Abnormal
factor IX variant
proteins were isolated from the plasmas of three unrelated severe
hemophilia-B families that had been previously shown to contain functionally impaired molecules immunologically similar to normal
factor IX. The families studied were: (1) a patient with markedly prolonged ox brain prothrombin time, designated
factor IX Bm Lake Elsinore (IXBmLE); (b) three patients (brothers) with moderately prolonged ox brain prothrombin time, designated
factor IX Long Beach (IXLB); and (c) a patient with normal ox brain prothrombin time designated
factor IX Los Angeles (IXLA). Each variant molecule comigrates with normal
factor IX (IXN) both in the
sodium dodecyl sulfate and in the nondenaturing alkaline gel electrophoresis. All three variant
proteins are indistinguishable from IXN in their
amino acid compositions, isoelectric points,
carbohydrate distributions and number of
gamma-carboxyglutamic acid residues. Each variant
protein undergoes a similar pattern of cleavage by
factor XIa/Ca2+ and by
factor VIIa/Ca2+/
tissue factor, and is activated at a rate similar to that observed for IXN. All of the three variant
proteins also react with an anti-IXN
monoclonal antibody that interferes with the binding of activated IXN(IXaN) to
thrombin-treated
factor VIIIC. However, in contrast to IXaN, the cleaved IXBmLE has negligible activity (approximately 0.2%), and cleaved forms of IXLA and IXLB have significantly reduced activity (approximately 5-6%) in binding to
antithrombin-III/
heparin, and in activating
factor VII (plus Ca2+ and
phospholipid) or
factor X (plus Ca2+ and
phospholipid) +/-
factor VIII. These data, taken together, strongly indicate that the defect in these three variant
proteins resides near or within the latent catalytic site. This results in virtually a complete loss of catalytic activity of the cleaved IXBmLE molecule and approximately 95% loss of catalytic activity of the cleaved IXLA and IXLB molecules.