Purification and characterization of factor VII 304-Gln: a variant molecule with reduced activity isolated from a clinically unaffected male

Abstract

Abstract Factor VII (FVII) is the plasma serine protease zymogen which, on binding to its cellular receptor tissue factor (TF), initiates blood coagulation. A 47-year-old man with no clinical bleeding tendency was found to have undetectable plasma FVII activity when tested in a one- stage assay using rabbit brain TF, but 0.3 U/mL using recombinant human TF and 1.04 U/mL FVII antigen. Variant FVII purified from his plasma showed an identical migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis to wild-type zymogen. By enzyme kinetic analysis the Km of the variant using FX as a substrate was 12-fold higher than that of normal FVII. Also, the variant FVII was unable to compete with wild-type FVII for limited rabbit TF binding sites. A ligand blot procedure was used to directly demonstrate reduced binding of recombinant human TF to the variant FVII compared with normal FVII. Genetic analysis of leukocyte DNA showed a G to A mutation in the propositus' gene at codon 304 that results in the substitution of a glutamine for an arginine residue in the catalytic domain of the protease. We conclude that this region of the FVII molecule is important for its function.