Indications for the presence of an atypical protease-activated receptor on rat platelets.

Activation of the protease-activated receptor (PAR)-1, one of four known PARs (PAR-1 to PAR-4), can be mimicked by thrombin receptor activating peptides (TRAPs) based on the PAR-1 tethered ligand. Interestingly, despite being activatable by thrombin, rodent platelets do not express PAR-1 and thus do not respond to PAR-1-derived TRAPs, indicating different activation mechanisms between human and rodent platelets. Using a rat platelet aggregation model, we determined that TRAPs based on the tethered ligand of PAR-1 fail to activate rat platelet aggregation at concentrations up to 1 mmol/l. In addition, TRAPs inhibit thrombin-mediated rat platelet aggregation, indicating the presence of a modified PAR-1 in this species. In order to determine characteristics of this putative receptor, we tested a panel of synthesized TRAPs based on the rat sequence (R) and human sequence (H) of the PAR-1 tethered ligand for their ability to inhibit thrombin-induced rat platelet aggregation. Peptides R1-9, R4-9, R4-10, and H4-10 inhibited rat platelet aggregation in response to alpha-thrombin [inhibitory concentration (IC) 50% 0.25-1.5 mmol/l]. None of these peptides blocked epinephrine-, collagen-, or arachidonic acid-induced platelet aggregation. Alanine substitution mapping of H4-10 indicated that both Leu4 and Arg5 are essential for inhibition. Inhibition of thrombin's catalytic activity required peptide concentrations tenfold higher than inhibition of platelet aggregation (IC50% 3-5 mmol/l). No prolongation of thrombin clotting time in response to TRAPs was detected at peptide concentrations up to 5 mmol/l. Our data suggest that (1) rat platelets express a PAR-1 subtype, (2) residues Leu4 and Arg5 of the tethered ligand peptide are required for binding to this new receptor, and (3) further analysis of peptide sequences might reveal a novel PAR-1 subtype.