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)

Macrophages in the tissues have been shown to express receptor for urokinase-type plasminogen activator (uPAR) on their cell surface which plays an important role in cell invasion and attachment. We examined the effects of inflammatory mediators on the expression of uPAR employing U937 cells which have monocyte/macrophage-like characteristics. U937 cells were incubated with various mediators such as interleukins (IL), tumor necrosis factors (TNF), dexamethasone, thrombin, fibrin fragment D, bradykinin, complement C5a, and components of the extracellular matrix. The uPAR expression on the cell surface was then analyzed by radio-ligand binding assay using 125I-scuPA. The strongest enhancement of uPAR was observed in the cells stimulated by TNF alpha and TNF beta. IL-1 beta, IL-6, and C5a also increased the uPA binding sites with various patterns of affinity change. Dexamethasone decreased the uPA binding sites without changing the affinity. Fibrin fragment D and IL-3 reduced the affinity without changing the number of receptors. These findings suggest that the expression of uPAR in inflammatory cells could be modulated by various inflammatory mediators.
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PMID:Modulation of the receptor for urokinase-type plasminogen activator in macrophage-like U937 cells by inflammatory mediators. 879 83

Using the Northern blot technique, we screened 6 human hepatoma cell lines to investigate the regulation mechanism of heparin cofactor II (HC II) biosynthesis. We found that HuH-7 and Hep G2 cells constitutively expressed the HC II gene. In conditioned medium, HuH-7 cells constantly produced HC II that was functionally active and formed a complex with thrombin in the presence of dermatan sulfate. HC II is thought be an acute phase reactant, and, therefore, we examined the effects of the major inflammatory cytokines, IL-6, IL-1 beta, and TNF-alpha, on the regulation of HC II production in HuH-7 and Hep G2 cells. In HuH-7 cells, the antigen and mRNA levels of plasminogen activator inhibitor type-1 (PAI-1), an acute phase protein produced by hepatocytes, were increased in response to stimulation with either IL-6 or IL-1 beta or both, but HC II antigen and mRNA levels were not changed by the same stimulation. Even when Hep G2 cells were treated with a combination of three cytokines, IL-6, IL-1 beta, and TNF-alpha, HC II antigen and mRNA levels were not changed; however, PAI-1 antigen and mRNA levels were clearly increased. These results suggest that the production of HC II in hepatoma cells is not regulated by the major inflammatory mediators, IL-6, IL-1 beta, and TNF-alpha.
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PMID:The production of heparin cofactor II is not regulated by inflammatory cytokines in human hepatoma cells: comparison with plasminogen activator inhibitor type-1. 881 80

The influence of cytokines on intracellular calcium concentration ([Ca2+]i) and the production of prostacyclin (prostaglandin l2; PGI2) by cultured human umbilical vein endothelial cells (HUVEC) were examined. HUVEC were incubated for 24 h in media containing interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN gamma), or interleukin-6 (IL-6), and thrombin-stimulated increases in [Ca2+]i and PGI2 production were then examined. Thrombin-stimulated PGI2 production by HUVEC pretreated with 10 U/mL of IL-1 beta or 200 U/mL of TNF-alpha for 24 h was potentiated, while increases in [Ca2+]i were suppressed. In contrast, HUVEC pretreated with 5000 U/mL of IFN-gamma for 24 h had both enhanced PGI2 production and increases in [Ca2+]i. IL-6 affected neither PGI2 production nor [Ca2+]i in HUVEC stimulated with thrombin. The burst increase in thrombin-stimulated PGI2 production by HUVEC pretreated with cytokines did not correlate with the increase in [Ca2+]i. Cytokines have been reported to induce enzymes involved in the arachidonic acid cascade, such as phospholipase A2 (PLA2) and cyclooxygenase-2 (COX-2). Therefore, the increase in [Ca2+]i does not appear to be as important for thrombin-stimulated PGI2 production as does the induction of these enzymes by cytokines.
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PMID:Effect of cytokines on thrombin-stimulated increases in intracellular calcium and PGI2 production by cultured human umbilical vein endothelial cells. 884 24

Thrombosis and disseminated intravascular coagulation (DIC) are common complications of infections. Abnormal activation of coagulation is due in part of expression of tissue factor on intravascular cells in response to cytokines, including interleukin-1 beta (IL1 beta ) and tumor necrosis factor (TNF). Both TNF and IL1 beta are thought to play significant roles in producing the pathologic manifestations of sepsis. Therefore, we examined the effects of thrombin on TNF and IL1 beta secretion of monocytes, and the ability of monocyte products to promote tissue factor expression by endothelial cells. Human monocytes were treated with thrombin or a thrombin receptor agonist peptide (SFLLRN), and/or bacterial lipopolysaccharide (LPS). The agonists were removed, and monocytes cultured 18 hours. The monocyte-conditioned supernatants were assayed for TNF and IL1 beta antigen, and for their ability to induce tissue factor expression on human umbilical vein endothelial cells and the Ea.hy endothelial cell line. Thrombin alone did not promote monocyte TNF or IL-1 beta secretion. However, thrombin enhanced LPS-induced TNF and IL1 secretion. Supernatants from monocytes exposed to LPS plus thrombin promoted greater tissue factor expression on endothelial cells than supernatants from those treated with LPS only. SFLLRN did not increase TNF secretion in response to LPS, but did enhance LPS-induced IL1 beta secretion and tissue factor-inducing activity. Neither SFLLRN nor active thrombin augmented the level of mRNA for TNF above that induced by LPS alone. However, both increased the LPS-induced level of IL1 beta message. Thus, thrombin enhanced LPS-induced TNF and IL1 beta secretion by monocytes. Unexpectedly, the effects on these two cytokines were mediated by different mechanisms. Enhancement of LPS-induced IL1 beta secretion was largely mediated via the tethered ligand type thrombin receptor and correlated with an increase in the steady state level of mRNA. By contrast, enhanced TNF required proteolytically active thrombin, but was not mediated by the tethered ligand receptor. These data demonstrate that physiologically relevant amounts of thrombin can synergize with endotoxin to stimulate monokine release. Thrombin could thereby play a role in the complex network of mediators involved in the pathophysiology of sepsis. We speculate that limiting thrombin activity during DIC could be a beneficial adjunct in the management of sepsis.
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PMID:Thrombin enhances monocyte secretion of tumor necrosis factor and interleukin-1 beta by two distinct mechanisms. 884 45

Fibrin formation within the glomeruli has been observed in various forms of human and experimental glomerulonephritis and it may play an important role in progressive glomerular injury. Furthermore it has been hypothesized that glomerular fibrin deposition may occur through activation of either the intrinsic or extrinsic coagulation pathway. It has been demonstrated that a procoagulant activity (PCA) which is compatible with tissue factor is present in the glomeruli and becomes increased in human proliferative glomerulonephritis and in animal models of nephritis. Tissue factor pathway inhibitor (TFPI) regulates the extrinsic pathway of blood coagulation through its ability to inhibit tissue factor activity. TFPI is present in plasma and in platelets, and it is now thought to be produced mainly by endothelial cells. We examined whether human mesangial cells (HMC) could produce TFPI and attempted to clarify regulatory factors which affect TFPI production. Cultured HMC were used and TFPI in the cell supernatants was measured by ELISA using a specific antibody. Cultured HMC showed the production of TFPI. Immunoblot analysis revealed 40 kD protein of TFPI. The concentration of TFPI was significantly increased following the incubation with thrombin and heparin, including low molecular weight heparin, in a dose- and time-dependent manner. However, fetal calf serum, phorbol myristate acetate, lipopolysaccharide, IL-1 beta and tissue factor did not stimulate TFPI synthesis. Our data show that cultured HMC have the ability to produce TFPI which inhibits fibrin formation. It is possible that thrombin-induced enhancement of TFPI synthesis may be caused by the autoregulatory system of blood coagulation and that with heparin it may represent another anticoagulatory effect of heparin.
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PMID:Tissue factor pathway inhibitor production by human mesangial cells in culture. 886 34

Canine endothelial cells express the adhesion molecule P-selectin to mediate the initial attachment of leukocytes to the vessel wall. Although it is known that agents like histamine and thrombin stimulate the surface expression of P-selectin, the effect of inflammatory mediators and cytokines such as lipopolysaccharides (LPS), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1 beta (IL-1 beta) on canine P-selectin expression has not been investigated. Therefore, the objective of this study was to analyze the regulation of P-selectin messenger RNA (mRNA) and protein by these cytokines in canine endothelial cells isolated from jugular veins. Analyses of cytoplasmic RNA by Northern blotting showed that stimulation of culture endothelial cells with either LPS (100 ng/ml) or recombinant human TNF-alpha (30 U/ml) for 3 or 6 hours significantly increased (P < 0.05) steady-state levels of mRNA for P-selectin (3.8- +/- 1.0- and 3.0- +/- 0.4-fold increase for LPS at 3 and 6 hours, respectively, and 2.5- +/- 0.8- and 2.7- +/- 0.9-fold increase for TNF-alpha at 3 and 6 hours, respectively). P-selectin mRNA had decreased by 48 hours to levels found in unstimulated cells. In contrast, human IL-1 beta had no effect on P-selectin mRNA. Increased levels of mRNA with LPS stimulation were associated with the synthesis of new protein, as demonstrated by the positive staining in LPS-stimulated cells using immunocytochemistry with a monoclonal antibody against canine P-selectin (MD3). These results reveal that important inflammatory mediators and cytokines such as LPS and TNF-alpha induce the synthesis of new P-selectin and suggest that this process could represent a means of sustaining local leukocyte recruitment for several hours during an acute inflammatory reaction.
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PMID:Regulation of P-selectin expression by inflammatory mediators in canine jugular endothelial cells. 895 25

We studied the effects of serine proteases on cytokine gene expression by cultured normal human keratinocytes. In resting keratinocytes, steady-state mRNA levels for interleukins IL-1 alpha, IL-1 beta, IL-7, and IL-8, transforming growth factors alpha and beta, and tumor necrosis alpha were sufficient to be detected by our reverse transcriptase-polymerase clozin reaction method. Incubation of keratinocytes with 25 nM trypsin or 1 unit/ml thrombin for 24 hr selectively upregulated mRNA levels for granulocyte-macrophage colony-stimulating factor (GM-CSF) and Il-6 to detectable levels. Keratinocytes secreted GM-CSF and IL-6 protein in response to these proteases. Monensin did not inhibit the gene expression for the cytokines, thereby excluding the possibility of intervention by secreted molecules. Aprotinin and argatroban inhibited the effects of the proteases. SFLLRN and SLIGRL, tethered ligand receptor peptides for thrombin receptor and for proteinase-activated receptor 2 (PAR-2), respectively, duplicated the effects of the proteases on keratinocytes, which expressed mRNA for both receptors. Trypsin increased tyrosine phosphorylated proteins and intracellular free calcium concentrations. Tyrphostin, pertussis toxin, or H-7 suppressed trypsin- and thrombin-induced GM-CSF gene expression. Our results demonstrate that the serine proteases activate thrombin receptors and PAR-2 on keratinocytes, triggering intracellular signaling and then inducing the synthesis of GM-CSF. We speculate that serine proteases modulate the course of physiological and pathological processes in the skin by stimulating keratinocytes to produce the cytokines.
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PMID:Thrombin and trypsin induce granulocyte-macrophage colony-stimulating factor and interleukin-6 gene expression in cultured normal human keratinocytes. 906 88

We evaluated the thrombin-stimulated production of prostacyclin (PGI2) by cultured human pulmonary artery smooth muscle cells (HPASMC) that were pretreated with cytokines (IL-1 beta, TNF alpha) and lipopolysaccharide (LPS). Cultured HPASMC, obtained from autopsied cases, were identified as smooth muscle cells by immune staining with mouse anti-human alpha-smooth muscle actin monoclonal IgG. A 3 hour incubation of HPASMC with LPS, IL-1 beta, or TNF alpha followed by a 10 min exposure to 2 U/ml thrombin was sufficient to generate a greater amount of PGI2 than observed in control cells. The increase in PGI2 production peaked after 8 h in the IL-1 beta- and TNF alpha-treated HPASMC, and continued to increase for 24 h in the LPS-treated HPASMC. We then investigated the effect of incubation time of thrombin on PGI2 production in HPASMC pretreated with cytokines or LPS for 24 h. PGI2 production by LPS- and cytokine-treated HPASMC peaked after a 15 min exposure to thrombin. In contrast, the exposure of LPS- or IL-1 beta-treated HPASMC to buffer seemed to increase the release of PGI2 for up to 30 min of incubation. However, the PGI2 released was less than that in the thrombin-stimulated HPASMC. After incubation with various concentrations of LPS or cytokines, the production of PGI2 by thrombin-stimulated HPASMC showed significant, dose-dependent increases beginning at 0.1 microgram/ml of LPS, 20 U/ml of IL-1 beta, and 50 U/ml of TNF alpha. We conclude that LPS, IL-1 beta, and TNF alpha enhanced both the basal and thrombin-stimulated production of PGI2 by HPASMC. This enhanced production of PGI2 might help in lowering the pulmonary vascular tone and modifying pulmonary hemodynamics in cytokine- or endotoxin-mediated inflammation and acute injury of the lung.
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PMID:Cytokines and lipopolysaccharide enhance basal and thrombin-stimulated production of PGI2 by cultured human pulmonary artery smooth muscle cells. 908 96

Hemodynamic forces modulate various endothelial cell functions even in the presence of cytokines under gene regulation. We have investigated the effect of shear stress on the coagulation and fibrinolysis systems in cultured human umbilical vein endothelial cells (HUVECs) perturbed by cytokines, using modified cone-plate viscometer. Thrombomodulin (TM), a surface glycoprotein receptor for thrombin that catalyzes the activation of the protein C anticoagulant pathway, and tissue factor (TF), a transmembrane glycoprotein that plays a central role in blood coagulation, are important regulators for coagulation in endothelium. Shear stress of 18 dynes/cm2 increased the expression of TM either in the presence or absence of TNF alpha (100 U/ml). In contrast, shear stresses of 6 approximately 24 dynes/cm2 decreased the expression of TNF alpha-induced TF in a shear intensity- and exposure time- dependent manner Tissue plasminogen activator(t-PA), which converts plasminogen to plasmin to degrade fibrin clot, and plasminogen activator inhibitor-1 (PAI-1), which inhibits t-PA function, play central roles in fibrinolysis in the endothelium. Treatment of the cells with IL-1 beta or TNF-alpha under static conditions had no effect on t-PA secretion, while release of PAI-1 increased. When cells were exposed to increasing shear stress up to 24 dynes/cm2, levels of t-PA significantly increased relative to shear stress, while PAI-1 secretion decreased gradually. In the presence of IL-1 beta or TNF-alpha, the increased production of t-PA was further augmented. These results clearly indicate that shear forces act as an important regulators of the coagulation and fibrinolysis systems in endothelium, to maintain antithrombogenicity of blood vessels.
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PMID:[Regulation of antithrombogenicity in endothelium by hemodynamic forces]. 913 94

We have examined the effects of N-acetyl-L-cysteine (NAC), a well-characterized, thiol-containing antioxidant, on agonist-induced monocytic cell adhesion to endothelial cells (EC). NAC inhibited interleukin-1 (IL-1 beta)-induced, but not basal, adhesion with 50% inhibition at approximately 20 mM. Monocytic cell adhesion to EC in response to tumor necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS), alpha-thrombin, or phorbol 12-myristate 13-acetate (PMA) was similarly inhibited by NAC. Unlike published studies with pyrrolidinedithiocarbamate, which specifically inhibited vascular cell adhesion molecule 1 (VCAM-1), NAC inhibited IL-1 beta-induced mRNA and cell surface expression of both E-selectin and VCAM-1. NAC had no effect on the half-life of E-selectin or VCAM-1 mRNA. Although NAC reduced nuclear factor-kappa B (NF-kappa B) activation in EC as measured by gel-shift assays using an oligonucleotide probe corresponding to the consensus NF-kappa B binding sites of the VCAM-1 gene (VCAM-NF-kappa B), the antioxidant had no appreciable effect when an oligomer corresponding to the consensus NF-kappa B binding site of the E-selectin gene (E-selectin-NF-kappa B) was used. Because NF-kappa B has been reported to be redox sensitive, we studied the effects of NAC on the EC redox environment. NAC caused an expected dramatic increase in the reduced glutathione (GSH) levels in EC. In vitro studies demonstrated that whereas the binding affinity of NF-kappa B to the VCAM-NF-kappa B oligomer peaked at a GSH-to-oxidized glutathione (GSSG) ratio of approximately 200 and decreased at higher ratios, the binding to the E-selectin-NF-kappa B oligomer appeared relatively unaffected even at ratios > 400, i.e., those achieved in EC treated with 40 mM NAC. These results suggest that NF-kappa B binding to its consensus sequences in the VCAM-1 and E-selectin gene exhibits marked differences in redox sensitivity, allowing for differential gene expression regulated by the same transcription factor. Our data also demonstrate that NAC increases the GSH-to-GSSG ratio within the EC suggesting one possible mechanism through which this antioxidant inhibits agonist-induced monocyte adhesion to EC.
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PMID:Distinct mechanisms for N-acetylcysteine inhibition of cytokine-induced E-selectin and VCAM-1 expression. 927 99


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