| In recent years, thrombosis disease are seriously endanger human health, not only the high incidence but also high mortality and disability. It mainly damages the blood systems of heart, brain and lung, resulting in death of disability in people. There are about 15 million people suffering from thrombotic diseases all over the world each year. In china, more than two million people die in cardio-cerebrovascular diseases, about five million people require thrombolytic therapy. Therefore, The research of thrombotic disease drug has been becoming the focus on the world medical profession.Thrombolytic therapy is the most important pathway in treatment of thrombotic disease. There are three generations of thrombolytic agents, but more or less side effects appear in each of them,such as the low capability of fibrin degradation, easy bleeding and so on. Due to these reasons, it is important to research and develop new ones currently. In the light of the unique advantages of marine lives, they are becoming more and more precious. For years of research in our lab, a novel fibrinolytic enzyme is investigated and isolated. In this paper, the main achievements about the enzymatic properties and pharmacodynamics of UFE are shown as follows.1. The fibrinolytic enzyme can be isolated and purified by ion-exchange chromatography (DEAE-52) and gel filtration (Sephacryl S-100). SDS-PAGE and HPLC show that only a single band for the purified enzyme ,and the molecular weight is estimated to be about 26kDa.2. The optimal temperature of enzyme is 50℃and the optimal pH is 8.0 respectively. The fibrinolytic enzyme of UFE activity is stable in the pH range of 6.0-9.0 and the optimal temperature between 20℃and 50℃. The enzyme activity is obviously activated by Mg2+, whereas Cu2+,Fe3+,Ca2+,Zn2+,Fe2+,Ba2+ can inhibit the enzyme activity. Using casein as substrate, the enzyme dynamic parameters calculated according to Lineweaver-Burk plot are shown as follows: Vmax= 41.15μg/min·mL,Km=8.33×10-5mol/L .3. UFE can not only directly degrade fibrin but also indirectly hydrolyze fibrin through transforming plasminogen into plasmin. The total specific activity of the enzyme is 2477.2 U/mg, while the specific kinase activity is 1605.6 U/mg ,accounted for about 64.8% when using urokinase as standard.4. The haemolytic test, hypodermic bleeding test and systemic allergic test are confirmed that the enzyme owns good bio-security. Acute toxicity test is calculated semi-lethal dose LD50 for 571.36mg/kg, 95% credibility limit of 531.83—623.21mg/kg, non-toxic.5. Based on analysis of fibrinogen degradation products, it is probably that UFE is aγ-plasmin.6. Thrombolysis in vitro experiments shows UFE has a direct ability to degrade fibrin, thrombolytic rate of 76.3% after 3 hours, with Lumbrokinase control group is significantly different (p <0.01).7. UFE could significantly prolong the APTT, PT, CT and TT values, with significant anticoagulant effect, but its anticoagulation don't inhibit the platelet aggregation. Carotid artery thrombosis in vivo experiments show that UFE can significantly shorten the time ELT, fibrin degradation product (FDP) are increased and D-dimer test shows positive reaction. All of these certify that UFE can enhance the body the overall fibrinolysis activity through secondary means. The above experiments results are confirmed that UFE can obviously enhance the systemic body on anticoagulant and fibrinolytic activity, with great value of clinical research. |
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