Study On Anticoagulant Mechanism Of Glycosaminoglycan From Urechis Unicinctus

Urechis unicinctus which commonly known as sea intestines belongs to Echiurioidea, Echiurida, Xenopneusta, Urchidae. It has rich protein, unsaturated fatty acids, polysacc harides, vitamins and trace elements. Previous research had shown that glycosaminoglycan from U. unicinctus had good anticoagulant effect. However, the anticoagulant mechanism of glycosaminoglycan from U. unicinctus is not very clear. In the thesis, the anticoagulant mechanism of glycosaminoglycan from U. unicinctus will have been revealed through studying the dependence of anticoagulation activity of glycosaminoglycan on AT-Ⅲ, HC-Ⅱ, the effect of glycosaminoglycan on several clotting factors and protein C system and the effect of glycosaminoglycans on the expression of tissue factor in human umbilical vein endothelial cells.The anticoagulation approaches of glycosaminoglycan was studied with AT-Ⅲ-deficient plasma, HC-Ⅱ-deficient plasma and both factor-deficient plasma in human. The activities of coagulation factor Ⅱ, Ⅴ, Ⅶ, Ⅷ, Ⅸ, Ⅹ, Ⅺ, Ⅻ and the fibrinogen content in human plasma were measured using the solidification method. The influence of glycosaminoglycan on blood recalcification time in rats was studied by using recalcification experiment. The Ca2+concentrations in rats and mice serum were measured through methyl thymol blue colorimetric method. The results showed that the antithrombin role of glycosaminoglycan from Urechis unicinctus mainly presented AT-Ⅲ dependency. Glycosaminoglycan could passivate the thrombin inhibitory activity of heparin cofactor Ⅱ. Glycosaminoglycan significantly reduced the activities of coagulation factor Ⅴ, Ⅶ, Ⅹ, Ⅷ, Ⅸ, Ⅺ, Ⅻ in the plasma (p<0.05, p<0.01). Besides, glycosaminoglycan could extend prolong recalcification time in rats by shielding Ca2+ in plasma, and significantly reduced Ca2+ concentration in rats and mice serum (p<0.01). Glycosaminoglycan reduced the Ca2+ concentration in a more intensive way than that of heparin sodium (p<0.01).The influence of glycosaminoglycan from U. unicinctus on the protein C system and relevant factors expression of human umbilical vein endothelial cells (HUVECs) were studied with enzyme linked immunosorbent and reverse transcript-polymerase chain reaction (RT-PCR). Results indicated that:glycosaminoglycan could significantly decrease content of activated protein C, activated protein S in blood of rats (P<0.05, P<0.01), significantly elevated content of activated protein C inhibitor (P<0.05, P<0.01), slightly increase the thrombomodulin level. Glycosaminoglycan could significantly reduce the expression of tissue factor gene, promote the expression of tissue factor pathway inhibitor and thrombomodulin gene (P<0.01).