Hemophilia B with mutations at glycine-48 of factor IX exhibited delayed activation by the factor VIIa-tissue factor complex

Gly-48 is in the conserved DGDQC sequence residues 47-51 of human factor IX ) of the first EGF (EGF-l)-like domain of factor IX. The importance of the Gly-48 is manifested by two hemophilia B patients; factor IXTainan and fact or IXMalmo27, with Gly-48 replaced by arginine (designated IXG48R) and vali ne (IXG48V), respectively. Both patients were CRM+ exhibiting mild hemophil ic 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 recom binant factor IX variants exhibited approximately 26%-27% normal factor IX' s clotting activities with G48R or G48V mutation. Both variants depicted no rmal quenching of the intrinsic fluorescence by increasing concentrations o f calcium ions and Tb3+, indicating that arginine and valine substitution f or 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 activit y of factors IXG48R and IXG48V was attributed to the failure of both mutant s to cleavage factor X: in the presence of only phospholipids and calcium i ons. both mutants showed a 4 similar to 7-fold elevation in K-m, and by add ing factor Villa to the system, although factor Villa potentiated the activ ation of factor X by the mutants factor IXaG48R and factor IXaG48V, a 2 sim ilar to 3-fold decrease in the catalytic function was observed with the mut ant factor IXa's, despite that they bound factor VIIIa on the phospholipid vesicles with only slightly reduced affinity when compared to wild-type fac tor IXa. The apparent K-d for factor VIIIa binding was 0.83 nM for normal f actor IXa, 1.74 nM for IXaG48R and 1.4 nM for IXaG48V. Strikingly, when int eraction with the factor VIIa-TF complex was examined, both mutations were barely activated by the VIIa-TF complex and they also showed abnormal inter action with VIIa-TF in bovine thromboplastin-based PT assays. Taken togethe r, our results suggest that mutations at Gly-48 altered the interaction of factor IX with its extrinsic pathway activator (VIIa-TF complex,, its macro molecular substrate (factor X), and its cofactor (factor Villa).