Key words Protease-activated receptor
Thrombin receptor activating peptides
Springer Online Journal Archives 1860-2000
Abstract 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 α-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.
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