Background and purpose: Protease-activated receptor-4 (PAR 4 ), the most recently discovered member of the PARs family, is activated by thrombin, trypsin and cathepsin G, but can also be selectively activated by small synthetic peptides (PAR 4 -activating peptide, PAR 4 -AP). PAR 4 is considered a potent mediator of platelet activation and inflammation. As both PAR 1 and PAR 2 have been implicated in the modulation of nociceptive mechanisms, we investigated the expression of PAR 4 in sensory neurons and the effects of its selective activation on nociception. Experimental approach and key results:We demonstrated the expression of PAR 4 in sensory neurons isolated from rat dorsal root ganglia by reverse transcription-polymerase chain reaction and immunofluorescence. We found that PAR 4 colocalized with calcitonin gene-related peptide and substance P. We also showed that a selective PAR 4 -AP was able to inhibit calcium mobilization evoked by KCl and capsaicin in rat sensory neurons. Moreover, the intraplantar injection of a PAR 4 -AP significantly increased nociceptive threshold in response to thermal and mechanical noxious stimuli, while a PAR 4 inactive control peptide had no effect. The anti-nociceptive effects of the PAR 4 -AP were dose-dependent and occurred at doses below the threshold needed to cause inflammation. Finally, co-injection of the PAR 4 -AP with carrageenan significantly reduced the carrageenaninduced inflammatory hyperalgesia and allodynia, but had no effect on inflammatory parameters such as oedema and granulocyte infiltration. Conclusions and implications: Taken together, these results identified PAR 4 as a novel potential endogenous analgesic factor, which can modulate nociceptive responses in normal and inflammatory conditions.

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 4GYPGKF-NH 2 4GYPGFK-NH 2 4GFPGKP-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. Table 1.); LM, gastric longitudinal muscle preparation; L-NAME, N o -nitro-L-arginine-methyl ester; P4pal-10, pepducin, N-palmitoyl-SGRRYGHALR-NH 2 ; PAR, proteinase-activated receptor, with subtypes denoted by subscripts; PAR-AP, PAR-activating peptide; PP1, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo [3,4-d]pyrimidine; RA, rat aorta ring preparation; Rev-P4pal-10, reverse pepducin N-palmitoyl-RLAHGYRRGS-NH 2 ; Tc, trans-cinnamoyl

1 We evaluated a potential role for proteinase-activated receptor 4 (PAR 4 ) in a rodent paw inflammation model, with a focus on two main features of inflammation: (1) oedema and (2) granulocyte recruitment. 2 A PAR 4 antagonist (Pepducin P4pal-10; palmitoyl-SGRRYGHALR-NH 2 ) reduced both the oedema and granulocyte recruitment induced by a localized administration of carrageenan in the rat hind paw, pointing to a key role for PAR 4 in this inflammation model. 3 Further, intraplantar injection in the mouse hind paw of a PAR 4 agonist (AYPGKF-NH 2 ), but not its standard PAR 4 -inactive peptide control (YAPGKF-NH 2 ), caused an inflammatory reaction characterized by oedema (increased paw thickness) and granulocyte recruitment (increased paw myeloperoxidase activity). The PAR 4 agonist-induced effects were inhibited in mice pretreated with pepducin P4pal10. 4 These PAR 4 agonist-mediated effects were not affected by pretreatment with inhibitors of either NO production or prostaglandin release (L-NAME and indomethacin, respectively). 5 However, selective immuno-depletion of neutrophils significantly reduced PAR 4 agonist-induced oedema formation. 6 Moreover, AYPGKF-NH 2 -induced oedema was also reduced by pretreatment with either a kinin B 2 receptor antagonist (icatibant) or a tissue or plasma kallikrein inhibitor (FE999024 and FE999026, respectively), but not with a kinin B 1 receptor antagonist (SSR240612). 7 We conclude: (1) that PAR 4 plays an important role in the inflammatory response as it mediates some of the hallmarks of inflammation and (2) that PAR 4 -mediated oedema is dependent on the recruitment of neutrophils and components of the kallikrein-kinin system.

Abstract-In a contractility assay based on the rabbit jugular vein, the structurally related drugs NPC 17731 or icatibant (1 to 3 nmol/L) were insurmountable antagonists of bradykinin (BK) B 2 receptors (B 2 Rs). After ample washing (3 hours), the antagonism exerted by these peptides was not reversible. By contrast, the antagonist LF 16.0687 (30 to 100 nmol/L) was competitive and reversible. A rabbit B 2 R-green fluorescent protein (B 2 R-GFP) conjugate was expressed in mammalian cells. In COS-1 cells, it exhibited an affinity for.61 nmol/L) similar to that of the wild-type rabbit B 2 R. The stably expressed construction in HEK-293 cells was functionally active (phospholipase A 2 assay), and the antagonists mentioned above retained their respective surmountable or insurmountable behavior. Competition of [3 H]BK binding to B 2 R-GFP by the antagonists or BK was largely reversible after a 3-hour washout period at 0°C; at 37°C, icatibant or NPC 17731 effects were not reversible. B 2 R-GFP was visualized in the plasma membranes of HEK-293 cells and rapidly internalized in response to BK. NPC 17731 or icatibant slowly translocated B 2 R-GFP into cells over 24 hours, whereas LF 16.0687 had no effect on the subcellular distribution of B 2 R-GFP. Cell extract immunoblotting with anti-GFP antibodies revealed a 101-to 105-kDa protein that was not significantly degraded on 24 hours of cell treatment with any of the ligands but was translocated in part to the 15 000-g pellet of the extract on treatment with BK or the noncompetitive antagonists. NPC 17731 and icatibant are noncompetitive, nonequilibrium antagonists that promote the cellular sequestration of rabbit B 2 R expressed in an heterologous system.