Interspecies Exchange Mutagenesis of the First Epidermal Growth Factor-Like Domain of Coagulation Factor VII

Abstract

A high degreee of structural and sequence homology exists between the EGF -1 domains of the vitamin K-dependent coagulation factors, as well as between the EGF-1 domains of individual vitamin K-dependent coagulation factors from various species. Through studies of protein evolution it has been determined the conserved amino acid residues observed are essential for protein structure while the variable residues have been implicated in specific protein-protein interactions. In the case of FVII, this interaction is the high affinity binding of its cofactor, TF, which initiates the extrinsic pathway of coagulation. 43% of the contact area of the FVII molecule in the FVIIa•TF complex is located within the FVII EGF-1 domain. A series of human FVII variants have been constructed in which the EGF-1 domain has been exchanged, either in its entirety or as single amino acid substitutions, with that of the mouse or rabbit. These species were chosen as it had previously been shown that plasma from mouse or rabbit, when combined with human TF, was able to clot at a significantly greater rate than homologous human plasma. We hypothesized that through these exchanges it might be possible to generate a human FVII variant with increased TF binding. Of the FVII variants generated, 2 human FVII point mutants have shown an increased affinity for human TF after transient expression. A75D and T83K exhibited TF binding at 200% and 150%, respectively, of that seen with wild-type human FVII. Both A75D and T83K exhibited clotting and amidolytic activity that was proportionally increased, with respect to TF binding. Computer-generated structures of these variants predict an additional hydrogen bond between the FVII and TF molecules likely to be responsible for the increased TF affinity in the T83K mutant. Intramolecular forces within the FVII molecule are predicted to have caused a conformational change with the A75D mutation which has lead to its increased TF binding. The greater affinity for TF exhibited by the human FVII point mutants A75D and T83K, as compared to wild-type human FVII, is an important step toward the creation of new class of competitive inhibitors of coagulation which are specifically directed towards its initial stage, rather than later in the cascade. However, more study is needed to determine the ability of either of these mutants to compete against each other for TF both 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰 and 𝘪𝘯 𝘷𝘪𝘷𝘰. If either mutant is successful, it can then be active site inhibited by site-directed mutagenesis or through the use of chloromethyl ketones, in order to decrease its clotting and amidolytic activity.