Borst Oliver, Walker Britta, Münzer Patrick, Russo Antonella, Schmid Evi, Faggio Caterina, Bigalke Boris, Laufer Stefan, Gawaz Meinrad, Lang Florian
Department of Physiology, University of Tübingen, Gmelinstrasse 5, Tübingen, Germany.
Cell Physiol Biochem. 2013;31(6):914-24. doi: 10.1159/000350110. Epub 2013 Jun 18.
BACKGROUND/AIMS: Platelets are critically important for primary haemostasis and the major players in thrombotic vascular occlusion. Platelets are activated by agonists, such as thrombin and collagen-related peptide as well as second-wave mediators including thromboxane A2 via different intracellular signaling pathways resulting in degranulation, aggregation and thrombus formation. Platelet activation is paralleled by phosphorylation and activation of p38 MAPK. The limited specificity of hitherto known p38 MAPK inhibitors precluded safe conclusions on the precise role of p38 MAPK in the regulation of platelet function. The present study examined the impact of Skepinone-L, a novel and highly selective inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), on platelet activation and thrombus formation.
Experiments were performed in freshly isolated human platelets. Protein phosphorylation was quantified by Western blotting, thromboxane B2 synthesis by enzyme immunoassay, ATP release by ChronoLume luciferin assay, cytosolic Ca(2+) concentration by Fura-2 fluorescence-measurements, platelet aggregation by a light transmissions measurement and in vitro thrombus formation by a flow chamber.
Skepinone-L (1 μM) virtually abrogated the phosphorylation of platelet p38 MAPK substrate Hsp27 following stimulation with CRP (1 μg/ml), thrombin (5 mU/ml) or thromboxane A2 analogue U-46619 (1 μM). Furthermore, Skepinone-L significantly blunted activation-dependent platelet secretion and aggregation following threshold concentrations of CRP, thrombin and thromboxane A2 analogue U-46619. Skepinone-L did not impair platelet Ca(2+) signaling but prevented agonist-induced thromboxane A2 synthesis through abrogation of p38 MAPK-dependent phosphorylation of platelet cytosolic phospholipase A2 (cPLA2). Skepinone-L further markedly blunted thrombus formation under low (500-s) and high (1700-s) arterial shear rates.
The present study discloses a powerful inhibiting effect of p38 MAPK-blocker Skepinone-L on platelet activation and thrombus formation.
背景/目的：血小板对于初级止血至关重要，是血栓性血管闭塞的主要参与者。血小板通过凝血酶和胶原相关肽等激动剂以及包括血栓素A2在内的第二波介质，经由不同的细胞内信号通路被激活，从而导致脱颗粒、聚集和血栓形成。血小板激活与p38丝裂原活化蛋白激酶（p38 MAPK）的磷酸化和激活同时发生。迄今已知的p38 MAPK抑制剂特异性有限，使得关于p38 MAPK在调节血小板功能中的确切作用难以得出可靠结论。本研究检测了新型高选择性p38丝裂原活化蛋白激酶（p38 MAPK）抑制剂Skepinone-L对血小板激活和血栓形成的影响。
实验在新鲜分离的人血小板中进行。通过蛋白质印迹法定量蛋白质磷酸化，通过酶免疫测定法检测血栓素B2合成，通过ChronoLume荧光素测定法检测ATP释放，通过Fura-2荧光测量法检测胞质Ca(2+)浓度，通过光透射测量法检测血小板聚集，并通过流动腔检测体外血栓形成。
Skepinone-L（1 μM）几乎完全消除了用CRP（1 μg/ml）、凝血酶（5 mU/ml）或血栓素A2类似物U-46619（1 μM）刺激后血小板p38 MAPK底物Hsp27的磷酸化。此外，在CRP、凝血酶和血栓素A2类似物U-46619的阈值浓度下，Skepinone-L显著减弱了激活依赖性血小板分泌和聚集。Skepinone-L不损害血小板Ca(2+)信号传导，但通过消除血小板胞质磷脂酶A2（cPLA2）的p38 MAPK依赖性磷酸化来阻止激动剂诱导的血栓素A2合成。Skepinone-L在低（500-s）和高（1700-s）动脉剪切速率下进一步显著减弱血栓形成。
本研究揭示了p38 MAPK阻滞剂Skepinone-L对血小板激活和血栓形成具有强大的抑制作用。
