Thrombin receptor: An endogenous inhibitor of inflammatory pain, activating opioid pathways Laurence Martin a,b , Céline Augé a,b , Jérôme Boué a,b , Michelle C. Buresi c , Kevin Chapman c , Samuel Asfaha c , Patricia Andrade-Gordon d , Martin Steinhoff e , Nicolas Cenac a,b , Gilles Dietrich a,b , Nathalie Vergnolle a,b,c, * a INSERM, U563, Centre de Physiopathologie de Toulouse Purpan, F-31000 Toulouse, France b Universite Paul Sabatier Toulouse III, F-31000 Toulouse, France c Department of Pharmacology and Therapeutics, University of Calgary, Calgary, Alta., Canada d R.W. Johnson Pharmaceutical Research Institute, Spring House, PA, USA e Department of Dermatology and Interdisciplinary Center for Clinical Research, University of Munster, Munster, Germany article info Article history: Received 10 December 2008 Received in revised form 8 July 2009 Accepted 13 July 2009 Keywords: Protease-activated receptors Opioids Pain Inflammation Analgesia Enkephalin abstract Serine proteases such as thrombin, trypsin and mast cell tryptase can act on different cell types through protease-activated receptors (PARs). These receptors have been shown to be implicated in several phe- nomena such as inflammation, platelet activation, immune response and atherosclerosis. Several studies recently reported PARs expression on neurons and some of them demonstrated that these receptors could interfere with nociception. The contribution of PAR 1 to inflammatory pain and the mechanism involved in this phenomenon were investigated. Intraplantar injection of PAR 1 agonist increased withdrawal latency and reduced response frequency to von Frey filaments, thus inhibiting nociceptive response to both mechanical and thermal stimuli in mice. PAR 1 agonist also reduced carrageenan-induced inflammatory hyperalgesia. The anti-nociceptive effects of PAR 1 agonist were mediated by endogenous opioids, as this effect was inhibited by local injection of naloxone methiodide, and because intraplantar injection of PAR 1 agonist increased mRNA expression of the endogenous opioid precursor proenkephalin. However, PAR 1 agonist was not able to inhibit calcium signals in isolated sensory neurons exposed to pro-nociceptive agents. Finally, despite similar inflammatory parameters, PAR 1 -deficient mice showed a strong potentia- tion of inflammatory hyperalgesia induced by the intraplantar injection of either formalin or carrageenan, or in the chronic model of collagen-induced arthritis, compared to wild-type mice. This study highlights a previously unknown endogenous mechanism of analgesia, showing a central role for the thrombin recep- tor PAR 1 in the regulation of inflammatory pain and as an activator of opioid pathways. Ó 2009 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. 1. Introduction Protease-activated receptors (PARs) belong to the G protein- coupled receptors family. They are activated by serine proteases through a special mechanism: proteases specifically cleave the N- terminal extracellular domain of PARs, releasing a new N-terminal sequence which acts as a tethered ligand to activate the receptor itself after binding on its second extracellular loop. Four PARs (PAR 1 to PAR 4 ) have been identified so far. PAR 1 , PAR 3 and PAR 4 are activated by thrombin and mediate the effects of thrombin on platelets [8]. Because of that specific role, they were called thrombin receptors but they can also be activated by other serine proteases such as trypsin and cathepsin G for PAR 4 , coagulation factor VIIa and Xa for PAR 1 [10,24]. Except for PAR 3 , PARs can phar- macologically be activated by synthetic peptides, PARs-APs (PARs- activating peptides), which sequence is based on that of the teth- ered ligand. PAR 1 was the first receptor of this family to be discovered and its role in cardio-vascular pathologies associated with thrombin re- lease has been largely studied, mostly because of its ability to re- spond to low concentrations of thrombin as compared to the other two thrombin receptors [8,9]. A role for PAR 1 as a pro-inflam- matory signal has also been identified, particularly in chronic inflammation of the joints [28] or of the gut [25], where it partic- ipates in the activation of monocytic cells. PAR 1 antagonists have 0304-3959/$36.00 Ó 2009 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.pain.2009.07.016 Abbreviations: BSA, bovine serum albumin; DC, dendritic cell; DRG, dorsal root ganglia; FBS, fetal bovine serum; GM-CSF, granulocyte–macrophage colony-stim- ulating factor; HBSS, Hanks’ balanced salt solution; HPRT, hypoxanthine phosphoribosyl transferase; M-CSF, macrophage colony-stimulating factor; PAR 1 , protease-activated receptor 1; PAR 1 -AP, PAR 1 -activating peptide; PAR 1 -RP, PAR 1 - reverse peptide; PBS, phosphate-buffered saline; PENK, proenkephalin; POMC, proopiomelanocortin; PDYN, prodynorphin. * Corresponding author. Address: INSERM U563, Centre de Physiopathologie Toulouse Purpan, BP3028, 31024 Toulouse Cedex 3, France. Tel.: +33 562 74 45 36; fax: +33 562 74 45 28. E-mail address: nathalie.vergnolle@inserm.fr (N. Vergnolle). PAIN Ò 146 (2009) 121–129 www.elsevier.com/locate/pain