单环刺螠纤溶酶的分离纯化及单环刺螠纤溶酶Ⅲ的药效学研究
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摘要
血栓性疾病（thrmobotic disease，TD）严重威胁着人类的生命健康，其发病率高居各种疾病之首，危害人体心、脑和肺的血管系统。据世界卫生组织统计，自2004年至今，全世界每年都有1700多万人死于心脑血管病，而我国心脑血管疾病患者已经超过2.7亿人，每年死于心脑血管疾病的患者多达300万以上。
溶栓疗法被认为是治疗血栓性疾病最为有效的方法，溶栓药物也早已经广泛应用于各种血栓性疾病的治疗。以尿激酶、t-PA为代表的临床溶栓剂效果显著，但也存在许多副作用，且价格不菲。因此，人们一直在努力从生物界寻找着理想溶栓物质的来源。基于海洋生物天然活性物质相比于陆生生物活性物质的诸多优势，本实验室经过多年积累，从海洋无脊椎动物单环刺螠（Urchis unicinctus）体内发现并分离纯化出了一系列具有纤溶活性的蛋白酶，并以其分子量由大及小命名为单环刺螠纤溶酶Ⅰ（45.1kDa）、Ⅱ（26.7kDa）、Ⅲ（20.8kDa）和Ⅳ（10-11kDa）。本研究利用离子交换色谱及凝胶过滤色谱等柱层析技术制备了纯度较高的单环刺螠纤溶酶Ⅰ（45.1kDa）和Ⅲ（20.8kDa），并以单环刺螠纤溶酶Ⅲ为研究对象，系统考察了其生化性质及酶学性质，评价了其生物安全性和体内外抗凝、溶栓活性。现将主要研究结果分述如下：
1.采用多种分离和纯化方法相结合，包括透析、冷冻干燥、凝胶柱层析和阴离子交换柱层析，获得了纯度较高的单环刺螠纤溶酶Ⅰ和Ⅲ，两种蛋白在Native-PAGE和SDS-PAGE中均显示单一条带，说明两者成分均一且均为单链蛋白，分子量分别为45.1kDa和20.8kDa。
2.对单环刺螠纤溶酶Ⅲ进行了系统地生化性质及酶学性质研究：UFEⅢ是一种丝氨酸蛋白酶，其N末端12个氨基酸依次为：IIGGSQAAITSY。UFEⅢ的等电点在7.2附近。每mg UFEⅢ的总纤溶酶活力为1461.5个尿激酶活力单位，其中包含692.3个直接降解纤维蛋白（原）的活力单位和769.2个纤溶酶原激酶的活力单位。UFEⅢ水解纤维蛋白原的方式为：A链> B链>链。UFEⅢ在60℃以下稳定，最适反应温度在55-60℃之间；UFEⅢ在pH6-10之间活力较高，最适pH值为8.5。Mn~(2+)和Mg~(2+)能提升UFEⅢ酶活力，Cu~(2+)能显著地抑制UFEⅢ酶活力。另外，丝氨酸蛋白酶抑制剂PMSF和SBTI能显著抑制UFEⅢ酶活力。以酪蛋白为底物，UFEⅢ的酶促反应动力学常数：Vmax=42.92μg/min·ml，Km=1.06mg/ml。
3.单环刺螠纤溶酶Ⅲ不引起机体溶血，无明显的出血反应，无小鼠全身急性毒性。
4.单环刺螠纤溶酶Ⅲ在体外有较好的抗凝、溶栓活性，对红细胞无害；在3h内，其体外溶栓率达60.5%。在动物体内，UFEⅢ能显著延长小鼠凝血时间，UFEⅢ高、中、低三个剂量组对大鼠颈动脉血栓形成的抑制率分别达到70.2%，54.0%和36.4%，表现出了良好的抗凝活性；在FeCl3诱导兔颈动脉血栓模型和结扎诱导大鼠下腔静脉血栓模型中，UFEⅢ能部分溶解造模动物血管堵塞处的血栓块，表现出较好的溶栓活性，延长给药时间可实现堵塞血管的再通。此外，UFEⅢ能显著降低受试动物的血浆FIB浓度、延长动物内源性凝血指标APTT和TT。
综上所述，单环刺螠纤溶酶Ⅲ同时具备直接降解纤维蛋白（原）和激活纤溶酶原的能力，具有较好的生物安全性，在动物体内外都表现出了显著的抗凝、溶栓活性，有潜力成为一种新的溶栓制剂来源。
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 Mn~(2+)and Mg~(2+)but inhibited by Cu~(2+). 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.
溶栓疗法被认为是治疗血栓性疾病最为有效的方法，溶栓药物也早已经广泛应用于各种血栓性疾病的治疗。以尿激酶、t-PA为代表的临床溶栓剂效果显著，但也存在许多副作用，且价格不菲。因此，人们一直在努力从生物界寻找着理想溶栓物质的来源。基于海洋生物天然活性物质相比于陆生生物活性物质的诸多优势，本实验室经过多年积累，从海洋无脊椎动物单环刺螠（Urchis unicinctus）体内发现并分离纯化出了一系列具有纤溶活性的蛋白酶，并以其分子量由大及小命名为单环刺螠纤溶酶Ⅰ（45.1kDa）、Ⅱ（26.7kDa）、Ⅲ（20.8kDa）和Ⅳ（10-11kDa）。本研究利用离子交换色谱及凝胶过滤色谱等柱层析技术制备了纯度较高的单环刺螠纤溶酶Ⅰ（45.1kDa）和Ⅲ（20.8kDa），并以单环刺螠纤溶酶Ⅲ为研究对象，系统考察了其生化性质及酶学性质，评价了其生物安全性和体内外抗凝、溶栓活性。现将主要研究结果分述如下：
1.采用多种分离和纯化方法相结合，包括透析、冷冻干燥、凝胶柱层析和阴离子交换柱层析，获得了纯度较高的单环刺螠纤溶酶Ⅰ和Ⅲ，两种蛋白在Native-PAGE和SDS-PAGE中均显示单一条带，说明两者成分均一且均为单链蛋白，分子量分别为45.1kDa和20.8kDa。
2.对单环刺螠纤溶酶Ⅲ进行了系统地生化性质及酶学性质研究：UFEⅢ是一种丝氨酸蛋白酶，其N末端12个氨基酸依次为：IIGGSQAAITSY。UFEⅢ的等电点在7.2附近。每mg UFEⅢ的总纤溶酶活力为1461.5个尿激酶活力单位，其中包含692.3个直接降解纤维蛋白（原）的活力单位和769.2个纤溶酶原激酶的活力单位。UFEⅢ水解纤维蛋白原的方式为：A链> B链>链。UFEⅢ在60℃以下稳定，最适反应温度在55-60℃之间；UFEⅢ在pH6-10之间活力较高，最适pH值为8.5。Mn~(2+)和Mg~(2+)能提升UFEⅢ酶活力，Cu~(2+)能显著地抑制UFEⅢ酶活力。另外，丝氨酸蛋白酶抑制剂PMSF和SBTI能显著抑制UFEⅢ酶活力。以酪蛋白为底物，UFEⅢ的酶促反应动力学常数：Vmax=42.92μg/min·ml，Km=1.06mg/ml。
3.单环刺螠纤溶酶Ⅲ不引起机体溶血，无明显的出血反应，无小鼠全身急性毒性。
4.单环刺螠纤溶酶Ⅲ在体外有较好的抗凝、溶栓活性，对红细胞无害；在3h内，其体外溶栓率达60.5%。在动物体内，UFEⅢ能显著延长小鼠凝血时间，UFEⅢ高、中、低三个剂量组对大鼠颈动脉血栓形成的抑制率分别达到70.2%，54.0%和36.4%，表现出了良好的抗凝活性；在FeCl3诱导兔颈动脉血栓模型和结扎诱导大鼠下腔静脉血栓模型中，UFEⅢ能部分溶解造模动物血管堵塞处的血栓块，表现出较好的溶栓活性，延长给药时间可实现堵塞血管的再通。此外，UFEⅢ能显著降低受试动物的血浆FIB浓度、延长动物内源性凝血指标APTT和TT。
综上所述，单环刺螠纤溶酶Ⅲ同时具备直接降解纤维蛋白（原）和激活纤溶酶原的能力，具有较好的生物安全性，在动物体内外都表现出了显著的抗凝、溶栓活性，有潜力成为一种新的溶栓制剂来源。
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 Mn~(2+)and Mg~(2+)but inhibited by Cu~(2+). 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.
引文
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