Animal experiment:

Mice[1]SCID mice Male SCID mice and their BALB/cBy controls are operated as C57BL/6J mice, and on the 4th postoperative day mice receive intracolonically (IC) infusion of 100 μg PAR-4-AP or vehicle. Visceral pain measurements started 1 h following the end of infusion[1].

AY-NH2 is a selective agonist of PAR4 with EC50 value of 11 μM [1].

Protease-activated receptor-4 (PAR4) is a member of PARs and plays an important role in mediating cellular effects of thrombin through acting G-proteins i, 12/13 (Rho and Ras activation) and q (calcium signaling) [2].

AY-NH2 is a potent PAR4 agonist and has a higher (~10 fold) activity than GYPGKF-NH2. Using rat platelet aggregation assay, it was shown that AY-NH2 had highly platelet aggregation ability than GY-NH2 and GF-NH2 [1]. When tested with platelet-rich plasma harvested from wild-type C57BL6 mice, AY-NH2 treatment exhibited highly agonist activity on PAR4 while had no effect on other PARs [3].

In male Wistar rats model of paw oedema, i.pl. injection of AY-NH2 markedly reduced the nociceptive score in response to both noxious and non-noxious mechanical stimuli, thus inhibiting carrageenan-induced mechanical hyperalgesia and allodynia [4].

References:
[1].  Hollenberg, M.D., et al., Proteinase-activated receptor-4: evaluation of tethered ligand-derived peptides as probes for receptor function and as inflammatory agonists in vivo. Br J Pharmacol, 2004. 143(4): p. 443-54.
[2].  Yu, G., et al., Increased expression of protease-activated receptor 4 and Trefoil factor 2 in human colorectal cancer. PLoS One, 2015. 10(4): p. e0122678.
[3].  Faruqi, T.R., et al., Structure-function analysis of protease-activated receptor 4 tethered ligand peptides. Determinants of specificity and utility in assays of receptor function. J Biol Chem, 2000. 275(26): p. 19728-34.
[4].   Asfaha, S., et al., Protease-activated receptor-4: a novel mechanism of inflammatory pain modulation. Br J Pharmacol, 2007. 150(2): p. 176-85.