宽体金线蛭与菲牛蛭抗凝血酶活性及抗凝机制比较研究

Comparative Studies on Anti-thrombin Activity and Anticoagulant Mechanism between Whitmania Pigra Whitman and Hirudinaria Manillensis

目的:比较宽体金线蛭和菲牛蛭抗凝血酶活性及对凝血途径的影响,为进一步揭示水蛭抗凝作用机制提供参考。方法:采用凝血酶滴定法、发色底物法-萃取-HPLC联合法测定水蛭对凝血酶的作用;同时采用凝固法测定其活化部分凝血活酶时间(APTT)、凝血酶原时间(PT)和凝血酶时间(TT),比较宽体金线蛭与菲牛蛭对不同凝血途径的影响。结果:凝血酶滴定法的结果表明,不同水蛭的抗凝活性顺序依次为:菲牛蛭活体冻干品>菲牛蛭清水吊干品>>宽体金线蛭清水吊干品;发色底物法-萃取-HPLC法测定不同水蛭对凝血酶抑制作用的结果表明:不同水蛭水提取物对凝血酶的作用均表现出低浓度促进,高浓度抑制,且菲牛蛭具有强烈凝血酶抑制活性,而宽体金线蛭在加入更高剂量时才表现出抑制作用,且抑制作用较弱,菲牛蛭的抗凝血酶活性远远高于宽体金线蛭;不同水蛭样品均使APTT、PT、TT延长,但是菲牛蛭活体冻干品主要延长TT,清水吊干品主要延长APTT,而宽体金线蛭使APTT、TT延长更加显著。结论:宽体金线蛭和菲牛蛭对凝血酶及凝血途径的作用很大区别。

Objective ： To compare the anti-thrombin activity and the effects on the coagulation pathway between Whitmania pigra Whitman and Hirudinaria manillensis to provide scientific reference for the anticoagulation mechanism revelation for Hirudo. Methods： Anti-thrombin titration and chromogenic substrate assay-extraction-HPLC were applied to study the anti-thrombin activity of Whitmania pigra Whitman and Hirudinaria manillensis. APTT, PT and TT were determined by a clotting assay to compare the effects on the path- way of blood clotting. Results： The anticoagulation activity order measured by anti-thr6mbin titration was living Hirudinaria manillensis 〉 dried Hirudinaria manillensis 〉 〉 dried Whitmania pigra Whitman. The results of chromogenic substrate assay-extraction-HPLC indicated that the low dose of aqueous extract promoted the thrombin activity, while the high dose inhibited the thrombin activity. Hirudi- Maria manillensis significantly inhibited the activity of thrombin, while Whitmania pigra Whitman showed weak anti-thrombin activity only at the higher dose. All leeches could prolong APTT, PT and TT. However, living Hirudinaria manillensis mainly affected TF, and dried Hirudinaria manillensis mainly affected APTT. Dried Whitmania pigra Whitman dramatically influenced APTT and TT. All the results indicated that the anticoagulant activity of Whitmania pigra Whitmanis was significantly higher than that of Hirudinaria manillensis. Conclusion： There are notable differences in the anti-thrombin activity and the effect on the pathway of blood clotting between Whitmania pigra Whitman and Hirudinaria manillensis.