Anti-thrombotic Activity Of The Anti-clotting Component, Acoagulatin In The Venom Of Agkistrodon

There are abundant proteases and polypeptides that are active in the process of blood coagulation, which have been widely used in anti-coagulation, anti-thrombosis and hemostasis. In our study, a protein component in the venom of Chinese Agkistrodon with a molecular weight of 31,400 dalton is found to have the anti-clotting activity. And we conduct a primary research on its physical and chemical properties as well as its anti-thrombotic activity.The experiment can be divided into two parts according to the purposes of our study.1. Purification of the anti-clotting component, acoagulatin in the venom of AgkistrodonThe venom of Agkistrodon was isolated and purified using ion-exchange chromatography of DEAE-Sepharose Fast Flow and CM-sepharose Fast Flow. After the DEAE-Sepharose Fast Flow, the component in the elution cusp of I-XII was collected and injected intravenously into rats respectively to record the bleeding time of the rat caudal vein by scissoring the tail. Then the component of each group was injected into the dorsal derma of rat to observe if there was bleeding toxicity. The results showed that the collected component from the cusp III could significantly prolong the bleeding time of the rat vein with no obvious bleeding toxicity. Therefore, we further isolated and purified the cusp III.The cusp III was performed gradient elution using CM-sepharose Fast Flow chromatography and the isolated and purified components in cusp I-IV were collected. Then the collected component was injected intravenously into rats respectively to record the bleeding time by scissoring the tail. Thecomponent was injected into the dorsal derma of rat to observe if there wasbleeding toxicity as well. Meanwhile, the thrombase-resembling activity of the component was tested as following: the additional 20ul PT was added into 20ul whole blood, followed by continuous observation for 1 hour to evaluate its anti-clotting activity. The observable blood coagulation was regarded as the criterion for its anti-coagulability. It was found that the cusp I could prolong the rat's bleeding time and PT with no obvious bleeding toxicity and thrombase-resembling activity, which indicated that the component was a safe and effective anti-clotting agent in the venom of Agkistrodon without thrombase-resembling activity. Therefore, the cusp I was collected for further experiment.Through procedures of DEAE-Sepharose Fast Flow and CM-sepharose Fast Flow, this new anti-clotting agent was successfully purified and was nominated as acoagulatin.In order to evaluate the purity of the acoagulatin, HPLC was performed for the purity evaluation. HPLC showed a symmetrical single cusp with relative percentage of the main content of 95.30%, which meant a high purity. Meanwhile, the SDS-PAGE electrophoresis (5% concentration gel, pH6.8, 12% separation gel, pH8.8, Tris-aminoacetic acid buffer of pH8.3 as the electrode buffer) was performed to determine the molecular weight of acoagulatin, which showed its molecular weight was about 31,400 dalton and it was composed of two subunits with respective molecular weight of 14,400 delton and 17,000 dalton. The standard curve showed that the concentration of the target protein, namely acoagulatin was 0.265mg/ml. 2.Anti-thrombotic activity of acoagulatinWe have conducted the experiments in vitro and the animal experiment in vivo to understand the anti-thrombotic activity of acoagulatin.I. experiment in vitroi. anti-clotting experiment in vitro: TT, PT and APTT20ul acoagulatin solution in concentration of 0.30ug/ul, 0.20ug/ul and 0.15ug/ul was evenly mixed with 100ul rabbit anti-clotting plasmarespectively to perform the anti-clotting experiment on LBY-NJ blood clotting device.The results showed that the acoagulatin could significantl -y prolong the PT and APTT, which reflected the endogenous and extr o-genous blood clotting activity. However, there were no effects on TT.ii.Fibrinolysis activity determination: hydrolysis fibrinogen electroph -oresis and fibrous protein plate test.Hydrolysis fibrino...