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

We evaluated the ability of a number of peptides based on the tethered ligand sequences of human, rat and murine proteinase‐activated receptor‐4 (PAR4), to serve as receptor‐activating probes or antagonists for bioassays carried out in vitro and for in vivo models of inflammation. In a rat PAR4‐depe...

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Published in:British journal of pharmacology 2004-10, Vol.143 (4), p.443-454
Main Authors: Hollenberg, Morley D, Saifeddine, Mahmoud, Sandhu, Sabrina, Houle, Steeve, Vergnolle, Nathalie
Format: Article
Language:English
Subjects:
Amino Acid Sequence - genetics
Animals
Biological and medical sciences
Dose-Response Relationship, Drug
In Vitro Techniques
inflammation
Inflammation - chemically induced
Inflammation - genetics
Ligands
Male
Medical sciences
Muscle Contraction - drug effects
Muscle Contraction - physiology
PAR4
Peptide Fragments - genetics
Peptide Fragments - pharmacology
Peptide Fragments - toxicity
Pharmacology. Drug treatments
platelets
proteases
Proteinase‐activated receptors
Rats
Rats, Sprague-Dawley
Rats, Wistar
Receptors, Thrombin - agonists
Receptors, Thrombin - genetics
Receptors, Thrombin - physiology
vascular and gastric smooth muscle
Vasodilation - drug effects
Vasodilation - physiology
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Summary:We evaluated the ability of a number of peptides based on the tethered ligand sequences of human, rat and murine proteinase‐activated receptor‐4 (PAR4), to serve as receptor‐activating probes or antagonists for bioassays carried out in vitro and for in vivo models of inflammation. In a rat PAR4‐dependent platelet aggregation assay, the relative potencies of the active sequences (AYPGKF‐NH2>GYPGKF‐NH2>GYPGFK‐NH2>GFPGKP‐NH2) were consistent with an activation of PAR4. In the aggregation assay, the reverse or partial reverse‐sequence peptides (VQGPYG‐NH2, YAPGKF‐NH2 and FKGPYA‐NH2) were inactive, while trans‐cinnamoyl (Tc)‐YPGKF‐NH2, Tc‐APGKF‐NH2 and N‐palmitoyl‐SGRRYGHALR‐NH2 (pepducin P4pal‐10) were antagonists. 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. We conclude that PAR4‐derived tethered ligand peptide agonists can act at receptors other than PAR4 and that a judicious choice of ligands is required to probe for PAR4 function in bioassay systems and in particular for in vivo models. By selecting from these peptides the ones most reliably reflecting PAR4 activation (AYPGKF‐NH2 as a standard agonist; YAPGKF‐NH2 as a PAR4‐inactive standard), we were able to establish an inflammatory role for the PAR4‐activating peptides acting via a non‐neurogenic mechanism in a rat paw oedema model. British Journal of Pharmacology (2004) 143, 443–454. doi:10.1038/sj.bjp.0705946
ISSN:0007-1188
1476-5381
DOI:10.1038/sj.bjp.0705946