MUTATIONS IN THE CATALYTIC DOMAIN OF FACTOR-IX THAT ARE RELATED TO THE SUBCLASS HEMOPHILIA BM

Hemophilia Bm, a variant of hemophilia B, results in a marked increase in the ox brain prothrombin time. Mutations known to cause hemophilia Bm occur at residue 180, 181, or 182 near the amino terminus of the heavy chain and at residue 311, 364, 368, 390, 396, or 397 near the activation site of factor IX (Giannelli et al.,1990). In this study were placed factor IX residues 181, 182, and 390 in separate experiments by site-directed mutagenesis. Valine 181 was replaced by isoleucine or alanine, and valine 182 was replaced by alanine or glycine. Alanine 390 was replaced by valine or aspartic acid. Recombinant factor IXs were expressed in human kidney 293 cells and purified by absorption and elution from a conformational specific monoclonal antibody column. The results show that factor IX Bm is a function not only of the position of the mutated amino acid but also of the particular amino acid substituted. For example, when valine 181 or 182 was replaced by small hydrophobic amino acids (alanine and glycine), factor IXs were found to have significantly decreased clotting activity. Unlike the naturally occurring mutations (Val181-->Phe181 or Val181-->Leu182), however, the small amino acid replacements did not result in prolonged ox brain prothrombin times. Surprisingly, the Ala390-->AsP390 exchange did not affect clotting activity or binding to the macromolecular inhibitor antithrombin III. The Ala390-->Val390 exchange resulted in loss of both clotting activity and binding to antithrombin III. These results suggest that residue 390 is not directly involved in binding to antithrombin III. Furthermore, our results suggest that residue 390 does not make significant intermolecular interactions. It is likely those factor IX Bm variants with mutations at 181, 182, or 390 are defective in the conformational change required for activation, which normally occurs upon cleavage between residues 180 and 181.