双齿围沙蚕抗凝血肽的制备及其抗血栓作用

摘要

The present study was intended to investigate the enzymatic preparation and antithrombotic activity of anticoagulant peptides from Perinereis aibuhitensis(PAAP).The optimum enzyme was determined based on anticoagulant activity. Ultrafiltration, anion-exchange chromatography, gel chromatography and reversed phase high-performance liquid chromatography were used sequentially to separate and purify peptides with the strongest anticoagulant activity from the protein hydrolysate of Perinereis aibuhitensis,named as PAAP.Then,the acute toxicity of PAAP was determined.Furthermore,the antithrombotic activity of PAAP was observed by carrageenan-induced tail thrombosis in mice, FeCl3-induced arterial thrombosis in rats and platelet aggregation induced by adenosine diphosphate (ADP). Alkaline protease was selected as the optimal protease to digest Perinereis aibuhitensis.An anticoagulant peptide was then obtained after separation and purification,and identified as Pro-Val-Glu-Arg-Lys. The anticoagulant activity of PAAP could reach up to (1 320.0 ± 20.3) U/g. The maximum tolerance dose of PAAP was 11.0 g/(kg·d)without the occurrence of abnormality and death in mice.The in vivo experiments also showed that PAAP could prevent the formation of carrageenan-induced tail thrombosis in mice, FeCl3-induced arterial thrombosis in rats and platelet aggregation induced by ADP. Thus PAAP has an antithrombotic effect.%目的:探讨双齿围沙蚕抗凝血肽(anticoagulant peptides from Perinereis aibuhitensis,PAAP)的制备及其抗血栓作用.方法:以抗凝活力为检测指标,筛选最佳酶对沙蚕进行酶解.酶解产物经超滤、阴离子交换层析、凝胶过滤层析和反相高效液相色谱法分离纯化,得到具有最强抗凝活力的沙蚕寡肽,将此肽命名为PAAP并测定其急性毒性作用.采用角叉菜胶所致的小鼠尾部血栓形成实验、三氯化铁所致的大鼠动脉血栓形成实验及二磷酸腺苷(adenonisine disphosphate,ADP)诱导的血小板聚集实验,研究PAAP的抗血栓作用.结果:选取碱性蛋白酶作为最佳酶对沙蚕进行酶解,经过分离纯化后得到一条氨基酸序列:脯氨酸-缬氨酸-谷氨酸-精氨酸-赖氨酸(Pro-Val-Glu-Arg-Lys).经测定,该寡肽的体外抗凝活力可达(1320.0±20.3)U/g.PAAP的最大耐受量为11.0 g/(kg·d)且未出现小鼠异常及死亡现象,体内实验表明PAAP对小鼠尾部血栓的形成、大鼠动脉血栓的形成及ADP诱导的血小板聚集均有明显抑制作用.结论:从双齿围沙蚕中提取出的抗凝肽PAAP具有抗血栓作用.