Rho-associated coiled-coil-containing protein kinase in platelet shape change
Platelet shape change is mediated by both calcium-dependent and -independent signaling pathways - Role of p160 Rho-associated coiled-coil-containing protein kinase in platelet shape change
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DOI：
10.1074/jbc.274.40.28293
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发表时间：
1999-10-01
影响因子：
4.8
通讯作者：
Kunapuli, SP
中科院分区：
文献类型：
--
作者：
Paul, BZS;Daniel, JL;Kunapuli, SP
血小板在激动剂激活后发生形状变化，在形状变化过程中，圆盘状血小板变成具有突出丝状伪足的针状球体，当使用胞质 Ca2+ 螯合剂 5,5'-二甲基双（邻氨基苯氧基）乙烷-N,N,N',N'-四乙酸（5,5'-二甲基-BAPTA）阻止激动剂诱导的胞质 Ca2+ 增加时，血小板仍然发生形状变化，尽管发作被延迟并且初始速率显着降低。在没有胞质 Ca2+ 的情况下，激动剂刺激的肌球蛋白轻链磷酸化被显着抑制。对照血小板中的肌球蛋白轻链在 2 秒时达到最大磷酸化，而经 5,5'-二甲基-BAPTA 处理的血小板则需要 30 秒。 ADP、凝血酶或 U46619 诱导的不依赖于 Ca2+ 的血小板形状变化可被十字孢菌素（一种非选择性激酶抑制剂）、含有选择性 p160 Rho 相关卷曲螺旋的蛋白激酶抑制剂 Y-27632 或 HA 1077 显着降低。Y-27632 和 HA 1077 均降低了 ADP 诱导的血小板形状变化和肌球蛋白轻链磷酸化的峰值水平。控制血小板。在 5,5'-二甲基-BAPTA 处理的血小板中，Y-27632 和 HA 1077 完全消除了 ADP 诱导的血小板形状变化和肌球蛋白轻链磷酸化。我们的结果表明，Ca2+/钙调蛋白刺激的肌球蛋白轻链激酶和 p160 Rho 相关卷曲螺旋蛋白激酶分别通过 Ca2+ 敏感和 Ca2+ 不敏感途径独立促进肌球蛋白轻链磷酸化和血小板形状变化。
Platelets undergo shape change upon activation with agonists, During shape change, disc-shaped platelets turn into spiculated spheres with protruding filopodia, When agonist-induced cytosolic Ca2+ increases were prevented using the cytosolic Ca2+ chelator, 5,5'-dimethyl-bis-(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (5,5'-dimethyl-BAPTA), platelets still underwent shape change, although the onset was delayed and the initial rate was dramatically decreased. In the absence of cytosolic Ca2+, agonist-stimulated myosin light chain phosphorylation was significantly inhibited. The myosin light chain was maximally phosphorylated at 2 s in control platelets compared with 30 s in 5,5'-dimethyl-BAPTA-treated platelets. ADP, thrombin, or U46619-induced Ca2+-independent platelet shape change was significantly reduced by staurosporine, a nonselective kinase inhibitor, by the selective p160 Rho-associated coiled-coil containing protein kinase inhibitor Y-27632, or by HA 1077. Both Y-27632 and HA 1077 reduced peak levels of ADP-induced platelet shape change and myosin light chain phosphorylation in control platelets. In 5,5'-dimethyl-BAPTA-treated platelets, Y-27632 and HA 1077 completely abolished both ADP-induced platelet shape change and myosin light chain phosphorylation. Our results indicate that Ca2+/calmodulin-stimulated myosin light chain kinase and p160 Rho-associated coiled-coil-containing protein kinase independently contribute to myosin light chain phosphorylation and platelet shape change, through Ca2+-sensitive and Ca2+-insensitive pathways, respectively.