AUTOACTIVATION OF HUMAN RECOMBINANT COAGULATION FACTOR-VII
AUTOACTIVATION OF HUMAN RECOMBINANT COAGULATION FACTOR-VII
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DOI：
10.1021/bi00450a013
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发表时间：
1989-11-28
期刊：
影响因子：
2.9
通讯作者：
PETERSEN, LC
中科院分区：
文献类型：
--
作者：
PEDERSEN, AH;LUNDHANSEN, T;PETERSEN, LC
在苯甲酰胺存在下，从转基因的幼年仓鼠肾细胞产生的单链人重组因子VII被纯化为均一。以氨基对硝基苯胺为底物的单链重组因子VII的酰胺化活性不到因子VIIa的1%。纯化的单链重组因子VII在没有抑制剂的情况下自发激活。在以阴离子交换基质或聚(D-赖氨酸)形式提供的带正电的表面的存在下，活化反应至少提高了2个数量级。VIIa因子生成的进展曲线呈S形。对重组因子VIIa活性和第VII因子激活有抑制作用，其抑制常数(KI)均为11 mM。相反，当Ki值分别为0.3 mM和0.5 mM时，对牛凝血因子Xa和凝血因子IIa的抑制作用被观察到。牛因子Xa和IIa是已知的凝血因子VII的激活剂，也是我们的重组凝血因子VII制剂中最有可能的污染物。从无牛血清培养的细胞中纯化的单链重组因子VII，其激活速度与有牛血清培养的细胞中的因子VII的活性相同。这也排除了激活反应是由污染的牛蛋白酶引起的可能性。在这些观察的基础上，我们认为因子VII在体外是在带正电的表面存在的情况下自动激活的。
Single-chain human recombinant factor VII produced by transfected baby hamster kidney cells was purified to homogeneity in the presence of benzamidine. The amidolytic activity of single-chain recombinant factor VII with a peptidylnitroanilide substrate, methoxycarbonyl-D-cyclohexanylglycyl-L-arginine-p-nitroanilide, was less than 1% of that obtained with factor VIIa. Purified single-chain recombinant factor VII spontaneously activated in the absence of inhibitor. The activation reaction was enhanced by at least 2 orders of magnitude in the presence of a positively charged surface, provided either as an anion-exchange matrix or as poly(D-lysine). The progress curve for factor VIIa generation was sigmoidal. Benzamidine inhibits recombinant factor VIIa activity and factor VII activation with identical inhibition constants (Ki) of 11 mM. In contrast, benzamidine inhibition of bovine factor Xa and bovine factor IIa was observed at Ki values equal to 0.3 and 0.5 mM, respectively. Bovine factors Xa and IIa are known activators of factor VII and the most likely contaminants of our recombinant factor VII preparations. Single-chain recombinant factor VII purified from cells cultured in the absence of bovine serum activated at the same rate as factor VII from cells cultured in the presence of bovine serum. This also excluded the possibility that the activation reaction was caused by contaminating bovine proteases. On the basis of these observations, we propose that factor VII is autoactivated in vitro in the presence of a positively charged surface.