毕赤酵母表达去氨普酶及其突变体的溶栓活性研究

目的：研究毕赤酵母表达野生型去氨普酶（DSPAwt）及其突变体F195和QNRR的溶栓活性,为开发新型溶栓类药物奠定基础。方法：以阿替普酶（rt-PA）和中国仓鼠卵巢细胞表达野生型DSPAα1为阳性对照,采用大鼠动-静脉旁路血栓模型,通过对血栓重量、出血时间、大鼠凝血指标凝血酶时间（TT）、凝血酶原时间（PT）、活化部分凝血酶原时间（APTT）和纤维蛋白降解产物D-二聚体的测定考察毕赤酵母表达DSPAwt及其两种突变体的溶栓活性。结果：DSPAwt及其突变体F195和QNRR在0.7mg/kg和1.5mg/kg剂量下均有一定的溶栓活性,其中DSPAwt溶栓作用较弱,有效动物数少,溶栓率较低,相同剂量突变体F195和QNRR溶栓作用显著增强,溶栓效果与DSPAα1接近。此外,不同剂量各样品均能在一定程度上延长大鼠出血时间,延长大鼠血浆TT、PT和APTT,提高血浆中D-二聚体含量。结论：通过酵母表达系统有望获得具有高溶栓活性的突变型DSPA,从而开发成新一代溶栓类药物。

Thrombolysis activities of desmoteplase and mutants expressed in yeast Pichia methanolica

AIM：To study the thrombolysis activities of wide type Desmodus rotundus Salivary Plasminogen Activivator （DSPAwt） and its mutations F195 and QNRR expressed in yeast Pichia methanolica.METHODS：The rat model of artery-vein bypass thrombosis was used to measure the thrombolysis activities of DSPAwt,F195 and QNRR,in which indexes such as weight of thrombus,bleeding time （BT）,thrombin time （TT）,prothrombin time （PT）,activated partial thromboplastin time （APTT） and D-dimer were investigated.DSPA was expressed in CHO cells （DSPAαl） and the launched thrombolytic drug rt-PA were used as positive controls.RESULTS：Positive controls as well as DSPAwt,F195 and QNRR at doses of 0.7 mg/kg and 1.5 mg/kg individually showed thrombolysis potency,which decreased the weight of thrombus,delayed the levels of BT,TT,PT,APTT and elevated the level of D-dimer.F195,QNRR and DSPAαl had the similar higher activities,while DSPAwt possessed limited thrombolysis effects.CONCLUSION：The results suggest a novel method to express DSPAs for further development as a novel type of drug for treatment of thrombotic diseases such as acute ischemic stoke.