Autoactivation of human recombinant coagulation factor VII.
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.
Pedersen
A H
AH
Novo Research Institute, Bagsvaerd, Denmark.
Lund-Hansen
T
T
Bisgaard-Frantzen
H
H
Olsen
F
F
Petersen
L C
LC
D000818:Animals; D001550:Benzamidines / Q000494:pharmacology; D015153:Blotting, Western; D002122:Calcium Chloride / Q000494:pharmacology; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D005167:Factor VII / Q000302:isolation & purification / Q000378:metabolism*; D015942:Factor VIIa / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D011107:Polylysine / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D014361:Trypsin Inhibitors / Q000494:pharmacology
D001550:Benzamidines; D011994:Recombinant Proteins; D014361:Trypsin Inhibitors; D011107:Polylysine; D005167:Factor VII; D015942:Factor VIIa; D002122:Calcium Chloride