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 fibroblasts express proteinase-activated receptor-1 (PAR1), PAR2 and PAR3, but not PAR4. Because PAR2 has inflammatory effects on human primary bronchial fibroblasts (HPBF), we asked 1) whether the inflammatory mediators TNF-alpha and LPS could modify HPBF PAR expression and 2) whether modified PAR expression altered HPBF responsiveness to PAR agonists in terms of calcium signaling and cell growth. TNF-alpha and LPS induced PAR4 mRNA expression (RT-PCR) at 6 h and 24 h, respectively. TNF-alpha and LPS also upregulated PAR2 mRNA expression with similar kinetics but had negligible effect on PAR1 and PAR3. Flow cytometry for PAR1, PAR2, and PAR3 also demonstrated selective PAR2 upregulation in response to TNF-alpha and LPS. Intracellular calcium signaling to SLIGKV-NH2 (a selective PAR2-activating peptide; PAR2-AP) and AYPGQV-NH2 (PAR4-AP) revealed that TNF-alpha and LPS induced maximal responses to these PAR agonists at 24 h and 48 h, respectively. Upregulation of PAR2 by TNF-alpha heightened HPBF responses to trypsin, while PAR4 induction enabled cathepsin-G-mediated calcium signaling. Cathepsin-G also disarmed PAR1 and PAR2 in HPBF, while tryptase disarmed PAR2. Induction of PAR4 also enabled thrombin to elicit a calcium signal through both PAR1 and PAR4, as determined by a desensitization assay. In cell growth assays the PAR4 agonists cathepsin-G and AYPGQV-NH2 reduced HPBF cell number only in TNF-alpha-treated HPBF. Moreover, the mitogenic effect of thrombin (a PAR1/PAR4 agonist) but not the PAR1-AP TFLLR-NH2, was ablated in TNF-alpha-treated HPBF. These findings point to an important mechanism, whereby cellular responses to thrombin and cathepsin-G can be modified during an inflammatory response.
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PMID:Inflammatory mediators modulate thrombin and cathepsin-G signaling in human bronchial fibroblasts by inducing expression of proteinase-activated receptor-4. 1714 51

We have previously described protective effects of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) on pulmonary endothelial cell (EC) barrier function and demonstrated the critical role of cyclopentenone-containing modifications of arachidonoyl moiety in OxPAPC protective effects. In this study we used oxidized phosphocholine (OxPAPC), phosphoserine (OxPAPS), and glycerophosphate (OxPAPA) to investigate the role of polar head groups in EC barrier-protective responses to oxidized phospholipids (OxPLs). OxPAPC and OxPAPS induced sustained barrier enhancement in pulmonary EC, whereas OxPAPA caused a transient protective response as judged by measurements of transendothelial electrical resistance (TER). Non-OxPLs showed no effects on TER levels. All three OxPLs caused enhancement of peripheral EC actin cytoskeleton. OxPAPC and OxPAPS completely abolished LPS-induced EC hyperpermeability in vitro, whereas OxPAPA showed only a partial protective effect. In vivo, intravenous injection of OxPAPS or OxPAPC (1.5 mg/kg) markedly attenuated increases in the protein content, cell counts, and myeloperoxidase activities detected in bronchoalveolar lavage fluid upon intratracheal LPS instillation in mice, although OxPAPC showed less potency. All three OxPLs partially attenuated EC barrier dysfunction induced by IL-6 and thrombin. Their protective effects against thrombin-induced EC barrier dysfunction were linked to the attenuation of the thrombin-induced Rho pathway of EC hyperpermeability and stimulation of Rac-mediated mechanisms of EC barrier recovery. These results demonstrate for the first time the essential role of polar OxPL groups in blunting the LPS-induced EC dysfunction in vitro and in vivo and suggest the mechanism of agonist-induced hyperpermeability attenuation by OxPLs via reduction of Rho and stimulation of Rac signaling.
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PMID:Polar head groups are important for barrier-protective effects of oxidized phospholipids on pulmonary endothelium. 1715

Nitric oxide ((*)NO) plays an important role in a number of physiologic processes. Evidence exists that (*)NO, which stimulates soluble guanylate cyclase and enhances cyclic guanosine monophosphate (cGMP) levels, may inhibit platelet activation. In contrast, during platelet activation induced by different agonists, synthesis of (*)NO in platelets occurs. In these studies, production of the stable end-products of (*)NO-nitrite and nitrate (NO(x)) in human platelets, stimulated by different doses of lipopolysaccharide from Proteus mirabilis (LPS; endotoxin), has been evaluated. LPS is a weak platelet agonist that may activate various steps of platelet activation with the generation of reactive oxygen species. The mechanism of platelet activation induced by the endotoxin is not known. The aim of the present study was to measure the level of nitrite and NO(x) in blood platelets treated with LPS and to examine the level of nitrotyrosine in platelet proteins caused by LPS. Our results show that LPS at a low concentration (6.8 ng/ml) caused a decrease (approximately 80%) in the NO(x) level, whereas at higher concentrations (13.6 and 25 ng/ml) it induced an increase in the NO(x) level (approximately 210% and 260%, respectively). Our results indicate that LPS, like other agonists (thrombin, platelet-activating factor), can stimulate (*)NO production in platelets. After incubating platelets with LPS, we also observed a distinct increase in platelet protein nitration (3-nitrotyrosine).
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PMID:The effect of lipopolysaccharide from Proteus mirabilis on the level of the stable end metabolic products of nitric oxide in blood platelets. 1716 Mar 61

The effects of steroids on the outcome of sepsis are dose dependent. Low doses appear to be beneficial, but high doses do not improve outcome for reasons that are insufficiently understood. The effects of steroids on systemic inflammation as a function of dose have not previously been studied in humans. To determine the effects of increasing doses of prednisolone on inflammation and coagulation in humans exposed to LPS, 32 healthy males received prednisolone orally at doses of 0, 3, 10, or 30 mg (n = 8 per group) at 2 h before i.v. injection of Escherichia coli LPS (4 ng/kg). Prednisolone dose-dependently inhibited the LPS-induced release of cytokines (TNF-alpha and IL-6) and chemokines (IL-8 and MCP-1), while enhancing the release of the anti-inflammatory cytokine IL-10. Prednisolone attenuated neutrophil activation (plasma elastase levels) and endothelial cell activation (von Willebrand factor). Most remarkably, prednisolone did not inhibit LPS-induced coagulation activation, measured by plasma concentrations of thrombin-antithrombin complexes, prothrombin fragment F1+2, and soluble tissue factor. In addition, activation of the fibrinolytic pathway (tissue-type plasminogen activator and plasmin-alpha(2)-antiplasmin complexes) was dose-dependently enhanced by prednisolone. These data indicate that prednisolone dose-dependently and differentially influences the systemic activation of different host response pathways during human endotoxemia.
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PMID:Prednisolone dose-dependently influences inflammation and coagulation during human endotoxemia. 1723 35

Reparixin antagonizes interleukin-8 (IL-8) on the level of signal transduction in vitro. We hypothesized that IL-8 mediates some of the reactions occurring during acute inflammation and specifically that IL-8 may be a mediator of endotoxin induced neutrophilia. We therefore tested the effects of reparixin on humoral and cellular parameters in LPS-induced acute systemic inflammation. The study is a randomized (3:2 active:placebo), double-blind, placebo-controlled parallel group trial. Twenty healthy male volunteers randomly received either reparixin (12) or placebo (8) intravenously. One hour after the start of reparixin/placebo infusion a bolus of 2 ng/kg endotoxin was infused over 1-2 min. Blood samples were obtained over 24 h. Reparixin, being metabolized to ibuprofen, suppressed serum thromboxane B2 levels by 78 percent compared to baseline and control at 8 h. LPS-induced neutrophilia was not significantly affected by reparixin in human volunteers. Consistently, reparixin did not alter the lymphocyte or monocyte counts and had no effect on LPS-induced systemic inflammation as measured by tumor necrosis factor alpha (TNF-alpha) or interleukin-6 (IL-6) release. Regulation of IL-8 receptors CXCR1 and 2 and the degranulation marker CD11b showed the expected kinetics. Reparixin had no effect on thrombin formation as measured by prothrombin fragment (F1+2). In conclusion, our study showed that reparixin was safe but had no impact on endotoxin induced inflammation. In contrast to previous studies with its metabolite ibuprofen, reparixin does not enhance inflammation in this model.
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PMID:Reparixin, a specific interleukin-8 inhibitor, has no effects on inflammation during endotoxemia. 1734 25

Endothelial cell (EC) barrier dysfunction (i.e., increased vascular permeability) is observed in inflammatory states, tumor angiogenesis, atherosclerosis, and both sepsis and acute lung injury. Therefore, agents that preserve vascular integrity have important clinical therapeutic implications. We examined the effects of methylnaltrexone (MNTX), a mu opioid receptor (mOP-R) antagonist, on human pulmonary EC barrier disruption produced by edemagenic agents including morphine, the endogenous mOP-R agonist DAMGO, thrombin, and LPS. Pretreatment of EC with MNTX (0.1 muM, 1 h) or the uncharged mOP-R antagonist naloxone attenuated morphine- and DAMGO-induced barrier disruption in vitro. However, MNTX, but not naloxone, pretreatment of EC inhibited thrombin- and LPS-induced barrier disruption, indicating potential mOP-R-independent effects of MNTX. In addition, intravenously delivered MNTX attenuated LPS-induced vascular hyperpermeability in the murine lung. We next examined the mechanistic basis for this MNTX barrier protection and observed that silencing of mOP-R attenuated the morphine- and DAMGO-induced EC barrier disruption, but not the permeability response to either thrombin or LPS. Because activation of the sphingosine 1-phosphate receptor, S1P(3), is key to a number of barrier-disruptive responses, we examined the role of this receptor in the permeability response to mOP-R ligation. Morphine, DAMGO, thrombin, and LPS induced RhoA/ROCK-mediated threonine phosphorylation of S1P(3), which was blocked by MNTX, suggesting S1P(3) transactivation. In addition, silencing of S1P(3) receptor expression (siRNA) abolished the permeability response to each edemagenic agonist. These results indicate that MNTX provides barrier protection against edemagenic agonists via inhibition of S1P(3) receptor activation and represents a potentially useful therapeutic agent for syndromes of increased vascular permeability.
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PMID:Attenuation of vascular permeability by methylnaltrexone: role of mOP-R and S1P3 transactivation. 1739 91

Retinoic acid (RA) is a well-known antiinflammatory agent. In this study, we show that RA has a dual effect on cyclooxygenase-2 (COX-2) expression in inflammatory activated microglia, the resident brain macrophages. After treatment of microglia with LPS or thrombin, COX-2 expression was induced in two phases, specifically, an initial increase at about 12 hr after stimulation followed by a decrease, and another increase at about 48-72 hr. However, PGE(2) and 15d-PGJ(2) were detected at about 12 hr, and the levels continuously increased thereafter. Interestingly, all-trans retinoic acid (ATRA) suppressed the expression of early-phase COX-2 but augmented late-phase COX-2 and inhibited iNOS in the whole time sequence. ATRA enhanced PGE(2) production but had little effect on 15d-PGJ(2). Moreover, ATRA selectively up-regulated the expression of a PGE(2) synthase, mPGES-1, but had little effect on the PGD(2) synthase, H-PGDS. The results collectively suggest that ATRA modulates microglial responses to inflammatory stimulators, particularly at the late phase, via enhancement of COX-2 expression and PGE(2) production.
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PMID:Retinoic acid enhances prostaglandin E2 production through increased expression of cyclooxygenase-2 and microsomal prostaglandin E synthase-1 in rat brain microglia. 1818 17

The endothelium is a semi-permeable barrier that regulates the flux of liquid and solutes, including plasma proteins, between the blood and surrounding tissue. The permeability of the vascular barrier can be modified in response to specific stimuli acting on endothelial cells. Transport across the endothelium can occur via two different pathways: through the endothelial cell (transcellular) or between adjacent cells, through interendothelial junctions (paracellular). This review focuses on the regulation of the paracellular pathway. The paracellular pathway is composed of adhesive junctions between endothelial cells, both tight junctions and adherens junctions. The actin cytoskeleton is bound to each junction and controls the integrity of each through actin remodeling. These interendothelial junctions can be disassembled or assembled to either increase or decrease paracellular permeability. Mediators, such as thrombin, TNF-alpha, and LPS, stimulate their respective receptor on endothelial cells to initiate signaling that increases cytosolic Ca2+ and activates myosin light chain kinase (MLCK), as well as monomeric GTPases RhoA, Rac1, and Cdc42. Ca2+ activation of MLCK and RhoA disrupts junctions, whereas Rac1 and Cdc42 promote junctional assembly. Increased endothelial permeability can be reversed with "barrier stabilizing agents," such as sphingosine-1-phosphate and cyclic adenosine monophosphate (cAMP). This review provides an overview of the mechanisms that regulate paracellular permeability.
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PMID:Regulation of endothelial junctional permeability. 1837 86

Protease-activated receptors (PARs) are a family of G protein-coupled receptors that are activated by serine protease-mediated proteolytic cleavage of their extracellular domain. We have previously characterized the expression and function of PARs in human monocytes and macrophages, yet information about PARs in dendritic cells (DC) is scarce. Monocyte-derived immature DC do not express PARs. Upon maturation with LPS, but not with TNF-alpha or CD40 ligand, DC express PAR1 and PAR3, but not PAR2 or PAR4. Stimulation of DC with the serine protease thrombin or PAR1-activating peptide elicits actin polymerization and concentration-dependent chemotactic responses in LPS-, but not in TNF-alpha-matured DC. The thrombin-induced migration is a true chemotaxis with only negligible chemokinesis. Stimulation of PARs with thrombin or the respective receptor-activating peptides activates ERK1/2 and Rho kinase as well as subsequent phosphorylation of the regulatory myosin L chain 2. The ERK1/2- and Rho kinase 1-mediated phosphorylation of myosin L chain 2 was indispensable for the PAR-mediated chemotaxis as shown by pharmacological inhibitors. Additionally, thrombin stimulated the Rho-dependent release of the CC chemokine CCL18/pulmonary and activation-regulated chemokine, which induces chemotaxis of lymphocytes and immature DC as well as fibroblast proliferation. The colocalization of CD83(+) DC with CCL18 in human atherosclerotic plaques revealed by immunofluorescence microscopy combined with the presence of functionally active thrombin receptors on mature DC point to a previously unrecognized functional role of thrombin in DC biology. The thrombin-induced stimulation of mature DC may be of particular relevance in atherosclerotic lesions, which harbor all components of this novel mechanism.
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PMID:Mature dendritic cells express functional thrombin receptors triggering chemotaxis and CCL18/pulmonary and activation-regulated chemokine induction. 1860 75

We recently demonstrated that the occupancy of endothelial protein C receptor (EPCR) by its natural ligand activated protein C (APC)/protein C switches the protease activated receptor 1 (PAR-1)-dependent signaling specificity of thrombin from a disruptive to a protective effect in cultured human umbilical vein endothelial cells. Given the phenotypic differences between endothelial cells in venular and arterial beds, in this study we evaluated the signaling function of thrombin in human pulmonary artery endothelial cells (HPAECs) before and after treating them with PC-S195A which lacks catalytic activity but exhibits a normal affinity for EPCR. As expected, both thrombin and thrombin receptor agonist peptide (TRAP) enhanced the permeability barrier of HPAECs; however, both PAR-1 agonists exhibited a potent barrier protective effect when the cells were treated with PC-S195A prior to stimulation by the agonists. Interestingly, similar to APC, thrombin exhibited a potent cytoprotective activity in the LPS-induced permeability and TNF-alpha-induced apoptosis and adhesion assays in the PC-S195A treated HPAECs. Treatment of HPAECs with the cholesterol depleting molecule methyl-beta-cyclodextrin eliminated the protective effect of both APC and thrombin. These results suggest that the occupancy of EPCR by its natural ligand recruits PAR-1 to a protective signaling pathway within lipid rafts of HPAECs. Based on these results we conclude that the activation of PAR-1 by thrombin would initiate a protective response in intact arterial vascular cells expressing EPCR. These findings may have important ramifications for understanding the mechanism of the participation of the vascular PAR-1 in pathophysiology of the inflammatory disorders.
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PMID:Protease activated receptor 1 (PAR-1) activation by thrombin is protective in human pulmonary artery endothelial cells if endothelial protein C receptor is occupied by its natural ligand. 1861 44


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