Coagulation factor VII Gln100 --> Arg. Amino acid substitution at the epidermal growth factor 2-protease domain interface results in severely reduced tissue factor binding and procoagulant function.
We have used recombinant mammalian expression and purification of the factor VII (FVII) variant Gln100 --> Arg (Q100RFVII) to study FVII deficiency in subjects with this mutation. Q100RFVII was secreted poorly in comparison with wild-type FVII (WTFVII) in a stable mammalian expression system, and purified variant protein was found to have undetectable clotting activity. Following activation by immobilized factor Xa, Q100RFVIIa had amidolytic activity similar to WTFVIIa in the absence of soluble tissue factor (sTF); however, unlike WTFVIIa no typical increase in activity was seen after addition of sTF. In a factor X activation assay using relipidated transmembrane truncated tissue factor (residues 1-243), Q100RFVIIa showed less than 5% of the ability of WTFVIIa to activate factor X. We performed direct binding analysis of WT and Q100RFVII/FVIIa to immobilized sTF using surface plasmon resonance, and severely reduced binding of both non-activated and activated Q100RFVII to sTF was seen, indicating a pronounced defect in tissue factor (TF) interaction with this variant. In the sTF-FVIIa crystal structure the candidate residue Gln100 is not in contact with TF but is at the epidermal growth factor 2-protease domain interface. We suggest that the mutation results in a global fold change severely reducing tissue factor interaction; mutation of FVII residues not directly involved in the interaction with TF may still result in variant FVII unable to take part in the initiation of coagulation.
Kemball-Cook
G
G
Haemostasis Research Group, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London W12 0NN, United Kingdom.
Johnson
D J
DJ
Takamiya
O
O
Banner
D W
DW
McVey
J H
JH
Tuddenham
E G
EG
D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine / Q000235:genetics; D001777:Blood Coagulation; D005167:Factor VII / Q000235:genetics* / Q000378:metabolism*; D005168:Factor VII Deficiency / Q000235:genetics; D005998:Glycine / Q000235:genetics; D006801:Humans; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013925:Thromboplastin / Q000378:metabolism*
D011994:Recombinant Proteins; D005167:Factor VII; D013925:Thromboplastin; D001120:Arginine; D005998:Glycine