Platelets play a key role in hemostasis and in the initiation and propagation of thrombus formation. New peptide pGlu-Asn-Trp (pENW), initially extracted from snake venom, shows a concentration-dependent antithrombotic activity, significantly attenuated thrombus formation in the arterial and venous vessel systems. This study was designed to further reveal the mechanisms underlying its antithrombotic effect by focusing on its in vitro antiplatelet effect after precluding its influence on coagulation factors. It showed that pENW concentration-dependently inhibited ADP-, collagen- and platelet activating factor (PAF)-induced platelet aggregation, inversely depending upon the intensity of stimulation induced by agonists. Furthermore, data obtained by ELISA and flow cytometry presented that pENW also suppressed ADP-mediated serotonin secretion and P-selectin expression in a concentration-dependent manner. As shown by Western blot assay, ADP-induced platelet Akt phosphorylation was attenuated by the priming incubation with pENW, demonstrating the influence on platelet intracellular signaling. It provided the explaining information for its activity of inhibiting platelet activation in vitro. These results suggested pENW attenuated thrombus formation in part by inhibiting platelet activation instead of coagulation factors, presented evidence of pENW interfering intracellular signaling system in the process of platelet activation and indicated the possibility that pENW could potentially be developed as a novel therapeutic agent in the prevention and treatment of thrombotic disorders.
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