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)

The regulation of prostacyclin (PGI2) synthesis by cultured human umbilical vein endothelium (HUVEC) was investigated. HUVEC monolayer generation of PGI2 was monitored by RIA of 6-keto PGF1 alpha and dose-dependent increases observed with human alpha- and gamma-thrombins, histamine, or arachidonate. Alpha thrombin (10 nM) produced levels of 6-keto PGF1 alpha approximating responses with 1 microM gamma-thrombin, 5 microM arachidonate, or 10 microM histamine. Diisopropyl phosphorofluoridate-inactivated alpha-thrombin did not stimulate PGI2 release, demonstrating that catalytic activity was required for thrombin-stimulated PGI2 release. Sodium fluoride (NaF), at concentrations known to activate guanine nucleotide regulatory proteins (G proteins), directly stimulated HUVEC PGI2 synthesis in a dose-dependent and time-dependent manner (20 mM NaF, 4.4 +/- 0.5-fold increase at 10 min, 11.9 +/- 1.5-fold increase at 30 min). Neither alpha-thrombin nor NaF-stimulated PGI2 release was dependent upon the availability of extracellular Ca++). The hypothesis that G proteins are involved in agonist-stimulated PGI2 synthesis was further supported by studies using digitonin-permeabilized HUVEC monolayers challenged with another G protein activator, guanosine 5'-0-3-thiotrisphosphate (GTP gamma S), which effected significant dose-dependent increases in PGI2 synthesis compared with control levels of 6-keto PGF1 alpha. In contrast, the G-protein inhibitor GDP beta S, (guanosine 5'-0-2-thiodiphosphate), attenuated alpha-thrombin-mediated prostaglandin generation. Treatment of HUVEC monolayers with pertussis toxin (1 microgram/ml) did not inhibit the PGI2 synthesis stimulated by either alpha-thrombin, NaF, or histamine but catalyzed the ADP ribosylation of a 40 kDa membrane protein which cross-reacted with antisera against a synthetic peptide corresponding to an amino acid sequence common to the alpha-subunit of other G-proteins. Preincubation of HUVEC microsomal membranes with alpha-thrombin diminished pertussis toxin-catalyzed ADP ribosylation in a time-dependent manner. These data suggest that thrombin stimulation of PGI2 synthesis by HUVEC monolayers requires the catalytically functional enzyme and further suggests that the thrombin-occupied receptor is coupled to phospholipase activities by a pertussis toxin-insensitive guanine nucleotide regulatory protein in human endothelial cell membranes.
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PMID:Thrombin-induced prostacyclin biosynthesis in human endothelium: role of guanine nucleotide regulatory proteins in stimulus/coupling responses. 210 25

1. Metabolically stable analogues of GTP, e.g. guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and guanosine 5'-[beta,gamma-imido]triphosphate (pp[NH]pG), enhance the extent of Ca2(+)-dependent secretion of beta-N-acetylglucosaminidase and beta-galactosidase from electropermeabilised human platelets in the presence of less than 5 microM Ca2+. A similar effect is observed on addition either of 1,2-dioctanoin or of GTP in in the presence or absence of thrombin. 2. In the presence of higher Ca2+ concentrations the extent of enhancement of lysosomal secretion declines and little, or no, enhancement is observed at a [Ca2+] of 30-40 microM. Addition of leupeptin or antipain prevents this decrease in lysosomal secretion and enhances the extent of Ca2(+)-dependent lysosomal secretion obtained in the presence or absence of guanine nucleotides, thrombin or 1,2-dioctanoin. 3. The concentration of GTP[S] or pp[NH]pG required to obtain half-maximal enhancement of lysosomal secretion is dependent on [Ca2+] for secretion of 5-hydroxytryptamine, beta-N-acetylglucosaminidase and beta-galactosidase. At two fixed [Ca2+] the median effective concentration (EC50) values for GTP[S] and pp[NH]pG which characterise enhancement of 5-hydroxytryptamine secretion are significantly different from those characterising enhancement of the secretion of beta-N-acetylglucosaminidase and beta-galactosidase. 4. In the presence of a saturating concentration of GTP[S] marked 5-hydroxytryptamine and beta-N-acetylglucosaminidase secretion is observed at nanomolar [Ca2+] and these responses show little dependence on [Ca2+] over the attainable range. Secretion of beta-N-acetylglucosaminidase is also induced at nanomolar Ca2+ concentrations by addition of activators of protein kinase C. 5. Guanosine 5'-[beta-thio]diphosphate inhibits enhancement of beta-N-acetylglucosaminidase secretion induced by GTP[S] but has no effect on secretion of this enzyme induced by Ca2+ when added alone. 6. Our data provide some support for a model in which addition of metabolically stable guanine nucleotides enhances Ca2(+)-dependent platelet lysosomal secretion by activating a guanine-nucleotide-binding protein (GE) located close to the exocytotic site. However, not all the data are consistent with this postulate.
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PMID:Guanine nucleotides and Ca2(+)-dependent lysosomal secretion in electropermeabilised human platelets. 211 63

Production of the potent lipid autacoid, platelet-activating factor (PAF), is a stimulated response of the endothelium which has important physiologic consequences including mediating adherence of inflammatory cells to the endothelium. Consequently, an understanding of the mechanisms that regulate PAF synthesis by the endothelium is important. To this end, we investigated the role of G proteins as a component of the signal transduction pathway that couples hormonal stimuli to PAF production. The addition of aluminum fluoride (AlF-4) to endothelial cells resulted in production of PAF with a maximal effect at 20 mM fluoride and within 20-60 min of exposure. Alf-4 also augmented the production of PAF which occurs in response to hormonal agonists. In addition, submaximal concentrations of AlF-4 converted an ineffective hormonal agonist (thrombin in bovine cells) to a maximally effective agonist. The adherence of neutrophils to endothelial cells that had been exposed previously to AlF-4 was increased in a manner that paralleled PAF production. PAF production in response to AlF-4 was not consistently affected by pertussis or cholera toxin. Introduction of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) into permeabilized endothelial cells also resulted in PAF production, with reversal by guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), consistent with an effect mediated by a G protein. G protein activation with AlF-4 or GTP gamma S resulted in entry of extracellular Ca2+ as determined using 45Ca2+ flux studies and Indo-1 spectrofluorometry. Our data are consistent with the hypothesis that G proteins couple hormone-receptor binding to opening of a membrane calcium channel, a key step in the initiation of PAF production in endothelial cells.
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PMID:Synthesis of platelet-activating factor by endothelial cells. The role of G proteins. 211 26

Noradrenaline (NA) stimulated the release of arachidonic acid (AA) from the [3H]AA-labelled rabbit platelets via alpha 2-adrenergic receptors, since the effect of NA was inhibited by yohimbine. The stimulatory effect of NA in digitonin-permeabilized platelets was completely dependent on the simultaneous presence of GTP and Ca2+. The NA- and thrombin-stimulated releases of AA were markedly decreased by the prior ADP-ribosylation of the permeabilized platelets with pertussis toxin. Antiserum directed against the pig brain Go (a GTP-binding protein of unknown function), recognizing both alpha 39 and beta 35,36 subunits, but not alpha 41, of pig brain, reacted with 41 kDa and 40 kDa bands, with not one of 39 kDa, in rabbit platelet membranes. Anti-Go antiserum inhibited guanosine 5'-[gamma-thio]triphosphate-, A1F4(-)-, NA- and thrombin-stimulated AA releases in the membranes. Although the effect of thrombin was inhibited by low concentrations of anti-Go antiserum, high concentrations of the antiserum was needed for inhibition of the NA effect. Antiserum directed against the pig brain G1 (inhibitory G-protein), recognizing both alpha 41 and beta 35,36 subunits, but not alpha 39, of pig brain, reacted with the 41 kDa band in platelets. Anti-G1 antiserum inhibited only the effect of NA. Reconstitution of the platelet membranes ADP-ribosylated by pertussis toxin with Go, not Gi, purified from pig brain restored the thrombin-stimulated release of AA. In contrast, reconstitution of those membranes with Gi, not Go, restored the NA-stimulated release of AA. These results indicate that different GTP-binding proteins, Gi- and Go-like proteins, may be involved in the mechanism of signal transduction from alpha 2-adrenergic receptors and thrombin receptors to phospholipase A2 in rabbit platelets.
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PMID:Possible involvement of different GTP-binding proteins in noradrenaline- and thrombin-stimulated release of arachidonic acid in rabbit platelets. 211 62

rap1B is a member of the ras superfamily of low molecular weight GTP binding proteins which constitutes a focal point of GTP and cAMP signal transduction systems. Like other members of this superfamily, rap1B is membrane-associated in resting platelets, presumably through polyisoprenylation. The studies presented here were undertaken to determine the subcellular changes in rap1B localization during cell activation. Activated and unactivated platelets were fractionated by Triton X-100 lysis followed by differential centrifugation to obtain a 10,000 x g cytoskeleton fraction, a 100,000 x g membrane skeleton fraction, and a 100,000 x g supernatant fraction containing solubilized proteins. In unactivated platelets, rap1B was present in the 100,000 x g supernatant fraction. In contrast, in platelets activated with 1 unit/ml alpha-thrombin or with the calcium ionophore, A23187, rap1B was quantitatively recovered in the 10,000 x g cytoskeleton fraction. rap1B was absent from the 100,000 x g fraction containing the membrane skeleton and could not be detected in the 100,000 x g supernatant containing cytosolic proteins and solubilized membrane components. These results indicate that rap1B associates with the cytoskeleton during cell activation.
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PMID:rap1B, a cAMP-dependent protein kinase substrate, associates with the platelet cytoskeleton. 212 87

The tumor-promoting phorbol ester 4 beta-phorbol 12-myristate 13-acetate (PMA) inhibited thrombin-stimulated arachidonic acid (AA) release in rabbit and human platelets. PMA was effective over the same concentration range that activates protein kinase C in intact rabbit platelets: IC50 vs thrombin = 0.5 nM, greater than 90% inhibition at 10 nM. Suppression of thrombin-stimulated AA release was evident within 5 min of pretreatment with 1 nM PMA. A non-tumor-promoting phorbol ester, 4-O-methyl PMA, showed a very weak ability to inhibit AA release. Thrombin-stimulated serotonin secretion was progressively inhibited by PMA pretreatment in platelets, while PMA was a stimulus for secretion at higher concentrations. 1-(5-Isoquinolinylsulfonyl)-2-methyl-piperazine (H-7), a selective inhibitor of protein kinase C, blocked PMA-induced inhibition of AA release. Furthermore, H-7 enhanced the effect of thrombin on AA release. PMA pretreatment reduced the inhibitory effect of thrombin on forskolin-stimulated cAMP accumulation, but had no effect on nonstimulated cAMP metabolism in the presence of thrombin. PMA did not inhibit AA release caused by A23187 or melittin. In digitonin-permeabilized platelets, thrombin plus guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)-stimulated AA release, but not GTP gamma S- and AIF4(-)-stimulated AA release, was abolished by PMA pretreatment. These results suggest that activation of protein kinase C may exert negative feedback on the receptor-mediated activation of phospholipase A2. A possible uncoupling of thrombin receptor to GTP-binding protein leading to activation of phospholipase A2 by PMA pretreatment is discussed.
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PMID:Modes of inhibitory action of 4 beta-phorbol 12-myristate 13-acetate in thrombin-stimulated arachidonic acid release in intact and permeabilized platelets. 215 60

Human alpha-thrombin is known to elicit bone resorption in vitro and has been proposed as a mediator of increased bone turnover in inflammatory diseases. We used UMR 106-H5 rat osteoblast-like osteosarcoma cells to explore the signal transduction mechanism utilized by thrombin in bone. Thrombin produced a dose-dependent increase in the accumulation of [3H]inositol phosphates (IPs) in UMR 106-H5 cells prelabeled with [3H]myo-inositol (EC50 15 U/ml). In saponin-permeabilized cells, GTP gamma S increased [3H]IP production, whereas GDP beta S inhibited the response to both GTP gamma S and thrombin, indicating involvement of a G-protein in thrombin action. Thrombin produced a dose-dependent increase in intracellular free calcium (Cai2+) in UMR 106-H5 cells (EC50 1 U/ml; maximal increase 4-fold), as well as a small (20%) increase in [3H]thymidine incorporation. Treatment of UMR 106-H5 membranes with pertussis toxin (PT) and [32P]NAD+ resulted in labeling of a 40-kDa protein. However, pretreatment of cells with a dose of PT sufficient to produce maximal endogenous labeling of this protein failed to influence thrombin action on IP accumulation, Cai2+, or [3H]thymidine incorporation. In contrast, PT treatment of CCL39 hamster lung fibroblasts significantly blunted thrombin-stimulated [3H]IP accumulation and [3H]thymidine incorporation. These results suggest that thrombin raises Cai2+ in UMR 106-H5 cells by activating polyphosphoinositide-specific phospholipase C. Whereas in fibroblasts and platelets, thrombin receptors appear to couple to both PT-sensitive and PT-insensitive G-proteins, only a PT-insensitive G-protein appears to mediate thrombin action in UMR 106-H5 cells. Either these cells lack the relevant PT-sensitive G-protein or they possess thrombin receptors that selectively couple to a pertussis toxin-insensitive G-protein.
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PMID:Thrombin stimulates inositol phosphate production and intracellular free calcium by a pertussis toxin-insensitive mechanism in osteosarcoma cells. 215 36

Histamine is known to be a mediator of inflammation. In order to understand the role of histamine in platelets, we have examined the effects of histamine on arachidonic acid (AA) release, cAMP accumulation, inositol trisphosphate production, and serotonin secretion. Incubation of rabbit (and human) platelets with histamine resulted in rapid increase of [3H]AA release from the platelets prelabeled with [3H]AA. The effect of histamine was blocked by the addition of H1 receptor antagonist mepyramine. Histamine did not substantially affect the cAMP content and inositol trisphosphate production. Histamine-stimulated AA release was not observed in digitonin-permeabilized platelets, whereas histamine acted synergistically with GTP or GTP analog, guanosine 5'-(3-O-thio)triphosphate. Histamine-stimulated, and GTP analog-dependent AA release was inhibited by guanosine 5'-(2-O-thio) diphosphate. The effects of three receptor stimulants, thrombin, norepinephrine, and histamine were both diminished by 1 microgram/ml of pertussis toxin treatment and by the antiserum against GTP-binding proteins (G proteins) treatment. However, the antiserum against beta gamma subunits of G proteins inhibited the histamine effect, not thrombin effect. 4 beta-Phorbol 12-myristate 13-acetate (PMA) treatment enhanced histamine-stimulated AA release and serotonin secretion but inhibited thrombin-stimulated reactions. The effect of PMA was dose dependent and was due to enhance the coupling of histamine receptors and G proteins. The results show the existence of H1 histamine receptors which couple phospholipase A2 activation via pertussis toxin-sensitive G proteins. Histamine actions differ in sensitivities to anti-beta gamma antiserum treatment and PMA treatment from thrombin actions.
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PMID:Histamine-stimulated and GTP-binding proteins-mediated phospholipase A2 activation in rabbit platelets. 215 20

Human platelets stimulated with alpha-thrombin or the thromboxane A2 analogue U46619 form the novel phosphatidylinositol (PtdIns) polyphosphate species PtdIns (3,4)P2 and PtdIns(3,4,5)P3 in a time- and dose-dependent manner. In [32P]o-phosphate-labeled platelets, PtdIns(3,4)P2 increases in 2 min to 613% of the basal level in response to 1 unit/ml alpha-thrombin and 295% in response to 5 microM U46619. A dramatic increase is also observed with myo-[3H]inositol-labeled platelets. [32P]PtdIns(3,4,5)P3 is increased with alpha-thrombin and U46619 stimulation by 261 and 183%, respectively. PtdIns(3)P quantities remain nearly equal to those under resting conditions. Neither the basal nor increased levels of PtdIns(3,4)P2 appear to be adequate to account for the rapid elevation of Ins(1,3,4)P3 that we have observed in alpha-thrombin-stimulated platelets. A23187 and/or phorbol 12,13-dibutyrate are not as potent as alpha-thrombin in stimulating changes in PtdIns(3,4)P2 or PtdIns(3,4,5)P3. GTP gamma S (10 microM), however, increases the level of PtdIns(3,4)P2 by 3736% and that of PtdIns(3,4,5)P3 by 456% in saponin-permeabilized platelets incubated with 0.5 mM [gamma-32P]ATP, implying a role for a GTP-binding protein in promoting phosphatidylinositide 3-kinase activity(ies). This is the first report of stimulated generation of 3-phosphorylated phosphoinositides in anucleate cells. A role for these novel phospholipid species in signal transduction, however, awaits elucidation.
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PMID:Human platelets form 3-phosphorylated phosphoinositides in response to alpha-thrombin, U46619, or GTP gamma S. 215 15

Addition of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) to intact Chinese hamster lung fibroblasts (CCL39) depolarized by high K+ concentrations results in activation of phosphoinositide-specific phospholipase C (PLC) (at GTP gamma S concentrations greater than 0.1 mM), inhibition of adenylate cyclase (between 10 microM and 0.5 mM), and activation of adenylate cyclase (above 0.5 mM). Since GTP gamma S-induced activation of PLC is dramatically enhanced upon receptor-mediated stimulation of PLC by alpha-thrombin, we conclude that in depolarized CCL39 cells GTP gamma S directly activates various guanine nucleotide-binding regulatory proteins (G proteins) coupled to PLC (Gp(s)) and to adenylate cyclase (Gi and Gs). Pretreatment of cells with pertussis toxin strongly inhibits GTP gamma S-induced activation of PLC and inhibition of adenylate cyclase. GTP gamma S cannot be replaced by other nucleotides, except by guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which mimics after a lag period of 15-20 min all the effects of GTP gamma S, with the same concentration dependence and the same sensitivity to pertussis toxin. We suggest that GDP beta S is converted in cells into GTP beta S, which acts as GTP gamma S. Since cell viability is not affected by a transient depolarization, these observations provide a simple method to examine long-term effects of G protein activation on DNA synthesis. We show that a transient exposure of G0-arrested CCL39 cells to GTP gamma S or GDP beta S under depolarizing conditions is not sufficient by itself to induce a significant mitogenic response, but markedly potentiates the mitogenic action of fibroblast growth factor, a mitogen known to activate a receptor-tyrosine kinase. The potentiating effect is maximal after 60 min of pretreatment with 2 mM GTP gamma S. GDP beta S is equally efficient but only after a lag period of 15-20 min. Mitogenic effects of both guanine nucleotide analogs are suppressed by pertussis toxin. Since the activation of G proteins by GTP gamma S under these conditions vanishes after a few hours, we conclude that a transient activation of G proteins facilitates the transition G0----G1 in CCL39 cells, whereas tyrosine kinase-induced signals are sufficient to mediate the progression into S phase.
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PMID:Guanosine 5'-O-(3-thiotriphosphate) and guanosine 5'-O-(2-thiodiphosphate) activate G proteins and potentiate fibroblast growth factor-induced DNA synthesis in hamster fibroblasts. 216 8


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