Purification and characterization of factor VII 304-Gln: a variant molecule with reduced activity isolated from a clinically unaffected male.
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.
O'Brien
D P
DP
Haemostasis Research Group, MRC Clinical Research Centre, Harrow, Middlesex, UK.
Gale
K M
KM
Anderson
J S
JS
McVey
J H
JH
Miller
G J
GJ
Meade
T W
TW
Tuddenham
E G
EG
D000595:Amino Acid Sequence; D000941:Antigens / Q000032:analysis / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western; D004247:DNA / Q000032:analysis / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005167:Factor VII / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010949:Plasma / Q000737:chemistry; D013925:Thromboplastin / Q000378:metabolism
D000941:Antigens; D005167:Factor VII; D004247:DNA; D013925:Thromboplastin