Isolation and Properties of the Abnormal Factor IX Molecule of Hemophilia B_M

Abnormal factor IX from a hemophilia B_m patient (F. IX-B_m) has been isolated to homogeneity on SDS Polyacrylamide gel electrophoresis by the same technique utilized for purifying normal F. IX. F. IX-B_m generated no measurable procoagulant activity when incubated with F. XI_a in a two-stage F. IX_a assay (normal F. IX, 55 sec; F. IX-B_m, >30 min).F. IX-B_m inhibited the activation of F. X by F. VII and ox brain thromboplastin as measured in an amidolytic assay for factor X_a. Normal F. IX also inhibited this reaction but to a five times lesser degree. F. IX-B_m has the same molecular weight on SDS gel electrophoresis as normal F. IX (55,000) and does not differ from normal F. IX in its amino acid composition. F. XIa cleaves F. IX-B_m in the presence of Ca ions at the same rate as it cleaves normal F. IX, yielding a heavy chain of 27,000 molecular weight and a light chain of 16,000 molecular weight. However, the cleavage does not give rise to procoagulant activity. Like normal F. IX_a, the cleaved forms of F. IX-B_m appear to bind phospholipid since F. IX-B_m protein was precipitated with phospholipid in the presence of Ca ions. These data support an hypothesis that the abnormality in the F. IX-B_m molecule stems from a defect at the active site.