Blood, Vol. 94 No. 1 (July 1), 1999:
pp. 156-163
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
Mark S. Hertzberg,
Sandra L. Facey, and
Philip J. Hogg
From the Department of Haematology, Westmead Hospital and University
of Sydney, Sydney, New South Wales; and the Centre for Thrombosis and
Vascular Research, School of Pathology, University of New South Wales,
Sydney, Australia.
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 
50-fold reduced rate and by
factor VIIa/tissue factor/phospholipid/calcium with an 20-fold
reduced rate compared with wild-type factor IX. The apparent molecular
mass of the light chain of factor IXaR94S was 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
kc/Km for activation of factor X by factor IXaR94S/factor VIIIa/phospholipid/calcium was 4% ± 1% of the
kc/Km 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 kcat with
little effect on Km. 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.
