Purification Of UFE And Pharmacodynamic Study Of UFEⅢ

Thrombotic diseases, involving cardiovascular disease, cerebrovascular disease andvenous thromboembolism, are becoming a leading cause of morbidity and mortalityworldwide. According to WHO, since2004, there were17millions of people diedfrom cardiovascular and cerebrovascular diseases every year. In China, there arealmost270millions cardiovascular and cerebrovascular patients and3millions ofthem died every year.Thrombolytic therapy is commonly acknowledged to be the most effective way torealize recanalization. But most of the current fibrinolytic agents available for clinicsuch as tissue plasminogen activator （t-PA）, urokinase and lumbrukinase havehemorrhagic side effects, short half-life in the body and expensive. Therefore,searching for ideal thrombolytic products has never been suspended. Marineorganisms need to adapt to the complex living environment and fierce competition forsurvival in the ocean, which makes them evolve different metabolic mechanismscompared with terrestrial lives. Thus, they are more abundant in many kinds ofbiologically active substances including fibrinolytic compounds. In this lab, a seriesof enzymes with fibrino（gen）lytic activity were discovered and purified from themarine invertebrate, Urchis unicinctus and they named after their molecular weightsorder from high to low: UFEⅠ（45.1kDa）, UFEⅡ（26.7kDa）, UFEⅢ（20.8kDa） andUFEⅣ（10-11kDa）. In this present study, UFEⅠ（45.1kDa） and UFEⅢ（20.8kDa）were purified to electrophoretic homogeneity via ion exchange and gel filtrationchromatography from this worm. Subsequently, UFE Ⅲ was picked out andinvestigated from many aspects involving biochemical characters, biosafety andpharmacological activities. The main achievements in this work are shown as follows.1. Urchis unicinctus fibrinolytic enzyme Ⅰ&Ⅲ were obtained after acombination of isolation procedures including dialysis, lyophillization, gelfiltration and anion exchange chromatography. According to Native-PAGE and SDS-PAGE, UFEⅠ and UFEⅢ were both monomeric proteins, withmolecular weights of45.1kDa and20.8kDa, respectively.2. UFEⅢ was a serine protease and its N-terminal amino acid sequence wasIIGGSQAAITSY. Isoelectric point of UFEⅢ was around7.2. In fibrin plateassays, UFEⅢ was found to contain1461.5U （urokinase units）/mg totalfibrinolytic activity, which consisted of692.3U/mg direct fibrinolyticactivity and769.2U/mg plasminogen-activator activity. The fibrinogendegrading pattern of UFEⅢ was A-chains> B-chains>-chain. Besides,UFEⅢ was stable at pH6-10below60℃with an optimal catalytic pH of8.5at approximately55℃. The activity of UFEⅢ was enhanced by Mn²⁺and Mg²⁺but inhibited by Cu²⁺. Besides, UFEⅢ was also inhibited by PMSFand SBTI. Further, Kmand Vmaxvalues for casein were1.06mg/ml and42.92g/min-1ml-1, respectively.3. UFEⅢ exhibited neither hemolysis nor hemorrhagic effect. In Kunming mice,UFEⅢ exhibited no acute toxicity, too.4. UFEⅢ exhibited good anticoagulant and thrombolytic effects in vitro, with athrombus dissolve rate of60.5%within3h. In vivo, UFEⅢ not onlyprolonged the clotting time of Kunming mice, also inhibited the carotidarterial thrombosis in Wistar rats, exhibiting a nice anticoagulant effect.Moreover, UFEⅢ showed thrombolytic effect in vivo. In FeCl3inducedcarotid arterial thrombus model and stasis induced vena caval thrombusmodel, UFEⅢ could partially degrade the clots in the modeling segment ofvessel. Further, UFEⅢ significantly decreased the fibrinogen content oftesting animals and prolonged the intrinsic coagulation parameters-APTT andTT.From the above, UFEⅢ exhibited both anticoagulant and thrombolytic activities invivo and in vitro, which make it potentially be a new source of thrombolytic agents.