Characterization of a new peptide agonist of the protease-activated receptor-1

Abstract

A new peptide (TFRRRLSRATR), from the c-terminal of human platelet P2Y 1 receptor, was synthesized and its biological function was evaluated. This peptide activated platelets in a concentration-dependent manner, causing shape change, aggregation, secretion and calcium mobilization. Of the several receptor antagonists tested, only BMS200261, a PAR-1 specific antagonist, totally abolished the peptide-induced platelet aggregation, secretion and calcium mobilization. The TFRRR-peptide-pretreated washed platelets failed to aggregate in response to SFLLRN (10 μM) but not to AYPGKF (500 μM). In addition, in mouse platelets, peptide concentrations up to 600 μM failed to cause platelet activation, indicating that the TFRRR-peptide activated platelets through the PAR-1 receptor rather than through the PAR-4 receptor. The shape change induced by 10 μM peptide was totally abolished by Y-27632, an inhibitor of p160 ROCK which is the downstream signal of G 12/13 pathways. The TFRRR-peptide, YFLLRNP, and the physiological agonist thrombin selectively activated G12/13 pathways at low concentrations and began to activate both Gq and G12/13 pathways with increased concentrations. Similar to SFLLRN, the TFRRRpeptide caused phosphorylation of Akt and Erk in a P2Y 12 receptor-dependent manner, and p-38 MAP kinase activation in a P2Y 12 -independent manner. The effects of this peptide are elicited by the first six amino acids (TFRRRL) whereas the remaining peptide (LSRATR), TFERRN, or TFEERN had no effects on platelets. We conclude that TFRRRL activates human platelets through the PAR-1 receptors.

Protease-Activated Receptors in the Intestine: Focus on Inflammation and Cancer

“…Indeed, it has been observed that the signaling generated via an agonist peptide is not identical in all respects to the one induced by proteolytic cleavage, confirming the biased activation. For example, several agonist peptides for PAR1 have shown various effects on signaling triggering platelet activation: no activation, little activation or complete activation (40). In addition, the MAPK pathway generated by the activation of PAR1 via thrombin is not triggered by the SFLLRN-NH 2 agonist peptide (41) unless the doses of agonist peptides used are significantly higher (100-fold) than the commonly used doses (42).…”

From Multiple PAR1 Receptor/Protein Interactions to their Multiple Therapeutic Implications

“…These and other modifications, including the incorporation of heterocyclic and macrocyclic moieties into the PAR1-APs, have provided several peptide PAR1 ligands with agonist, antagonist and mixed agonist-antagonist activity, but often with low potency and selectivity [21,391]. The peptide TFRRRLSRATR derived from the C-terminal sequence of the human platelet P2Y1 receptor has been characterized as a peptide PAR1 agonist in which the pentapeptide TFRRRL is the minimum fragment able to directly activate human platelets through the PAR1 receptor [58].…”

Oral Antithrombotic Effects of Acylhydrazone Derivatives

“…PAR-1 and PAR-4 are present in human platelets, whereas PAR-3 and PAR-4 exist in mouse and rat platelets. PAR-3 serves as a co-receptor for PAR-4, and thrombin binds to PAR-3 and PAR-4 and cleaves the amino terminus of PAR-4 6,25) . Again using rat platelets, the ability of these compounds to inhibit thrombin-induced aggregation was confirmed.…”