Medizinische Klinik III, Dept. of Cardiology and Cardiovascular Diseases, Eberhard Karls University, Tübingen, Germany.
Cell Signal. 2012 Sep;24(9):1743-52. doi: 10.1016/j.cellsig.2012.04.018. Epub 2012 May 3.
Platelet aggregation, secretion and thrombus formation play a critical role in primary hemostasis to prevent excessive blood loss. On the other hand, uncontrolled platelet activation leads to pathological thrombus formation resulting in myocardial infarction or stroke. Stimulation of heterotrimeric G-proteins by soluble agonists or immunoreceptor tyrosine based activation motif-coupled receptors that interact with immobilized ligands such as the collagen receptor glycoprotein (GP) VI lead to the activation of phospholipases that cleave membrane phospholipids to generate soluble second messengers. Platelets contain the phospholipases (PL) D1 and D2 which catalyze the hydrolysis of phosphatidylcholine to generate the second messenger phosphatidic acid (PA). The production of PA is abrogated by primary alcohols that have been widely used for the analysis of PLD-mediated processes. However, it is not clear if primary alcohols effectively reduce PA generation or if they induce PLD-independent cellular effects. In the present study we made use of the specific PLD inhibitor 5-fluoro-2-indolyl des-chlorohalopemide (FIPI) and show for the first time, that FIPI enhances platelet dense granule secretion and aggregation of human platelets. Further, FIPI has no effect on cytosolic Ca(2+) activity but needs proper Rho kinase signaling to mediate FIPI-induced effects on platelet activation. Upon FIPI treatment the phosphorylation of the PKC substrate pleckstrin was prominently enhanced suggesting that FIPI affects PKC-mediated secretion and aggregation in platelets. Similar effects of FIPI were observed in platelets from mouse wild-type and Pld1(-/-) mice pointing to a new role for PLD2 as a negative regulator of platelet sensitivity.
血小板聚集、分泌和血栓形成在初级止血中起着至关重要的作用，以防止过度失血。另一方面，不受控制的血小板激活导致病理性血栓形成，导致心肌梗死或中风。可溶性激动剂或免疫受体酪氨酸基激活基序偶联受体与固定化配体（如胶原受体糖蛋白[GP] VI）相互作用刺激异三聚体 G 蛋白，导致磷脂酶的激活，将膜磷脂水解产生可溶性第二信使。血小板含有磷脂酶（PL）D1 和 D2，它们催化磷脂酰胆碱的水解生成第二信使磷脂酸（PA）。初级醇广泛用于 PLD 介导的过程分析，可阻断 PA 的产生。然而，目前尚不清楚初级醇是否能有效地减少 PA 的生成，或者它们是否会诱导 PLD 非依赖性的细胞效应。在本研究中，我们利用特定的 PLD 抑制剂 5-氟-2-吲哚基去氯卤代苯并恶嗪（FIPI），并首次表明 FIPI 增强了人血小板致密颗粒的分泌和聚集。此外，FIPI 对胞质 Ca(2+)活性没有影响，但需要适当的 Rho 激酶信号转导来介导 FIPI 对血小板激活的影响。在 FIPI 处理后，PKC 底物 pleckstrin 的磷酸化明显增强，表明 FIPI 影响 PKC 介导的血小板分泌和聚集。在来自野生型小鼠和 Pld1(-/-)小鼠的血小板中观察到 FIPI 的类似作用，表明 PLD2 作为血小板敏感性的负调节剂具有新的作用。
