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Department of Dermatology and Ludwig Boltzmann Institute for Cell and Immunobiology of the Skin (M.S., J.B., V.S., A.R., C.M., T.A.L.), University of Münster, 48149 Münster, Germany; and Department of Pharmacology (N.V., M.D.H.), University of Calgary, Calgary, Canada T2N 4N1
Correspondence: Address all correspondence and requests for reprints to: Martin Steinhoff, M.D., Ph.D., Department of Dermatology and Boltzmann Institute for Immunobiology of the Skin, University of Münster, von-Esmarch-Strasse 58, 48149 Münster, Germany. E-mail: msteinho{at}uni-muenster.de.
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 Top Abstract I. IntroductionII. PAR1 in Inflammation...III. PAR2 in Inflammation...IV. PAR3 and PAR4V. ConclusionsReferences |
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I. Introduction |
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TopAbstractI. IntroductionII. PAR1 in Inflammation...III. PAR2 in Inflammation...IV. PAR3 and PAR4V. ConclusionsReferences |
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Recent studies elucidated the ability of certain serine proteinases to regulate cell function via G protein-coupled receptors (GPCRs). At least two different types of proteinase receptors have been identified involving proteolytic cleavage in their activation mechanism: urokinase receptors and proteinase-activated receptors (PARs) (12, 13, 14).
PARs belong to a new subfamily of GPCRs with seven transmembrane domains activated via proteolytic cleavage by serine proteinases (13, 14, 15, 16). PAR1, PAR3, and PAR4 are targets for thrombin, trypsin, or cathepsin G (17, 18, 19, 20). In contrast, PAR2 is resistant to thrombin, but can be activated by trypsin, mast cell tryptase, factor Xa, acrosin, gingipain, and neuronal serine proteinases (21, 22, 23, 24, 25, 26, 27, 28) (Table 1
). Interestingly, PARs are activated by a unique mechanism: proteinases activate PARs by proteolytic cleavage within the extracellular N terminus of their receptors, thereby exposing a novel "cryptic" receptor-activating N-terminal sequence that, remaining tethered, binds to and activates the receptor (Fig. 1) within the same receptor (17, 21, 22). Specific residues (about six amino acids) within this tethered ligand domain are believed to interact with extracellular loop 2 and other domains of the cleaved receptor (29), resulting in activation. This intramolecular activation process is followed by coupling to G proteins and the triggering of a variety of downstream signal transduction pathways (see Sections II.H, III.H, and IV.B; also see Table 2 and Fig. 2, and Refs.13 , 14 , and 16). Thus, PARs are not activated like "classical" receptors because the specific receptor-activating ligand is part of the receptor, whereas the circulating agonist is a relatively nonspecific serine proteinase that does not behave like a traditional hormonal regulator akin to insulin.
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). Some PAR-APs activate more than one PAR, and they activate receptors at concentrations in the micromolar range as compared with nanomolar potencies of the proteinases themselves (31, 32). Although the PAR1-AP, SFLLRN-NH2 also activates PAR2, PAR2-APs, like SLIGRL-NH2 are not capable of activating other PARs. Unfortunately, the relatively low potency (10 to 100 µM EC50) and susceptibility to aminopeptidases (33) limit the utility of the PAR-APs in some bioassay systems. Recently, modified synthetic agonist peptides with higher potency, resistance to aminopeptidases, and greater receptor selectivity have been developed and characterized. These receptor-selective agonists are of use to study the consequences of activating PARs in vivo (Table 1) (13, 14, 16, 34). So far, antagonists for PAR1 (35, 36, 37) and PAR4 (38) have been synthesized, but are not yet available for PAR2. PAR3, as will be elaborated upon in Section IV, remains a puzzle, in that studies with the murine receptor indicate that it cannot be activated either by its cognate synthetic tethered ligand peptide or by thrombin (39, 40). Rather than acting as an independent cell regulator, PAR3 appears to function as a cofactor for the activation of PAR4 (39). Complementary data documenting PAR-mediated effects in various tissues have been obtained using PAR-deficient (PAR–/–) mice. Taken together, data obtained using the enzyme activators themselves (trypsin, thrombin), the PAR-APs and using PAR gene-deficient mice provide compelling evidence that PARs play a critical role in the regulation of various physiological and pathophysiological functions in mammals, including humans. This review focuses on the biology and signaling properties of PARs in various mammalian tissues and highlights the current knowledge about the role of PARs during inflammation and immune response. To complement the information summarized in the sections that follow, the reader is encouraged to consult a number of other recent reviews concerning the activation mechanisms and cell biological aspects of PARs (13, 14, 16, 34, 41, 42, 43, 44, 45).
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II. PAR1 in Inflammation and Immune Response |
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TopAbstractI. IntroductionII. PAR1 in Inflammation...III. PAR2 in Inflammation...IV. PAR3 and PAR4V. ConclusionsReferences |
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However, recent evidence revealed that thrombin is not only a clotting proteinase serving as both a pro- and anticoagulant molecule but also appears to play multifunctional roles related to inflammation, allergy, tumor growth, metastasis, tissue remodeling, thrombosis, and probably wound healing (13, 15, 42, 43). Subsequent to the cloning of PAR1 (17, 46), it was realized that many of the cellular actions of thrombin (e.g., platelet aggregation, angiogenesis, endothelial cell permeability, vasoregulation, gene regulation, leukocyte trafficking, immunomodulation) could be attributed to the activation of its GPCR (40, 47, 48, 49, 50, 51, 52, 53).
PAR1 has been detected in a variety of tissues, including platelets; endothelial cells; fibroblasts; monocytes; T cells positive for CD 8, CD 16, and either CD 56 or CD 57 (54, 55); natural killer (NK) cells (48); CD 34+ hematopoietic progenitor cells (56); dental pulp cells (57); smooth muscle cells (SMC); epithelial cells; neurons; glial cells; mast cells; and certain tumor cell lines (17, 54, 58, 59, 60, 61). A receptor with high affinity for thrombin has also been detected in rat peritoneal macrophages (62). It should be pointed out, however, that PAR1 very likely does not represent the only target for thrombin, in that other (non-PAR) high-affinity binding sites for thrombin [e.g., on platelets or macrophages (62)] and other conserved thrombin sequences apart from the catalytic domain (63) may also play a role in the cellular actions of thrombin in a variety of target tissues.
A. Vasculature and heart: PAR1 antagonists—a novel potential approach for the treatment of cardiovascular diseases
PAR1 can potentially regulate vascular function under both physiological as well as pathophysiological conditions (15, 42). A number of studies have revealed that thrombin and other agonists of PAR1 can affect the vascular tone. For example, before the discovery of PAR1, it was observed that thrombin can regulate vascular tone by an endothelial-dependent mechanism involving the release of nitric oxide (NO) (64). It is now recognized that this effect of thrombin is due to the activation of PAR1. Moreover, thrombin and PAR1 agonists can contract vascular SMC (VSMC) by a direct effect that requires extracellular Ca2+ (64). Thus, in isolated coronary artery and aorta preparations, PAR1 mediates relaxation (64, 65). In contrast, PAR1 agonists contract human placental and umbilical arteries (66). Furthermore, PAR1 mediates prostanoid generation and secretion, cellular contraction and barrier dysfunction, and enhanced expression of platelet-derived growth factor (PDGF) in human and bovine endothelial cells (67). Interestingly, progestins such as progesterone or gestodene up-regulate PAR1 expression and thereby stimulate thrombin-induced tissue factor-dependent surface procoagulant activity in the rat vascular system, suggesting a role of thrombin in hormone-induced thrombosis via PAR1 activation (68).
Recent observations support a role of thrombin and PAR1 in regulation of functions normal (69, 70, 71, 73) and atherosclerotic (75) endothelium. In normal human arteries, PAR1 is mostly confined to the endothelium, whereas during atherogenesis, its expression is enhanced in regions of inflammation associated with macrophage influx, smooth muscle cell proliferation, and an increase in mesenchymal-like intimal cells (75). In vivo, a neutralizing antibody to PAR1 has been observed to reduce expression of mRNA for the proliferating cell nuclear antigen, an index of intimal and neointimal smooth muscle cell accumulation in rat arteries during balloon angioplasty. These data suggest that PAR1 regulates proliferation and accumulation of neointimal SMC during tissue repair (76).
Thrombin and PAR1 agonists cause a rapid but transient contraction of endothelial cells in various tissues resulting in gap formation and increased permeability of plasma proteins and inflammatory cells. Several mediators are involved in this PAR1 modulated process such as cytokines, kinins, and biogenic amines (67, 72, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95).
It was also revealed that PAR1 plays a crucial role during vascular ontogenesis. Interestingly, about 50% of the PAR1-deficient mouse embryos die at midgestation with bleeding from multiple sites. However, a PAR1 transgene driven by an endothelial-specific promoter prevented death of PAR1-deficient embryos (96), indicating that PAR1 modulates endothelial cell function in developing blood vessels, thereby contributing to vascular development and homeostasis in mice (88, 96).
Vascular wall cells respond to the procoagulant factor Xa by an increase in intracellular Ca2+ ([Ca2+]i) and by assembly of this factor into prothrombinase complexes that even enhance this effect. Additionally, factor Xa stimulation of PAR1 leads to an increased production of tissue factor, a prothrombotic agent, underlining the important role of PAR1 for thrombosis (86). Together, these results point to a pivotal role of PAR1 in vascular homeostasis and thrombosis.
PAR1 agonists are also mitogenic, stimulating proliferation of endothelial cells (97, 98), mediating endothelium-dependent relaxation to thrombin and trypsin in human pulmonary arteries (99), and causing the release of IL-6 from human microvascular endothelial cells (HMEC) (72). Because PAR1 up-regulates 
1(I)-procollagen synthesis in VSMC, one may speculate that PAR1 plays a role in vascular wound healing (100).
Finally, factors that regulate PAR1 function on endothelial cells have also been studied. For example, PAR1 is down-regulated by shear stress (101) and inflammatory mediators such as TNF- (102), and is directly modulated by thrombomodulin (103) in human endothelial cells. On the other hand, cyclic mechanical stress leads to an up-regulation of PAR1 in VSMC (104). In contrast to PAR2, little is known about the inflammatory mediators that regulate PAR1 expression in unstimulated or stimulated endothelial cells.
As of yet, only a few studies exist about the role of GRKs in regulating PAR1 in endothelial cells. Only recently, Tiruppathi et al. (105) found a crucial role of the isoform GRK-5 on thrombin-induced desensitization of PAR1.
It is also necessary to mention the recently demonstrated role of PAR1 in vascular associated pathological processes in which thrombin is involved. The role of PAR1 in thrombus formation was recently investigated in an animal model. Cook et al. (106) examined the role of PAR1 in intravascular thrombus formation in an experimental model of arterial thrombosis in the African green monkey. Using a blocking antibody to PAR1, this group demonstrated a dramatically diminished thrombin-stimulated aggregation and secretion of human platelets, whereas platelet activation induced by the PAR1 agonist SFLLR-NH2 was not affected. These results demonstrated that a specific blockade of PAR1 activation by thrombin can prevent arterial thrombosis in this animal model without significantly altering hemostatic parameters. The data suggest that PAR1 is crucially involved in this disease and is an attractive antithrombotic therapeutic target, although it probably has to be inhibited in concert with the low-affinity thrombin receptor PAR4 to fully prevent platelet activation (see Section II.A).
A demonstrated role of PAR1 in intravascular thrombus formation and knowledge about side effects of thrombin inhibitors made appealing an idea to investigate the potency of PAR1 antagonists as antithrombotic therapeutic drugs. Indirect thrombin inhibitors like heparin and warfarin, which prevent the circulation of thrombin, or the newly developed direct thrombin inhibitor ximelagatran (465) are currently used for the treatment of cardiovascular diseases, e.g., myocardial infarction, atherosclerosis, and thrombosis. However, they carry potential bleeding liabilities as an undesirable side effect when administered in chronic patients. Because it is now well known that human platelet responses to thrombin are mainly mediated via PAR1 (19), direct inhibition of this receptor instead of the proteinase represents an attractive way to circumvent possible side effects by thrombin inhibitors.
PAR1 possesses several extra- and intracellular sites that are crucial for its function and that might represent possible targets for antagonists (107). On the extracellular side, blocking of the cleavage site or the hirudin-like domain could inhibit N-terminal cleavage. Bradykinin or peptides derived thereof, also known as thrombostatins, have been shown to prevent thrombin-induced platelet aggregation (108). In addition, antibodies directed against both the cleavage site and the hirudin-binding site have been generated (106, 109). Binding of the antagonist to either the tethered ligand or extracellular loop 2 prevents interaction of the novel N terminus with the extracellular loop. Several small synthetic antagonists have been developed that block the binding site of the tethered ligand. However, peptide antagonists based on modifications of the tethered ligand were relatively unstable or showed only limited inhibitory activity (35). Thus, a second generation of indole or indazole-based peptide-mimetic antagonists was created that block binding of both the tethered ligand and the proteinase (110). One of these was recently shown to prevent thrombus formation in nonhuman primates (107). In addition, several nonpeptide inhibitors were developed. However, it was not reported whether these compounds were PAR1-specific (111). On the intracellular side, the G protein binding sites are possible targets for pharmacological intervention. One approach used the transfection of endothelial cells with a G minigene (112). However, this might suppress all signaling pathways that involve G. Another more specific approach used so-called "pepducins," peptides that were derived from the third intracellular loop of PAR1. These peptides were able to permeate the cells and also carried a membrane anchor (113, 114).
In summary, two preclinical studies with primates demonstrated that PAR1 antagonists have indeed a therapeutic potential for the treatment of cardiovascular diseases (106, 107). However, extensive research needs to be done for the development of orally administered PAR1 antagonists because only those drugs are thought to be suitable for chronic patients (111).
B. Platelets
Activation of platelets by thrombin or APs specific for PAR1 is characterized by calcium influx; cytoskeletal reorganization; platelet aggregation; degranulation (17, 115, 116); thromboxane production (117); mobilization of the adhesion molecule P-selectin and the CD40 ligand to the platelet surface (119); stimulation of serotonin and epinephrine release (120); enhanced expression of CD62, PDGF (AB) and PDGF(BB) (121); and exposure of anionic phospholipids (phosphatidylserine, phosphatidylethanolamine) that support blood clotting (122). Thrombin has also been demonstrated to stimulate vascular endothelial growth factor release in megakaryocytes and platelets (123). The growth factor in turn is able to induce proliferation of endothelial cells. In addition, tissue inhibitor of matrix metalloproteinase (MMP)-4 that colocalized with MMP-2 in resting platelets was released upon platelet aggregation induced by collagen and thrombin. However, a direct effect using specific PAR1 agonists has not been shown yet (124). The density of human PAR1 on the platelet cell surface was estimated to be about 1500 molecules per resting platelet (125). A recently reported intronic polymorphism in the PAR1 gene leads to decreased expression on the platelet surface and thus to a lower response to the AP (126, 127). PAR1 is a high-affinity thrombin receptor in humans: receptor blocking using an antagonist, domain specific antibodies, or simply by desensitization led to inhibition of responses at 1 nM thrombin but only to attenuated responses at 30 nM (128).
High-affinity thrombin binding of platelets is possibly associated via the membrane glycoprotein (GP)Ib-IX-V complex. This interaction is lost in patients suffering from the Bernard-Soulier syndrome, a disease where GPIb-IX-V is not expressed (129). The same condition can be mimicked using either monoclonal antibodies directed against GPIb or proteolytic removal of this protein from the platelet surface (130). Under these conditions, platelet aggregation is either delayed or requires higher thrombin concentrations.
Recently, Schmidt et al. (131) reported that the gene encoding IQ motif containing GTPase activating protein-2, a scaffolding protein for filopodal extension during platelet activation, was identified within the human PAR gene cluster at 5q13, flanked by the PAR1 gene and encompassing the PAR3 gene. Only thrombin- or PAR1-AP-activated platelets showed a rapid translocation of IQ motif containing GTPase activating protein-2 to the platelet cytoskeleton. This suggests that a functional genomic unit evolved to mediate thrombin signaling events in humans (132).
PAR1 on platelets also seems to play an important role during bacterial invasion: the cysteine proteinase gingipain from Porphyromonas gingivalis was reported to activate the receptor leading to uncontrolled activation of the host cells (28). The enzyme streptokinase derived from Streptococcus sp. is widely used in the treatment of coronary thrombosis. It functions as a plasminogen activator that forms an active complex with plasminogen of the host that is able to cleave PAR1 (133). In mouse platelets, PAR1 does not seem to play a role: PAR1 expression was hardly detectable, and a specific AP did not activate rodent platelets (23, 134, 135). Moreover, platelets derived from PAR1-deficient mice responded to thrombin-like wild-type platelets (135).
C. Immune cells
For some time, an important role for serine proteinases and PARs for the modulation of leukocyte effector functions has been proposed (136). As of yet, only limited data about the regulation of immune and inflammatory responses by PARs are available. It is well documented that PARs influence monocyte motility and chemotaxis, modulate pleiotropic cytokine responses, contribute to mononuclear cell proliferation, and induce apoptosis in various immune cells (84, 137). Recently, it was shown that PAR1 is capable of stimulating elastase secretion from macrophages (138). Moreover, functional thrombin receptors are expressed on human T lymphoblastoid cells (139). However, the receptor subtype(s) (PAR1, PAR3, and PAR4) have not been characterized as of yet. Human -thrombin stimulated five different T lymphoblastoid cell lines to increase intracellular free Ca2+ concentrations and to activate protein kinase C (PKC), whereas thrombin receptors were absent in B cell lines. Thus, PARs may regulate T cell function during inflammation and immune responses, but the precise mechanism is still unknown. Granzyme A from cytotoxic and helper T lymphocytes appears to interact with PAR1 in astrocytes to regulate thrombin function (140). Interestingly, granzyme A blocked the thrombin-induced platelet aggregation in a dose-dependent manner by cleaving PAR1, presumably downstream from its thrombin targeted activation site, thereby reducing the response to subsequent challenge with thrombin; but granzyme A itself did not induce a signal in thrombin-stimulated platelets. Thus, granzyme A may interact with PAR1 in a manner that is insufficient to cause aggregation, but sufficient to disarm the ability of the receptor to respond to thrombin. Finally, these observations demonstrate that granzyme A release occurring during immune responses within blood vessels would not directly cause platelet aggregation. Thus, the T cell-derived proteinase granzyme A seems to inhibit responses triggered by thrombin during inflammation or tissue injury.
PAR1 modulates chemotaxis in inflammatory cells. Besides IL-8 secretion (71), thrombin induces production of monocyte chemoattractant protein-1 (MCP-1) in monocytes, probably via PAR1 (58). However, other PAR1-specific agonists were not used in this study. Therefore, it cannot be excluded that other thrombin receptors are involved. IL-8 secretion is up-regulated by interferon-
(IFN-) and diminished by prostaglandin (PG)E2 (51), suggesting a role in cytokine modulation. Furthermore, PAR1 is capable of inducing IL-1 as well as IL-6 production in monocytes (82). These cytokines are known to be proangiogenic, implicating PAR1 in angiogenesis and tissue repair. However, IFN--differentiated growth-arrested U937 cells also respond to PAR1 agonist administration by overcoming cell arrest and revert to a high proliferation rate via regulation of p21CIP1/WAF1 and cyclin D1. Together, these results may help to explain how thrombin promotes tissue repair and unrestricted proliferation in malignant tissues (137, 141).
Because thrombin, via PAR1, is chemotactic for large granular lymphocytes in humans (55), large granular lymphocytes can enhance the effects of PAR1 in patients with inflammatory disorders (142, 143, 144). Moreover, thrombin modulates activity of NK cells (48). For example, thrombin can enhance NK cell-mediated cytotoxicity and IL-2 production in rheumatoid arthritis, and PAR1 may therefore play an important role in this inflammatory process, as well as in NK cell responsiveness to IL-2.
D. Airways
Various cells in mammalian airways such as epithelial cells, SMC, and fibroblasts abundantly express PAR1 on the cell surface. Several recent studies also suggest that PAR1 may play an important role in inflammatory lung diseases such as neutrophilic alveolitis, pulmonary fibrosis, and asthma (50, 77, 91, 145, 146, 147, 148, 149).
In the airways of different species, PAR1 exerts a dual role leading to stimulation of contraction on the one hand and relaxation on the other hand. For example, thrombin stimulates contraction of human bronchial rings (150) and constriction in guinea pig bronchi (as does AP) (151), but activates constriction as well as relaxation in mouse tracheal SMC (152, 153). In these mouse studies, trypsin, thrombin, and peptide agonists of PAR1, PAR2, and PAR4 induced relaxant responses of isolated tracheal smooth muscle preparations, which were mediated by a prostanoid, probably PGE2. This effect was abolished by indomethacin, the cyclooxygenase (COX)-2 inhibitor, nimesulide, and a prostanoid receptor antagonist (AH6809). PAR1 and PAR4 synthetic peptides induced a rapid, transient, contractile response that preceded the relaxant response. Interestingly, the relaxations but not the contractions were inhibited by indomethacin, indicating that this response is mediated by cyclooxygenase products.
E. Gastrointestinal tract
PAR1 was detected in the lamina propria, the submucosa, endothelial cells, and nerves of the gastrointestinal (GI) tract (154, 155, 156, 157). The GI tract expresses relatively high levels of PAR1 mRNA compared with other tissues, both in mice and humans. The functional role of PAR1 as a receptor mediating thrombin-induced effects in the GI tract is far from being resolved because thrombin can also interact with PAR3 and PAR4 (18, 20, 74). Several studies suggest a role for PAR1 in regulating GI motility. In guinea pigs, PAR1 mediates contraction of longitudinal smooth muscle tissue, dependent on extracellular Ca2+ (158, 159). In mouse intestine, PAR1 agonists modulate the function of L-type Ca-channels and also cause contraction (161). Similar results have been observed in rats (162, 163). Irradiation by fractionated X-radiation leads to up-regulation of PAR1 at the protein level, indicating a regulatory role of external trigger factors such as irradiation on PAR1 expression (164). Stimulation of PAR1 upon adhesion of pancreatic carcinoma cells to extracellular matrix proteins such as laminin, collagen IV, and fibronectin has been observed in a pancreatic carcinoma cell line (MIA PaCa-2) (165, 166). Other studies have pointed out a role for PAR1 in intestinal secretory pathways. Buresi et al. (156) have shown that selective PAR1 agonists stimulated Ca2+-dependent chloride secretion in intestinal epithelial cells. This PAR1-induced intestinal chloride secretion involves protein tyrosin kinase Src (Src), epidermal growth factor (EGF) receptor (EGFR) transactivation, activation of a MAPK pathway, phosphorylation of cytosolic phospholipase A2 (cPLA2), and cyclooxygenase activity. The presence of functional PAR1 on intestinal epithelium and the fact that PAR1 activation leads to ion secretion suggest that PAR1 might have important implications for intestinal barrier functions. PAR1 activation on intestinal surfaces could lead to a secretory response, thus contributing to diarrhea, a symptom of intestinal inflammation. More recent observations further suggest a role of PAR1 in intestinal inflammation. We have shown that intracolonic administration of PAR1 agonists caused inflammation and disruption of intestinal barrier integrity (53). Taken together, these results suggest a proinflammatory role of PAR1 in the gut (reviewed in Ref.167). However, additional studies using PAR1 antagonists and/or PAR1-deficient mice will have to verify the role of PAR1 in the pathogenesis of inflammatory bowel diseases.
F. Kidneys and urogenital tract
Recently, a crucial role of PAR1 in the cell-mediated renal inflammation of crescentic glomerulonephritis has been demonstrated in vivo (49). In wild-type mice treated with hirudin (a direct thrombin inhibitor, characterized by a bifunctional mechanism of inhibition, exclusive specificity and strong ability to bind the enzyme) and in PAR1-deficient animals, the proinflammatory effect of thrombin was significantly reduced. Moreover, treatment of wild-type mice with the PAR1 peptide agonist (SFLLRN-NH2) augmented the inflammatory response, suggesting that PAR1 plays an important role in renal inflammation in vivo. Unfortunately, as mentioned above, the peptide SFLLRN-NH2 can also activate PAR2 with a comparable potency, and therefore a role of PAR2 in renal inflammation cannot be ruled out by this study. Surprisingly, although highly expressed in human kidneys, data clarifying a role for PAR1 in inflammation of human kidneys are still lacking.
Recent data revealed that PAR1 is involved in cellular invasion of a transfected canine kidney cell line. PAR1 agonists abrogated G(olf)-mediated invasion of MDCKts.src cells in collagen gels, indicating an important role for PAR1 in tumor metastasis (168, 169). Using the human urogenital cell line RT4, studies done in vitro have revealed that PAR1 and PAR2 agonists are capable of activating iPLA2 accompanied by release of PGE2, which may provide cytoprotection during an acute inflammatory reaction (170).
G. Brain and peripheral nervous system
Recent studies are in favor of an important role of thrombin and PARs in the brain under normal and pathophysiological conditions such as trauma, inflammation, or tumorigenesis (171, 172). Under pathophysiological conditions, i.e., during breakdown of the blood-brain barrier, circulating thrombin in the bloodstream may enter the central nervous system (CNS), leading to activation of PAR1 or PAR4. Additionally, neurons and glia cells are capable of generating prothrombin (173). Most data so far have been achieved by investigating PAR1 (13, 14, 174). In rat brain, PAR1 is expressed by neurons of the neocortex, cingulate cortex, subsets of thalamic and hypothalamic nuclei, discrete layers of the hippocampus, cerebellum, and olfactory bulb, as well as by astroglia (60). Striggow et al. (175) showed that all four PAR subtypes are expressed in rat brain. PAR1 expression was most abundant in the hippocampus, amygdala, and cortex. Interestingly, the expression of PAR1, PAR2, and PAR3 was up-regulated during experimentally induced ischemia.
In human brain, PAR1 is expressed in neurons and astrocytes (175). Cultured rat glia cells (C6) express both functional PAR1 and PAR2 (176, 177). Moreover, PAR1 agonists induce up-regulation of inducible NO synthase (iNOS) in these cells (85) and promote neuronal survival after ischemia (178) or brain trauma (179). PAR1 also protects astrocytes and neurons from apoptosis induced by hypoglycemia and oxidative stress during inflammation (180). In contrast, thrombin may also exert cytotoxic effects on neurons and induce neurite retraction (181, 182), which may be at least in part due to PAR1. Functional studies further revealed that PAR1 agonists cause retraction of neurites by neuroblastoma cells (181) and induce Ca2+ mobilization by hippocampal neurons (178).
In astrocytes, thrombin stimulates aggregation, morphological changes, and proliferation via PAR1 and induces intracellular caspase pathways leading to apoptosis in a cultured motor neuron cell line (NSC19) (183, 184). Both PAR1 and PAR2 stimulate enhanced proliferation of astrocytes (185). Friedmann et al. (186) reported a key role of thrombin in PAR1-mediated postinjury neuron survival. They demonstrated that the toxicity of thrombin on neurons can be controlled by down-regulation of PAR1 and/or release of antithrombin III by T cells. Interestingly, prothrombin expression was enhanced 24 h after injury, whereas PAR1, PAR3, and nexin-1 mRNA expression was unchanged (187). Nexin-1 is a thrombin inhibitor that also appears to control thrombin-PAR1 interactions in a rat trauma model (171, 187, 188). Thus, the whole factory needed to regulate and modulate thrombin function, including receptor as well as activating and inhibiting proteinases, can be generated in the brain. However, PAR1 also induces the reversal of astrocyte stellation in mice and rat (189, 190, 191). A comparable process is caused by exogenous or endogenous injuries of the CNS, triggering astrogliosis.
Taken together, these data clearly indicate a functional role of PAR1 during inflammation and injury in the CNS. However, in some studies using thrombin as an agonist, activation of other PARs and a role of other molecules like thrombomodulin cannot be excluded. For example, thrombin itself up-regulates thrombomodulin in astrocytes in a dose-dependent manner (192). Thus, future studies taking into account all CNS-derived PARs, their proteinases, and proteinase inhibitors are necessary to fully explore the role of PARs in the brain. Finally, some authors suggest a role of thrombin in Alzheimer’s disease, amyotrophic lateral sclerosis, or HIV encephalitis, probably via activation of PARs (182, 193, 194).
Accumulating data also indicate the role of PAR1 in the peripheral nervous system. In rat peripheral nerves, PAR1 is expressed by primary afferent neurons (195). From these observations, one may speculate that thrombin may also regulate inflammatory responses in the peripheral nervous system via PAR1. Indeed, very recently it has been demonstrated that PAR1 is expressed by a large proportion of primary spinal afferent neurons (155). Thus, thrombin directly signals to sensory neurons by cleaving PAR1. Moreover, administration of PAR1 agonists can induce plasma extravasation and edema, which were blocked by ablation of sensory nerves and administration of antagonists to the neurokinin-1 receptor, supporting the idea that thrombin cleaves PAR1 on sensory nerves to stimulate release of SP, which in turn interacts with the NK1 receptor to induce neurogenic inflammation. Thus, thrombin triggers neurogenic inflammation via PAR1 (155). However, because PAR2 and PAR4 mRNA are also expressed in the CNS, it cannot be excluded that proinflammatory effects of thrombin in the nervous system may, in addition, be mediated by other PARs. Functional studies further revealed that PAR1 agonists induce Ca2+ mobilization in enteric neurons, and regulate both excitatory and inhibitory neurons of the myenteric plexus in guinea pig small intestine, e.g., by releasing neuropeptides (196).
Recently, Vergnolle and co-workers (197) as well as other groups (198) examined the effects of PAR1 agonists on nociceptive responses to mechanical and thermal noxious stimuli. Interestingly, intraplantar injection of selective PAR1 agonists induced an enhanced nociceptive threshold and withdrawal latency resulting in mechanical and thermal analgesia. However, thrombin was analgesic in response to mechanical, but not to thermal, stimuli. Moreover, application of PAR1-AP with carrageenan significantly reduced the hyperalgesia. Thus, thrombin may play a dual nociceptive-analgesic role, and future studies will be required to determine whether PAR1 agonists might be of therapeutic use for the treatment of pain.
H. Signaling by proteinases via PAR1
Investigations during the last few years revealed that the above-mentioned PAR1-mediated effects are transduced by various signaling pathways leading to diverse functions of PAR1 under physiological and pathophysiological conditions. These findings were recently reviewed (13). However, in the current work we focus on the signaling events mediated via PAR1 in different tissues and cell types (Table 2).
1. Platelets.
After its generation from prothrombin, thrombin plays multiple roles in the blood coagulation cascade that are mediated by interaction with a number of physiological substrates, effectors, and inhibitors. The accumulation of thrombin at sites of vascular injury provides the recruitment of platelets into a growing hemostatic plug. When added to human platelets in vitro, thrombin causes platelets to change shape, stick to each other, and secrete the contents of their storage granules. How this is accomplished is still not fully understood, but a major step forward occurred since the identification of PAR1 in the 1990s. This finding shed light on the way by which an extracellular protease may initiate intracellular events.
Mammalian GTP-binding proteins (G proteins) fall into four families that are typically referred to by the designation of the -subunit: Gs, Gi, Gq/11, and G12/13. Human platelets express at least one member of the Gs family and four members of the Gi family (Gi1, Gi2, Gi3, and Gz), which, among other functions, stimulate or inhibit cAMP formation by adenylyl cyclase (199). In addition, platelets express one or more members of the Gq/11 family and members of the G12/13 family (G12 and G13) (199).
A number of different G protein -subunits have been shown to bind to PAR1, including members of the Gi, Gq/11 and G12/13 families (200). However, the knowledge of these interactions in humans under physiological and pathophysiological conditions is still far from completion.
Kim et al. (201) have demonstrated that thrombin and PAR1-AP, as well as PAR4-AP, induce Gi pathway stimulation in human platelets. Additionally, the authors demonstrated that thrombin, PAR1-AP, and PAR4-AP cause platelet aggregation independently of Gi stimulation (201). Offermanns et al. (202) also demonstrated a direct interaction between G12, G13, and PAR1. Additionally, it was reported that thrombin-mediated cleavage of the PAR1 (and PAR4; see Section IV.B) receptor leads to calcium-dependent and calcium-independent shape changes of human platelets in consequence of direct activation of both Gq and G12/13 pathways, respectively (115, 202). Therefore, current evidence suggests that PAR1 interacts with Gq, G12/13, and possibly Gi protein family members in human platelets. In turn, these data suggest an activation of downstream signaling cascade members after PAR1 stimulation on human platelets. Among such members are phospholipase C (PLC)-ß, phosphoinositide 3 (PI3)-kinase, and monomeric G proteins.
Indeed, PI3 kinase was demonstrated to play an important role in some PAR1 or thrombin-mediated cellular effects such as cytoskeletal reorganization, alterations in cell motility, cell survival, and mitogenesis. For example, thrombin is able to activate multiple PI3-kinase isoforms, including the recently discovered 110-kDa isoforms that can be directly activated by G protein ß-subunits (145, 150, 203). Stimulation of platelets by PAR1 leads to the activation of PI3 kinase, which is dependent on the small G protein Rho (204). Recently, Trumel et al. (205) have demonstrated that PI3 kinase plays an important role in the PAR1-dependent reorganization of the platelet cytoskeleton via myosin heavy chain translocation and stable association of signaling complexes with the actin cytoskeleton. Interestingly, the activity of small G proteins such as Rac and cdc42 in platelets may be regulated through PAR1 stimulation, but the role of PI3 kinase in these events remains to be determined (206). In their recent study, Vaidyula and Rao (207) provided evidence that in human platelets PLC-ß2 plays a major role in responses to PAR1 and PAR4 activation, and that PLC-ß2 is required for the sustained rise in [Ca2+]i concentration upon thrombin activation.
Putting all of this together, current data suggest that thrombin activates human platelets by cleaving and activating PAR1 and PAR4. In turn, PAR1 activates the members of Gq/11, G12/13, and perhaps Gi families, leading to the activation of PI3 kinase, PLC-ß, and monomeric G proteins (Rho, Rac, and possibly RapI), and also causes an increase of cytosolic Ca2+ concentration and inhibition of cAMP formation.
Additionally, it is important to note that despite the fact that human platelets express both PAR1 and PAR4, these receptors appear to be activated by different thrombin concentrations. Cleavage of human PAR4 requires a higher concentration of thrombin than does cleavage of PAR1, and it is likely that PAR1 is the predominant signaling receptor at low thrombin concentrations (208, 209).
Mouse platelets provide an interesting contrast to human platelets: whereas human platelets express functional PAR1 and PAR4, mouse platelets express PAR3 and PAR4, although in the latter case signaling appears to be mediated entirely by PAR4, with PAR3 serving to facilitate PAR4 cleavage at low thrombin concentrations (for details, see Section IV.A) (39, 40).
2. Cells in the nervous system.
PAR1, as mentioned above, appears to affect various processes in both the central and peripheral nervous systems (45, 210). Among such processes are: neuroinflammation and neurodegeneration, neuritogenesis, astrocyte proliferation, and synaptic plasticity. However, here we would like to focus on data concerning the involvement of PAR1 in intracellular signaling cascades in neuronal cells.
PAR1 may have an effect on various intracellular signaling cascades within neuronal cells (171, 181, 189). By coupling to different G proteins, PAR1 affects a wide range of neuronal cell functions. For example, the necessity of PAR1 coupling to G12 was demonstrated for thrombin-stimulated DNA synthesis in 1321N1 astrocytoma cells (211). Interestingly, injection of antibodies directed against G12 abolished the thrombin-stimulated DNA synthesis (212). Additional studies performed on 1321N1 astrocytoma cells revealed that thrombin treatment causes a concentration-dependent rounding. One may speculate that such an effect of thrombin could be Rho-dependent. The Rho family of small GTPases is known to be involved in the control of cytoskeletal changes via modulation of actin polymerization. Indeed, this thrombin-induced rounding of 1321N1 astrocytoma cells was Rho-dependent and mediated via G12 (213).
Moreover, LaMorte et al. (214) demonstrated binding of PAR1 to Gq by using the same cell line. The authors also showed that thrombin stimulation induces Ras-GTP complex formation and that Ras is required for PAR1-mediated activation of PLC in 1321N1 astrocytoma cells (214). This is especially interesting because thrombin is known to be a factor regulating the proliferation of astrocytes. This effect of thrombin appears to be associated with Go/i- and Gq-mediated pathways. Wang and coworkers recently shed some light on the downstream cascade events of PAR1 signaling (174, 210). Gq-Mediated PLC activity results in Ca2+ mobilization and activation of PKC, which phosphorylates a nonreceptor tyrosine kinase, proline-rich tyrosine kinase (Pyk2). Interestingly, the activation of other nonreceptor tyrosine kinases like Src and focal adhesion kinase (FAK) was also demonstrated after thrombin stimulation. Pyk2 is a factor that is able to connect GPCRs to ERK1/2 activation. Therefore, Pyk2, acting together with Src-tyrosine kinase, causes the activation of the ERK/MAPK pathway, which mediates the proliferative effect of thrombin. Additionally, the same group of authors demonstrated that the Go/i-mediated PI3 kinase pathway is also involved in thrombin-induced astrocyte proliferation (174).
Thrombin is also known to exhibit both beneficial and unfavorable effects in hippocampal neurons and astrocytes (178, 180, 215). Donovan and colleagues (184, 216) found that tyrosine kinases, serine/threonine kinases, and the actin cytoskeleton are involved in both pro- and antiapoptotic effects of thrombin in neuronal cells. However, there was no involvement of Go/i and the PI3 kinase pathway observed in these thrombin effects.
Additionally, Zieger et al. (217) demonstrated the existence of a novel PAR1-associated signaling pathway in the nervous system. According to these data, PAR1 participates in cAMP-independent PKA activation in SNB-19 glioblastoma cells. Moreover, PAR1 stimulation causes the activation of transcriptional factor nuclear factor 
B (NFB) in this cell type (217).
In summary, besides morphological and proliferative effects described above that are mediated via G12/13, Gq, and Go/i, respectively, thrombin also affects activation of transcriptional factors such as activator protein-1 (AP-1) in neuronal cells (214).
3. SMC.
Thrombin exerts direct effects on vascular cells such as SMC and endothelial cells via interactions with PARs (218). As mentioned in Sections II.H.1 and II.H.2, PAR1 interacts with G12/13, Gq, and Gi to elicit diverse downstream signaling events. For example, thrombin-stimulated DNA synthesis and cell migration are associated with activation of the G13 signaling pathway in SMC. The G13 signaling cascade includes the activation of Rho and thus induction of cytoskeletal changes affecting cell migration (219). The Gq-dependent signaling pathway includes the activation of PLC, which in turn leads to MAPK phosphorylation and receptor tyrosine kinase transactivation, both necessary events in thrombin-mediated proliferation. It is interesting that the thrombin-mediated activation of MAPKs such as ERK1/2 was recently demonstrated in SMC. As noticed, PAR1 caused rapid phosphorylation, whereas PAR4 induced prolonged phosphorylation of these kinases in SMC (220). Additionally, Ghosh et al. (221) have shown that thrombin activates p38 MAPK in a time-dependent manner in VSMC. Furthermore, Sabri et al. (222) demonstrated that PAR1 agonists induce activation of Jun N-terminal kinase (JNK) and Akt/PKB in rat ventricular cardiomyocytes.
After dissociation of the G protein heterodimer, Gß interactions activate phosphoinositide 3-kinase, which promotes [Ca2+]i release that is required for SMC growth in response to thrombin stimulation (223).
It is also interesting that PAR1 signaling can modulate gene transcription induced by cytokines in SMC. Thrombin, acting via PAR1, can block IL-6-induced signal transducer and activator of transcription 3/Sis-inducible factor-A (Stat3/SIF-A) activation (224).
In summary, since the discovery of PARs, considerable progress in our understanding of thrombin signaling in SMC was achieved. This allowed some light to be shed on signaling events associated with SMC proliferation, migration, and synthesis of extracellular matrix proteins (e.g., collagen) after thrombin stimulation. However, additional studies are necessary to reveal the role of thrombin in SMC apoptosis, vascular lesion formation, and wound-healing associated pathways.
4. Endothelial cells.
Thrombin is known to affect various functions of endothelial cells. Among these are cell rounding, changes of cell-cell junctions, proliferation, barrier function, and permeability. These thrombin-induced effects appear to be mediated via PARs, particularly PAR1 (225, 226). A major step forward was reached in recent studies, which revealed important intracellular signaling events underlying PAR1-mediated effects in endothelial cells (227).
PAR1 was demonstrated to interact with Go/i, Gq, and probably G12/13 in endothelial cells (228, 229). As well as it was demonstrated for neuronal cells, PAR1-mediated cytoskeletal changes in endothelial cells (cell rounding) are associated with the activation of RhoA (225). Additionally, in the same work it was demonstrated that PAR1-AP stimulation rapidly enhanced vascular permeability in a mouse skin assay (225). Moreover, it was found that binding of PAR1 to pertussis toxin (PTX)-sensitive G proteins (however only to Go, but not to Gi) is also necessary for thrombin-induced changes of endothelial barrier permeability (229). The activation of the MAPK signaling pathway by thrombin in endothelial cells was also demonstrated. This activation plays a crucial role in thrombin-induced effects of endothelial cell functions such as chemokine and cytokine production as well as the expression of cell adhesion molecules (89, 230, 231).
The role of thrombin and PAR1 in the activation of transcriptional factor networks was intensively investigated in recent publications. Malik and coauthors (228, 231, 232) studied the involvement of PAR1 in the activation of NFB in endothelial cells. Their studies showed that Gq and the Gß dimer are responsible for NFB activation and intercellular adhesion molecule-1 (ICAM-1) transcription in endothelial cells induced by the PAR1 agonist thrombin and the PAR1-specific AP TFLLRNPNDK. Furthermore, transfection experiments strongly supported simultaneous activation of Gß/PKC-
/p38 and Gß/PI3-kinase pathways that converge into the Akt pathway, leading to subsequent NFB activation and ICAM-1 expression (228, 231, 232). Moreover, thrombin-induced stimulation of vascular cell adhesion molecule-1 (VCAM-1) production involves the inducible binding of p65 NFB to a tandem NFB motif in the 5' flanking region (233). Taken together, these findings suggest that in the case of ICAM-1 and VCAM-1, p65 NFB is necessary for transducing the thrombin response in endothelial cells.
Additionally, thrombin-mediated induction of VCAM-1 was shown to involve the inducible binding of GATA-2 to a tandem GATA motif in the upstream promoter region (234). Interestingly, the effect of thrombin on GATA-2 DNA binding and transcriptional activity was found to be mediated by a PI3K, PKC-
-dependent signaling pathway (233).
It is important to note that thrombin effects on transcriptional factor networks have been more deeply investigated in endothelial cells than in other cell types. These data explain, at least in part, thrombin-mediated effects on the production of cell adhesion molecules and some chemokines in endothelial cells. Subsequently, this accounts for thrombin effects at leukocyte migration via endothelial barrier (ICAM-1 serves as a ligand for leukocyte ß2 integrins and promotes leukocyte adhesion) and leukocyte recruitment.
5. Immune cells.
The participation of PARs in T cell signaling pathways has also been demonstrated (235). Recently, Bar-Shavit et al. (236) have examined a possible involvement of Vav 1 in PAR-mediated signaling in human Jurkat T cells. The Vav family has three known members in mammalian cells (Vav, Vav2, and Vav3) and one in nematodes (CelVav) (237). Tyrosine phosphorylation of Vav1 regulates its activity as a guanine-nucleotide exchange factor for the Rho-like small GTPases RhoA, Rac1, and cdc 42, which affect cytoskeletal reorganization and activation of stress-activated protein kinases/JNKs. Bar-Shavit et al. (236) clearly showed that activation of PARs induces tyrosine phosphorylation of Vav1 in Jurkat T-leukemic cells. Because 
-chain-associated protein kinase of 70 kDa (ZAP-70) and SH2-domain containing leukocyte-specific phosphoprotein of 76 kDa (SLP-76) have been shown to associate physically with Vav1 and because this association is critical for normal functioning of T cells, it was tempting to investigate whether ZAP-70 and SLP-76 are involved in PAR-induced signaling cascades. Indeed, an increase of tyrosine phosphorylation of ZAP-70 and SLP-76 was observed after activation of Jurkat cells with PAR-APs. Moreover, phosphorylation of Vav1 in response to PAR stimulation was dependent on p56lck (236). Unfortunately, because nonselective PAR-APs were used in the studies done with the Jurkat cells (236), it is difficult to distinguish between the effects of PAR1 and PAR2 in this T cell model system. Furthermore, because of the lack of the ability of PAR3-derived peptides to activate PAR3 (39), the role of PAR3 in T cell signaling remains unknown.
6. Factor Xa signaling mediated via PAR1.
Most of the PAR1-associated signaling events have been observed subsequent to cell stimulation by thrombin. However, recently a possible role of PAR1 in coagulation factor Xa-mediated signaling has been demonstrated (238, 239, 240). The coagulation proteinase factor Xa is generated at sites of vascular injury and inflammation after formation of the tissue factor/VIIa (TF/VIIa) complex. Coagulation factor Xa has been shown to be mitogenic for SMC (238) and elicits inflammatory responses in endothelial cells. Riewald and Ruf (241) have presented new data indicating the involvement of PAR1 in Xa-mediated signaling. They used HeLa cells expressing only PAR1 and demonstrated that factor Xa induces NFB activation and MAPK phosphorylation in these cells. Inhibition studies with specific antibodies revealed that factor Xa responses were mediated via PAR1 (241). Thus, factor Xa (or potentially other serine proteinases) may substitute for thrombin in proteolytic signaling via PAR activation. In endothelial cells, which also express PAR2, Camerer et al. (240) have shown that factor Xa signaling was mediated not only by PAR1, but also to a large extent via PAR2. Together, PAR-1 and PAR-2 appear to account for more than 90% of factor Xa signaling in endothelial cells (240).
In summary, the multiple biological as well as inflammatory and immune responses of thrombin that are mediated by PAR1 [including 1) vasoregulation; 2) increased vascular permeability; 3) cellular adhesion and infiltration of leukocytes; 4) angiogenesis; 5) stimulation of the production of inflammatory mediators such as cytokines, neuropeptides, NO and prostanoids, for example; 6) regulation of extracellular matrix proteins; and 7) induction of signal transduction pathways which are involved in immunomodulation] suggest an important role of PAR1 during inflammation and immune response.
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III. PAR2 in Inflammation and Immune Response |
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TopAbstractI. IntroductionII. PAR1 in Inflammation...III. PAR2 in Inflammation...IV. PAR3 and PAR4V. ConclusionsReferences |
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). However, the endogenous enzymes responsible for activating PAR2 in many tissues remain to be determined. Many endogenous or exogenous trypsin-like enzymes may cleave and activate PAR2. Expression of trypsinogen-2 mRNA and its translation product has been demonstrated in endothelial cells (255). Interestingly, various types of human cancer cells secrete enzymes with trypsin-like specificity (255) that may activate PAR2. In human skin, trypsinogen-4 generated by keratinocytes and trypsinogen-2 from human dermal microvascular endothelial cells can activate PAR2 in vitro (M. Steinhoff, unpublished observation). Another candidate is mast cell tryptase, a major secretory protein of human mast cells. Mast cells from mice and rats are more heterogeneous regarding their protease content, although they also produce proteases with tryptic specificity. Tryptase has been shown to activate PAR2 on epithelial as well as endothelial cells and neurons (23, 196, 243, 251, 256). The observation that tryptase can activate PAR2 suggests a role of this receptor in humans under circumstances when mast cells are involved, e.g., during inflammation, hypersensitivity reactions, and wound repair (24, 257). However, the ability of human tryptase to activate PAR2 appears to be restricted by receptor glycosylation at an N-terminal residue just proximal to the receptor’s cleavage activation site (258, 259). Notwithstanding, the effects of tryptase on cells in vitro often mimic those of PAR2 activation: tryptase up-regulates IL-1ß and IL-8 secretion, enhances the presence of intracellular adhesion molecules/selectins on endothelial cells, mediates accumulation of neutrophils and eosinophils, produces vascular leakage, and is mitogenic for epithelial cells, fibroblasts, and SMC (260, 261, 262, 263). That said, direct proof that PAR2 mediates the effects of tryptase during inflammation in vivo is still lacking.
A. Vasculature
In the vasculature, PAR2 has many effects that are proinflammatory. Agonists of PAR2 induce relaxation in the rings of rat aorta or porcine coronary artery dependent on endothelial NO synthase activity (246, 248, 250). This effect is abolished in the absence of endothelium (246, 250). In contrast, trypsin stimulates contraction of the rabbit aorta in the absence of endothelium (264). In the intact rat, iv injection of SLIGRL-NH2 produces a marked fall in blood pressure, consistent with release of NO from endothelial cells (248). Furthermore, PAR2 agonists increase IL-6 production (72), induce von Willebrand factor release, and serve as a mitogen for human umbilical vein endothelial cells (HUVEC) (97, 265, 266).
Moreover, it was demonstrated that some inflammatory mediators are able to affect the expression of PAR2 in endothelial cells. In HUVEC, PAR2 mRNA is up-regulated by TNF- and IL-1, cytokines that act together to orchestrate the acute inflammatory response (25, 265).
In summary, PAR2 mediates vasodilation, plasma protein extravasation, as well as endothelial cell proliferation in the cardiovascular system. Thus, this receptor can be regarded as an important factor in neovascularization. Moreover, PAR2 can be considered as a vascular sensor for trypsin-like proteinases of the coagulation cascade, which play an important role in cardiovascular medicine.
B. Immune cells
Although information has been acquired about the role of PAR2 in epithelial and endothelial function, relatively little is known so far about the role of this receptor in the immune system. As was recently demonstrated, PAR2 is expressed by various cells involved in immune response, such as T cell lines, eosinophils, neutrophils, and mast cells (147, 160, 249, 252, 267). Accumulating evidence points to a role of PAR2 in the regulation of leukocyte function. Some of the PAR2- mediated effects on leukocyte behavior have been observed in vivo in rodents (262). In this model, it has been reported that the PAR2-AP (SLIGRL-NH2) caused a significant increase in leukocyte migration into the peritoneal cavity after ip injection. Furthermore, PAR2-APs induced a significant increase in leukocyte rolling and adherence by a mechanism depending on the r
