Study On Antiplatelet Mechanism Of Glycosaminoglycan From Urechis Unicinctus

Thrombosis is a common disease in human beings,which seriously endangers human health.Platelet activation and aggregation play an important role in the formation of thrombus.Thus,antiplatelet therapy is still an important means to prevent and treat thrombotic diseases clinically.Although the antiplatelet drugs have achieved clinical efficacy widely,the fatalities and disability rates are still high.Therefore,it is still in great demand and very difficult to search for new targets for developing new drugs to reduce the occurrence of bleeding risk.In this experiment,we studied the effects of GAG from Urechis unicinctus on rat platelet aggregation induced by ADP in vivo/vitro.The results showed that GAG attenuated ADP induced platelet aggregation and adhesion dose-dependently in vitro(P<0.05,P<0.05).The effects of GAG on platelet signaling pathway of rats were investigated using methods such as enzyme-linked immunosorbent assay,fluorescence probe,immunoblotting,flow cytometry and real-time quantitative PCR.The results showed that GAG could significantly inhibit the release of TXA2 and the phosphorylation of cPLA2 in ADP-activated platelets(P<0.01,P<0.05),but no effects on the expression of COX-1 in platelets induced by ADP(P>0.05).Suggesting that GAG inhibits cPLA2 activity which may be the initial cause of the reduction of TXA2 production and release during platelet activation.Rises in cytosolic Ca2+ concentration were significantly inhibited by GAG in ADP-induced platelets in the absence of extracellular Ca2+(P<0.05).However,the cytosolic Ca2+ increased rapidly in the GAG group when Ca2+ was provided extracellularly,there was no significant difference compared to the control group(P>0.05).It is suggested that GAG mainly inhibits Ca2+release,but does not involved in ion channel-mediated Ca2+ influx.GAG significantly inhibited ADP-induced platelet P-selectin expression and phosphorylation of PLC(P<0.01,P<0.05),and resulted in a significant decrease in downstream IP3 concentration(P<0.05).These results suggest that GAG mainly inhibits the activation of platelets by inhibiting release of Ca2+ from the intracellular stores via the classical PLC signaling pathway.GAG had no significant effect on the expression of P2Y1 and P2X1 receptor mRNA(P>0.05,P>0.05),neither on the PI3K expression and the Akt phosphorylation ADP-induced platelets(P>0.05,P>0.05),indicating that GAG does not play a role in PI3K/Akt signaling pathway.GAG had a great inhibitory effect on phosphorylation of p38 MAPK and ERK in ADP-induced platelets(P<0.01,P<0.01),suggesting that GAG may play a inmportant role in and inhibit the multi-signal cascade of MAPK signaling pathway,Its inhibition on platelet MAPK signaling pathways may serve as an important target for antiplatelet activation.GAG significantly attenuated the phosphorylation of PKC and the expression of GP?b/?a in ADP-activated platelets.It confirms that GAG can inhibit platelet adhesion receptor activation and then inhibit platelet aggregation?...