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)

Human lung epithelial (Calu-3) cells were used to investigate the effects of protease-activated receptor (PAR) stimulation on Cl(-) secretion. Quantitative RT-PCR (QRT-PCR) showed that Calu-3 cells express PAR-1, -2, and -3 receptor mRNAs, with PAR-2 mRNA in greatest abundance. Addition of either thrombin or the PAR-2 agonist peptide SLIGRL to the basolateral solution of monolayers mounted in Ussing chambers produced a rapid increase in short-circuit current (I(sc): thrombin, 21 +/- 2 microA; SLIGRL, 83 +/- 22 microA), which returned to baseline within 5 min after stimulation. Pretreatment of monolayers with the cell-permeant Ca(2+)-chelating agent BAPTA-AM (50 microM) abolished the increase in I(sc) produced by SLIGRL. When monolayers were treated with the cyclooxygenase inhibitor indomethacin (10 microM), nearly complete inhibition of both the thrombin- and SLIGRL-stimulated I(sc) was observed. In addition, basolateral treatment with the PGE(2) receptor antagonist AH-6809 (25 microM) significantly inhibited the effects of SLIGRL on I(sc). QRT-PCR revealed that Calu-3 cells express mRNAs for CFTR, the Ca(2+)-activated KCNN4 K(+) channel, and the KCNQ1 K(+) channel subunit, which, in association with KCNE3, is known to be regulated by cAMP. Stimulation with SLIGRL produced an increase in apical Cl(-) conductance that was blocked in cells expressing short hairpin RNAs designed to target CFTR. These results support the conclusion that PAR stimulation of Cl(-) secretion occurs by an indirect mechanism involving the synthesis and release of prostaglandins. In addition, PAR-stimulated Cl(-) secretion requires activation of CFTR and at least two distinct K(+) channels located in the basolateral membrane.
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PMID:Protease-activated receptor regulation of Cl- secretion in Calu-3 cells requires prostaglandin release and CFTR activation. 1653 69

Endothelial cells react to factor Xa and thrombin by proinflammatory responses. It is unclear how these cells respond under physiological conditions, where the serine proteases factor VIIa, factor Xa and thrombin are all simultaneously generated, as in tissue factor-driven blood coagulation. We studied the Ca(2+) signaling and downstream release of interleukins (ILs), induced by these proteases in monolayers of human umbilical vein endothelial cells. In single cells, factor Xa, but not factor VIIa, complexed with tissue factor, evoked a greatly delayed, oscillatory Ca(2+) response, which relied on its catalytic activity and resembled that of SLIGRL, a peptide specifically activating the protease-activated receptor 2 (PAR2). Thrombin even at low concentrations evoked a rapid, mostly non-oscillating Ca(2+) response through activation of PAR1, which reinforced the factor Xa response. The additive Ca(2+) signals persisted, when factor X and prothrombin were activated in situ, or in the presence of plasma that was triggered to coagulate with tissue factor. Further, thrombin reinforced the factor Xa-induced production of IL-8, but not of IL-6. Both interleukins were produced in the presence of coagulating plasma. In conclusion, under coagulant conditions, factor Xa and thrombin appear to contribute in different and additive ways to the Ca(2+)-mobilizing and proinflammatory reactions of endothelial cells. These data provide first evidence that these serine proteases trigger distinct signaling modules in endothelium that is activated by plasma coagulation.
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PMID:Factor Xa and thrombin evoke additive calcium and proinflammatory responses in endothelial cells subjected to coagulation. 1676 66

Tryptic enzymes such as tryptase, trypsin and thrombin are reportedly able to alter neutrophil behavior. However, little is known of the influence of these proteinases on lactoferrin or IL-8 release from neutrophils. In the present study, we investigated the effects of tryptase, trypsin, thrombin and elastase, and agonist peptides of PAR-1 SFLLR-NH(2) and PAR-2 SLIGKV-NH(2) and tc-LIGRLO-NH(2) on lactoferrin and IL-8 release from highly purified human neutrophils. Flow cytometry shows CD16(+) neutrophils express PAR-1 and PAR-2, but not PAR-3 and PAR-4 proteins. RT-PCR analysis reveals that neutrophils express only PAR-2 genes. Tryptase and trypsin, but not thrombin and elastase, induced significant lactoferrin and IL-8 secretion from neutrophils. SLIGKV-NH(2) and tc-LIGRLO-NH(2), but not SFLLR-NH(2), also stimulated lactoferrin and IL-8 secretion from neutrophils. In conclusion, only a proportion of neutrophils express PAR-1 and/or PAR-2. Tryptase and trypsin-induced lactoferrin and IL-8 secretion from neutrophils most likely occur through activation of PAR-2.
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PMID:Induction of lactoferrin and IL-8 release from human neutrophils by tryptic enzymes via proteinase activated receptor-2. 1682 Mar 7

Thrombin and other proteinases exert vascular effects by activating the proteinase-activated receptors (PARs). The expression of PARs has been shown to be upregulated after balloon injury and in human arteriosclerosis. However, the relationship between the receptor upregulation and the alteration of vasomotor function remains to be elucidated. We herein demonstrated that the contractile responses to the PAR-1 and PAR-2 agonist were markedly enhanced in the rabbit femoral arteries after balloon injury. Neointimal thickening was established 4 wk after the injury. No histological change was observed in the sham operation, where the saphenous artery was ligated without any balloon injury. The contractile response to K(+) depolarization was significantly attenuated 1 wk after the injury and then partly recovered after 4 wk. Thrombin, PAR-1-activating peptide, trypsin, and PAR-2-activating peptide induced no significant contraction in the control. All these stimulants induced enhanced responses 1 wk after balloon injury. Such enhanced responses were seen 4 wk after the injury, except for thrombin. There was no change in the Ca(2+) sensitivity of the contractile apparatus as evaluated in the permeabilized preparations. PAR-1-activating peptide (100 mumol/l), but no other stimulants, induced an enhanced contraction in the sham operation. The expression of PAR-1 and PAR-2 slightly increased after the sham operation, whereas it markedly and significantly increased after balloon injury. Our observations suggest that balloon injury induced the receptor upregulation, thereby enhancing the contractile response before the establishment of vascular lesions. The local inflammation associated with the sham operation may also contribute to the receptor upregulation.
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PMID:Upregulation of proteinase-activated receptors and hypercontractile responses precede development of arterial lesions after balloon injury. 1684 9

Proteinase-activated receptors (PAR) have been recognized as playing an important role in inflammation and immune response. However, little is known of the expression and function of PAR on human T cells. In this study, the expression of PAR on highly purified human T cells was determined and the secretion of IL-6 from cultured T cells in response to serine proteinases and agonist peptides of PAR was examined. The results showed that T cells express PAR-1, PAR-2 and PAR-3 proteins and genes. Thrombin, trypsin and tryptase, but not elastase, were able to stimulate concentration-dependent secretion of IL-6 from T cells following a 16 h incubation period. The specific inhibitors of thrombin, trypsin and tryptase inhibited the actions of these proteinases on T cells, indicating that the enzymatic activity is essential for their actions. Agonist peptides of PAR SFLLR-NH2, TFLLRN-NH2 and SLIGKV-NH2, but not TFRGAP-NH2, GYPGQV-NH2 and AYPGKF-NH2, are also capable of inducing IL-6 release from T cells. In conclusion, induction of IL-6 secretion from T cells by thrombin, trypsin and tryptase is probably through the activation of PAR, suggesting that serine proteinases are involved in the regulation of immune response of the body.
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PMID:Induction of IL-6 release from human T cells by PAR-1 and PAR-2 agonists. 1686 43

Proteinase-activated receptors (PARs), a subfamily of G protein-coupled receptors, which are activated by serine proteases, such as trypsin, play pivotal roles in the CNS. Mesotrypsin (trypsin IV) has been identified as a brain-specific trypsin isoform. However, its potential physiological role concerning PAR activation in the brain is largely unknown. Here, we show for the first time that mesotrypsin, encoded by the PRSS3 (proteinase, serine) gene, evokes a transient and pronounced Ca(2+) mobilization in both primary rat astrocytes and retinal ganglion RGC-5 cells, suggesting a physiological role of mesotrypsin in brain cells. Mesotrypsin mediates Ca(2+) responses in rat astrocytes in a concentration-dependent manner, with a 50% effective concentration (EC(50)) value of 25 nm. The maximal effect of mesotrypsin on Ca(2+) mobilization in rat astrocytes is much higher than that observed in 1321N1 human astrocytoma cells, indicating that the activity of mesotrypsin is species-specific. The pre-treatment of cells with thrombin or the PAR-1-specific peptide TRag (Ala-pFluoro-Phe-Arg-Cha-HomoArg-Tyr-NH(2), synthetic thrombin receptor agonist peptide), but not the PAR-2-specific peptide, reduces significantly the mesotrypsin-induced Ca(2+) response. Treatment with the PAR-1 antagonist SCH79797 confirms that mesotrypsin selectively activates PAR-1 in rat astrocytes. Unlike mesotrypsin, the two other trypsin isoforms, cationic and anionic trypsin, activate multiple PARs in rat astrocytes. Therefore, our data suggest that brain-specific mesotrypsin, via the regulation of PAR-1, is likely to be involved in multiple physiological/pathological processes in the brain.
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PMID:Mesotrypsin, a brain trypsin, activates selectively proteinase-activated receptor-1, but not proteinase-activated receptor-2, in rat astrocytes. 1690 72

Ischemia/reperfusion (IR) injury is a leading cause of acute renal failure and an important contributor to allograft damage. Tissue factor (TF) is up-regulated during IR, and TF inhibition reduces renal injury. However, the underlying mechanisms by which TF contributes to injury have not been elucidated. We postulated that TF contributes to IR injury by production of coagulation proteases and subsequent signaling by protease activated receptor (PARs). We compared renal injury after 25 minutes of bilateral renal ischemia and varying periods of reperfusion in C57BL/6 mice, those expressing low levels of TF (low-TF), hirudin-treated C57BL/6, and mice lacking either PAR-1 or PAR-2. C57BL/6 mice developed severe renal failure and died within 48 hours of reperfusion. In contrast, low-TF, hirudin-treated C57BL/6, and PAR-1-/- mice were protected from renal failure and had reduced mortality, tubular injury, neutrophil accumulation, and lower levels of the chemokines KC and MIP-2. Importantly, PAR-1-/- mice had lower chemokine levels despite up-regulation of TF and fibrin deposition. In addition, treating PAR-1-/- mice with hirudin conferred no additional benefit. Somewhat surprisingly, PAR-2 deficiency did not protect from renal failure. These experiments indicate that increased TF activity after renal IR leads to increased CXC chemokine expression and subsequent neutrophil-mediated injury predominantly by thrombin-dependent PAR-1 signaling.
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PMID:Tissue factor deficiency and PAR-1 deficiency are protective against renal ischemia reperfusion injury. 1699 Jun 8

Leukocyte recruitment and the expression of pro-inflammatory cytokines are prevalent characteristics of early atherogenesis. Recently, several inflammatory mediators have been linked to atheroma formation and inflammatory pathways have been shown to promote thrombosis. The discovery of mast cells, activated T lymphocytes and macrophages in atherosclerotic lesions, the detection of human leukocyte antigen class II expression, and the finding of local secretion of several cytokines all suggest the involvement of immune and inflammatory mechanisms in the pathogenesis of atherosclerosis. Recent research suggests activation of protease activated receptors (PAR) on the surface of endothelial cells may play a role in general mechanisms of inflammation. In previous studies, our laboratory has demonstrated that thrombin (which activates PAR-1) and tryptase (which activates PAR-2) stimulation of endothelial cells results in activation of calcium-independent phospholipase A(2) (iPLA(2)). iPLA(2) plays a critical role in the synthesis of membrane phospholipid-derived inflammatory mediators such as arachidonic acid, platelet activating factor (PAF), and prostaglandins, all demonstrated to be central in both the initiation and propagation of the inflammatory response. Activation of iPLA(2) results in release of choline lysophospholipids from endothelial cells, these metabolites may contribute to the initiation of ventricular arrhythmias following myocardial ischemia as a direct result of incorporation into the myocyte sarcolemma. This biochemical event represents a direct link between occlusion of a coronary vessel and the nearly immediate initiation of arrhythmogenesis often seen in myocardial ischemia.
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PMID:The therapeutic potential of phospholipase A2 inhibitors in cardiovascular disease. 1726 51

Hypersecretion of cytokines and serine proteases has been observed in asthma. However, the influence of proteases and protease-activated receptors (PARs) on monocyte chemoattractant protein-1 (MCP-1) release from airway epithelial cells remains largely unknown. In the present study, A549 cells were challenged with agonists of PARs, and levels of MCP-1 released in the supernatant and mRNA expression were examined by ELISA and real time polymerase chain reaction (PCR), respectively. The results show that thrombin, tryptase, elastase and trypsin induced an up to 6.5-, 1.8-, 1.6-, and 3.1-fold increase in MCP-1 release from A549 cells, respectively, following a 16-h incubation period. The protease-induced secretion of MCP-1 can be abolished by specific protease inhibitors. Agonist peptides of PAR-1 and PAR-2 stimulate MCP-1 secretion up to 15- and 12.7-fold, respectively. Real-time PCR showed that MCP-1 mRNA is up-regulated by the serine proteases tested and by agonist peptides of PAR-1 and PAR-2. In conclusion, serine proteases can stimulate MCP-1 release from A549 cells possibly through a PARs-related mechanism, suggesting that they are likely to contribute to MCP-1-related airway inflammatory disorders in man.
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PMID:Induction of monocyte chemoattractant protein-1 release from A549 cells by agonists of protease-activated receptor-1 and -2. 1728 Jul 38

Our recent data showed that activation of protease-activated receptor (PAR)-1 and PAR-2 in rat astrocytes not only evokes calcium signaling, but also regulates the release of the chemokine growth-regulated oncogene/cytokine-induced neutrophil chemoattractant-1 (GRO/CINC-1), a counterpart of the human GRO. This chemokine provides a feedback to protect astrocytes from toxic insults. Activated PAR-1 and PAR-2 were strong stimuli to induce the release of GRO/CINC-1. The effect was comparable to that induced by TNF-alpha. However, the role of calcium in the PAR-induced GRO/CINC-1 secretion remains unknown. Here, we found that the pharmacological blockade of either calcium release from the intracellular stores, or influx from the extracellular space, increased PAR-1- and PAR-2-induced GRO/CINC-1 secretion. Under calcium-free conditions, the basal mRNA level of GRO/CINC-1 was clearly increased. Further studies revealed that the intracellular GRO/CINC-1 protein level was slightly increased by treatment with thrombin or TRag in calcium-free conditions. However, the amount of protein synthesized was largely reduced in the absence of extracellular calcium as compared to that under normal calcium conditions. Importantly, we found that the intracellularly formed GRO/CINC-1 was not secreted into the cell culture supernatant under calcium-free conditions. These data suggest a dual role of calcium. On the one side, an increase in cytosolic calcium negatively regulates PAR-induced GRO/CINC-1 gene expression in rat astrocytes, but on the other side, the basal level of calcium is the pre-requisite for GRO/CINC-1 protein synthesis and secretion.
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PMID:The role of calcium in protease-activated receptor-induced secretion of chemokine GRO/CINC-1 in rat brain astrocytes. 1766 44


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