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)

Cyclic GMP accumulation in endothelial cells-smooth muscle cells (EC-SMC) coculture induced by both receptor-dependent (thrombin, bradykinin, BK) and receptor-independent (Ca(2+)-ionophore A23187) stimulation, was inhibited by preincubation with low-density lipoprotein (LDL) in time- and concentration-dependent manner. At least 5 min was necessary for the complete blockade with LDL (protein 1 mg/ml). LDL did not affect cyclic GMP-increase by sodium nitroprusside (SNP), a direct stimulator of SMC, but oxidized (ox)LDL (50-250 micrograms/ml) markedly reduced it. An increase of cyclic GMP accumulation in SMC by eluate from stimulated EC columns was completely blocked by 10-min pre-incubation of the column with LDL with or without superoxide dismutase (SOD). In contrast, preincubation of the SMC dish with LDL did not affect cyclic GMP accumulation by the eluate from the stimulated EC column, but preincubation with oxLDL (protein 50-100 micrograms/ml) greatly reduced it. Exposure time of released EDRF to LDL in both coculture and column experiments was < 40-45 s. These results suggest that a brief exposure of EC to pathophysiologic concentration of LDL exclusively affects EC functions, attenuating endothelium-derived relaxing factor (EDRF) release through intracellular mechanisms, and consumption of released EDRF by LDL does not appear to be involved in this LDL inhibitory effect. Possible inhibitory mechanisms of LDL are discussed.
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PMID:Inhibition of EDRF release by native low-density lipoprotein from cultured porcine endothelial cells through intracellular mechanisms. 752 37

Investigation of the regulation of permeability properties of the endothelium has yielded evidence to support the concept of a dual regulation of EC gap formation and barrier function. In this model, the primary determinants of EC permeability are tethering/adhesive properties (Figure 1) and tensile centripetal force generation (Figure 2). The importance of actin-myosin interactions and active cellular contraction and force generation has been reviewed. In the model of thrombin-induced EC barrier dysfunction, there is a strong shift in the MLC species from the unphosphorylated to the diphosphorylated form, indicating activation of MLCK, a key enzyme whose importance in EC contraction has been well established. Although important differences between EC and SMC exist, endothelial cell gap formation involves actomyosin-dependent contractile mechanisms similar to SMC, a cellular system in which MLC phosphorylation correlates with the initial rate of tension development. The increase in MLC phosphorylation and isometric tension is consistent with the hypothesis that activation of signal transduction mediates an increase in isometric tension to a new level of "latch state" through the cytoskeleton. Thus, the available evidence implicates a strong role for cellular force generation and contraction in the evolution of thrombin-induced barrier dysfunction. Accumulating evidence also indicates that modulation of tethering properties, primarily those involving cell-matrix and cell-cell adhesion, is also a key determinant of basal EC barrier properties as well as agonist-mediated barrier dysfunction. Because each of these focal adhesion constituents may be involved in establishing tethering properties in endothelium, they each may be involved in determining barrier permeability and may be involved in the evolution of agonist-mediated barrier dysfunction. Therefore, in addition to MLCK-dependent active tensile force generation, agonist-induced barrier dysfunction may occur via MLCK-independent pathways that rely on basal levels of MLC phosphorylation or by affecting proteins involved in tethering properties of endothelium that contribute to barrier function. Further examination of tethering force properties, combined with elucidation of EC relaxation via MLC dephosphorylation may yield clues as to how this important vascular barrier is maintained and restored after vascular insult.
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PMID:Regulation of endothelial cell gap formation and paracellular permeability. 773 15

Plasminogen activator inhibitor-1 (PAI-1) is the major inhibitor for plasmin formation promoted by tissue and urokinase plasminogen activators. The present study demonstrates that thrombin increase PAI-1 antigen, biological activity, and gene expression in cultured baboon aortic smooth muscle cells (BASMC). Thrombin elevates PAI-1 antigen in conditioned medium of BASMC within 10 min of the treatment, with the peak increase after 30 min of the treatment. Overexpression of PAI-1 gene was detected in the cultures exposed to thrombin for at least 60 min. PAI activity in conditioned medium increased in the cultures treated with thrombin for at least 4 h. The thrombin-induced early increase of PAI-1 antigen (up to 30 min of the stimulation) was blocked by hirudin (a specific inhibitor of thrombin), mimicked by trypsin and not suppressed by cycloheximide (a protein synthesis inhibitor). The majority of metabolically labeled PAI-1 associated with BASMC was present in extracellular matrix. The level of extracellular matrix-associated PAI-1 was reduced 40% by 30 min of thrombin treatment. Our results suggest that thrombin not only increases PAI-1 transcription but also proteolytically cleaves PAI-1 from the extracellular matrix of vascular SMC. PAI-1 released by thrombin from the extracellular matrix may not alter PAI activity in extracellular fluid but may reduce the storage of PAI-1 in the extracellular matrix of vascular smooth muscle cells.
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PMID:Effect of thrombin on release of plasminogen activator inhibitor-1 from cultured primate arterial smooth muscle cells. 774 May 4

The present study investigated transcellular signalling mechanism involved in thrombin-induced production of plasminogen activator inhibitor-1 (PAI-1) in cultured vascular baboon aortic smooth muscle cells (BASMC). Treatments with thrombin dose-dependently increased the steady state levels of PAI-1 mRNA and the generation of PAI-1 antigen from BASMC. Thrombin receptor-activating peptide mimicked the effect of thrombin on the generation of PAI-1. Sodium fluoride (1 mM) stimulated PAI-1 generation from BASMC. Pertussis toxin dose-dependently suppressed thrombin-induced increase of PAI-1 generation. Treatment with 5 mM neomycin, 10 microM U73122 or 1 microM calphostin C blocked thrombin-induced PAI-1 generation. Phorbol myristate acetate at 10 nM for 3 h strongly stimulated the generation of PAI-1 from BASMC. Forskolin (100 microM) or 8-bromo-cAMP (100 microM) suppressed thrombin-induced PAI-1 generation. The responses of quiescent BASMC to thrombin or the inhibitors on PAI-1 generation were comparable to that of growing cells. The results of the present study suggest that pertussis toxin-sensitive G proteins and a phospholipase C are involved in thrombin-induced generation of PAI-1 in BASMC, which may transmit signals from occupied thrombin receptor to protein kinase C and thereby increase the generation of PAI-1. Elevated levels of intracellular cAMP may negatively regulate the generation of PAI-1 from vascular SMC.
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PMID:G proteins and phospholipase C mediate thrombin-induced generation of plasminogen activator inhibitor-1 from vascular smooth muscle cells. 916 40

3-Hydroxy-3-methylglutaryl Coenzyme A (HMG-CoA) reductase inhibitors (statins) are therapeutically used to lower plasma cholesterol levels. In addition, these drugs can block vascular smooth muscle cell (VSMC) proliferation. The present study addressed the question whether the inhibitory effect of lovastatin on premitotic DNA synthesis correlates with a downregulation of c-fos mRNA levels, a marker of signaling efficiency, in human SMC. Here we show that in human SMC exposed to individual growth factors (platelet-derived growth factor, epidermal growth factor, alpha-thrombin, insulin, insulin-like growth factor I (IGF-I)) and human serum, the maximal [3H]thymidine incorporation and c-fos mRNA expression are closely correlated. Only alpha-thrombin elicited overexpression of c-fos as compared with its effect on [3H]thymidine incorporation. Lovastatin efficiently inhibited [3H]thymidine uptake promoted by all mitogens tested (76-87%); however, it significantly inhibited upregulation of c-fos mRNA levels induced only by insulin (33-67%, P < 0.05) and IGF-I (31 57%, P < 0.05). This inhibition was overcome by mevalonate and geranylgeraniol, and partially by farnesol. c-fos mRNA expression induced by 4-beta-phorbol-12-myristate-13-acetate, an activator of protein kinase C, was insensitive to lovastatin treatment. Thus, in human vascular SMC, lovastatin impairs premitotic DNA synthesis induced by growth factors, but only c-fos expression promoted by insulin and IGF-I. These data indicate that statin-sensitive and -insensitive pathways seem to be involved in the regulation of c-fos in the response of human SMC to proliferative stimuli, and suggest a prominent role of isoprenylated proteins in the activation of VSMC through the IGF-I/insulin dependent pathways.
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PMID:Mevalonate deprivation impairs IGF-I/insulin signaling in human vascular smooth muscle cells. 943 Mar 71

Activated platelets are instrumental in restenosis due to their role in thrombus formation. Aurintricarboxylic acid (ATA) has been reported to prevent platelet activation by inhibiting von Willebrand factor binding to platelet glycoprotein (GP)Ib. We investigated the effects of ATA in vitro and in vivo in hamsters. ATA inhibited the in vitro platelet aggregation induced by ADP, botrocetin and thrombin, but not by collagen. The IC50 values during the ex vivo platelet aggregation by ADP, botrocetin and thrombin were 8.2 +/- 1.8 microM, 0.9 +/- 0.4 microg/ml and 2.4 +/- 0.8 unit/ml, respectively. The platelet retention time to collagen-coated beads of hamster blood samples was inhibited by ATA (0.1, 0.3 and 1.0 mg/kg per hour) in a dose-dependent manner. Continuous administration of ATA (0, 0.1, 0.3, 1.0, 3.0 and 10.0 mg/kg per h) via an infusion pump produced dose-dependent antithrombotic effects: the time to occlude the carotid artery after vascular injury with a modified catheter was prolonged. Only when infused at doses of 3.0 and 10.0 mg/kg per hour, bleeding times were significantly prolonged. The continuous treatment with ATA (1.0 mg/kg per h) using a 2ML1 Alzet infusion pump for 2 weeks, resulted in a decrease in neointimal area by 22.2 +/- 6.8% when measured 2 weeks after injury induction. DNA synthesis using DDT1MF2 hamster SMCs was decreased by ATA in a dose-dependent manner. ATA reduced the number of platelets adhering on the injured area, as detected by electron microscopy. These results indicated that treatment with ATA inhibited platelet adhesion but also SMC proliferation. These observations may explain the effect of ATA on neointima formation.
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PMID:Multiple inhibition of platelet activation by aurintricarboxylic acid prevents vascular stenosis after endothelial injury in hamster carotid artery. 956 6

While effects of alpha-thrombin have been well characterized in different cell types, the biological function of intermediates of prothrombin activation is still undefined. Meizothrombin could be shown to be a potent agonist for vascular contraction which seems to be mediated by an interaction with the vascular smooth muscle. To explore this effect at intracellular signaling level, we used rat aortic smooth muscle cells and investigated the effect of the intermediates formed by cleavage of human prothrombin with ecarin, the prothrombin activator from Echis carinatus venom, on mobilization of free intracellular calcium. The ecarin-activated prothrombin induced very rapidly transient calcium mobilization in SMC's comparable to that observed with alpha-thrombin. We conclude that meizothrombin/meizothrombin desF1 (MT/MT desF1) are the most likely candidates for this effect. Furthermore, our results suggest the involvement of PAR-1-type thrombin receptors in MT/MT desF1-induced calcium signaling in rat aortic smooth muscle cells.
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PMID:Intermediates of prothrombin activation induce intracellular calcium mobilization in rat aortic smooth muscle cells. 986 77

Thrombin mediates acute vascular thrombosis following mechanical denuding injury or spontaneous rupture of atherosclerotic plaques. In the process of generating thrombin, factor VII/VIIa binds avidly with tissue factor exposed on cellular membranes, leading to sequential activation of coagulation serine proteases via macromolecular catalytic complexes on phospholipid surfaces. At sites of disrupted arteries thrombin activates platelets, blood leukocytes, endothelium, and vascular SMCs by cleaving G protein-coupled TRs, mediating SMC intimal proliferation in the formation of neointimal vascular lesions. Therapeutic strategies targeting thrombin include inactivation of bound thrombin, inhibition of TR activation by thrombin, and interruption of thrombin production. In patients having orthopedic surgery, inactivating bound thrombin with direct antithrombins markedly reduces venous thromboembolism as compared with heparin or its derivatives, without significant impairment of hemostasis. Antithrombotic effects in arterial thromboembolism, such as acute coronary syndrome, are not conclusively benefitted by systemic direct antithrombins when administered at safe levels, because interrupting TR-dependent platelet thrombosis demands systemic levels of direct antithrombins that compromise hemostatic function. Alternative safer strategies evolving from preclinical studies include (1) inhibiting thrombin activation of TRs, thereby abolishing platelet recruitment in arterial thrombogenesis, while sparing fibrin formation in hemostatic plugs; (2) enhancing the formation of endogenous activated protein C by protein C-selective thrombin mutants; and (3) preventing thrombin production by inhibiting precursor serine protease function and interrupting the formation of both acute thrombosis and vascular lesion formation. Tissue factor pathway antagonists are particularly promising because they exhibit both efficacy and safety in the prevention of thrombosis and vascular lesions.
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PMID:Therapeutic inhibition of thrombin activities, receptors, and production. 992 33

Several groups have demonstrated apoptotic cell death in atherosclerotic plaques. The significance of apoptosis in atherosclerosis depends on the stage of the plaque, localization and the cell types involved. Both macrophages and smooth muscle cells undergo apoptosis in atherosclerotic plaques. Apoptosis of macrophages is mainly present in regions showing signs of DNA synthesis/repair. Smooth muscle cell apoptosis is mainly present in less cellular regions and is not associated with DNA synthesis/repair. Even in early stages of atherosclerosis smooth muscle cells become susceptible to undergoing apoptosis since they increase different pro-apoptotic factors. Moreover, recent data indicate that smooth muscle cells may be killed by activated macrophages. The loss of the smooth muscle cells can be detrimental for plaque stability since most of the interstitial collagen fibers, which are important for the tensile strength of the fibrous cap, are produced by SMC. Apoptosis of macrophages could be beneficial for plaque stability if apoptotic bodies are removed. Apoptotic cells that are not scavenged in the plaque activate thrombin which could further induce intraplaque thrombosis. It can be concluded that apoptosis in the primary atherosclerosis is detrimental since it could lead to plaque rupture and thrombosis. Recent data of our group indicate that apoptosis decreases after lipid lowering which could be important in our understanding of the cell biology of plaque stabilization.
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PMID:Apoptosis in atherosclerosis: beneficial or detrimental? 1072 96

Vascular smooth muscle cells (VSMCs) migration and proliferation play a key role in the pathophysiology of cardiovascular disease. However, the transcription factors that regulate VSMC activation are not completely characterized. By a mRNA-differential display approach, we have identified neuron-derived orphan receptor-1 (NOR-1), a transcription factor within the NGFI-B subfamily of nuclear receptors, as a immediate-early gene in VSMCs. Two NOR-1 isoforms (alpha and beta) were identified and cloned from serum-induced porcine VSMC that shared high homology with the human isoforms. Northern blot analysis revealed a strong and transient (1 to 6 hours) upregulation of NOR-1 in both porcine and human coronary SMCs by growth factors (serum, platelet-derived growth factor-BB, and epidermal growth factor) and alpha-thrombin but not by cytokines. NOR-1 upregulation is processed through G protein-coupled receptors and tyrosine kinase receptors, and involves Ca2+ mobilization, protein kinase C activation, and the mitogen-activated protein kinase pathway. This induction was closely dependent of the cAMP response elements present in NOR-1 promoter as transfection assays indicate. Human coronary atherosclerotic lesions overexpress NOR-1, and balloon angioplasty transiently induces NOR-1 in porcine coronary arteries with a pattern similar to that observed in VSMCs in culture. Antisense oligonucleotides against NOR-1 inhibited human coronary SMC proliferation (reduced de novo DNA synthesis, cell cycle progression, and VSMC wound repair) as efficiently as antisense against the protooncogene c-fos. These results show that NOR-1 modulates VSMC proliferation, and suggest that this transcription factor may play a role in both spontaneous and accelerated atherosclerosis.
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PMID:Neuron-derived orphan receptor-1 (NOR-1) modulates vascular smooth muscle cell proliferation. 1252 26


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