Molecular recognition in the activation of human blood coagulation factor X

Factor X can be activated by the extrinsic activation complex (factor VIIa:tissue factor), the intrinsic activation complex (factor IXa:factor VIIIa) and by an enzyme from Russell's viper venom (RVV-X). To identify the regions on the surface of factor X that mediate its association with these three activators, we have prepared 21 synthetic peptides representing 65% of the primary structure of factor X. Only 3 of the 21 peptides inhibited the rate of factor X activation, indicating the regions represented by these three peptides are involved in factor X association. Using purified components, the rate of factor Xa formation was inhibited in a dose-dependent manner by these three peptides with the same relative potency of inhibition in each of the activation systems. The observed relative potencies were: peptide 267-283 greater than or equal to peptide 284-303 greater than peptide 417-431. Kinetic analyses indicated that the three peptides inhibited factor X activation in a non-competitive manner, and in mixed inhibitor assays the peptides were shown to be mutually exclusive of one another. In coagulation-based assays, the potency of inhibition by each peptide was decreased. However, in Russell's viper venom-X-initiated assays peptide 417-431 was the best inhibitor. Fab fragments of antibodies raised to these peptides and affinity purified on factor X-agarose columns inhibited both the purified and coagulation-based assays in a dose-dependent manner. Using the x-ray crystal structure of chymotrypsinogen as a model, these three peptides were found to be located spatially close to one another on the surface of factor X and opposite to the region where factor X is cleaved for activation. These data are consistent with a model in which the three activators combine with factor X through a recognition site composed of multiple loci that is distal to the potential cleavage site. This interaction aligns the active sites of these three enzymes in the correct orientation to cleave factor X at the same arginyl-isoleucyl bond.