Stimulation of nitric oxide production contributes to the antiplatelet and antithrombotic effect of new peptide pENW (pGlu-Asn-Trp)

Introduction: New peptide pGlu-Asn-Trp (pENW), initially extracted from snake venom, significantly attenuates the formation of arterial and venous thrombi in vivo, and has modest in-vitro antiplatelet activity. This study was designed to investigate the underlying mechanisms.

Methods: The rat carotid thrombosis model induced by FeCl3 was established to evaluate the antithrombotic activity of pENW. The effects of pENW on the production of nitric oxide (NO), as well as the expression and activity of endothelial nitric oxide synthase (eNOS), were determined. The vasorelaxant effect of pENW was evaluated using isolated rat aortic rings in the absence or presence of N(G)-nitro-L-arginine methyl ester (L-NAME, eNOS inhibitor). Furthermore, the in-vitro antiplatelet activity of pENW was investigated with the addition of sodium nitroprusside (SNP, NO donor) and/or L-NAME to further prove the role of NO and eNOS in the inhibitory effect of pENW on platelet aggregation.

Results: In vivo, pENW inhibited thrombus formation induced by endothelial injury in a dose-dependent manner, with a significantly prolonged time to the occurrence of arterial occlusion. It was shown that pENW offered protection for blood vessels from oxidative injury. pENW significantly increased NO production in rats treated with pENW at 4 or 2mg/kg body weight. Furthermore, the production of NO from the cultured vascular endothelial cells was increased with the treatment of 10(-4)M and 10(-5)M pENW; pENW also enhanced eNOS expression and activity both in vivo and in vitro, and elicited a concentration-dependent vasorelaxation which was significantly inhibited by L-NAME. Notably, pENW inhibited ADP-induced platelet aggregation, and the inhibition was more significant in the presence of NO. The inhibition of platelet aggregation by pENW was significantly abolished by L-NAME.

Conclusions: The in-vivo antiplatelet and antithrombotic effects of pENW are at least partly mediated by the increased production of endogenous NO via up-regulation and stimulation of eNOS. The findings suggest that pENW could potentially be developed as a novel therapeutic agent in the treatment of platelet-driven disorders.