Pandey Dharmendra, Goyal Pankaj, Dwivedi Suman, Siess Wolfgang
Institute for Prevention of Cardiovascular Diseases, University of Munich, Munich, Germany.
Blood. 2009 Jul 9;114(2):415-24. doi: 10.1182/blood-2008-10-183582. Epub 2009 May 8.
In platelets stimulated by thrombin to secrete and aggregate, cofilin is rapidly dephosphorylated leading to its activation. Cofilin by severing existing actin filaments and stimulating F-actin polymerization on newly created barbed ends dynamizes the actin cytoskeleton. We previously found that cofilin dephosphorylation is Ca(2+)-dependent and occurs upstream of degranulation in stimulated platelets. We report now in thrombin-stimulated platelets that Rac1 and class II PAKs (PAK4/5/6) were rapidly (within 5 seconds) activated, whereas PAK1/2 (class I PAKs) phosphorylation was slower. The Rac1-specific inhibitor NSC23766 blocked phosphorylation of class II PAKs, but not PAK1/2. Moreover, inhibition of the Ca(2+)/calmodulin-dependent phosphatase calcineurin inhibited Rac1 activation and class II PAKs phosphorylation. Prevention of Rac1 activation by calcineurin inhibition or NSC23766 also blocked cofilin dephosphorylation and platelet granule secretion indicating that a calcineurin/Rac1/class II PAKs pathway regulates cofilin dephosphorylation leading to secretion. We further found that PI3-kinases were activated downstream of Rac1, but were not involved in regulating cofilin dephosphorylation and secretion in thrombin-stimulated platelets. Our study unravels a Ca(2+)-dependent pathway of secretion in stimulated platelets as a signaling pathway linking Rac1 activation to actin dynamics: calcineurin-->Rac1-->class II PAKs-->cofilin activation. We further demonstrate that this pathway is separate and independent of the protein kinase C (PKC) pathway mediating secretion.
在凝血酶刺激下分泌和聚集的血小板中，丝切蛋白迅速去磷酸化从而被激活。丝切蛋白通过切断现有的肌动蛋白丝并在新形成的带刺末端刺激F-肌动蛋白聚合，使肌动蛋白细胞骨架动态化。我们之前发现丝切蛋白去磷酸化是Ca(2+)依赖性的，且发生在受刺激血小板脱颗粒的上游。我们现在报告，在凝血酶刺激的血小板中，Rac1和II类PAK（PAK4/5/6）迅速（在5秒内）被激活，而PAK1/2（I类PAK）的磷酸化较慢。Rac1特异性抑制剂NSC23766阻断了II类PAK的磷酸化，但不影响PAK1/2。此外，抑制Ca(2+)/钙调蛋白依赖性磷酸酶钙调神经磷酸酶可抑制Rac1激活和II类PAK磷酸化。通过抑制钙调神经磷酸酶或NSC23766来阻止Rac1激活，也会阻断丝切蛋白去磷酸化和血小板颗粒分泌，这表明钙调神经磷酸酶/Rac1/II类PAK途径调节丝切蛋白去磷酸化从而导致分泌。我们进一步发现PI3激酶在Rac1下游被激活，但不参与调节凝血酶刺激的血小板中丝切蛋白去磷酸化和分泌。我们的研究揭示了受刺激血小板中一种Ca(2+)依赖性分泌途径，作为一种将Rac1激活与肌动蛋白动态变化联系起来的信号通路：钙调神经磷酸酶-->Rac1-->II类PAK-->丝切蛋白激活。我们进一步证明该途径与介导分泌的蛋白激酶C（PKC）途径是分开且独立的。
