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)

Guanosine 5'-O-thiotriphosphate (GTP gamma S) and thrombin stimulate the activity of phospholipase C in platelets that have been permeabilized with saponin and whose inositol phospholipids have been prelabeled with [3H]inositol. Ca2+ has opposite effects on the formation of [3H]inositol phosphates induced by thrombin or GTP gamma S. While the action of GTP gamma S on the formation of [3H]inositol phosphates is inhibited by Ca2+, action of thrombin is stimulated by Ca2+. Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which inhibits the function of GTP-binding proteins, also inhibits the effect of GTP gamma S on phospholipase C stimulation but, surprisingly, increases the effect of thrombin. Ca2+ increases the inhibitory effect of GDP beta S on GTP gamma S activation of phospholipase C, but Ca2+ further enhances the stimulatory effect of GDP beta S on the thrombin activation of phospholipase C. This indicates that two mechanisms are responsible for the activation of phospholipase C in platelets. A GTP-binding protein is responsible for regulation of phospholipase C induced by GTP gamma S, while the effect of thrombin on the stimulation of phospholipase C is independent of GTP-binding proteins. However, the effect of thrombin may be modulated by the action of an inhibitory GTP-binding protein.
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PMID:GDP beta S enhances the activation of phospholipase C caused by thrombin in human platelets: evidence for involvement of an inhibitory GTP-binding protein. 310 63

GTP or GTP gamma S alone caused low but significant liberation of arachidonic acid in saponin-permeabilized human platelets but not in intact platelets. GTP or GTP gamma S also enhanced thrombin-induced [3H]arachidonic acid release in permeabilized platelets. Inhibitors of the phospholipase C (neomycin)/diacylglycerol lipase (RHC 80267) pathway for arachidonate liberation did not reduce the [3H]arachidonic acid release. The loss of [3H]arachidonate radioactivity from phosphatidylcholine was almost equivalent to the increase in released [3H]arachidonic acid, suggesting the hydrolysis of phosphatidylcholine by phospholipase A2. The effect of GTP gamma S was greater at lower Ca2+ concentrations. These data indicate that the release of arachidonic acid by phospholipase A2 in saponin-treated platelets may be linked to a GTP-binding protein.
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PMID:Guanine nucleotides stimulate arachidonic acid release by phospholipase A2 in saponin-permeabilized human platelets. 311 31

The effects of guanine nucleotides on arachidonic acid (AA) release were studied in intact and saponin-permeabilized human platelets. While GTP[S] itself caused a stimulation of AA release in permeabilized cells, GTP[S], GDP[S], GTP, ATP and other nucleotides inhibited AA release in response to thrombin and other agonists in intact, as well as permeabilized platelets. Inhibition of agonist-stimulated AA release by nucleotides was partially attenuated by addition of ADP, and was abolished by prior stimulation of platelets to discharge the ADP-containing dense granules. These results suggest: (i) that released ADP plays an important contributory role in agonist-stimulated platelet AA release, and (ii) that guanine nucleotides can modulate platelet activation through an extracellular action which is distinct from their effects on G-proteins.
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PMID:Guanine nucleotides inhibit agonist-stimulated arachidonic acid release in both intact and saponin-permeabilized human platelets. 313 60

In previous studies we have demonstrated that ethanol activates hormone-sensitive phospholipase C in intact human platelets, resulting in the mobilization of intracellular Ca2+ and platelet shape change. The present study aims to localize further this effect of ethanol by examining its interaction with the regulation of phospholipase C in a permeabilized cell system. In platelets permeabilized with a minimal concentration (18 micrograms/ml) of saponin, ethanol by itself did not activate phospholipase C. However, ethanol potentiated the activation of phospholipase C in response to the non-hydrolysable GTP analogue GTP[S] (guanosine 5'-[gamma-thio]triphosphate), an effect similar to that observed with thrombin. Ethanol also potentiated the response to fluoride, which acts directly on G-proteins. Other short-chain alcohols also stimulated phospholipase C in a synergistic manner with GTP[S]. The ability of specific alcohols to stimulate phospholipase C was directly related to their respective lipid-solubilities, as determined by their partition coefficients. Moreover, the potencies of each alcohol correlated with their ability to elicit Ca2+ mobilization and shape change in intact platelets. These effects of ethanol were eliminated by a disruption of receptor-phospholipase C coupling induced by the addition of higher concentrations of saponin. These data indicate that the activation of phospholipase C by ethanol may occur by affecting protein-protein interactions in the signal-transduction complex involving GTP-binding regulatory proteins.
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PMID:Alcohol-induced stimulation of phospholipase C in human platelets requires G-protein activation. 314 Jul 95

The effects of trypsin, acrosin and a recently described trypsin-like protease from bovine sperm were studied on adenylate cyclase activity in membranes of human platelets. These proteases caused an immediate decrease in adenylate cyclase activity, which was independent of the platelet membrane concentration used and which was constant for up to 20 min of incubation at 25 degrees C. When the incubation was prolonged, the proteases eliminated their own inhibitory action as well as that of the inhibitory hormone epinephrine. The adenylate cyclase inhibition caused by the proteases was strictly dependent on the presence of GTP (EC50 approximately 0.1 microM), whereas in the absence of GTP only minor changes in enzyme activity were observed at the conditions and protease concentrations used. Maximal inhibition caused by the proteases was between 40% and 60%. Half-maximal inhibition by the purified proteases trypsin and acrosin was observed at about 30 ng/ml and 2 micrograms/ml respectively. Inhibition of platelet adenylate cyclase by the proteases was partially additive with that caused by epinephrine, while with thrombin no additivity was observed. The serine protease inhibitor leupeptin blocked the actions of the proteases when added simultaneously with the enzymes, but was ineffective when added later on. Treatment of platelet membranes with the alkylating N-ethylmaleimide at low concentrations and Mn2+ ions (greater than or equal to 1 mM), both agents known to abolish inhibition of adenylate cyclase via the inhibitory guanine-nucleotide-binding protein Gi, eliminated the inhibitory action of the proteases. The data indicate that trypsin and trypsin-like proteases have two opposite effects on the platelet adenylate cyclase system, the well-documented elimination of Gi action and, as shown here, an immediate activation of Gi with subsequent adenylate cyclase inhibition. The data are consistent with the hypothesis that the activation of Gi caused by the proteases is due to an interaction of the proteases with specific cell-surface receptor sites in a manner similar to thrombin.
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PMID:Thrombin-like inhibitory action of trypsin and trypsin-like proteases on human platelet adenylate cyclase. 327 7

The effect of guanosine 5'-[beta-thio]diphosphate (GDP[beta S]), reported to be an antagonist of GTP at the G-protein-binding site, on human platelet activation was examined. GDP[beta S] (0.3-3 mM) had significant inhibitory effects on platelet aggregation and 5-hydroxytryptamine (5HT) secretion induced by thrombin, collagen, the thromboxane mimetic U46619 and 1,2-dioctanoylglycerol (diC8) in intact platelets, as well as in saponin-permeabilized platelets. Similar inhibitory effects in intact platelets were also observed with ATP (over similar concentration ranges) and GDP and GTP (at 2- and 10-fold higher concentrations respectively). All four nucleotides also inhibited ADP-induced platelet aggregation in indomethacin-treated platelets under conditions where no 5HT secretion occurred. Inhibition of thrombin-induced aggregation and secretion by GDP[beta S] and ATP in intact platelets was accompanied by a reduction in the thrombin-induced rise in intracellular Ca2+ levels and 45 kDa-protein phosphorylation. The results suggest that at least some of the effects of GDP[beta S] may be unrelated to inhibition of G-protein-GTP interaction, but, instead, may be mediated via an extracellular site, common to all the nucleotides tested and perhaps via inhibition of the effects of endogenous/released ADP. The usefulness of GDP[beta S] as a tool in studying G-protein-GTP interactions in platelets is thus questionable.
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PMID:Inhibition of agonist-induced platelet aggregation, Ca2+ mobilization and granule secretion by guanosine 5'-[beta-thio]diphosphate and GDP in intact platelets. Evidence for an inhibitory mechanism unrelated to the inhibition of G-protein-GTP interaction. 335 12

We have assessed the binding of [alpha-32P]GTP to platelet proteins from cytosolic and membrane fractions. Proteins were separated by NaDodSO4/PAGE and electrophoretically transferred to nitrocellulose. Incubation of the nitrocellulose blots with [alpha-32P]GTP indicated the presence of specific and distinct GTP-binding proteins in cytosol and membranes. Binding was prevented by 10-100 nM GTP and by 100 nM guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]) or GDP; binding was unaffected by 1 nM-1 microM ATP. One main GTP-binding protein (29.5 kDa) was detected in the membrane fraction, while three others (29, 27, and 21 kDa) were detected in the soluble fraction. Two cytosolic GTP-binding proteins (29 and 27 kDa) were degraded by trypsin; another cytosolic protein (21 kDa) and the membrane-bound protein (29.5 kDa) were resistant to the action of trypsin. Treatment of intact platelets with trypsin or thrombin, followed by lysis and fractionation, did not affect the binding of [alpha-32P]GTP to the membrane-bound protein. GTP[gamma S] still stimulated phospholipase C in permeabilized platelets already preincubated with trypsin. This suggests that trypsin-resistant GTP-binding proteins might regulate phospholipase C stimulated by GTP[gamma S].
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PMID:Specific binding of [alpha-32P]GTP to cytosolic and membrane-bound proteins of human platelets correlates with the activation of phospholipase C. 347 Jul 89

Amine secretion from electropermeabilized bovine chromaffin cells and human platelets requires Ca2+ and MgATP. There appears to be little correlation between the pH or potential of the interior of the amine storage granules of the chromaffin cells and the Ca2+ sensitivity or extent of secretion. The Ca2+ sensitivities of secretion for both preparations are increased by activators of protein kinase C. In the platelet, thrombin also increases the Ca2+ sensitivity. The thrombin-induced response is further enhanced by micromolar levels of GTP. The non-hydrolysable analogue GTP gamma S also potentiates the Ca2+-dependent secretory response, but this effect is additive to that seen by thrombin rather than synergistic, as is the case with GTP. GTP gamma S inhibits catecholamine secretion from bovine chromaffin cells. In both preparations the effects of GTP gamma S are inhibited by 10 microM GTP, even though GTP concentrations up to 1 mM are without effect when added alone. These results are consistent with there being two sites of action for the guanine nucleotides, one at the level of the agonist receptor and activated by GTP or one of its breakdown products, and the other one activated by GTP gamma S--possibly at the level of protein kinase C itself.
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PMID:Calcium and exocytosis. 349 44

Addition of GTP markedly enhances the ability of thrombin to cause a leftward shift in the Ca2+ dose/response curve for 5-hydroxytryptamine secretion from permeabilised human platelets. Little effect is observed on addition of GTP in the absence of thrombin. Neither ADP nor adrenaline, in the presence or absence of GTP, causes such a shift, whereas 5-hydroxytryptamine does so to a small extent but only in the presence of GTP. The leftward shift in the Ca2+ dose/response curve induced by 12-O-tetradecanoyl-phorbol-13-acetate or 1-oleyl-2-acetylglycerol is not enhanced by addition of GTP. The thrombin concentration required for half-maximal enhancement of the response to Ca2+ is markedly reduced by addition of GTP. The results support the postulate that the effects of excitatory agonists in this system correlate with their ability to activate phospholipase C and provide further evidence for a role for GTP in signal transduction between the receptor and phospholipase C.
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PMID:Effect of various excitatory agonists on the secretion of 5-hydroxytryptamine from permeabilised human platelets induced by Ca2+ in the presence or absence of GTP. 387 12

The influence of the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), a direct activator of the Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C), was studied on regulation of human platelet adenylate cyclase. Intact platelets were pretreated with the phorbol ester and, thereafter, membranes were prepared and the regulation of the hormone-sensitive adenylate cyclase in these membranes was studied. The following data were obtained: The TPA treatment applied had apparently no effect on the activity of the catalytic moiety of the platelet adenylate cyclase nor on the stimulatory NS protein nor on stimulatory hormone receptors (prostaglandin E1) and the mutual interactions of these components of the stimulatory hormone-sensitive pathway. However, the TPA treatment of intact platelets largely impaired the GTP-dependent, hormone-sensitive inhibitory pathway to the adenylate cyclase, involving the inhibitory Ni protein. The pretreatment led to a large reduction or loss of adenylate cyclase inhibition by GTP itself and by the inhibitory agonists, epinephrine and thrombin, inhibiting the untreated enzyme via separate receptors by an Ni-mediated process. In contrast, platelet adenylate cyclase inhibition not involving the Ni protein was not affected by the TPA treatment. The observed effects of TPA were very rapid in onset and were not shared by a derivative of TPA which did not activate protein kinase C. The data obtained suggest than protein kinase C activated by the phorbol ester interferes with the platelet adenylate cyclase system, leading to a specific alteration of the Ni-protein-mediated signal transduction to the adenylate cyclase.
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PMID:Modulation of adenylate cyclase of human platelets by phorbol ester. Impairment of the hormone-sensitive inhibitory pathway. 404 Aug 56


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