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)

We prepared anti-platelet 20-kDa myosin light chain (MLC-20) antibody and demonstrated diphosphorylation of MLC-20 in platelets ex vivo in the initial phase of activation by thrombin. Our results are as follows. (1) By Western blotting, using anti-MLC-20 antibody, both mono- and diphosphorylated myosin were seen in the initial phase of aggregation of platelets by thrombin. The peak of the diphosphorylation was later than that of monophosphorylation and the degree of both mono- and diphosphorylation reduced in the process of aggregation. (2) ML-7 (a synthetic inhibitor of MLCK) inhibited both mono- and diphosphorylation of myosin and also blocked aggregation of thrombin-activated platelets. However, H-7 (an inhibitor of protein kinase C) had little effect on either the (di)phosphorylation of myosin or the aggregation of thrombin-activated platelets. (3) Arg-Gly-Asp-Ser (RGDS) peptide, a synthetic anti-adhesive peptide, inhibited aggregation of thrombin-activated platelets in a dose-dependent manner (100-200 microM). However, it had little effect on either mono- or diphosphorylation of myosin in the process of the platelet aggregation stimulated by thrombin. From these results, we conclude that mono- and diphosphorylation of myosin by MLCK play a role in the initial phase of activation of thrombin-stimulated platelets in vivo and that mono- and diphosphorylation of myosin by MLCK precedes the secondary signal mediated by GPIIb/IIIa.
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PMID:Diphosphorylation of platelet myosin ex vivo in the initial phase of activation by thrombin. 164 15

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

Thrombin-induced Ca2+ mobilization, activation of Ca2+/calmodulin-dependent myosin light chain (MLC) kinase (MLCK), and increased phosphorylation of MLCs precede and are critical to endothelial cell (EC) barrier dysfunction. Net MLC dephosphorylation after thrombin is nearly complete by 60 min and involves type 1 phosphatase (PPase 1) activity. We now report that thrombin does not alter total PPase 1 activity in EC homogenates but rather decreases myosin-associated PPase 1 activity. The PPase 1 inhibitor calyculin fails to prevent thrombin-induced MLC dephosphorylation. However, thrombin significantly increased the activity of Ca2+-dependent PPase 2B in EC homogenates (approximately 1.5- to 2-fold), with PPase 2B activation correlating with phosphorylation of the PPase 2B catalytic subunit. Western immunoblotting revealed PPase 2B to be present in cytoskeletal EC fractions, with specific PPase 2B inhibitors such as cyclosporin (200 nM) and deltamethrin (100 nM to 1 microM) attenuating thrombin-induced cytoskeletal protein dephosphorylation, including EC MLC dephosphorylation. These results suggest a model whereby thrombin-inducible contraction is determined by the phosphorylation status of EC MLC regulated by the balance between EC MLCK, PPase 1 (constitutive), and PPase 2B (inducible) activities.
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PMID:Role of Ca2+/calmodulin-dependent phosphatase 2B in thrombin-induced endothelial cell contractile responses. 975 12

Thrombin and proteinase-activated receptors (PAR) specifically regulate several functions that markedly enhance the transformation phenotype such as inflammation, cell proliferation, tumor growth, and metastasis. We recently reported that thrombin inhibits cellular invasion induced by src, hepatocyte growth factor (HGF), and leptin in kidney and colonic epithelial cells via predominant activation of the pertussis toxin (PTx) -sensitive G-proteins Galphao/Galphai. We provide pharmacological and biochemical evidence that in the presence of PTx, PAR-1 induced cellular invasion through Galpha12/Galpha13- and RhoA/Rho kinase (ROCK) -dependent signaling. However, inhibition of the endogenous small GTPase RhoA by the C3 exoenzyme, dominant-negative N19-RhoA, activated G26V-RhoD, and activators of the nitric oxide/cGMP pathways conferred invasive activity to PAR-1 via a signaling cascade using Galphaq, phospholipase C (PLC), Ca(2+)/calmodulin myosin light chain kinase (CaM-MLCK), and phosphorylation of MLC. We found that cellular invasion induced by the src oncogene is abrogated by inhibitors of the RhoA/ROCK pathway and is independent of PLC/CaM-MLCK signaling. Our data demonstrate that the RhoA and RhoD small GTPases are acting as a molecular switch of cellular invasion and reveal a novel critical mechanism by which PAR-1 bypass Galphao/i and RhoA inhibition via differential coupling to heterotrimeric G-proteins linked to divergent or convergent biological responses. Our data also indicate that Rho GTPases and ROCK mediate a src-dependent invasion signal in kidney and colonic cancer cells. We conclude that dynamic regulation of Rho GTPases activation and inactivation by oncogenes, growth factors, cGMP-inducing agents, and adhesion molecules can initiate convergent invasion signals controlled by the thrombin PAR-1 in cancer cells.-Nguyen, Q.-D., Faivre, S., Bruyneel, E., Rivat, C., Seto, M., Endo, T., Mareel, M., Emami, S., Gespach, C. RhoA- and RhoD-dependent regulatory switch of Galpha subunit signaling by PAR-1 receptors in cellular invasion.
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PMID:RhoA- and RhoD-dependent regulatory switch of Galpha subunit signaling by PAR-1 receptors in cellular invasion. 1191 59

The endothelial cell Ca2+/calmodulin (CaM)-dependent myosin light chain kinase isoform (EC MLCK) is a multifunctional contractile effector involved in vascular barrier regulation, leukocyte diapedesis, apoptosis, and angiogenesis. The EC MLCK isoform and its splice variants contain a unique N-terminal sequence not present in the smooth muscle MLCK isoform (SM MLCK), which allows novel upregulation of MLCK activation by signaling cascades including p60src. The yeast two-hybrid assay system using the entire EC MLCK1 N-terminus (922 aa) as bait, identified additional stable MLCK binding partners including the 12 KDa macrophage migration inhibitory factor (MIF). This finding was confirmed by cross immunoprecipitation assays under non-denaturing conditions and by GST pull down experiments using GST-N-terminal MLCK (#1-923) and MLCK N-terminal deletion mutants in TNFalpha- and thrombin-stimulated endothelium. This EC MLCK-MIF interaction was shown biochemically and by immunofluorescent microscopy to be enhanced in TNFalpha- and thrombin-stimulated endothelium, both of which induce increased MLCK activity. Thrombin induced the colocalization of an epitope-tagged, full-length MIF fusion protein with phosphorylated MLC along peripheral actin stress fibers. Together these studies suggest that the novel interaction between MIF and MLCK may have important implications for the regulation of both non-muscle cytoskeletal dynamics as well as pathobiologic vascular events that involve MLCK.
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PMID:Intracellular interaction of myosin light chain kinase with macrophage migration inhibition factor (MIF) in endothelium. 1583 79

Calcium/calmodulin-dependent protein kinase II (CaM Kinase II) is a known modulator of cardiac pathophysiology. The present review uniquely focuses on novel CaM Kinase II-mediated endothelial cell signalling which, under pathophysiological conditions, may indirectly modulate cardiac functions via alterations in endothelial or endocardial responses. CaM Kinase II has four different isoforms and various splicing variants for each isoform. The endothelial cell CaM Kinase II isoforms are sensitive to KN93 and a threonine 286-mutated inhibitory peptide. In macrovascular endothelial cells derived from aortas, CaM Kinase II mediates redox-sensitive upregulation of endothelial nitric oxide synthase (eNOS) gene expression by hydrogen peroxide (H2O2) and oscillatory shear stress, and a rapid activation of eNOS in response to bradykinin. In endothelial cells derived from lung microvessels, CaM Kinase II mediates barrier dysfunction, particularly when activated by thrombin. In brain capillary endothelial cells, CaM Kinase II lies upstream of voltage-gated potassium channels and hypoxia-induced cell swelling. In both macrovascular and microvascular endothelial cells, CaM Kinase II mediates actin cytoskeleton reorganization via distinct p38 MAPK/HSP27 and ERK1/2/MLCK signalling pathways, respectively. Although understanding of endothelium-specific CaM Kinase II signalling is nascent, data accumulated so far have demonstrated a potentially significant role of CaM Kinase II in endothelial cell pathophysiology.
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PMID:CaM Kinase II-dependent pathophysiological signalling in endothelial cells. 1800 82

Acute lung injury represents the result of multiple pathways initiated by local or systemic insults and is characterized by profound vascular permeability, pulmonary edema, and life-threatening respiratory failure. Permeability-reducing therapies are of potential clinical utility but are currently unavailable. We hypothesized that polyethylene glycol (PEG) compounds, inert and non-toxic polymers that serve as a surrogate mucin lining in intestinal epithelium, may attenuate agonist-mediated lung endothelial cell (EC) barrier dysfunction. High molecular weight PEG (PEG15-20) produced rapid, dose-dependent increases in transendothelial electrical resistance (TER) in human lung endothelium cultured on gold microelectrodes, reflecting increased paracellular integrity. The maximal effective concentration of 8% PEG induced a sustained 125% increase in TER (40 h), results similar to barrier-enhancing agonists such as sphingosine 1-phosphate (40% increase in TER). Maximal PEG barrier enhancement was achieved at 45-60 min and PEG effectively reversed both thrombin- and LPS-induced EC barrier dysfunction. Consistent with the increase in TER, immunofluorescent studies demonstrated that PEG produced significant cytoskeletal rearrangement with formation of well-defined cortical actin rings and lamellipodia containing the actin-binding proteins, cortactin and MLCK, known participants in cell-matrix and cell-cell junctional adhesion. Finally, PEG challenge induced rapid alterations in levels of MAP kinase and MLC phosphorylation. In summary, PEG joins a number of EC barrier-regulatory agents which rapidly activate barrier-enhancing signal transduction pathways which target the cytoskeleton and provides a potential therapeutic strategy in inflammatory lung injury.
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PMID:Protective effects of high-molecular weight polyethylene glycol (PEG) in human lung endothelial cell barrier regulation: role of actin cytoskeletal rearrangement. 1912 27

Inflammatory mediators like thrombin disrupt endothelial adherens junctions (AJs) and barrier integrity leading to oedema formation followed by resealing of AJs and a slow recovery of the barrier function. The molecular mechanisms of this process have not yet been fully delineated. The aim of the present study was to analyse the molecular mechanism of endothelial barrier recovery and thrombin was used as model inflammatory mediator. Thrombin caused a strong increase in endothelial permeability within 10 min accompanied by loss of Rac1 but not cdc42 activity, drop in cellular cAMP contents, and a strong activation of the endothelial contractile machinery mainly via RhoA/Rock signalling. Activation of RhoA/Rock signalling precedes and is dependent upon a rise in the cytosolic Ca(2+) concentration. Inhibition of cytosolic Ca(2+) rise but not MLCK or Rock enhances the recovery of endothelial barrier function. The cellular cAMP contents increased gradually during the barrier recovery phase (30-60 min after thrombin challenge) accompanied by an increase in Rac1 activity. Inhibition of Rac1 activity using a specific pharmacological inhibitor (NSC23766) abrogated the endothelial barrier recovery process, suggesting a Rac1-dependent phenomenon. Likewise, inhibition of either adenylyl cyclase or the cAMP-effectors PKA and Epac (with PKI and ESI-09, respectively) caused an abrogation of Rac1 activation, resealing of endothelial AJs and recovery of endothelial barrier function. The data demonstrate that endothelial barrier recovery after thrombin challenge is regulated by Rac1 GTPase activation. This Rac1 activation is due to increased levels of cellular cAMP and activation of downstream signalling during the barrier recovery phase.
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PMID:cAMP controls the restoration of endothelial barrier function after thrombin-induced hyperpermeability via Rac1 activation. 2534 77

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