The Antithrombotic And Fibrinolytic Effects Of A Newly Derived Crude Enzyme From Spirulina Called Spirulinakinase

OBJECTIVE: To investigate the anticoagulant and thrombolytic effects of Spirulinakinase(SPK) extract solution and Crude Enzyme in vivo and in vitro .So it made foundation for the development of new anticoagulant or thrombolytic drugs.METHODS:1.The experimental in vitro (1)The preparation of crude enzyme solution. The SPK power(activity: 7000IU/ml, GuangXi testing center) was immersed in saline and mixed for one hour, placed at﹣4 degrees Celsius refrigerator overnight, then go 10–minute Centrifugal, the sedimentation was disposed and the supernatant was the SPK extract. The SPK extract was sub-separated into several components by Ammonium sulfate Salting-out. The content of protein was determined by Coomassie Brilliant Blue method.And the activity by the content was the relative activity. The component with higher relative activity was the crude enzyme solution. And the fibrinolytic activity was determined by Agarose-Fibrin plate method. (2)The anticoagulant effect of SPK extract solution. The anticoagulant effect of SPK extract solution was tested in vitro using plastic tubes by determining the whole blood clotting time. (3)The thrombolytic effect of SPK extract solution. The thrombolytic effect of SPK extract solution was tested in vitro using plastic tubes by determining the thrombolytic rate of the rabbit blood. (4)The effect of SPK extract solution on the activated partial thromboplastin time(APTT) , prothrombin time (PT) and thrombin time (TT) In vitro. After adding certain concentration of SPK extract solution to the plasma in vitro, the APTT , PT and TT were tested . Then the Correction Experiment was done determining the possible mechanism. 2.The experimental In vivo. (1)The effect of crude enzyme on CT of mice. The mice were randomly divided into 4 groups: high dose, low dose, saline control group and heparin positive control group. The Coagulation time(CT) was determined by experiment in vivo to test the effect of the crude enzyme by capillary method after tail vein administration. (2)The impact of crude enzyme on the PT, APTT and TT in rats. The rats were randomly divided into 4 groups: high dose, low dose, saline control group and heparin positive control group. After femoral vein administration the blood was gotten from abdominal aorta, and the APTT, PT and TT were tested using sodium citrate solution anticoagulant plasma. (3)The impact of crude enzyme on the weight of venous thrombolus(VT) in rats. The rats were randomly divided into 4 groups: high dose, low dose, saline control group and heparin positive control group. The thrombus was weighed after sublingual vein administration. (4)The effect of crude enzyme on the weight of arterio-venous loop(AVL) thrombus made in rats and the content of D–dimer and t–PA. The rats were randomly divided into 4 groups: high dose, low dose, saline control group and urokinase positive control group. The arterio–venous loop thrombosis model was made delivering drug by femoral veinl and the thrombus was weighed. The blood was gotten from abdominal aorta, the content of D–dimer was tested in Clinic Laboratory Department of the First Affiliated Hospital of Guang Xi Medical University, and the content of t–PA was tested using sodium citrate solution anticoagulant plasma.RESULTS: 1.The experimental In vitro (1)The preparation of crude enzyme solution. The component with relative higher activity was collected with 45%～75% saturation of ammonium sulfate, It was the crude enzyme needed after dialysis and freeze-drying. (2)The anticoagulant effect of SPK extract solution. The SPK extract solution could significantly elongate the exsisting time of the whole blood clotting time(P<0.01). (3)The thrombolytic effect of SPK extract solution.The weight of the clot was significantly alleviated by SPK extract solution(P<0.01).(4)The effect of SPK extract solution on the activated partial hromboplastin time(APTT), prothrombin time (PT) and thrombin time (TT) In vitro. Due to the use of high dose SPK extract solution, the PT and APTT were all elongated (P<0.01), and the high dose and low dose SPK extract solution could significantly extend the TT(The high dose P<0.01; The low dose P<0.05). 2.The experimental In vivo. (1)The effect of crude enzyme on CT of mice. The crude enzyme could significantly prolong the CT of mice(P<0.01). (2)The impact of crude enzyme on the PT, APTT and TT in rats. There was a significant difference between the crude enzym groups and the saline control group of the PT, APTT and TT(low dose P<0.05, high dose P<0.01). (3) The impact of crude enzyme on the weight of venous thrombolus in rats. The high dose and the low dose group could both low the weight of venous thrombus(P<0.01), campared to the saline control group.(4)The effect of crude enzyme on the weight of Arterio-Venous Loop thrombus made in rats and the content of D–dimer and the t–PA . Campared to the saline control group, The high dose and the low dose group could both low the weight of arterio–venous loop thrombus(P<0.05) and increase the content of D–dimer(P<0.05) and the t–PA(P<0.05).CONCLUSION: The precipitation product with 45%～75% saturation of ammonium sulfate holds higher relative activity. The SPK extract solution holds obvious effects of anticoagulation and thrombolysis in vitro. The crude enzyme shows obvious anticoagulant and fibrinolytic effect, it inhibits the formation of obstructive venous thrombolus and platelet Arterio–Venous Loop thrombus. The crude enzyme dissolves the fibrin directly. The content of D-dimer increases by delivering the SPK enzyme. And it also promotes the secretion of t–PA.