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 have previously demonstrated that platelets in polycythaemia vera (PV) exhibit decreased aggregation after stimulation with platelet activating factor (PAF) and reduced expression of GPIIIa on both resting and stimulated platelets. In the present study, we investigated if these results were related to changes in the mobilization of intracellular calcium, activation of phospholipase D (PLD) or amounts of GPIIIa and the intracellular tyrosine kinases Fak, Syk, Grb2, Shc and rhoA. Intracellular calcium levels were not different in resting platelets from 14 PV patients and 15 healthy controls (median 43 nmol/L, range 10-114, vs. 36 nmol/L, range 10-119). After stimulation with PAF (1 micromol/L) an equal increase was seen (125 nmol/L for PV platelets, range 67-257, vs. 113 nmol/L for controls, range 60-250). Also formation of phosphatidyl ethanol (PEt) was similar after exposure to 0.5 U/ml thrombin (0.28% PEt of total phospholipid, range 0.16-1.10, vs. 0.24 for controls, range 0.11-2.3) and 1 micromol/L PMA (0.25, range 0.16-0.32, vs. 0.14, range 0.09-0.6). In contrast to the reduced amount of GPIIIa on the surface of PV platelets, immunoblotting on whole cell lysates showed no reduction in PV patients compared to controls, indicating the possibility of an impaired incorporation of GPIIIa to the cell membrane. Levels of Fak, Syk, Shc, Grb2 and rhoA appeared equal in patients and controls. Similar intracellular proteins were tyrosine phosphorylated after stimulation with thrombin, PAF and PMA. In summary, defective platelet aggregation after stimulation with PAF is caused by neither defective mobilization of intracellular calcium nor, in contrast to the situation in PV granulocytes, an impaired activation of PLD. Moreover, no apparent differences in the intracellular amounts of Fak, Syk Shc, Grb2 and rhoA could be detected between PV and control platelets.
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PMID:Defective platelet aggregation in polycythaemia vera is not caused by impaired calcium signaling, phospholipase D activation or decreased amounts of focal adhesion proteins. 1109 63

In the present study, the roles of the small GTPase RhoA and its target Rho kinase in endothelial permeability were investigated in vitro. We have shown previously that, in addition to a rise in the intracellular Ca(2+) concentration ([Ca(2+)](i)), RhoA is involved in the prolonged thrombin-induced barrier dysfunction. To study the role of RhoA and Rho kinase more specifically, endothelial cells were stimulated with lysophosphatidic acid (LPA), a commonly used RhoA activator. LPA induced a 2- to 3-fold increase in the passage of horseradish peroxidase (HRP) across endothelial monolayers that lasted for several hours, whereas thrombin induced a 5- to 10-fold increase. Comparable to the thrombin-induced barrier dysfunction, the LPA-induced barrier dysfunction was accompanied by a reorganization of the F-actin cytoskeleton and the formation of focal attachment sites. LPA induced only a transient increase in myosin light-chain (MLC) phosphorylation, which returned to basal level within 10 minutes. In endothelial cells, [Ca(2+)](i) was not elevated by LPA. Chelation of Ca(2+)(i) ions by 1, 2-bis(2-aminophenoxy)ethane-N:,N:,N:',N:'-tetraacetic acid did not prevent the LPA-induced passage of HRP. Apparently, a low degree of MLC kinase activation occurred, because the MLC kinase inhibitor KT5926 reduced the levels of both basal and LPA-stimulated HRP passage. Inhibition of RhoA by the C3 transferase from Clostridium botulinum inhibited the LPA-induced cytoskeletal changes and prevented the LPA-induced HRP passage. Inhibition of Rho kinase by Y-27632 completely prevented the LPA-induced increase in HRP passage without affecting basal permeability. These data indicate that LPA-induced endothelial hyperpermeability occurs without a change in [Ca(2+)](i) and requires activation of RhoA and Rho kinase.
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PMID:Role of RhoA and Rho kinase in lysophosphatidic acid-induced endothelial barrier dysfunction. 1111 77

Loss of membrane potential (membrane depolarization) is one of the earliest and most striking responses of quiescent cells to stimulation with serum or G protein-coupled receptor (GPCR) agonists such as lysophosphatidic acid and thrombin. Membrane depolarization is due to the activation of a chloride conductance. While this response has received relatively little attention in the past, it is clear that the acute loss of membrane potential may have important physiological consequences. However, the dissection of the underlying G protein pathway and the establishment of cause-effect relationships have remained elusive to date. Here we report that, in neuronal cells, the depolarizing chloride current invariably accompanies GPCR-induced activation of RhoA and subsequent neurite retraction, and neither of these events requires phosphoinositide hydrolysis or Ca2+ mobilization. Through antibody microinjections and a genetic approach, we demonstrate that activation of the chloride conductance is mediated by Galpha(13) in a RhoA-independent manner in both neuronal cells and fibroblasts. We further show that, in neuronal cells, this newly described Galpha(13) pathway may profoundly modulate membrane excitability during RhoA-regulated neurite remodeling.
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PMID:Galpha(13) mediates activation of a depolarizing chloride current that accompanies RhoA activation in both neuronal and nonneuronal cells. 1123 Nov 29

Endothelial permeability induced by thrombin and histamine is accompanied by actin stress fibre assembly and intercellular gap formation. Here, we investigate the roles of the Rho family GTPases Rho1, Rac1 and Cdc42 in regulating endothelial barrier function, and correlate this with their effects on F-actin organization and intercellular junctions. RhoA, Rac1 and Cdc42 proteins were expressed efficiently in human umbilical vein endothelial cells by adenovirus-mediated gene transfer. We show that inhibition of Rho prevents both thrombin- and histamine-induced increases in endothelial permeability and decreases in transendothelial resistance. Dominant-negative RhoA and a Rho kinase inhibitor, Y-27632, not only inhibit stress fibre assembly and contractility but also prevent thrombin- and histamine-induced disassembly of adherens and tight junctions in endothelial cells, providing an explanation for their effects on permeability. In contrast, dominant-negative Rac1 induces permeability in unstimulated cells and enhances thrombin-induced permeability, yet inhibits stress fibre assembly, indicating that increased stress fibre formation is not essential for endothelial permeability. Dominant-negative Cdc42 reduces thrombin-induced stress fibre formation and contractility but does not affect endothelial cell permeability or responses to histamine. These results demonstrate that Rho and Rac act in different ways to alter endothelial barrier function, whereas Cdc42 does not affect barrier function.
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PMID:Rho and Rac but not Cdc42 regulate endothelial cell permeability. 1125

Yersinia spp. inject effector proteins (Yersinia outer proteins, Yops) into target cells via a type III secretion apparatus. The effector YopE was recently shown to possess GAP activity towards the Rho GTPases RhoA, Rac and CDC42 in vitro. To investigate the intracellular, 'in vivo' targets of YopE we generated a Yersinia enterocolitica strain [WA(pYLCR+E)] that injects 'life-like' amounts of YopE as only effector. Primary human umbilical vein endothelial cells (HUVEC) were infected with WA(pYLCR+E) and were then stimulated with: (i) bradykinin to induce actin microspikes followed by ruffles as an assay for CDC42 activity followed by CDC42 stimulated Rac activity; (ii) sphingosine-1-phosphate to form ruffles by direct Rac activation; or (iii) thrombin to generate actin stress fibres through Rho activation. In WA(pYLCR+E)-infected HUVEC microspike formation stimulated with bradykinin remained intact but the subsequent development of ruffles was abolished. Furthermore, ruffle formation after stimulation with sphingosine-1-phosphate or thrombin induced production of stress fibres was unaltered in the infected cells. These data suggest that YopE is able to inhibit Rac- but not Rho- or CDC42-regulated actin structures and, more specifically, that YopE is capable of blocking CDC42Hs dependent Rac activation but not direct Rac activation in HUVEC. This provides evidence for a considerable specificity of YopE towards selective Rac-mediated signalling pathways in primary target cells of Yersinia.
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PMID:YopE of Yersinia, a GAP for Rho GTPases, selectively modulates Rac-dependent actin structures in endothelial cells. 1129 53

Currently, there is substantial evidence that nuclear lipid metabolism plays a critical role in a number of signal transduction cascades. Previous work from our laboratory showed that stimulation of quiescent fibroblasts with alpha-thrombin leads to the production of two lipid second messengers in the nucleus: an increase in nuclear diacylglycerol mass and an activation of phospholipase D, which catalyzes the hydrolysis of phosphatidylcholine to generate phosphatidic acid. Diacylglycerol kinase (DGK) catalyzes the conversion of diacylglycerol to phosphatidic acid, making it an attractive candidate for a signal transduction component. There is substantial evidence that this activity is indeed regulated in a number of signaling cascades (reviewed by van Blitterswijk, W. J., and Houssa, B. (1999) Chem. Phys. Lipids 98, 95-108). In this report, we show that the addition of alpha-thrombin to quiescent IIC9 fibroblasts results in an increase in nuclear DGK activity. The examination of nuclei isolated from quiescent IIC9 cells indicates that DGK-theta and DGK-delta are both present. We took advantage of the previous observations that phosphatidylserine inhibits DGK-delta (reviewed by Sakane, F., Imai, S., Kai, M., Wada, I., and Kanoh, H. (1996) J. Biol. Chem. 271, 8394-8401), and constitutively active RhoA inhibits DGK-theta (reviewed by Houssa, B., de Widt, J., Kranenburg, O., Moolenaar, W. H., and van Blitterswijk, W. J. (1999) J. Biol. Chem. 274, 6820-6822) to identify the activity induced by alpha-thrombin. Constitutively active RhoA inhibited the nuclear stimulated activity, whereas phosphatidylserine did not have an inhibitory effect. In addition, a monoclonal anti-DGK-theta antibody inhibited the alpha-thrombin-stimulated nuclear activity in vitro. These results demonstrate that DGK-theta is the isoform responsive to alpha-thrombin stimulation. Western blot and immunofluorescence microscopy analyses showed that alpha-thrombin induced the translocation of DGK-theta to the nucleus, implicating that this translocation is at least partly responsible for the increased nuclear activity. Taken together, these data are the first to demonstrate an agonist-induced activity of nuclear DGK-theta activity and a nuclear localization of DGK-delta.
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PMID:Nuclear diacylglycerol kinase-theta is activated in response to alpha-thrombin. 1130 92

We utilized a cDNA expression library derived from the B6SutA(1) mouse myeloid progenitor cell line to search for novel oncogenes that promote growth transformation of NIH3T3 cells. A 2.2 kb transforming cDNA was recovered that encodes the wild type thrombin-stimulated G protein-coupled receptor PAR-1. In addition to its potent focus forming activity, constitutive overexpression of PAR-1 in NIH3T3 cells promoted the loss of anchorage- and serum-dependent growth. Although inhibitors of thrombin failed to block PAR-1 transforming activity, a PAR-1 mutant that cannot be cleaved by thrombin was nontransforming. Since the foci of transformed cells induced by PAR-1 bear a striking resemblance to those induced by activated RhoA, we determined if PAR-1 transformation was due to the aberrant activation of a specific Rho family member. Like RhoA, PAR-1 cooperated with activated Raf-1 and caused synergistic enhancement of transforming activity, induced stress fibers when microinjected into porcine aortic endothelial cells, stimulated the activity of the serum response factor and NF-kappaB transcription factors, and PAR-1 transformation was blocked by co-expression of dominant negative RhoA. Finally, PAR-1 transforming activity was blocked by pertussis toxin and by co-expression of the RGS domain of Lsc, implicating Galpha(i) and Galpha(12)/Galpha(13) subunits, respectively, as mediators of PAR-1 transformation. Taken together, these observations suggest that PAR-1 growth transformation is mediated, in part, by activation of RhoA.
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PMID:The thrombin receptor, PAR-1, causes transformation by activation of Rho-mediated signaling pathways. 1136 Jan 79

Thrombin is a serine protease that evokes various cellular responses involved in injury and repair of the nervous system through the activation of protease-activated receptor-1 (PAR-1). Signals that modulate cell morphology precede most PAR-1 effects, but the initial signal transduction molecules are not known. Using the yeast two-hybrid system, we identified Hsp90, a chaperone with known signaling properties, as a binding partner of PAR-1. The interaction was confirmed by glutathione S-transferase pull-down, overlay, and co-immunoprecipitation assays. Morphological assays in mouse astrocytes were carried out to evaluate the importance of Hsp90 during cytoskeletal signaling. Reducing Hsp90 levels by antisense treatment or disruption of the Hsp90.PAR-1 complex by the Hsp90-specific drug geldanamycin attenuated thrombin-mediated astrocyte shape changes. Furthermore, overexpression of the PAR-1 cytoplasmic tail abrogated thrombin-induced cytoskeletal changes in neuronal cells. Treatment with geldanamycin specifically inhibited activation of RhoA without affecting thrombin-mediated intracellular calcium release, revealing the regulation of a distinct signaling pathway by Hsp90. Taken together, these studies demonstrate that Hsp90 may be essential for PAR-1-mediated signaling to the cytoskeleton.
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PMID:Thrombin receptor signaling to cytoskeleton requires Hsp90. 1141 45

Thrombin, the ultimate protease in the blood coagulation cascade, mediates its known cellular effects by unique proteolytic activation of G-protein-coupled protease-activated receptors (PARs), such as PAR1, PAR3, and PAR4, and a "tethered ligand" mechanism. PAR1 is variably expressed in subpopulations of neurons and largely determines thrombin's effects on morphology, calcium mobilization, and caspase-mediated apoptosis. In spinal cord motoneurons, PAR1 expression correlates with transient thrombin-mediated [Ca(2+)](i) flux, receptor cleavage, and elevation of rest [Ca(2+)](i) activating intracellular proteases. At nanomolar concentrations, thrombin retracts neurites via PAR1 activation of the monomeric, 21 kDa Ras G-protein RhoA, which is also involved in neuroprotection at lower thrombin concentrations. Such results suggest potential downstream targets for thrombin's injurious effects. Consequently, we employed several G-protein-specific modulators prior to thrombin exposure in an attempt to uncouple both heterotrimeric and monomeric G-proteins from motoneuronal PAR1. Cholera toxin, stimulating Gs, and lovastatin, which blocks isoprenylation of Rho, reduced thrombin-induced calcium mobilization. In contrast, pertussis toxin and mastoparan, inhibiting or stimulating G(o)/G(i), were found to exacerbate thrombin action. Effects on neuronal rounding and apoptosis were also detected, suggesting therapeutic utility may result from interference with downstream components of thrombin signaling pathways in human motor neuron disorders, and possibly other neurodegenerative diseases. Published 2001 John Wiley & Sons, Inc.
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PMID:Neuroprotective signal transduction in model motor neurons exposed to thrombin: G-protein modulation effects on neurite outgrowth, Ca(2+) mobilization, and apoptosis. 1143 39

Our laboratory has recently demonstrated that insulin induces relaxation of vascular smooth muscle cells (VSMCs) by activating myosin-bound phosphatase (MBP) and by inhibiting Rho kinase (Begum N, Duddy N, Sandu OA, Reinzie J, and Ragolia L. Mol Endocrinol 14: 1365-1376, 2000). In this study, we tested the hypothesis that insulin via the nitric oxide (NO)/cGMP pathway may inactivate Rho, resulting in a decrease in phosphorylation of the myosin-bound subunit (MBS(Thr695)) of MBP and in its activation. Treatment of confluent serum-starved VSMCs with insulin prevented thrombin-induced increases in membrane-associated RhoA, Rho kinase activation, and site-specific phosphorylation of MBS(Thr695) of MBP and caused MBP activation. Preexposure to N(G)-monomethyl-L-arginine, a NO synthase inhibitor, and R-p-8-(4-chlorophenylthio)cGMP, a cGMP antagonist, attenuated insulin's inhibitory effect on Rho translocation and restored thrombin-mediated Rho kinase activation and site-specific MBS(Thr695) phosphorylation, resulting in MBP inactivation. In contrast, 8-bromo-cGMP, a cGMP agonist, mimicked insulin's inhibitory effects by abolishing thrombin-mediated Rho signaling and promoted dephosphorylation of MBS(Thr695). Furthermore, expression of a dominant-negative RhoA decreased basal as well as thrombin-induced MBS(Thr695) phosphorylation and caused insulin activation of MBP. Collectively, these results indicate that insulin inhibits Rho signaling by decreasing RhoA translocation via the NO/cGMP signaling pathway to cause MBP activation via site-specific dephosphorylation of its regulatory subunit MBS.
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PMID:Selected contribution: insulin utilizes NO/cGMP pathway to activate myosin phosphatase via Rho inhibition in vascular smooth muscle. 1150 51


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