Peptides were synthesized corresponding to those of the tethered ligand following the thrombin activation cleavage of the cloned human thrombin receptor. To determine affinities of synthetic peptides, the hydrolysis of a tripeptide chromogenic substrate by both human alpha-thrombin (with high fibrinogen clotting activity) and gamma-thrombin (essentially lacking this activity) was examined. For the longest peptide (22 residues), alpha- and gamma-thrombins gave similar but significantly different inhibition constants (i.e. 16 and 27 microM, respectively). However, the first 14-residue peptide did not behave significantly differently with either form of thrombin, nor did peptides containing the agonist ligand (e.g. SFLLRNP), suggesting that these peptides interacted with common regions in both thrombin forms. In contrast, peptide 8-22 (following the agonist peptide) was an activator of alpha- and an inhibitor of gamma-thrombin. Peptide 11-20 bracketed the enhancement activity with alpha-thrombin, while neither peptide 10-14 nor 15-22 displayed this activity. Such enhancement is believed to result from interactions with the fibrinogen recognition exosite (exosite I), which is present in alpha- but missing or disrupted in gamma-thrombin.