Identification of molecular sites on factor VII which mediate its assembly and function in the extrinsic pathway...

Factor VII-VIIa, in association with tissue factor, participates in the complex which initiates blood coagulation through the extrinsic pathway. To identify functional domains on factor VII which mediate the activation of factor X, 16 synthetic peptides corresponding to 55% of the primary structure were assayed for their ability to inhibit factor VII function. Factor Xa formation was inhibited by eight of the peptides in a dose-dependent manner. Kinetic analyses indicated noncompetitive inhibition of factor X activation by seven of these peptides. Peptide-(347-361) inhibited factor Xa cleavage of a chromogenic substrate by a competitive mechanism and was excluded from further analysis in this study. Among the seven inhibitory peptides which have the ability to prevent the factor VIIa-tissue factor-mediated conversion of factor X to factor Xa, peptide-(285-305) was most inhibitory, with a Ki value of 2.4 microM. The Ki values were in the range of 42-65 microM for peptides-(44-50), -(194-214), -(208-229), and -(376-390). The least inhibitory peptides were at positions 170-178 and 330-340, with a Ki value greater than 200 microM. Polyclonal antibodies were raised against four of these peptides; and when antisera were assayed by a solid-phase radioimmunoassay, they bound not only to their respective immunizing peptides, but also to factor VII. The Fab fragments of specific IgG preparations, affinity-purified on a factor VII-agarose column, inhibited the rate of factor X activation in a dose-dependent manner. Six of the seven inhibitory peptides represent amino acid sequences within the heavy chain of factor VII, and the remaining one corresponds to a sequence within the light chain. The corresponding regions in the x-ray crystal structure of chymotrypsin represented by the six heavy chain inhibitory peptides are found to be located in three distinct regions, one region located spatially distal to the active site and the other two regions located relatively closer to the active site and the substrate-binding pocket. The results suggest that at least three specific regions in the heavy chain and one region in the light chain of factor VII mediate its interaction with the factor X activation complex.