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

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.

Worldwide diffusion of FVII Arg304Gln coagulation defect (FVII Padua)*

“…In the same year, the same mutation was reported by O’Brien et al. (8) in an isolated patient. The main features of the defect are a low or a normal FVII activity when rabbit brain or ox‐brain thromboplastins are used in the assay system, respectively.…”

“…Altogether, 27 proven homozygous patients with the FVII Padua defect, belonging to 21 kindreds, were discovered in the world literature (4–20). At least about 15 additional probable cases but without the molecular biology confirmation were also found (25, 26).…”

“…The rarity or lack of this abnormality in northern European countries is also difficult to explain (3, 29). The only case described in England and by us assigned to that country was a patient part of a trial on the primary prevention of ischemic heart disease which gathered patients with different extractions (8, 30). In addition to the previously mentioned sure cases for FVII Padua, several others are probable.…”

Factor VII Gene Defects: Review of Functional Studies and Their Clinical Implications

“…4B). Although F7 mutations are very heterogeneous, some are common in particular populations [51] . On the other hand, many patients with a specific mutation in the F7 gene may have no significant clinical manifestation.…”

Structural requirements for the interaction between tissue factor and factor VII: characterization of chymotrypsin-derived tissue factor polypeptides

“…FVII was purified as previously described from freeze-dried FVII concentrate [15]. The purified protein had a specific activity of 2500 units of FVII clotting activity/mg by one-stage FVII Abbreviations used: FVII etc., Factor VII etc.…”