| Background&ObjectiveHaemocoagulase agkistrodon is a snake venom thrombin-like enzymes obtained from Agkistrodon.Acutus. The current research shows that the haemocoagulase agkistrodon has good hemostatic effects, with a little adverse reaction and well tolerated.As a new hemostatic drug, it can be used for some hemorrhagic diseases clinically, such as the bleeding caused by trauma, surgery, or gastrointestinal bleeding, respiratory tract hemorrhage and gynaecologist bleeding. Haemocoagulase Agkistrodon was marketed as a national first class medicine by KangChen pharmaceutical Co., LTD in March 2009.The trade name is suling.It is a new generation of clinical hemostatic medicine extracted and separated from Chinese endemic snake kinds, Agkistrodon.Acutus. It uses the global leading venom monomer purification technology and makes the single component purity over 99%. As so far, Haemocoagulase Agkistrodon is the sole snake venom thrombin-like enzymes which had completed all amino acid sequence determined. The current research shows that HCA can cause a high level of soluble fiber protein inside blood-vessel and can not activate blood coagulation factorⅩⅢand prothrombin. It could accelerate hemostatic in the wound. But in normal vessels, it won't cause thrombosis. It is a good hemostatic medicine.As a new discovery of clinical medicine, HCA's efficacy and mechanisms has a preliminary understanding. But compared with other clinically similar products, HCA's efficacy and mechanisms need a further research. This experiment was start from HCA's hemostatic effects in vivo and vitro. Research the role of HCA on various human blood coagulation factors. Comprehensive understanding HCA's curative effect and its mechanisms provide the experiment evidence for the clinical application of SuLing, and ensure the safety of drugs.Methods1,Hemostatic Effects Evaluation of HCA in vitro1.1 The research of HCA's optimal experimental concentration in vitroTake 4 units of HCA, preparation of different concentrations of HCA solution with water for injection, and then incubationed with human plasma at 37℃. After about 30 minutes, weigh and record the blood clots.1.2 The experiments which contained blood Coagulation factorⅩⅢTake HCA or reptilase or thrombin incubationed with human plasma at 37℃. After about 30 minutes, weigh and record the blood clots.1.3 The experiments which excluded blood Coagulation factorⅩⅢTake HCA or reptilase or thrombin incubationed with human fibrinogen at 37℃. After about 30 minutes, weighing and record the blood clots.2,Hemostatic Effects Evaluation of HCA in vivo2.1 Determination of bleeding time in mice tails testKM mice were intravenous injected to the experimental drug, after 30 minutes, pointing upwards in rat tail about 0.5 cm place quickly cut tail, immediately press the stopwatch, use filter to exhaust the blood, then calculated the length of bleeding time to evaluate the effect of the experimental drug.2.2 Rats in vivo thrombosis experimentSD rats were intravenous injected to the experimental drug, then were given intraperitoneal injection of sodium pentobarbital to anesthetize them, supine fixed on operating table in longitudinal incision, belly line about 3 cm, open abdominal cavity along white line of the abdomen, blunt separation of the inferior vena cava, left renal vein at the bottom of the 4th suture ligation with inferior vena cava, while two main branch vein beneath were ligated, suture abdominal wall, after 3 hr, re-open the abdominal cavity at the bottom of about 2cm in the ligation with the 4th at the suture Cord ligation, exhausting section of the lumen of blood, cut longitudinally to observe whether the thrombosis and, if have, remove blood clots known as wet heavy. Calculating the dissolving rate.3,Mechanisms3.1 The role of HC A for Coagulation FactorⅠ3.1.1 HPLC analysis HC A release fibrinopeptideA sample of the purified fibrinogen solution was incubated with either 4 U of HCA,4 U of reptilase or 8 U of thrombin at 37℃. After incubation, take the products in boiling water for 5 min, then 13000 rpm centrifuge for 10 min, Supernatant was analyzed by high performance liquid chromatography, check the cleavage of peptides.3.1.2 SDS-PAGE analysis HCA splitting fibrinopeptideThe purified fibrinogen solution was incubated with 4 U of HCA for 10,30min, 1,2,4 and 12h at 37℃. The clots were dissolved in 0.5 ml of 5 M urea. After overnight incubation at room temperature, the samples were electrophoresed on SDS gels.3.2 The role of HCA for Coagulation FactorⅩⅢ3.2.1 In vitro clot solubility research 0.5 ml of plasma was mixed with either preheated 1 U of HCA, reptilase or thrombin until the clot formed, blotted on Whaterman 1 filter paper to removed as much fluid as possible, weighing the clots, and then dissolved in 5 ml 5 M urea After 18 hr incubation, observe and record the clots dissolution, weighing the weight to calculate dissolving rate.3.2.2 In vivo clot solubility researchSD rats were intravenous injected to the experimental drug, then were given intraperitoneal injection of sodium pentobarbital to anesthetize them, supine fixed on operating table in longitudinal incision, belly line about 3 cm, open abdominal cavity along white line of the abdomen, blunt separation of the inferior vena cava, left renal vein at the bottom of the 4th suture ligation with inferior vena cava, while two main branch vein beneath were ligated,suture abdominal wall, after 3 hr, re-open the abdominal cavity at the bottom of about 2cm in the ligation with the 4th at the suture Cord ligation, exhausting section of the lumen of blood, cut longitudinally to observe whether the thrombosis and, if have, remove blood clots known as wet heavy. Calculating the dissolving rate.3.2.3 Formation of fibrin from plasma by HCA1-ml portions of heparinized plasma were clotted by either 0.5 ml of tris buffer(pH7.4), HCA, reptilase or thrombin, and allowed to stand for 4 hr at 37℃. At the end of the incubation times the fibrin was washed with large volume of 0.15M sodium chloride,blotted,,and dissolved in lml of 5M urea-2% SDS-2% P-mercaptoethanol in 0.01M sodium phosphate buffer,pH 7.15. After an overnight incubationat room temperature, the samples were electrophoresed on SDS gels.3.2.4 Incubation of FactorⅩⅢ250μl of FSF was incubated with either 250μl of tris buffer(pH7.4), HCA, reptilase or thrombin for 30min,1,2 and 4 hr at 37℃.150-ul portions were removed from the preincubation mixtures and added to 100μl of 9 M urea-2% SDS-2% P-mercaptoethanol in 0.01 M sodium phosphate buffer,pH7.15 in order to stop the reaction and to prepare the samples for SDS-polyacrylamide gel electrophoresis.3.3 The role of HCA for Coagulation Factor X10μl of FX was incubated with either 50μl of tris buffer(pH7.4), HCA, reptilase or thrombin for 2 hr at 37℃. Then added to 60μl of 10 M urea-2% SDS-2%β-mercaptoethanol in 0.01 M sodium phosphate buffer,pH7.15 in order to stop the reaction and to prepare the samples for SDS-polyacrylamide gel electrophoresis.Results1,Respectively in the concentration of 0.5 U/ml,0.75 U/ml,1.0 U/ml, the clots weight is 30.6 mg,29.8 mg and 30.1 mg. So the optimal concentration of HCA for test is 0.5 U/ml.2,The clot formed by HCA is 24.4±1.5mg. Compared with reptilase and thrombin, the weight of clots are almost equal. It shows that HCA and reptilase and thrombin have the equal hemostatic effects.3,The clot formed by HCA is 28.5±1.7mg. Compared with reptilase, the weight of clots are almost equal. It shows that HCA and reptilase have the equal hemostatic effects.4,The bleeding time of HCA high dose group is 71.7±21.2s. Compared with the control group, difference was statistically significant. The positive control medicine also can significantly shortened the bleeding time.5,The thrombosis weight formed by HCA at the dose level of 3U/kg is 20.2±3.3mg. Significantly greater than negative control group (P<0.001). Compared with reptilase, difference was not statistically significant. It shows that HCA and reptilase have the equal hemostatic effects in rats. 6,High performance liquid chromatography shows that when HCA acted on fibrinogen only released FpA.The retention time is 8.8min.And when thrombin acted on fibrinogen released FpA and FpB simultaneously. The retention time are 8.7min and 18.5min, respectively.7,SDS-polyacrylamide gel electrophoresis shows that theα-chain of fibrinogen disappeared after 12h incubation with HCA.That means HCA could degrade theα-chain of fibrinogen.8,The clot formed by HCA effected on the human plasma could dissolve completely.And the colt formed by reptilase or thrombin can only partly dissolve. Compared with HCA group,the differences between groups are statistically significant (P<0.001).That proves HCA could not activate blood coagulation factorⅩⅢ.9,The dissolution ratio of thrombosis formed by HCA in vivo is 75.3±2.8%。Significantly greater than other groups (P<0.001). That proves HCA could not activate blood coagulation factorⅩⅢ.10,Heparin can inhibit thrombin, but has no influence to HCA and reptilase.11,HCA could not degrade blood coagulation factorⅩⅢ.12,HCA could degrade blood coagulation factorⅩ, so HCA could not activate blood coagulation factorⅩ.Conclusion1,HCA has a good hemostatic effect in vivo and in vitro.It has the equal hemostatic effects with reptilase.2,HCA could not activate blood coagulation factorⅩⅢ, and won't cause disseminated intravascular coagulation.3,HCA could not activate blood coagulation factorⅩ, won't amplify the clotting cascade and cause thrombosis.4,Heparin has no influence to HCA's hemostatic effects.5,HCA could degrade theα-chain of fibrinogen and release A peptide fragment(FpA) to generate the fibrin monomers (αBβγ) 2 and polymerizing them for hemostasis. |
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