Question:

Platelets are critically important for primary haemostasis and the major players in thrombotic vascular occlusion. Platelets become activated by agonists as thrombin, collagen-related peptide and second-wave mediators including ADP and thromboxane A₂ resulting in degranulation, aggregation and thrombus formation. The limited specificity of hitherto known p38 kinase inhibitors precluded safe conclusions on the precise role of p38 kinase in the regulation of platelet function. The present study examines 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.

Methods and Results:

Skepinone-L (1 µM) virtually abrogated the stimulation of platelet p38 kinase substrate Hsp27 phosphorylation by thrombin (5 mU/ml), CRP (1 µg/ml), thromboxane A₂ analog U-46619 (1 µM) and ADP (5 µM). Skepinone-L blunted agonist-induced release of dense (ATP) granules at low agonist concentrations. Skepinone-L impaired activation-dependent phosphorylation of platelet phosopholipase A₂ and platelet thromboxane A₂ synthesis. Moreover, confocal microcopy revealed that platelet spreading on collagen was significantly affected by Skepinone-L. While platelet Ca²⁺ signaling was not impaired by Skepinone-L, platelet aggregation following stimulation with low dose agonists was significantly reduced in the presence of Skepinone-L (1 µM). Finally, Skepinone-L markedly blunted in vitro thrombus formation under low (500^-s) and high (1700^-s) arterial shear rates.

Conclusions:

The present study discloses a powerful inhibiting effect of p38 MAPK-inhibitor Skepinone-L on platelet activation and thrombus formation.