Molecular defect in factor IX Tokyo: substitution of valine-182 by alanine at position P2' in the second cleavage site by factor XIa resulting in impaired activation.

Utilizing polymerase chain reaction and directly sequencing the amplified exon 6 of the factor IX gene derived from a mild hemophilia Bm patient, we have identified a T to C mutation at nucleotide 20,525. This point mutation predicted a Val182 to Ala substitution in the abnormal factor IX molecule, designated as factor IX Tokyo. The patient manifested a low factor IX activity and a moderately prolonged ox-brain prothrombin time but a normal factor IX antigen level in plasma. Immunopurified factor IX derived from the patient was found to have a normal molecular weight but a reduced specific activity (23% of normal). Limited proteolysis by activated factor XI or by a snake venom-derived factor X-activating enzyme was considerably delayed, indicating the presence of structural alteration(s) most probably at or near the second enzyme-cleavage site. Once activated, however, factor IXa Tokyo was able to activate factor X normally and was inactivated by antithrombin III also in a normal fashion. The structural model of factor IXa and a docking model of factor IX and activated factor VII (factor VIIa) suggested that the Val182 to Ala substitution would not affect the local conformation of the catalytic domain. This mutation would rather loosen the fitness of the molecule into the substrate-binding pocket of factor VIIa due to a shorter side chain of the Ala substitution at the P2' position of the second cleavage site.