STRUCTURAL INTEGRITY OF THE GAMMA-CARBOXYGLUTAMIC ACID DOMAIN OF HUMAN BLOOD-COAGULATION FACTOR IXA IS REQUIRED FOR ITS BINDING TO COFACTORVIIIA

This report describes the analysis of a novel mutant human factor IX p rotein from a patient with hemophilia B (factor IX activity <1%; facto r IX antigen 45%), Enzymatic amplification of all eight exons of the f actor IX gene followed by direct sequence analysis reveals a single nu cleotide change (a guanine --> adenine transition) in exon 2 at nucleo tide 6409 which results in a glycine --> arginine substitution at amin o acid 12 in the gamma-carboxyglutamic acid rich (Gla) domain of the m ature protein. Factor IX was isolated by immunoaffinity chromatography from plasma obtained from the proband, The purified protein is indist inguishable from normal factor IX by polyacrylamide gel electrophoresi s, Characterization of the variant in purified component assays reveal s that it is activated normally by its physiologic activator factor XI a, but its phospholipid-dependent activation by the factor VIIa-tissue factor complex is diminished, In the presence of phospholipid and 5 m ar Ca2+, the activities of variant and normal plasma-derived factor IX are similar; however, in the presence of activated factor VIIIa (intr insic tenase complex), the normal augmentation of the cleavage of the specific substrate of factor IX factor X, is not observed. The determi nation of the association constants for normal and variant factor IXa with factor VIIIa shows that the affinity of the activated variant fac tor IX for the cofactor factor VIIIa is 172-fold lower than normal, Co mpetition studies using active site-inactivated factor IXas in the int rinsic tenase complex confirm that the defect in the variant protein i s in its binding to factor VIIIa. We conclude that the structural inte grity 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 activ ation of factor IX by the factor VIIa-tissue factor complex.