1种新型合成水蛭肽的纤溶动力学研究

Kinetic study on the fibrinolytic reaction of a novel synthetic hirudin

目的研究水蛭肽Hirulog-s影响单链尿激酶型纤溶酶原激活剂和纤溶酶原相互活化作用考察新型合成水蛭肽的纤溶作用特性。方法构筑单链尿激酶型纤溶酶原激活剂(pro-uPA)和纤溶酶原(plg)相互活化反应体系,添加Hirulog-s成反应体系不同浓度,比较pro-uPA和plg相互活化反应体系中纤溶酶(plm)作用于pro-uPA反应的酶促反应动力学常数(Km,kcat),分析Hirulog-s对plm作用于pro-uPA反应的催化速率及亲和性。纤溶活性作用检测采用底物酶促反应法,酶促反应动力学常数的测定基于单链尿激酶型纤溶酶原激活剂激活纤溶酶原的反应。结果在plm作用于pro-uPA的反应体系中,30μmol·L^(-1) Hirulog-s与对照相比kcat值增大了3.1倍,Km值减小了1.6倍,表明Hirulog-s可以显著提高plm的最大催化速率同时增强亲和性。30μmol·L^(-1) Hirulog-s与对照相比kcat/Km值增大了4.75倍,表明Hirulog-s明显提高了plm的催化效率。通过活性对比表明Hirulog-s的纤溶活性强于水蛭肽Hirulog-1。结论 Hirulog-s促进单链尿激酶型纤溶酶原激活剂和纤溶酶原相互活化作用而发挥纤溶作用,新型合成水蛭肽从酶促反应动力学参数显示出优良的纤溶作用特性。

Objective To study the influence of Hirulog-s in mutual activation reaction system constituted by single chain urokinase-type plasminogen activator（pro-uPA）and plasminogen（plg）.Methods The enzymatic reaction kinetic constant（Km,kcat）of plasmin（plm）acts on pro-uPA in mutual activation reaction system influenced by pro-uPA and plg was measured.And the impact of Hirulog-s on the catalytic reaction activity and affinity by comparing kinetic constants of the enzymatic reaction in different concentrations of Hirulog-s was investigated as well.The role of fibrinolytic activity was detected by enzyme substrate reaction.And the method was used to determine the kinetic constant of the reaction of plasminogen activation which was catalyzed by single chain urokinase-type plasminogen activator.Results The kcatvalue of Hirulog-s（30μmol·L^（-1））was 3.1times more than the control group in the prouPA reaction system influenced by plm.The Kmvalue of Hirulog-s（30μmol·L^（-1））was 1.6times lower than that of control group.The results indicated that Hirulog-s had increased the maximum rate of catalysis and affinity significantly.Compared to the control group,the kcat/Kmvalue of Hirulog-s（30μmol·L^（-1））was 4.75 times increased.That meaned Hirulog-s greatly improved the catalytic efficiency of the plm and had a stronger reactivity than Hirulog-1.Conclusion The Hirulog-s could influence fibrinolysis process by promoting mutual activation reaction between pro-uPA and plg.The novel synthetic hirudin significantly enhanced the kinetic constant of fibrinolytic activation.