Characterization of Thrombin-Induced Leukocyte Rolling and Adherence: A Potential Proinflammatory Role for Proteinase-Activated Receptor-4 1 Nathalie Vergnolle, 2 * Claudia K. Derian, ‡ Michael R. D’Andrea, ‡ Martin Steinhoff, † and Patricia Andrade-Gordon ‡ It is commonly accepted that thrombin exerts its proinflammatory properties through the activation of proteinase-activated receptor (PAR)-1, although two other thrombin receptors have been discovered: PAR-3 and PAR-4. In this study, we have investigated the mechanisms and the receptors involved in thrombin-induced leukocyte/endothelial cell interactions by using selective agonists and antagonists of thrombin receptors in an in vivo intravital microscopy system. Topical addition of selective PAR-1 agonists to rat mesenteric venules failed to reproduce the increased leukocyte rolling and adhesion observed after thrombin topical addition. When added together with the selective PAR-1 antagonist RWJ-56110, thrombin was still able to provoke increased leukocyte rolling and adherence. The thrombin-induced leukocyte rolling and adherence was not affected by pretreat- ment of rats with an anti-platelet serum. Selective PAR-4-activating peptide was able to reproduce the effects of thrombin on leukocyte rolling and adhesion. Intraperitoneal injection of PAR-4-activating peptide also caused a significant increase in leuko- cyte migration into the peritoneal cavity. In rat tissues, PAR-4 expression was detected both on endothelium and isolated leuko- cytes. Taken together, these results showed that in rat mesenteric venules, thrombin exerts proinflammatory properties inducing leukocyte rolling and adherence, by a mechanism independent of PAR-1 activation or platelet activation. However, PAR-4 acti- vation either on endothelial cells or on leukocytes might be responsible for the thrombin-induced effects. These findings suggest that PAR-4 activation could contribute to several early events in the inflammatory reaction, including leukocyte rolling, adherence and recruitment, and that in addition to PAR-1, PAR-4 could be involved in proinflammatory properties of thrombin. The Journal of Immunology, 2002, 169: 1467–1473. T he mechanism of action of thrombin has been shown to be mediated in part, via the proteolytic activation of cell sur- face G-protein-coupled receptors (1– 4). Thrombin acts as a proteinase, cleaving and unmasking the N-terminal amino acid sequence of the G-protein-coupled receptors and this new N-ter- minal domain acts as a tethered self-activating ligand. Three mem- bers of the proteinase-activated receptor (PAR) 3 family have been described as receptors activated by thrombin: PAR-1, PAR-3, and PAR-4. These thrombin receptors have distinct tethered ligand se- quences (SFLLRNPN. . . for human PAR-1, TFRGAPPN. . . for human PAR-3, and GYPGQV. . . for human PAR-4) (1, 3–5). A remarkable property for PAR-1 and PAR-4 (but not PAR-3) is that synthetic peptides based on the proteolytically revealed sequence (e.g., SFLLR-NH 2 for PAR-1 and GYPGQV-NH 2 for PAR-4) are able to activate the receptor, mimicking the actions of thrombin (3, 6). Nonetheless, it has been shown that the PAR-1-activating pep- tide (AP) SFLLR-NH 2 (SF-NH 2 ) is not selective for PAR-1 but is also able to activate PAR-2, a PAR triggered by trypsin or tryptase, but not thrombin (3, 6 – 8). Because the synthetic peptide corre- sponding to the tethered ligand of PAR-1 (SF-NH 2 ) is not a se- lective agonist for PAR-1 but also activates PAR-2, selective PAR- 1-APs have been developed: TFLLR-NH 2 (TF-NH 2 ) (3, 6, 8) and aparafluoroFRCyclohexylACitY-NH 2 (Cit-NH 2 ) (3, 9), which are both highly selective for PAR-1. These latter two peptides can be used to reproduce the PAR-1-mediated effects of thrombin in vivo, without concurrently activating PAR-2. The discovery of PAR-1, PAR-3, and PAR-4 resulted from a successful search for the receptors responsible for the cellular ac- tions of thrombin on platelet aggregation (2–5). PAR-1 and PAR-4 were identified as the receptors responsible for thrombin-induced platelet activation in humans (5), while in rodents PAR-3 and PAR-4 exert the same role (10). Therefore, it is widely accepted that PAR-1, PAR-3, and PAR-4 play a key physiological role in hemostatis. Although other physiological roles have been sug- gested for PAR-1, especially in the setting of inflammation, no potential functions other than platelet aggregation have been iden- tified for PAR-3 and PAR-4. However, PAR-3 appears to act only as a cofactor in the activation of PAR-4 by thrombin (11). The teth- ered ligand for PAR-4, GYPGKF-NH 2 , selectively activates PAR-4, but its lack of potency renders it of limited utility (12). A structure analysis of PAR-4 peptides has highlighted the peptide AYPGKF- NH 2 as a potent and selective PAR-4 agonist, identifying this peptide as a useful tool for probing PAR-4 functions (12). *Mucosal Inflammation Research Group, Department of Pharmacology and Thera- peutics, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada; † De- partment of Dermatology, University of Mu ¨nster, Mu ¨nster, Germany; and ‡ Drug Dis- covery, Johnson and Johnson Pharmaceutical Research and Development, Spring House, PA 19477 Received for publication March 19, 2002. Accepted for publication May 22, 2002. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by Canadian Institute of Health Research and NicOx SA grants. N.V. is a Canadian Institute of Health Research Scholar. M.S. is supported by grants from the Bundesministerium fu ¨r Bildung, Wissenschaft, Forschung und Tech- nologie (Fo ¨ 01KS9604/0) and Interdisziplina ¨res Zentrum fu ¨r Klinische Forschung (D-16). 2 Address correspondence and reprint requests to Dr. Nathalie Vergnolle, Department of Pharmacology, Faculty of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, T2N4N1, Canada. E-mail address: nvergnol@ucalgary.ca 3 Abbreviations used in this paper: PAR, proteinase-activated receptor; AP, activating peptide; PMN, polymorphonuclear leukocyte; SF-NH 2 , SFLLR-NH 2 ; TF-NH 2 , TFLLR-NH 2 ; Cit-NH 2 , aparafluoroFRCyclohexylACitY-NH 2 . The Journal of Immunology Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00