SPECIFIC MOLECULAR INTERACTION SITES ON FACTOR-VII INVOLVED IN FACTOR-X ACTIVATION

Factor VII, a serine-protease zymogen, and tissue factor, the cellular receptor/coenzyme, are the protein components of the macromolecular complex which initiates the extrinsic pathway of the coagulation cascade. Previous studies were directed to the identification of functional sites on factor VII which mediate factor X activation, employing a series of potentially inhibitory synthetic peptides representing the primary structure of factor VII and antibodies to selected peptides. The involvement of at least four high-affinity interactive regions [factor VII (44-50), (196-229), (285-305) and (376-396) peptides] on the surface of factor VII was clearly demonstrated. The minimal sequences for the expression of inhibitory activity of these four molecular recognition domains on factor VII were identified using short and overlapping peptides. The short factor VII-(206-218)-peptide (most inhibitory peptide in the sequence 196-229 on factor VII) inhibited the binding of factor VII to the tisssue-factor-expressing J82 cell line. Furthermore, radiolabeled [Tyr201] factor VII-(199-221)-peptide, with a tyrosine substituted for the normal tryptophan residue, was specifically bound to J82 cells, and also the binding of the radiolabeled peptide to this cell line was specifically inhibited by a monoclonal antibody to tissue factor, confirming that the interaction site for tissue factor on factor VII is present within the peptide sequence 196-229. Kinetic analyses suggested that the regions represented by factor VII-(285-305)- and factor VII-(376-396)-peptides are involved in factor X recognition and the chemical cross-linking of the radiolabeled peptides resulted in specific binding to factor X, confirming that these two regions on factor VII represent the substrate-recognition site. Furthermore, these radiolabeled peptides specifically interact with the heavy chain of factor X, suggesting that the complementary binding region for the substrate-recognition site on factor VII are present on the heavy chain of factor X.