Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Inhibition of the tissue factor-factor VIIa complex reduces coagulation and inflammation in animal models of endotoxemia and sepsis and in patients with severe sepsis. However, the mechanism by which tissue factor-dependent activation of the coagulation cascade enhances inflammation is not known. We tested the hypothesis that coagulation proteases enhance inflammation during endotoxemia by activating protease-activated receptors (PARs) within the vasculature. We found that genetically modified mice expressing low levels of tissue factor exhibited reduced interleukin-6 expression and increased survival in a mouse model of endotoxemia compared with control mice. In contrast, hirudin inhibition of thrombin or a deficiency in either PAR-1 or PAR-2 did not affect interleukin-6 expression or mortality. However, combining hirudin treatment to inhibit thrombin signaling through PAR-1 and PAR-4 with PAR-2 deficiency reduced lipopolysaccharide-induced interleukin-6 expression and increased survival. Taken together, our results suggest that activation of multiple PARs by coagulation proteases enhances inflammation during endotoxemia.
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PMID:Tissue factor, coagulation proteases, and protease-activated receptors in endotoxemia and sepsis. 1511 33

Protease-activated receptors (PARs) are a group of four members of the superfamily of G protein-coupled receptors that transduce cell signaling by proteolytic activity of extracellular serine proteases, such as thrombin. Possible expression and functions of PARs in oligodendrocytes, the myelin forming cells of the CNS, are still unclear. Here, the oligodendrocyte cell line OLN-93 was used to investigate the signaling of PARs. By reverse transcription-polymerase chain reaction (RT-PCR), immunostaining and Ca(2+) imaging studies, we demonstrate that OLN-93 cells functionally express PAR-1. PAR-3 seems to be expressed without apparent activity, and PAR-2 and PAR-4 cannot be detected. Short-term stimulation of the OLN-93 cells with PAR-1 agonists, such as thrombin, trypsin and PAR-1 activating peptide, dose-dependently induced a transient rise of [Ca(2+)](i). Concentration-effect curves display a sigmoidal concentration dependence. Elevation of [Ca(2+)](i) induced by PAR-1 mainly resulted from Ca(2+) release from intracellular stores. Studies on the effects of pertussis toxin (PTX), phospholipase C antagonist and 2-APB, showed that in OLN-93 cells (i). the calcium signaling cascade from PAR-1 was mediated through PTX-insensitive G proteins, (ii). activation of phospholipase C and liberation of InsP(3) were events upstream of the Ca(2+) release from the stores. In addition, the present study analyzed PAR-1 desensitization caused by exposure to thrombin, trypsin, and PAR-1 activating peptide, elucidated the influence of the protease cathepsin G on PAR-1 activation, and also characterized PAR-1 desensitization. This is the first study, which shows that OLN-93 oligodendrocytes functionally express PAR-1, and identifies the receptor coupling to mobilization of intracellular calcium. Moreover, the expression of PAR-1 was demonstrated by RT-PCR in primary oligodendrocytes from rat brain.
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PMID:Expression of protease-activated receptors (PARs) in OLN-93 oligodendroglial cells and mechanism of PAR-1-induced calcium signaling. 1514 74

Protease-activated receptors (PARs) represent a unique family of seven-transmembrane G-protein-coupled receptors, which are enzymatically cleaved to expose a new 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. By using a de novo design approach, we have discovered a series of potent heterocycle-based peptide-miimetic antagonists of PAR-1, exemplified by advanced leads RWJ-56110 (22) and RWJ-58259 (32). These compounds are potent, selective PAR-1 antagonists, devoid of PAR-1 agonist and thrombin inhibitory activity: they bind to PAR-1, interfere with calcium mobilization and cellular functions associated with PAR-1, and do not affect PAR-2, PAR-3, or PAR-4. RWJ-56110 was determined to be a direct inhibitor of PAR-1 activation and internalization, without affecting PAR-1 N-terminal cleavage. At high concentrations of alpha-thrombin, RWJ-56110 fully blocked activation responses in human vascular cells, but not in human platelets; whereas, at high concentrations of TRAP-6, RWJ-56110 blocked activation responses in both cell types. This result is consistent with the presence of another thrombin receptor on human platelets, namely PAR-4. RWJ-56110 and RWJ-58259 clearly interrupt the binding of a tethered ligand to its receptor. RWJ-58259 demonstrated antirestenotic activity in a rat balloon angioplasty model and antithrombotic activity in a cynomolgus monkey arterial injury model. Such PAR-1 antagonists should not only serve as useful tools to delineate the physiological and pathophysiological roles of PAR-1, but also may have therapeutic potential for treating thrombosis and restenosis in humans.
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PMID:Discovery of potent peptide-mimetic antagonists for the human thrombin receptor, protease-activated receptor-1 (PAR-1). 1531 88

Recent studies have shown that a novel class of protease activated receptors (PARs), which are composed of seven transmembrane G protein-coupled domains, are activated by serine proteases such as thrombin, trypsin and tryptase. Although four types (PAR 1, PAR 2, PAR 3 and PAR 4) of this class of receptors have been identified, their discrete physiological and pathological roles are still being unraveled. Extracellular proteolytic activation of PARs results in the cleavage of specific sites in the extracellular domain and formation of a new N-terminus which functions as a tethered ligand. The newly formed tethered ligand binds intramolecularly to an exposed site in the second transmembrane loop and triggers G-protein binding and intracellular signaling. Recent studies have shown that PAR-1, PAR-2 and PAR-4 have been involved in vascular development and a variety of other biological processes including apoptosis and remodeling. The use of animal model systems, mainly transgenic mice and synthetic tethered ligand domains, have contributed enormously to our knowledge of molecular signaling and the regulatory properties of various PARs in cardiomyocytes. This review focuses on the role of PARs in cardiovascular function and disease.
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PMID:Protease activated receptors in cardiovascular function and disease. 1552 83

Recently a new concept has emerged implicating thrombin signaling as the "bridge" that connects tissue damage to hemostatic and inflammatory responses. In view of this concept, we hypothesized that induction of protease-activated receptor (PAR) expression may play a critical role in endotoxin-induced tissue injury through the cellular actions of thrombin. Thus, in this study, temporal changes in expression of key precoagulant molecules, including PARs, in lungs from rabbits rendered endotoxemic by 100 microg/kg lipopolysaccharide (LPS) were examined with measurements of variables reflecting acute lung injury (ALI). ALI induction by LPS was confirmed by blood gas derangement, lung vascular hyperpermeability, and histopathological changes, and was characterized by the deposition of fibrin in the alveolar spaces, bronchioles and vessels. Plasminogen activator inhibitor-1 (PAI-1) and tissue factor (TF) were highly expressed in lungs after LPS injection. While the peaks in levels of PAI-1 and TF were comparable (12 approximately 13-fold from control), their expression time-courses were different: PAI-1 exhibited a bell-shaped expression pattern and peak at 6 h, whereas TF level reached maximum at 10 h. Of note, LPS induced a rapid and significant increase in levels of PAR-1 compared to control, with a peak level at 1 h (3.3-fold). Although declining thereafter, it remained significantly higher than the control level throughout the study period. Expressions of PAR-2, -3, and -4 were also increased by LPS with different time courses from PAR-1 expression. Immunofluorescence staining for PAR-1 were localized in blood vessels, bronchial epithelium, and alveolar pneumocytes after LPS. These results suggest that the increased expression levels of PARs, in addition to PAI-1 and TF, may, in part, underlie the development of ALI occurring during endotoxemia.
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PMID:Temporal changes in pulmonary expression of key procoagulant molecules in rabbits with endotoxin-induced acute lung injury: elevated expression levels of protease-activated receptors. 1554 22

Among the four protease-activated receptors (PARs), PAR-1 plays an important role in normal lung functioning and in the development of lung diseases, including fibrosis. We compared the expression and functional activity of PARs in normal and fibrotic human lung fibroblasts. Both normal and fibrotic cells express PAR-1, -2, and -3, with PAR-2 showing the lowest level. There was no significant difference between normal and fibrotic fibroblasts in expression levels of PAR-1 and PAR-3, whereas a fourfold higher expression level of PAR-2 was observed in fibrotic cells compared with normal cells. Ca(2+) imaging studies revealed apparently only PAR-1-induced Ca(2+) signaling in lung fibroblasts. PAR-1 agonists, thrombin and synthetic activating peptide, induced concentration-dependent Ca(2+) mobilization with EC(50) values of 5 nM and 1 microM, respectively. The neutrophil protease cathepsin G produced a transient Ca(2+) response followed by disabling PAR-1, whereas elastase did not affect Ca(2+) level. PAR-1 activation by thrombin or receptor-activating peptide downregulated expression of all three PARs in lung fibroblasts, with maximal effect at 3-6 h, whereas expression returned toward basal level after 24 h. Furthermore, PAR-1 agonists dose dependently increased PGE(2) secretion from lung fibroblasts and induction of cyclooxygenase-2 expression. We then found that PGE(2) downregulated expression of all three PARs. The effect of PGE(2) was continuously growing with time. Furthermore, PGE(2) exerts its effect through the EP2 receptor that was confirmed using the selective EP2 agonist butaprost. This novel autocrine feedback mechanism of PGE(2) in lung fibroblasts seems to be an important regulator in lung physiology and pathology.
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PMID:Protease-activated receptor-1 in human lung fibroblasts mediates a negative feedback downregulation via prostaglandin E2. 1582 Oct 19

The plasminogen activator/plasmin system is believed to play an important role in diverse pathophysiological processes, including wound healing, vascular remodeling and pulmonary fibrosis. Our recent studies show that plasmin upregulates the expression of Cyr61, a growth factor-like gene that has been implicated in cell proliferation and migration. In the present study, we investigated whether plasmin promotes fibroblast proliferation and, if so, determine the role of Cyr61 in the plasmin-induced response. Human lung fibroblasts were exposed to varying concentrations of plasmin and DNA synthesis was monitored by measuring the incorporation of 3H-thymidine into DNA. Plasmin increased DNA synthesis of fibroblasts in a dose-dependent manner. Protease-activated receptor-1 (PAR-1)-specific antibodies, but not PAR-2-specific antibodies, reduced the plasmin-induced DNA synthesis. Consistent with this, plasmin had no substantial effect on the DNA synthesis in PAR-1-deficient mouse fibroblasts. Plasmin activated both p38 and p44/42 MAPKs and specific inhibitors of these pathways inhibited the plasmin-induced DNA synthesis. Plasmin-induced increase in the DNA synthesis was completely abrogated by anti-Cyr61 antibodies. Interestingly, thrombin, which is a potent inducer of Cyr61, had only a minimal effect on fibroblast proliferation. Additional experiments suggested that plasmin cleaved cell/extracellular matrix-associated Cyr61 and the conditioned media from plasmin-treated cells could support the cell proliferation. Overall, these data suggest that plasmin promotes fibroblast proliferation by a novel pathway, involving two independent steps. In the first step, plasmin induces Cyr61 expression via activation of PAR-1, and in the second step, plasmin releases Cyr61 deposited in the extracellular matrix, thus making it accessible to act on cells.
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PMID:A novel mechanism of plasmin-induced mitogenesis in fibroblasts. 1563 80

We investigate the role of resting tension on thrombin (THR) induced relaxation of guinea-pig tracheas precontracted with acetylcholine (ACh). Isometric contractions of isolated guinea-pig tracheas were recorded at 4 and 6 g resting tension; and ACh dose-response curves were performed. THR relaxed ACh-precontracted tracheas and this effect was mimicked by the type 2 protease activating receptor agonist peptide (PAR-2 AP) and trypsin. The relaxant effect of 3 U ml(-1) THR and 100 nmol ml(-1) PAR-2 AP was prevented at 4 g by preincubation with the nitric oxide synthase (NOS) inhibitor l-NAME and at 6g resting tension by ibuprofen and diclofenac. However, adenosine trisphospahate (ATP) relaxation was totally prevented by cyclooxygenase (COX) inhibitors but not by NOS inhibitors at both resting tensions. Resting tension influenced the effect of PGE2 on contractile tone of isolated guinea-pig tracheas, the maximal relaxation being -11.1+/-2.97 and -2.0+/-0.4 6 mg mg(-1) tissue wet weight at 6 and 4 g, respectively. Moreover, 30 nmol ml(-1) PGE2 can relax ACh-precontracted tracheas, being the effect up to 91 and 30% at 6 and 4 g, respectively. These data demonstrate that trachea responsiveness is highly dependent on the smooth muscle length, revealing new aspects of stretch-activated receptors that can influence trachea responsiveness in vivo.
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PMID:Influence of resting tension on protease-activated receptor-mediated relaxation in guinea-pig tracheas. 1564 56

Hemostatic factors play a crucial role in generating thrombotic plugs at sites of vascular damage (atherothrombosis). However, whether hemostatic factors contribute directly or indirectly to the pathogenesis of atherosclerosis remains uncertain. Autopsy studies have revealed that intimal thickening represents the first stage of atherosclerosis and that lipid-rich plaque arises from such lesions. Several factors contribute to the start of intimal thickening. Platelets release several growth factors and bioactive agents that play a central role in development of not only thrombus but also of intimal thickening. We have been investigating which coagulation factors simultaneously, or subsequently with platelet aggregation, participate in thrombus formation. Tissue factor (TF) is an essential initiator of blood coagulation that is expressed in various stages of atherosclerotic lesions in humans and other animals. Factors including thrombin and fibrin, which are downstream of the coagulation cascade activated by TF, also contribute to atherosclerosis. TF is involved in cell migration, embryogenesis and angiogenesis. Thus TF, in addition to factors downstream of the coagulation cascade and the protease-activated receptor 2 activation system, would be a multifactorial regulator of atherogenesis.
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PMID:Role of thrombogenic factors in the development of atherosclerosis. 1572 89

Human mononuclear phagocytes have recently been shown to express constitutively and even more so, upon stimulation with bacteria, fungi, lipopolysaccharide (LPS), zymosan, or thrombin platelet basic protein (PBP). This CXC chemokine as well as platelet factor 4 (PF4), which is located genomically at a short distance from the PBP, were previously considered to be specific markers for the megakaryocyte cell lineage. Both chemokines have signaling and antimicrobial activity. In the present studies, transcriptional and expressional regulation of PF4 and related chemokines was studied in human monocytes. As shown by quantitative mRNA analysis, Western blots, radioimmunoprecipitation of cell extracts, and immunofluorescence and quantitatively with enzyme-linked immunosorbent assay, human monocytes express PF4 in the same order of magnitude as the known, regulated CXC chemokine interleukin (IL)-8. Expression of PF4 is up-regulated at the mRNA and protein level by thrombin and mediated by proteinase-activated receptors (PARs), resulting in a 32- to 128-fold higher mRNA level and leading to an up-to-sixfold increase of the peptide concentration in monocyte culture supernatants. Thrombin and the synthetic ligand of PAR-1 and PAR-2, SFLLRN, also induced comparable increases in the levels of mRNA for PBP, IL-8, regulated on activation, normal T expressed and secreted (RANTES), monocyte chemoattractant protein-1, and macrophage-inflammatory protein-1alpha and increased synthesis of these chemokines as shown by immunofluorescence or a quantitative immunobead-based method. The induction of increased mRNA levels for all chemokines by SFLLRN was unsurpassed by LPS, zymosan, interferon-gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and IL-1. Activation of monocytes through PARs represents an alternate activation mechanism, independent from IFN-gamma, TNF-alpha, or other signaling pathways.
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PMID:Regulated expression of platelet factor 4 in human monocytes--role of PARs as a quantitatively important monocyte activation pathway. 1578 41


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