AIM:To study the quality standard of Agacutin.Methods:By using the identification test,human plasma and bovine fibrinogen clotting activity,gel-filtration HPLC,SDS-PAGE,and Lachypleus amebocyte lysate,the quality standard of Agacutin was obtained.Results:The experimental results show 98% of average purity on HPLC,hetrodimer bands and whole molecular weight 31733±1157 D on SDS-PAGE.0.2 KU of Agacutin solution makes 0.2 ml of human plasma and 0.8 ml of bovine fibrinogen coagulating in 60±20 and 188±20 seconds.0.25 U of Agacutin showed a negative abnormal toxicity and allergic reaction.The dose 0.2 U/0.2 ml showed negative hemorrhagin reaction.1 KU of Agacutin product contained less than 10 EU of endotoxin.The experimental results also showed that Agacutin has no alkaline phosphatase,L-amino acid oxidase,phosphalipase-A activity.Conclusion According to the above results,the quality standard parameters,purity≥95%,molecular weight 31±2 kDa,endotoxin 10 EU/KU,human plasma and bovine fibrinogen clotting time 60±20 and 188 ± 20 seconds,abnormal toxicity and allergic test dose 0.25 U,and hemorrhagin test 0.2 U/0.2 ml,were determined.

Thrombin-like enzyme (TLE) plays a significant role in vessel injury hemostasis. A novel snake venom TLE (Agacutin) was purified from Agkistrodon Acutus snake venom. Structural analysis indicated that Agacutin is a heterodimer that has a MW of 29,402 Da, a pI value of 5.39, and optimum activity at 35 degrees C and pH 7.5. The N-terminal 15 amino acid sequences of Agacutin are DSSGWSSYEGHEYYV (small subunit) and DCSSGWSSYEEHQYY (large subunit). In vitro studies indicated that the coagulation activity of Agacutin was activated by Ca(+2) or inhibited by phenylmethanesulfonyl fluoride, but not influenced by heparin or hirudin. The arginine esterase activity and fibrinogen hydrolysis result showed that Agacutin only cleaves alpha-subunit and releases fibrinopeptide A. In vivo studies indicated that Agacutin iv (0.01-0.05 U/kg) shortened 30.2-49% of the rabbit blood clotting time, or ip (0.5-2.0 U/kg) shortened 29.7-73.1% of the mouse tail bleeding time. Agacutin does not influence APTT, platelet or euglobulin clotting time, and activate Factor II or XIII. It converts fibrinogen into the soluble fibrin that accelerates hemostasis at wound.