Activation and active site occupation alter conformation in the region of the first epidermal growth factor-like domain of human factor VII

The first epidermal growth factor-like domain (EGF-1) of factor VII (FVII) provides the region of greatest contact during the interaction of FVIIa wit h tissue factor. To understand this interaction better, the conformation-se nsitive FVII EGF-l-specific monoclonal antibody (mAb) 231-7 was used to inv estigate the conformational effects occurring in this region upon both FVII activation and active site occupation. The binding affinity of mAb 231-7 w as approximately 3-fold greater for the zymogen state than for the active s tate; a result affected by the presence of both calcium and the adjacent Gl a domain. Once activated, active site inhibition of FVIIa with:a variety of chloromethyl ketone inhibitors resulted in a 10-fold range of affinities o f FVIIai molecules to mAb 231-7. Gla domain removal eliminated this variati on in affinity, suggesting the involvement of a Gla/EGF-I interaction in th is conformational effect. In addition, the binding of mAb 231-7 to FVIIa EG F-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 str ucture. Thus, these present studies indicate that the conformational change associated with FVII activation and active site occupation involves the EG F-1 domain and suggest potential functional consequences of these changes.