Mechanism of action of thrombin on fibrinogen. II. Kinetics of hydrolysis of fibrinogen-like peptides by thrombin and trypsin

A series of oligopeptide substrates for bovine thrombin and bovine trypsin has been prepared. These peptides have similar amino acid sequences as the α (A) chain of bovine fibrinogen around the arg-gly bond which is hydrolyzed by thrombin, and are of increasing length. The longest peptide synthesized is the octapeptide gly-gly-gly-val-arg-gly-pro-ala-NH₂. The rates of hydrolysis of the arg-gly linkage in these peptides have been measured. In addition, the relative inhibition of fibrinogen clotting by these peptides was determined; also, constants for the inhibition of the thrombin- and trypsin-catalyzed hydrolysis of p-tosyl-l-arginine methyl ester by these peptides were measured.The data indicate that, for both thrombin and trypsin, the enzyme contains at least three subsites, corresponding to the carboxyl side of the hydrolyzed linkage of the substrate, which are involved in substrate binding. A comparison is also made with the rate of proteolysis of the α (A) chain of native bovine fibrinogen by bovine thrombin. This comparison shows that none of the synthetic peptides studied here is as good a substrate as fibrinogen, indicating that some additional factor outside of the eight amino acids in our largest peptide must be involved, in order to account for the high specificity of thrombin.