| Factor X activator could activate factor X in injuried vascular, promote thrombin generation, accelerate blood clotting. It has great potential to be developed as a hemostatic. This study was designed to isolate and purify factor X activator (RVV-VA) from venom of Vipera russellii in Guangxi,and investigate factors which affect RVV-VA coagulation activity. We also tested its hemostasis effects in animals.1. Purification of factor X activator from venom of Vipera russelliiRVV-VA was purified from the venom of Vipera russellii by ion-exchange chromatography on a DEAE FF column and by Superdex G-75 gel filtration. It could shorten the coagulation time in the presence of Ca2+ ions. Homogenicity of RVV-VA was tested by SDS-PAGE and meanwhile molecular weight of it was estimating also. The image of SDS-PAGE showed one band in native sample of RVV-VA with molecular weight of 97,800Da. But for the denaturing sample of RVV-VA, two bands were showed with a 76,300Da heavy chain and a 21,500Da light chain .2. Factors which affect RVV-VA coagulation activityFactors which affect RVV-VA coagulation activity was investigated by activated partial thromboplastin time (APTT) coagulation assay. We first assigned a plot of clotting time against dose of RVV-VA. On the basis of this relationship, we determined the remaining activities after different treatments. The results showed that RVV-VA was stable when pH of buffers[0] was between 6 and 9, and temperatures were below 40℃.RVV-VA was rapidly and irreversibly inactivated by L- cysteine and EDTA.The reagents of Aprotinin and PMSF failed to inhibit RVV-VA.3. Effects of RVV-VA on mice bleeding time and coagulation timeBleeding time (BT) was determined by cutting-tail method in mice. Coagulation time (CT) was tested by test tube method in mice. The results showed that bleeding time and coagulation time of mice was shortened by RVV-VA in concentration between 0.625mU/kg and 10mU/kg and behavior was dose dependent. BT was 3.53±0.25min, 2.88±0.24min, 2.95±0.20min, 2.35±0.27min and 2.58±0.26min respectively. CT was 2.45±0.26min, 2.48±0.28min, 1.78±0.25min, 1.83±0.21min and 1.55±0.20min respectively. We chose 0.625mU/kg, 2.5mU/kg and 10mU/kg as the experimental dose.4. Effects of RVV-VA on coagulation time in blood coagulation disorder miceInjection of heparin or oral administration of warfarin both could induce coagulation disorders in mice. Then coagulation time was tested by test tube method in mice after injecting RVV-VA. The results showed that coagulation time of heparinized mice was prolonged to 5.25±0.33 min from 1.53±0.30 min(control). After administration RVV-VA 10mU/kg and 2.5mU/kg, CT of the heparinized mice was 3.70±0.28min and 4.88±0.27min respectively. CT of mice which administered with warfarin was prolonged to 4.43±0.41min from 1.58±0.24min(control). After administration RVV-VA 10mU/kg,2.5mU/kg and 0.625mU/kg, CT of the mice was 2.20±0.26min, 3.23±0.32min and 3.98±0.30min respectively. RVV-VA could reverse the hemorrhagic tendency induced by heparin or warfarin.5.Effects of RVV-VA on rabbit coagulation timeTesting rabbit coagulation time before and after intravenous injection of RVV-VA within 24h, in order to investigate the effect of RVV-VA on the rabbit coagulation function . The results showed that after administration RVV-VA 20mU/kg, 5mU/kg and 1.25mU/kg for 10min, CT was 2.75±0.35min, 3.13±0.34min and 3.46±0.49 min respectively. CT was significantly shorter than without administration of RVV-VA (4.08±0.30min, 4.17±0.41min and 4.21±0.37min)(p<0.01). The effect of RVV-VA reached the peak after administration 30 minute. CT was 2.33±0.26min, 2.54±0.29min and 2.88±0.41min respectively. Procoagulant of RVV-VA could last for 12 hours when its dose was 20mU/kg or 5mU/kg.6. Effects of RVV-VA on model of hepatic trauma of rabbitThe model of hepatic trauma of rabbit was made. After administration RVV-VA 20mU/kg, 5mU/kg or 1.25mU/kg, BT was 1.19±0.05min, 1.39±0.10min and 1.58±0.20min respectively. Meanwhile volume of the bleeding was 34.5±3.7mg, 35.9±2.6mg and 41.0±2.1mg respectively. Both BT and volume of the bleeding were significantly decreased compared to without administration RVV-VA(p<0.01). The BT without RVV-VA was 2.15±0.25min, 2.18±0.21min and 2.24±0.21min; The volume of bleeding without RVV-VA was 77.0±7.8mg, 79.0±2.0mg and 71.4±2.4mg. RVV-VA possessed of the effectiveness in shorting haemostatic duration and reducing bleeding, and this effect was dose-dependent manner.7. Effects of RVV-VA on APTT, PT and TT of rabbitAPTT, PT and TT were evaluated by test tube method in rabbit. RVV-VA couldn't affect on APTT, PT and TT in rabbit. It indicated that RVV-VA could not activate factor X in normal blood vessels.Conclusion: RVV-VA was obtained from the venom of Vipera Russellii in Guangxi by chromatography of DEAE FF ion-exchange and SuperdexG-75 gel filtration[0]. It possessed considerable hemostatic activities. |
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