The first epidermal growth factor-like domain (EGF-1) of factor VII (FVII) provides the region of greatest contact during the interaction of FVIIa with tissue factor. To understand this interaction better, the conformation-sensitive FVII EGF-1-specific monoclonal antibody (mAb) 231-7 was used to investigate the conformational effects occurring in this region upon both FVII activation and active site occupation. The binding affinity of mAb 231-7 was approximately 3-fold greater for the zymogen state than for the active state; a result affected by the presence of both calcium and the adjacent Gla domain. Once activated, active site inhibition of FVIIa with a variety of chloromethyl ketone inhibitors resulted in a 10-fold range of affinities of FVIIai molecules to mAb 231-7. Gla domain removal eliminated this variation in affinity, suggesting the involvement of a Gla/ EGF-1 interaction in this conformational effect. In addition, the binding of mAb 231-7 to FVIIa EGF-1 stimulated the amidolytic activity of free FVIIa. Taken together, these results imply an allosteric interaction between the FVIIa active site and the EGF-1 domain that is sensitive to variation in active site occupant structure. Thus, these present studies indicate that the conformational change associated with FVII activation and active site occupation involves the EGF-1 domain and suggest potential functional consequences of these changes.Blood coagulation is initiated when circulating FVII 1 in plasma binds to its essential cofactor, the trans-membrane lipoprotein receptor TF. Upon binding to cell surface TF, zymogen FVII can be activated to FVIIa by factor IXa (FIXa) (1), factor Xa (FXa) (2), or in an autocatalytic manner by endogenous FVIIa (3), thus allowing propagation of the coagulation cascade. Activation of FVII occurs upon proteolytic cleavage of the Arg 152 -Ile 153 bond, giving rise to a 152-amino acid light chain (ϳ20 kDa) linked by a disulfide bridge to a 254-amino acid heavy chain (ϳ30 kDa) (4). It is the light chain of FVII that contains the first epidermal growth factor-like domain (EGF-1), a stretch of 37 amino acids with characteristic structure that has been implicated as the principal site of FVII interaction with TF (5-8). The FVII molecule also requires calcium for the expression of optimum activity, one molecule of which is bound in each of the protease domains (9, 10) and the EGF-1 domain at a high affinity site (11), with seven more Ca 2ϩ molecules bound with variable affinity by the Gla domain (12).It has been shown recently that the activation of FVII to FVIIa, as well as occupation of the active site by pseudosubstrate inhibitors, results in conformational changes within the heavy chain with implications on cofactor and substrate interaction (13-15). To date, the conformational effects of these events on the TF-binding EGF-1 domain of the native FVII molecule have not been reported. Relevantly, experiments by Ambrosini et al. (16) have described conformational changes in the region of the EGF-1 domain of the highly homolo...

We have previously described a kindred with factor VII (FVII) deficiency whose members exhibited reduced procoagulant activity relative to FVII antigen concentration. In this report, the molecular genetic basis of the FVII defect has been determined to be a heterozygous substitution of Asp for Asn at position 57 in the first epidermal growth factor (EGF) domain. Recombinant FVII (N57D) cDNA was created by site-directed mutagenesis and transiently expressed in human 293 cells. The transfected cells synthesized an immunoprecipitable protein with an apparent molecular weight of 50 kD. Quantitation of expression by FVII enzyme-linked immunosorbent assay indicated that mutant protein yields were consistently low, typically 10% to 30% of wild-type FVII. FVII (N57D) protein did not accumulate intracellularly, and Northern blot analysis indicated equivalent FVII mRNA levels in 293 cells expressing either wild-type FVII or FVII (N57D). Secreted FVII (N57D) protein did not bind tissue factor, exhibited no procoagulant activity, and failed to bind a conformation-dependent monoclonal antibody specific for the first EGF domain of FVII. Molecular modeling of the first EGF domain of FVII predicted that the N57D amino acid substitution would disrupt tertiary bonding structure. We conclude that the N57D mutation affects folding of the first EGF domain of FVII resulting in decreased cellular secretion of a mutant FVII molecule, which is unable to bind tissue factor and is therefore biologically inactive.