Characterization, Pharmacodynamics And Bio-security Research Of Fibrinolytic Enzyme From Urechis Unicinctus

Thrombosis disease (TD) is a serious public health hazard, with high incidence of morbidity. It mainly damages the blood systems of heart, brain and lung, resulting in death and disability of people. Currently, the thrombolytic therapy is still the main treatment of thrombotic disease. Although there are three generations of thrombolytic agents, more or less side effects appearing in each of them, such as the low capability of fibrin degradation, easy bleeding and so on. New thrombolytic agents with higher safety and better effects need to be research.For years of research in our laboratory, a fibrinolytic enzyme with a molecular of 26 kDa was investigated and isolated from a marine invertebrate, Urechis unicinctus.In this paper, we will report another component (UFEⅠ) from Urechis unicinctus to enrich the knowledge of the marine bioactive substances. The main results are shown as follows:1. SDS-PAGE and IEF show that the purified enzyme (UFEⅠ) is a single protein with a molecular weight of 45 kDa and pI value of 4.582. The optimum temperature and pH of UFEⅠis 50℃and 8.5, respectively. The enzyme activity was stable with temperatures below 50℃and within a pH range of 7.0-9.0. The enzyme activity was inhibited by metal ions, such as Fe3+ and Mn2+ ions, and significantly inhibited by metal-chelating of EDTA. According to Lineweaver-Burk plot, the enzyme dynamic parameters calculated are shown as follows: Vmax= 34.5μg/min·mL,Km=1.02 mg/mL .3. UFEⅠcan both directly degrade fibrin and indirectly hydrolyze fibrin. The total fibrinolytic activity of the enzyme is 302.9 U/mg, of which the kinase activity is 177.6 U/mg, about 41.4% when using urokinase as standard.4. It is determined that UFEⅠis effective at anticagulability in vitro, and thrombolysis in vitro experiments shows UFEⅠhas a direct ability to degrade fibrin. 5. According to fibrinogen degradation products, UFEⅠis probably aγ-plasmin.6. In experimental coagulation test of mice, UFEⅠdramatically prolonged the blood cloting time. As the results of antithrombotic effect, which the thrombus formation time was significantly prolonged was considered to be due to the proteolytic enzyme absorbed into blood steam prolonging the APTT, and decreasing the FIB. All of these show that UFEⅠevoked in a complex way, the coagulation and fibrinolytic system in blood, via an antithrombotic effect in our carotid thrombosis rat model.7. UFEⅠowns good bio-security by haemolytic test.8. In case of the injury experiment in mice, UFEⅠinduces a temporary rise of specific antibodies in mice, but does not affect adversely on animal bodies.In summary, UFEⅠhas significant effect on anticagulability and antithrombotic activitywith good bio-security in research.