Fischer B E, Schlokat U, Mitterer A, Savidis-Dacho H, Grillberger L, Reiter M, Mundt W, Dorner F, Eibl J
IMMUNO AG, Biomedical Research Center, Uferstrasse 15, A-2304 Orth/Danau, Austria.
J Biol Chem. 1996 Sep 27;271(39):23737-42. doi: 10.1074/jbc.271.39.23737.
A mutant derivative of human prothrombin in which active site aspartate at position 419 is replaced by an asparagine (D419N-prothrombin) has been designed, expressed in recombinant Chinese hamster ovary cells, and purified to homogeneity. D419N-prothrombin was converted to the related molecules D419N-meizothrombin and D419N-thrombin by limited proteolysis by Echis carinatus and Oxyuranus scutellatus venom protease, respectively, and affinity-purified using an immobilized modified C-terminal hirudin-derived peptide. Neither D419N-thrombin nor D419N-meizothrombin exhibited thrombin activity. Titration resulted in no detection of the active site, but binding to the most specific thrombin inhibitor, hirudin, was conserved in both proteins. In vitro examinations showed that D419N-thrombin and D419N-meizothrombin bind to immobilized hirudin, neutralize hirudin in human blood plasma as well as in the purified system, and reactivate the thrombin-hirudin complex. Animal model studies confirmed that D419N-thrombin and D419N-meizothrombin act as hirudin antagonist in blood circulation without detectable effects on the coagulation system. Thus, both D419N-thrombin and D419N-meizothrombin combine for the first time hirudin-neutralizing properties with the advantages of recombinant production of human coagulation factors.
已设计出一种人凝血酶原的突变衍生物，其中第419位的活性位点天冬氨酸被天冬酰胺取代（D419N-凝血酶原），在重组中国仓鼠卵巢细胞中表达，并纯化至同质。分别用锯鳞蝰蛇毒蛋白酶和盾鳞棘背蛇毒蛋白酶进行有限的蛋白水解，将D419N-凝血酶原转化为相关分子D419N-中凝血酶和D419N-凝血酶，并用固定化的修饰C末端水蛭素衍生肽进行亲和纯化。D419N-凝血酶和D419N-中凝血酶均未表现出凝血酶活性。滴定未检测到活性位点，但两种蛋白质与最特异性的凝血酶抑制剂水蛭素的结合均得以保留。体外实验表明，D419N-凝血酶和D419N-中凝血酶与固定化水蛭素结合，在人血浆以及纯化系统中中和水蛭素，并使凝血酶-水蛭素复合物重新激活。动物模型研究证实，D419N-凝血酶和D419N-中凝血酶在血液循环中作为水蛭素拮抗剂发挥作用，对凝血系统无明显影响。因此，D419N-凝血酶和D419N-中凝血酶首次将水蛭素中和特性与重组生产人凝血因子的优势结合起来。
