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为解释以凝血因子Xa(FXa)的识别序列为连接肽的葡激酶和水蛭素的融合蛋白(命名为SFH)在体内的强溶栓和低出血的特征,研究分析了SFH的两个血栓靶向性作用机理。首先采用ELISA和免疫组化的方法在体外分析了由水蛭素游离的C末端赋予的SFH对血栓的靶向性,结果显示SFH对凝血酶和富含凝血酶的血栓具有更高的亲和力。为阐明SFH抗凝活性在血栓部位的靶向性释放,构建表达了仅在水蛭素N末端连接FXa识别序列的水蛭素衍生物(命名为FH)。体外试验结果表明完整的FH无抗凝活性,在体内FH可以发挥抗栓作用,且出血副作用较低,这些结果说明FXa的识别序列可以封闭水蛭素的抗凝活性,在体内FH可以由于FXa的成功裂解而释放其抗凝活性,且其抗凝活性可能仅局限于血栓局部。这就间接说明了SFH的抗凝活性可以在血栓局部进行靶向性释放。以上两个血栓靶向性作用机理是SFH在体内发挥更高溶栓效率和降低出血副作用的重要机制。
To elucidate the thrombolytic and hypoallergenic characteristics of fusion protein (named as SFH) of staphylokinase and hirudin fused with the recognition sequence of factor Xa (FXa) as linker, two thrombotic targets of SFH The role of sexual mechanism. First, the targeting of SFH by the free-end C-terminal of hirudin to thrombus was analyzed by ELISA and immunohistochemistry. The results showed that SFH had higher affinity for thrombin and thrombin-rich thrombus. In order to elucidate the targeted release of SFH anticoagulant activity at the thrombus site, a hirudin derivative (designated FH) expressing the FXa recognition sequence linked only to the N-terminus of hirudin was constructed. The results of in vitro tests showed that intact FH had no anticoagulant activity, and FH could exert antithrombotic effect in vivo with low bleeding side effects. These results indicate that the recognition sequence of FXa can block the anticoagulant activity of hirudin. In vivo FH can be inhibited by FXa Successfully cleaved to release its anticoagulant activity, and its anticoagulant activity may be localized only to the thrombus. This indirectly shows that the anticoagulant activity of SFH can be targeted release of local thrombosis. The above two thrombus-targeted mechanism of action is an important mechanism of SFH in vivo to play a higher thrombolytic efficiency and reduce bleeding side effects.