Proteinase-activated receptor-4: evaluation of tethered ligand-derived peptides as probes for receptor function and as inflammatory agonists in vivo.

1. We evaluated the ability of a number of peptides based on the tethered ligand sequences of human, rat and murine proteinase-activated receptor-4 (PAR(4)), to serve as receptor-activating probes or antagonists for bioassays carried out in vitro and for in vivo models of inflammation. 2. In a rat PAR(4)-dependent platelet aggregation assay, the relative potencies of the active sequences (AYPGKF-NH(2)>GYPGKF-NH(2)>GYPGFK-NH(2)>GFPGKP-NH(2)) were consistent with an activation of PAR(4). 3. In the aggregation assay, the reverse or partial reverse-sequence peptides (VQGPYG-NH(2), YAPGKF-NH(2) and FKGPYA-NH(2)) were inactive, while trans-cinnamoyl (Tc)-YPGKF-NH(2), Tc-APGKF-NH(2) and N-palmitoyl-SGRRYGHALR-NH(2) (pepducin P4pal-10) were antagonists. 4. However, in an endothelium-dependent NO-mediated rat aorta (RA) relaxation assay and in a gastric longitudinal muscle (LM) contraction assay, these antagonist peptides were agonists as were most other peptides, with distinct orders of potencies that differed for both the RA and LM assays and from the platelet assay. 5. We conclude that PAR(4)-derived tethered ligand peptide agonists can act at receptors other than PAR(4) and that a judicious choice of ligands is required to probe for PAR(4) function in bioassay systems and in particular for in vivo models. 6. By selecting from these peptides the ones most reliably reflecting PAR(4) activation (AYPGKF-NH(2) as a standard agonist; YAPGKF-NH(2) as a PAR(4)-inactive standard), we were able to establish an inflammatory role for the PAR(4)-activating peptides acting via a non-neurogenic mechanism in a rat paw oedema model.