Summary
Gly-48 is in the conserved DGDQC sequence (residues 47-51 of human factor IX) of the
first EGF (EGF-1)-like domain of factor IX. The importance of the Gly-48 is manifested
by two hemophilia B patients; factor IXTainan and factor IX>Malmö27, with Gly-48 replaced by arginine (designated IXG48R) and valine (IXG48V), respectively.
Both patients were CRM+ exhibiting mild hemophilic episodes with 25% (former) and 19% (latter) normal clotting
activities. We characterize both factor IX variants to show the roles of Gly-48 and
the conservation of the DGDQC sequence in factor IX. Purified plasma and recombinant
factor IX variants exhibited approximately 26%–27% normal factor IX’s clotting activities
with G48R or G48V mutation. Both variants depicted normal quenching of the intrinsic
fluorescence by increasing concentrations of calcium ions and Tb3+, indicating that arginine and valine substitution for Gly-48 did not perturb the
calcium site in the EGF-1 domain. Activation of both mutants by factor XIa appeared
normal. The reduced clotting activity of factors IXG48R and IXG48V was attributed
to the failure of both mutants to cleavage factor X; in the presence of only phospholipids
and calcium ions, both mutants showed a 4∼7-fold elevation in K
m, and by adding factor VIIIa to the system, although factor VIIIa potentiated the
activation of factor X by the mutants factor IXaG48R and factor IXaG48V, a 2∼3-fold
decrease in the catalytic function was observed with the mutant factor IXa’s, despite
that they bound factor VIIIa on the phospholipid vesicles with only slightly reduced
affinity when compared to wild-type factor IXa. The apparent K
d for factor VIIIa binding was 0.83 nM for normal factor IXa, 1.74 nM for IXaG48R and
1.4 nM for IXaG48V. Strikingly, when interaction with the factor VIIa-TF complex was
examined, both mutations were barely activated by the VIIa-TF complex and they also
showed abnormal interaction with VIIa-TF in bovine thromboplastinbased PT assays.
Taken together, our results suggest that mutations at Gly-48 altered the interaction
of factor IX with its extrinsic pathway activator (VIIa-TF complex), its macromolecular
substrate (factor X), and its cofactor (factor VIIIa).
Key words
Hemophilia B - factor IX - Gly-48 - mutagenesis