Surface plasmon resonance studies of the interaction between factor VII and tissue factor. Demonstration of defective tissue factor binding in a variant FVII molecule (FVII-R79Q).
The blood coagulation cascade is initiated when vessel injury allows factor VII (FVII) to form a complex with tissue factor (TF). Complete deficiency of FVII causes a lethal bleeding diathesis, but individuals with moderately reduced FVII levels are often asymptomatic. Some of these individuals have circulating partially functional FVII, as a result of point missense mutations in critical parts of the molecule. One such mutation has been reported at position 79 in the first epidermal growth factor-like (EGF) domain of FVII, where an arginine residue has been replaced by glutamine. There is controversy as to whether or not this mutation reduces the affinity of the FVII/TF interaction compared to wild-type FVII. To address this problem, we have expressed recombinant FVII-R79Q and subjected it to detailed biochemical analysis. One-stage FVII:C assays show the variant FVII to have reduced activity with respect to the wild type. Rates of autoactivation and activation by FXa to the two-chain molecule were identical for wild-type and variant FVII. The Vmax for FX activation was lower for the mutant as measured using an amidolytic assay for FX activity. In contrast, the Km for FX was lower for the variant than the wild-type molecule. Peptidyl substrate hydrolysis was virtually identical for both variant and normal FVIIa in the presence and absence of TF. The variant has reduced affinity for TF as measured by surface plasmon resonance. FVII-R79Q has an association rate constant (kassoc) one-fifth of that of normal FVII, but a similar kdiss, resulting in a decrease in the affinity of the enzyme for its cofactor.(ABSTRACT TRUNCATED AT 250 WORDS)
O'Brien
D P
DP
Haemostasis Research Group, Clinical Research Centre, Harrow, Middlesex, U.K.
Kemball-Cook
G
G
Hutchinson
A M
AM
Martin
D M
DM
Johnson
D J
DJ
Byfield
P G
PG
Takamiya
O
O
Tuddenham
E G
EG
McVey
J H
JH
D000818:Animals; D001665:Binding Sites; D015374:Biosensing Techniques; D016466:CHO Cells; D006224:Cricetinae; D017077:Culture Media, Conditioned; D004815:Epidermal Growth Factor / Q000378:metabolism; D005167:Factor VII / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015942:Factor VIIa / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D013925:Thromboplastin / Q000378:metabolism*; D014162:Transfection
D017077:Culture Media, Conditioned; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D005167:Factor VII; D013925:Thromboplastin; D015942:Factor VIIa