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Thrombin, a key mediator of blood coagulation, exerts a large number of cellular actions via activation of a specific G-protein-coupled receptor, named protease-activated receptor 1 (PAR1). Several studies in experimental animals have demonstrated a therapeutic potential of small molecules with PAR1 antagonistic properties for treatment of diseases such as vascular thrombosis and arterial restenosis. We have studied the biological actions of one highly potent, selective PAR1 antagonist, SCH79797 (in vitro , and found that this compound was able to interfere with the growth of several human and mouse cell lines, in a concentration-dependent manner. The ED 50 for growth inhibition was 75 nM, 81 nM and 116 nM for NIH 3T3, HEK 293 and A375 cells, respectively. Moreover, in NIH 3T3 cells, SCH79797 inhibited serum-stimulated activation of p44/p42 mitogen-activated protein kinases (MAPK) at low concentrations and induced apoptosis at higher concentrations. However, the antiproliferative and pro-apoptotic effects of SCH79797 are likely not mediated by PAR1 antagonism, as they were also observed in embryonic fibroblasts derived from PAR1 null mice. These data suggest that, in view of the development of PAR1-selective antagonists as therapeutic agents, effects potentially unrelated to PAR1 inhibition should be carefully scrutinized.Thrombin, a trypsin-like serine protease, is the most potent agonist for platelet aggregation and plays a central role in haemostatic processes [1]. Thrombin catalyses the conversion of fibrinogen to fibrin by cleaving the peptide bond between an arginine and a glycine residue in the fibrinogen sequence [2]; it is also responsible for proteolytic activation of factors V, VIII, XI, XIII and protein C [1]. However, in addition to its role in blood coagulation, thrombin also stimulates mitogenic events in several cell types including fibroblasts, smooth muscle cells and astrocytes [3], therefore playing a central role in tissue repair, fibrosis, inflammation, neurodegeneration, atherosclerosis and restenosis [4][5][6][7].All cellular actions of α -thrombin are mediated by specific G-protein-coupled receptors, named protease-activated receptors (PAR). Activation of PARs by thrombin and other trypsin-like serine proteases is based on a novel mechanism: the protease cleaves part of the N-terminal domain of the receptor, releasing a 'tethered ligand' that subsequently binds to an extracellular loop of the receptor and activates the G-protein-coupled signal transduction [8]. Four PARs have now been cloned [9]; in humans, PAR1 is considered the primary α -thrombin receptor, although thrombin can also activate PAR3 and PAR4 [10]. Thrombin cleaves PAR1 between Arg 41 and Ser 42 , unmasking the N-terminal recognition motif 'SFLLRN' [11].Several small molecules capable of blocking the α -thrombin active site have been characterized over the years as antithrombotic agents, starting with hirudin, a natural leech-derived peptide [12]. However, the identification of the many biological actions of α -t...

1. Kynurenic (KYNA) and quinolinic (QUIN) acids are neuroactive tryptophan metabolites formed along the kynurenine pathway: the first is considered a non-competitive antagonist and the second an agonist of glutamate receptors of NMDA type. The affinity of these compounds for glutamate receptors is, however, relatively low and does not explain KYNA neuroprotective actions in models of post-ischemic brain damage. 2. We evaluated KYNA effects on the release of fibroblast growth factor (FGF)-1, a potent neurotrophic cytokine. Because KYNA exhibits a neuroprotective profile in vitro and in vivo, we anticipated that it could function as an autocrine/paracrine inducer of FGF-1 release. Studies were performed in several models of FGF-1 secretion (FGF-1 transfected NIH 3T3 cells exposed to heat shock, A375 melanoma cells exposed to serum starvation, growth factor deprived human endothelial cells). To our surprise, KYNA, at low concentration, inhibited FGF-1 release in all cellular models. QUIN, a compound having opposite effects on glutamate receptors, also reduced this release, but its potency was significantly lower than that of KYNA. 3. KYNA and QUIN also displayed a major stimulatory effect on the proliferation rate of mouse microglia and human glioblastoma cells, in vitro. 4. Our data suggest that minor changes of local KYNA concentration may modulate FGF-1 release, cell proliferation, and ultimately tissue damage in different pathological conditions.

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Protease-activated receptor (PAR)-1 and PAR-2 are reported to contribute to the fibrotic process in a number of organs, including lung, liver, pancreas, and kidney. The aim of this study was to localize expression and biological activity of PAR-1 and PAR-2 in normal and pathological cutaneous scars. First, we investigated the immunohistochemical expression of PAR-1 and PAR-2 proteins in a series of human normal scars (NS, n = 10), hypertrophic scars (HS, n = 10), and keloids (K, n = 10). Expression of PAR-1 and PAR-2 was observed in all types of scar. Specifically, in HS and K, diffuse PAR-1 and PAR-2 positivity was found in dermal cellular areas composed of myofibroblasts, while no or minor staining was observed in the scattered fibroblasts embedded in abundant extracellular matrix in the context of the more collagenous nodules, irrespective of the type of scar. The hyperplastic epidermis overlying K was also found to be strongly PAR-1 and PAR-2 positive, whilst in most NS and HS the epidermis was faintly to moderately stained. Second, ribonuclease protection assay on paraffin-embedded specimens showed overexpression of PAR-1 and PAR-2 mRNA in K compared to NS and HS. Third, cultured human fibroblasts exposed to TGF-beta1 expressed a myofibroblast phenotype associated with overexpression of PAR-2, while PAR-1 expression was unaffected. Intracellular Ca(2+) mobilization by PAR-2 agonists in myofibroblasts was increased as compared to fibroblasts, whereas the effect of PAR-1 agonists was unchanged. Our in vivo study indicates that PAR-1 and PAR-2 are expressed in cells involved in physiological and pathological scar formation and suggests that in vitro overexpression and exaggerated functional response of PAR-2 may play a role in the function of myofibroblasts in scar evolution from a physiological repair process to a pathological tissue response.

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