Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.5 (thrombin)
 33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Protease-activated receptors (PARs) represent a unique family of seven-transmembrane G protein-coupled receptors, which are enzymatically cleaved to expose a truncated extracellular N terminus that acts as a tethered activating ligand. PAR-1 is cleaved and activated by the serine protease alpha-thrombin, is expressed in various tissues (e.g., platelets and vascular cells), and is involved in cellular responses associated with hemostasis, proliferation, and tissue injury. We have discovered a series of potent peptide-mimetic antagonists of PAR-1, exemplified by RWJ-56110. Spatial relationships between important functional groups of the PAR-1 agonist peptide epitope SFLLRN were employed to design and synthesize candidate ligands with appropriate groups attached to a rigid molecular scaffold. Prototype RWJ-53052 was identified and optimized via solid-phase parallel synthesis of chemical libraries. RWJ-56110 emerged as a potent, selective PAR-1 antagonist, devoid of PAR-1 agonist and thrombin inhibitory activity. It binds to PAR-1, interferes with PAR-1 calcium mobilization and cellular function (platelet aggregation; cell proliferation), and has no effect on PAR-2, PAR-3, or PAR-4. By flow cytometry, RWJ-56110 was confirmed as a direct inhibitor of PAR-1 activation and internalization, without affecting N-terminal cleavage. At high concentrations of alpha-thrombin, RWJ-56110 fully blocked activation responses in human vascular cells, albeit not in human platelets; whereas, at high concentrations of SFLLRN-NH(2), RWJ-56110 blocked activation responses in both cell types. Thus, thrombin activates human platelets independently of PAR-1, i.e., through PAR-4, which we confirmed by PCR analysis. Selective PAR-1 antagonists, such as RWJ-56110, should serve as useful tools to study PARs and may have therapeutic potential for treating thrombosis and restenosis.
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PMID:Design, synthesis, and biological characterization of a peptide-mimetic antagonist for a tethered-ligand receptor. 1053 8

Both thrombin and plasmin induce contraction of brain endothelial cells, which may increase capillary permeability thereby leading to disruption of the blood-brain barrier. Identification of thrombin receptors, as well as the influence of plasmin on their activation, in capillary endothelial cells and astrocytes are therefore essential for understanding injury-related actions of thrombin in the brain. Using the reverse transcriptase-polymerase chain reaction method, the present study shows that primary cultures of rat brain capillary endothelial (RBCE) cells and astrocytes derived from rat brain express two different thrombin receptors. The first is proteolytically activated receptor (PAR)-1, the receptor responsible for the vast majority of the thrombin's cellular activation functions; the second is PAR-3, a receptor described to be essential for normal responsiveness to thrombin in mouse platelets. In addition to these thrombin receptors, the mRNA (messenger RNA) for PAR-2, a possible trypsin receptor, was also identified. Functional significance of thrombin receptors was indicated by changes in [Ca2+]i in response to thrombin, as measured by FURA-2 fluorescence in RBCE cells. Thrombin as low as 4 nmol/L induced an abrupt increase in [Ca2+]i whereas, upon addition of active site-blocked thrombin or plasmin, [Ca2+]i remained unchanged. The [Ca2+]i signal attributable to thrombin was smaller in a low Ca2+-containing medium, indicating that an influx of Ca2+ from the extracellular medium makes a contribution to the overall [Ca2+]i rise. The amplitude of the transient [Ca2+]i signal was dependent on the concentration of thrombin, and repeated application of the enzyme caused an essentially complete and long-term desensitization of the receptor. The PAR-1 agonist peptide SFLLRN also elicited a transient increase in [Ca2+]i. After activation by SFLLRN, cells showed a diminished response to thrombin, but the response was not absent, indicating that PAR-3 might contribute to the generation of the [Ca2+]i signal. Pretreatment of RBCE cells with 100 nmol/L plasmin completely prevented [Ca2+]i rise attributable to thrombin. These data show that RBCE cells and astrocytes express at least two receptors for thrombin, PAR-1 and PAR-3, and probably both receptors are involved in thrombin-induced [Ca2+]i signals. Plasmin itself does not elevate [Ca2+]i but prevents the activation of receptors by thrombin.
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PMID:Identification of thrombin receptors in rat brain capillary endothelial cells. 1061 6

The protease-activated receptor (PAR), a G protein-coupled receptor present on cell surface, mediates cellular actions of extracellular proteases. Proteases cleave the extracellular N-terminal of PAR molecules at a specific site, unmasking and exposing a novel N-terminal, a tethered ligand, that binds to the body of receptor molecules resulting in receptor activation. Amongst four distinct PARs that have been cloned, PARs 1, 3 and 4 are activated by thrombin, but PAR-2 is activated by trypsin or mast cell tryptase. Human platelets express two distinct thrombin receptors, PAR-1 and PAR-4, while murine platelets express PAR-3 and PAR-4. Apart from roles of PARs in platelet activation, PARs are distributed to a number of organs in various species, predicting their physiological importance. We have been evaluating agonists specific for each PAR, using multiple procedures including a HEK cell calcium signal receptor desensitization assay. Using specific agonists that we developed, we found the following: 1) the salivary glands express PAR-2 mRNA and secret saliva in response to PAR-2 activation; 2) pancreatic juice secretion occurs following in vivo PAR-2 activation; 3) PAR-1 and PAR-2 modulate duodenal motility. Collectively, PAR plays various physiological and/or pathophysiological roles, especially in the digestive systems, and could be a novel target for drug development.
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PMID:[Physiology of protease-activated receptors (PARs): involvement of PARs in digestive functions]. 1062 76

Protease-activated receptor (PAR)-1 is a cellular receptor for thrombin that is activated after proteolytic cleavage. The contribution of PAR-1 to inflammatory cell-mediated renal injury was assessed in murine crescentic glomerulonephritis (GN). A pivotal role for thrombin in this model was demonstrated by the capacity of hirudin, a selective thrombin antagonist, to attenuate renal injury. Compared with control treatment, hirudin significantly reduced glomerular crescent formation, T cell and macrophage infiltration, fibrin deposition, and elevated serum creatinine, which are prominent features of GN. PAR-1-deficient (PAR-1(-/-)) mice, which have normal coagulation, also showed significant protection from crescentic GN compared with wild-type mice. The reductions in crescent formation, inflammatory cell infiltration, and serum creatinine were similar in PAR-1(-/-) and hirudin-treated mice, but hirudin afforded significantly greater protection from fibrin deposition. Treatment of wild-type mice with a selective PAR-1-activating peptide (TRAP) augmented histological and functional indices of GN, but TRAP treatment did not alter the severity of GN in PAR(-/-) mice. These results indicate that activation of PAR-1 by thrombin or TRAP amplifies crescentic GN. Thus, in addition to its procoagulant role, thrombin has proinflammatory, PAR-1-dependent effects that augment inflammatory renal injury.
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PMID:Protease-activated receptor 1 mediates thrombin-dependent, cell-mediated renal inflammation in crescentic glomerulonephritis. 1066 91

Putative binding sites in a platelet thrombin receptor (PAR-1) for the activating peptide SFLLRNPNDKYEPF (AP) and for the bradykinin analogue MKRPPGFSPFRSSRIG were revealed using a computer program for identifying complementary peptide segments. The program is based on the assumption that interactions of agonist's peptides and protein's receptors can be elucidated by complementary average hydropathies as much as possible equal by size and opposite by sign. Some of the computer-found putative binding sites were close to the supposed AP-PAR-1 contacts in the amino-terminal exodomain and in the second extracellulary loop of PAR-1. Peptides complementary to these binding sites were also computer-designed and were synthesized. They mostly inhibited the aggregation of gel filtered platelets by thrombin (0.025 U/mL) with IC50 in a high micromolar range of concentrations. The peptide complementary to site L258-Y269 of PAR-1 induced aggregation of gel filtered platelets with EC50 = 98 [micromol/L] related to thrombin (0.025 U/mL) aggregation response.
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PMID:Determination of the putative binding sites for thrombin receptor activating peptide through a hydropathic complementary approach. 1066 71

Thrombin induces well-characterized effects on normal and neoplastic brain cells by interaction with protease-activated receptor (PAR)-type thrombin receptors. However, nothing is known about the function of intermediate enzymes of prothrombin activation recently shown to evoke PAR-1-mediated signaling in smooth muscle cells. Therefore, we investigated the effect of recombinant human meizothrombin (rMT), one of thrombin's catalytically active precursor enzymes in the prothrombin cleavage cascade, on calcium mobilization in human SNB-19 glioblastoma cells. By using reverse-transcription polymerase chain reaction, immunofluorescence studies with a monoclonal anti-PAR-1 antibody and calcium measurements, SNB-19 cells were shown to express functional PAR-1-type thrombin receptors. PAR-1 is not only a receptor for thrombin in SNB-19 cells but was also activated by rMT very effectively. Under the conditions used in our experiments, SNB-19 cells stimulated with thrombin after rMT challenge were unable to elicit a new calcium response and vice versa. In addition, both rMT and thrombin induced no further calcium signal after that observed with the PAR-1-activating peptide SFLLRN. Therefore, rMT and thrombin seem to activate calcium signaling by similar mechanisms including PAR-1. Our results demonstrate rMT as a potent activator of PAR-1-type thrombin receptors in SNB-19 glioblastoma cells, suggesting a function of catalytically active thrombin precursor enzymes in cells of glial origin.
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PMID:Meizothrombin, an intermediate of prothrombin activation, stimulates human glioblastoma cells by interaction with PAR-1-type thrombin receptors. 1068 92

Thrombin, a multifunctional protein, has been found to be involved in cellular mitogenesis, tumor growth, and metastasis, in addition to its well known effects on the initiation of platelet aggregation and secretion and the conversion of fibrinogen to fibrin to form blood clots. These properties of thrombin rely on its action as a serine protease, which cleaves the N-terminal region of a 7-transmembrane G protein receptor (protease-activated receptor, PAR-1), thus exposing a tethered end hexapeptide sequence capable of activating its receptor. Little is known about its effect on genes that regulate the cell cycle. This study was undertaken to investigate the possible mechanisms by which thrombin regulates tumor cell growth in several tumor cell lines: human CHRF megakaryocyte, DU145 prostate, MDAMB231 and MCF7 breast, U3A fibrosarcoma, and 2 murine fibroblast cell lines, MEFp53(-/-) and CD STAT(-/-). We have found that thrombin under the conditions of culture employed inhibits cell growth by both up-regulation of p21(waf/cip1) and induction of caspases via its PAR-1 receptor. The increased expression of p21(waf/cip1) by thrombin was p53 independent, STAT1 dependent, and protein synthesis independent. This was associated with tyrosine phosphorylation of JAK2 and STAT1, and nuclear translocation of STAT1. Induction of apoptosis is also PAR-1-specific, STAT1-dependent, and associated with up-regulation of caspases 1, 2, and 3. Our study establishes, for the first time, a link between PAR-1 receptor activation with the STAT signal pathway, which leads to cell cycle control and apoptosis. This observation broadens our understanding of the mechanism of PAR-1 activation and its effect on cell growth, and could possibly lead to therapeutic approaches for the treatment of cancer.
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PMID:Thrombin inhibits tumor cell growth in association with up-regulation of p21(waf/cip1) and caspases via a p53-independent, STAT-1-dependent pathway. 1069 50

Relaxant and contractile effects of the tethered ligand domain sequences of murine PAR-1, PAR-2, PAR-3 and PAR-4, and of the proteases thrombin and trypsin were examined in mouse isolated tracheal preparations. The epithelium- and cyclo-oxygenase-dependence of these effects and the potential modulatory effects of respiratory tract viral infection were also investigated. In carbachol-contracted preparations, trypsin, thrombin, and the tethered ligand domain sequences of murine PAR-1 (SFFLRN-NH(2)), PAR-2 (SLIGRL-NH(2)) and PAR-4 (GYPGKF-NH(2)), but not PAR-3 (SFNGGP-NH(2)), induced transient, relaxant responses that were abolished by the cyclo-oxygenase inhibitor indomethacin. Repeated administration of SFFLRN-NH(2), SLIGRL-NH(2) or GYPGKF-NH(2) (30 microM) was associated with markedly diminished relaxation responses (homologous desensitization), although there was no evidence of cross-desensitization between these peptides. The tethered ligand domain sequences for PAR-1 and PAR-4 induced a rapid, transient contractile response that preceded the relaxant response. Contractions were not inhibited by indomethacin and were not induced by either thrombin or trypsin. Influenza A virus infection did not significantly affect the responses induced by either the proteases or peptides. Furthermore, epithelial disruption caused by mechanical rubbing had no significant effect on responses to these PAR activators in preparations from either virus- or sham-infected mice. In summary, the proteases trypsin and thrombin, and peptide activators of PAR-1, PAR-2 and PAR-4 induced relaxant responses of mouse isolated tracheal smooth muscle preparations, which were mediated by a prostanoid, probably PGE(2). Interestingly, PAR-mediated relaxations were not significantly diminished following acute damage to the epithelium caused by mechanical rubbing and/or the respiratory tract viral pathogen, influenza A. British Journal of Pharmacology (2000) 129, 63 - 70.
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PMID:Modulation of airway smooth muscle tone by protease activated receptor-1,-2,-3 and -4 in trachea isolated from influenza A virus-infected mice. 1069 3

Proteinase-activated receptors (PARs) have the common property of being activated by the proteolytic cleavage of their extracellular N-terminal domain. The new NH2-terminus acts as a 'tethered ligand' binding and activating the receptor itself. Four members of this family have been cloned, three of which are activated by thrombin (PAR-1, PAR-3 and PAR-4) while the fourth (PAR-2) is activated by trypsin or mast cell tryptase. In physiological or pathophysiological conditions, the gastrointestinal tract is exposed more than other tissues to proteinases (digestive enzymes, proteinases from pathogens or proteinases from inflammatory cells) that can activate PARs. Since PARs are highly expressed throughout the gastrointestinal tract, the study of the role of PARs in these tissues appears to be particularly important. It has already been shown that PAR-2 activation induces calcium mobilization and eicosanoid production in enterocytes as well as changes in ion transport in jejunal tissue segments. PAR-2 activation also causes calcium mobilization and stimulates amylase release from pancreatic acini. Moreover, both PAR-1 and PAR-2 activation can alter the gastrointestinal motility. In inflammatory or allergic conditions, the proteinases that constitute the major agonists for PARs (thrombin, trypsin and mast cell tryptase) are usually released. The activation of PARs by these proteinases might contribute to the gastrointestinal disorders associated with these pathologies. A complete understanding of the role of PARs in the gastrointestinal tract will require the development of selective receptor antagonists that are not yet available. Nonetheless, the use of PAR agonists has already highlighted new potential functions for proteinases in the gastrointestinal tract, thus the control of PAR activation might represent a promising therapeutic target.
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PMID:Review article: proteinase-activated receptors - novel signals for gastrointestinal pathophysiology. 1073 17

The vasodilator effects of thrombin depend on activation of proteinase-activated receptor (PAR)-1 and the subsequent release of endothelin (ET)-1, which stimulates the generation of nitric oxide and PGs. We recently showed that thrombin released matrix metalloproteinase-2 (MMP-2) from rat arteries. We have now studied the significance of this release for the vasodilator effects of thrombin. Thrombin (>/=100 pmol), but not a PAR-1-activating peptide (TFLLR-NH(2)), produced a long-lasting (>10 min) vasorelaxation of rat mesenteric arteries, as detected by a microperfusion bioassay. Thrombin induced a simultaneous release of vascular MMP-2 into arterial perfusates, as revealed by zymography. Interestingly, the vasodilator effects of thrombin were inhibited by a tissue inhibitor of MMP-2 (TIMP-2, 10 pmol). Moreover, infusion of exogenous MMP-2 (5 pmol) resulted in vasorelaxation. These vasodilatory effects of thrombin and MMP-2 were significantly (P < 0.05) inhibited by endothelium denudation and by PD-142893 (2 nmol), an antagonist of ET receptors. Furthermore, both thrombin and MMP-2 constricted endothelium-denuded arteries. These results show that the vasodilator effects of thrombin may depend, in part, on a release of vascular MMP-2 and downstream activation of ETs. Thus MMP-2-dependent signaling may complement the PAR-1-dependent pathway of vasodilator action of thrombin.
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PMID:Role of matrix metalloproteinase-2 in thrombin-induced vasorelaxation of rat mesenteric arteries. 1077 24


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