An Arg Ser substitution in the second epidermal growth factor-like module of factor IX introduces an O-linked carbohydrate and markedly impairs activation by factor XIa and factor VIIa tissue factor and catalytic efficiency of factor IXa

Factor IXR94S is a naturally occurring hemophilia B defect, which results from an Arg 94 to Ser mutation in the second epidermal growth factor (EGF)-like module of factor IX, Recombinant factor IXR94S was activated by factor XIa/ calcium with an approximate to 50-fold reduced rate and by factor VIIa/ tissue factor/phospholipid/calcium with an approximate to 20-fold reduced rate compared with wild-type factor IX, The apparent molecular mass of the light chain of factor IXaR94S was approximate to 6 kD higher than that of plasma or wild-type factor IX, which was not corrected by N-glycosidase F digestion. This result indicated the presence of additional O-linked carbohydrate in the mutant light chain, probably at new Ser 94. The initial rate of activation of factor X by factor IXaR94S in the presence of polylysine was 7% +/- 1% of the initial rate of activation of factor X by plasma factor IXa, and the k(c)/K-M for activation of factor X by factor IXaR94S/factor VIIIa/ phospholipid/calcium was 4% +/- 1% of the k(c)/K-m for activation of factor X by plasma factor IXa/factor VIIIa/phospholipid/calcium. The reduced efficiency of activation of factor X by factor IXaR94S in the tenase enzyme complex was due to a 58-fold +/- 12-fold decrease in k(cat) with little effect on K-m. In conclusion, the R94S mutation had introduced an O-linked carbohydrate, which markedly impaired both activation by factor XIa and turnover of factor X in the tenase enzyme complex. (C) 1999 by The American Society of Hematology.