Abstract
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.
Highlights
Blood coagulation is initiated when circulating FVII1 in plasma binds to its essential cofactor, the trans-membrane lipoprotein receptor TF
We provide evidence demonstrating that both modifications are associated with conformational changes involving the FVII EGF-1 region, as well as providing evidence of functional consequences for these changes on cofactor binding and the catalytic activity of factor VII
The conformation specificity of monoclonal antibody (mAb) 231-7 was used to characterize the conformational behavior of the FVII EGF-1 domain upon zymogen activation, as well as the occupation of the active site of FVIIa with various active site-specific chloromethyl ketones
Summary
Reagents—Recombinant human FVIIa was purified from 293 cell culture supernatant using a protocol adapted from Ref. 28. Serial dilutions of mAb 231-7 or rabbit anti-human FVII polyclonal sera in TBS-T/Ca2ϩ buffer, containing 5 mg/ml BSA, were incubated in triplicate in the sample wells for 3 h at room temperature. Sample wells were washed (3 times) with TBS-T/Ca2ϩ buffer followed by incubation with either goat anti-mouse IgG-conjugated AP for mAb 231-7 or goat anti-rabbit IgG-conjugated AP for the rabbit anti-FVII polyclonal sera, for 1 h at room temperature. After washing 5 times with TBS-T/Ca2ϩ buffer, serial dilutions of FVII(R152Q), FVIIa, FPRckFVIIa, and D1⁄7EGRckFVIIa molecules in TBS-T/Ca2ϩ buffer containing 2 mg/ml BSA were incubated in duplicate in the microtiter wells for 2 h at room temperature. Sample wells were washed 5 times with TBS-T/Ca2ϩ buffer and incubated with biotinylated rabbit anti-human FVII polyclonal antibody (100 l of 1 g/ml antibody concentration) in TBS-T/Ca2ϩ buffer containing 2 mg/ml BSA for 1 h at room temperature. Aliquots of the mAb 231-7/FVIIa/substrate mix (100 l) were incubated in triplicate in the 96-well microtiter plate, and the color was allowed to develop for 6 h at room temperature, at which time the absorbance at 405 nm was determined using the automated microplate reader
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