Abstract: 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.

Key words: Perinereis aibuhitensis, anticoagulant peptides, alkaline protease, separation and purification, antithrombosis