FACTOR-IX BM KIRYU - A VAL-313-TO-ASP SUBSTITUTION IN THE CATALYTIC DOMAIN RESULTS IN LOSS OF FUNCTION DUE TO A CONFORMATIONAL CHANGE OF THE SURFACE LOOP - EVIDENCE OBTAINED BY CHIMERIC MODELING

Factor IX Kiryu is a naturally occurring mutant of factor M that has 2 .5% coagulant activity, even though normal plasma levels of factor IX antigen are detected. Factor M Kiryu was purified from a patient's pla sma by immunoaffinity chromatography with a calcium-dependent anti-fac tor IX monoclonal antibody column. It was cleaved normally by factor M a in the presence of Ca2+, yielding a two-chain factor IXa. However, t he resulting factor Ma showed only 1.5% of the normal factor Ma in ter ms of factor X activation in the presence of factor VIII, phospholipid s, and Ca2+, and had 20% of the normal esterase activity for Z-Arg p-n itrobenzyl ester. Therefore factor IXa Kiryu showed the defect of the catalytic triad or primary substrate binding site as well as defective interaction with factors VIII/X. Single-strand conformational polymor phism analysis and DNA sequencing of the amplified DNA revealed a miss ense point mutation, a T-to-A substitution at nucleotide number 31059 of the factor M Kiryu gene. This mutation resulted in the amino acid s ubstitution of Val-313 by Asp in the catalytic domain. Restriction enz yme analysis of the amplified DNA showed that the mutation was inherit ed from the patient's mother. The chimaeric method was employed to con struct a model of the serine protease domain of factor Ma, and the res ultant model suggested that the Val-313 to Asp substitution altered th e conformation of the substrate-binding site. These data combined with our previous findings on a Gly-311-to-Glu mutant of factor IX suggest that the loop conformation from Gly-311 to Arg-318 is important for t he expression of coagulant activity.