Effect Of Glycosaminoglycan From Urechis Unicinctus On Platelet Membrane P₂Y₁₂ Receptor Signaling Pathway

Urechis unicinctus,which belongs to Echiurioidea,Echiurida,Xenopneusta,Urchidae.It has rich protein and purtenance contains rich amino acids,polysaccharides and trace elements.As we all kown,U.unicinctus has very high nutritional value and medicinal value.Previous research had shown that glycosaminoglycan from U.unicinctus had good anticoagulant effect.In this paper,platelet cAMP,thromboxane B2,6-keto-prostaglandin and platelet membrane glycoprotein ?b/?a(GP?b/?a)concentrations were detected,and phosphorylation of vasodilation stimulated phosphoprotein(VASP),protein kinase A(PKA),protein kinase B(Akt),and phosphatidylinositol kinase(PI3K)receptor P2Y12 mRNA expression was analyzed.The effect of glycosaminoglycans on the platelet P2Y12 receptor signaling pathway was investigated.The SD rats were divided into three groups randomly:Control group;Glycosaminoglycan group(4 mg/kg);Positive control group(4 mg/kg).Administration and preparation of platelet-rich plasma(PRP):Femoral artery blood was taken after tail vein injection 30 minutes with 3.8%sodium citrate anticoagulation.Conventional method was used for platelet-rich plasma(PRP)preparation(in vivo experiments)and collectting rat blood to preparate PRP(in vitro experiment)directly.The PRP was incubated with ADP at 37 ? for 5 min and quenched on ice,then centrifuged at 4000 rpm for 10 min.The supernatant PPP was used to determinate TXB2 and 6-keto-PGF1? content through enzyme linked immunosorbent assay(ELISA).The platelet precipitation would be freezed and thawed repeately to determinate cAMP,GP II b/III a content through enzyme linked immunosorbent assay.Western blot was used to detect phosphorylation of vasodilation stimulated phosphoprotein(VASP),phosphorylation of protein kinase A,phosphorylation of platelet phosphatidylinositol kinase(PI3K)and protein kinase B(Akt).Real-time fluorescent quantitative PCR and RT-PCR were used to detect the effect of GAG on platelet P2Y12 receptor expression.The results show that:GAG could increase the platelet concentration of cAMP significantly(p<0.01)?increase the rat plasma concentration of TXB2 ignificantly(p<0.05),reduce the rat plasma concentration of 6-keto-PGF1?ignificantly(p<0.01),promote the PKA phosphorylation level(p<0.01)and VASP phosphorylation level(p<0.05),reduce platelet membrane glycoproteins GP II b/?a complex level(p<0.05),inhibit platelet P2Y12 receptor mRNA expression(p<0.01).It shows that GAG has no significant effect(p>0.05)on PI3K and Akt phosphorylation level.It shows that GAG and P2Y12 receptor antagonist MRS2395 have synergistic effect.Conclusion:The inhibition of platelet aggregation by GAG is achieved by increasing the concentration of platelet cAMP and the concentration of 6-keto-PGFl? in plasma,reducing the concentration of TXB2 and the level of the GP II b/? complex and promoting the phosphorylation level of PKA and VASP.