Structural Integrity of the γ-Carboxyglutamic Acid Domain of Human Blood Coagulation Factor IXa Is Required for Its Binding to Cofactor VIIIa

Abstract

This report describes the analysis of a novel mutant human factor IX protein from a patient with hemophilia B (factor IX activity <1%; factor IX antigen 45%). Enzymatic amplification of all eight exons of the factor IX gene followed by direct sequence analysis reveals a single nucleotide change (a guanine 3 adenine transition) in exon 2 at nucleotide 6409 which results in a glycine 3 arginine substitution at amino acid 12 in the ␥-carboxyglutamic acid rich (Gla) domain of the mature protein.Factor IX was isolated by immunoaffinity chromatography from plasma obtained from the proband. The purified protein is indistinguishable from normal factor IX by polyacrylamide gel electrophoresis. Characterization of the variant in purified component assays reveals that it is activated normally by its physiologic activator factor XIa, but its phospholipid-dependent activation by the factor VIIa-tissue factor complex is diminished. In the presence of phospholipid and 5 mM Ca 2؉ , the activities of variant and normal plasma-derived factor IX are similar; however, in the presence of activated factor VIIIa (intrinsic tenase complex), the normal augmentation of the cleavage of the specific substrate of factor IX, factor X, is not observed. The determination of the association constants for normal and variant factor IXa with factor VIIIa shows that the affinity of the activated variant factor IX for the cofactor factor VIIIa is 172-fold lower than normal. Competition studies using active site-inactivated factor IXas in the intrinsic tenase complex confirm that the defect in the variant protein is in its binding to factor VIIIa. We conclude that the structural integrity of the Gla domain of human factor IX is critical for the normal binding of factor IXa to factor VIIIa in the intrinsic tenase complex. In addition, a glycine at amino acid 12 is necessary for normal activation of factor IX by the factor VIIa-tissue factor complex.

Replacing the first epidermal growth factor-like domain of factor IX with that of factor VII enhances activity in vitro and in canine hemophilia B.

“…where [FIXa/VIIIa] is determined as described in Larson et al (22) in equations 4-10 in a minor modification of the equations reported by Krishnaswamy (23).…”

Identification of Functionally Important Residues of the Epidermal Growth Factor-2 Domain of Factor IX by Alanine-scanning Mutagenesis

“…Binding of Factor IXa to Factor VIIIa-Binding experiments were performed by monitoring the intrinsic factor tenase activity at limiting concentrations of factor VIIIa as described previously (26,33). Freshly prepared factor VIIIa (1.6 nM, 25 l) was incubated with 25 l of different concentrations of wild-type or mutant factor IXa at room temperature for 5 min to form a factor tenase complex.…”

“…Preparation of the Active Site-modified Wild-type and Mutant Factor IXa-Wild-type and mutant factor IXa (N89A, N92A, and G93A) were inactivated with DEGR-CK as described previously (33,36,37). Recombinant factor IXa, at a concentration of 1 M in 50 l of TBS, was incubated with a 3-fold molar excess of DEGR-CK for 7 h at room temperature and then for 17 h at 4°C.…”

“…The factor IXa activity was then calcu-lated according to an active site as described previously (24,30) by the following equation: A 405 ϭ a t 2 ϩ b t ϩ c (30). Factor VIIIa was prepared freshly by incubation with thrombin as described previously (24,33). To analyze the interaction of factor IXa with factor X in the presence of factor VIIIa, 25 l of the factor VIIIa preparation was mixed with an equal volume of factor IXa preincubated with PCPS in TBS/Ca/bovine serum albumin.…”

Localization of the Heparin Binding Exosite of Factor IXa

“…The catalytic domain of FIXa, with a trypsin-like primary specificity pocket, is located on the C-terminal heavy chain of the molecule (7). The Gla and EGF1 domains of FIXa are involved in the Ca 2ϩ -dependent interaction with factor VIIIa on membrane surfaces (6,8,9). Recent mutagenesis data have indicated that the protease domain of FIXa also interacts with factor VIIIa (9).…”