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 previously reported that thrombin stimulates Ca2+ influx and activates phosphatidylcholine-hydrolyzing phospholipase D in osteoblast-like MC3T3-E1 cells. In this study, we investigated the effect of thrombin on interleukin-6 (IL-6) synthesis in these cells. Thrombin stimulated IL-6 synthesis dose-dependently in the range between 0.01 and 1 U/ml. The depletion of extracellular Ca2+ by EGTA suppressed the thrombin-induced IL-6 synthesis. TMB-8, an inhibitor of intracellular Ca2+ mobilization, also inhibited the IL-6 synthesis by thrombin. Propranolol, a phosphatidic acid phosphohydrolase inhibitor, enhanced the IL-6 synthesis by thrombin. Calphostin C, a highly potent and specific inhibitor for protein kinase C, significantly amplified the IL-6 synthesis by thrombin. The thrombin-induced IL-6 synthesis was enhanced in PKC down-regulated MC3T3-E1 cells. These results strongly suggest that thrombin stimulates IL-6 synthesis, which depends on intracellular Ca2+ mobilization mainly from extracellular space in osteoblasts, and that the IL-6 synthesis by thrombin is regulated due to thrombin-activated protein kinase C through phosphatidylcholine-hydrolyzing phospholipase D.
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PMID:Thrombin regulates interleukin-6 synthesis through phosphatidylcholine hydrolysis by phospholipase D in osteoblasts. 928 6

In the present study evidence is provided for a rapid activation of lipid signalling pathways induced by thrombin and bradykinin (BK) in C2C12 myoblasts. Both agonists were able to increase [3H]inositol phosphates (InsP), 1,2-[3H]diacylglycerol (DAG) and [3H]phosphatidic acid (PtdOH) levels. In particular [3H]PtdOH values were rapidly increased and maintained at significantly high values at prolonged times of incubation. BK and thrombin were able to activate phospholipase D (PLD) in vivo as demonstrated by the accumulation of [3H]phosphatidylethanol (PtdEtOH) through the transphoshatidylation reaction catalyzed by the enzyme in the presence of ethanol. The observation that ethanol could significantly reduce [3H]PtdOH formation in myoblasts stimulated with BK and thrombin indicates that stimulation of PLD has a major role. The two agonists appear to stimulate PLD activity through a common molecular mechanism, involving the activation of protein kinase C (PKC). In addition, BK and thrombin appear able to activate DAG kinase at early times of incubation and also this pathway may contribute to determine the increase in [3H]PtdOH levels. This is the first report which describes activation of lipid signalling pathways by BK and thrombin in myoblast cells and it is possible that these early signals may have an important role in mediating the biological effects of the two agonists.
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PMID:A role for phospholipase D activation in the lipid signalling cascade generated by bradykinin and thrombin in C2C12 myoblasts. 955 59

Thrombin and other agonists that induce secretion and aggregation in human platelets also activate phospholipase D (PLD), but the signaling cascade leading to activation of PLD in human platelets is not yet clear. We have determined that apyrase, which scavenges ADP secreted during platelet activation, is able to block or reduce the PLD activation stimulated by low (0.1 U/ml or less) or high (0.3- 1.0 U/ml) concentrations of thrombin, respectively. Neither ADP (up to 100 microM) nor its more potent analogue 2-methylthio-ADP (up to 100 microM), however, are able to stimulate PLD alone, and even the addition of fibrinogen, which results in platelet aggregation, is not sufficient for PLD activation. In contrast, ADP is able to stimulate PLD in the presence of low concentrations of thrombin that alone have little or no effect, suggesting ADP may play an amplifying role in platelet PLD activation. This hypothesis is supported by the finding that the purinergic receptor antagonist ARL 66096, an ATP analogue, reduces in a concentration-dependent fashion the PLD response to thrombin (IC50=28 nM with 0.1 U/ml thrombin). ARL 66096 also abolishes the PLD activation by ADP observed in the presence of low concentrations of thrombin, confirming that the antagonist inhibits an ADP-dependent component of the response. In addition, the thromboxane A2 receptor agonist U46619 activates PLD, and this response is markedly reduced by ARL 66096. Concomitantly, phosphorylation of the protein kinase C substrate pleckstrin in response to thrombin or U46619 is partially or totally inhibited by ARL 66096, respectively, consistent with ADP stimulation of protein kinase C being involved in the PLD response to these agonists. Based on these findings, we conclude that ADP secretion and activation of purinergic ADP receptors is an important amplification mechanism in the signal transduction pathways leading to PLD activation in human platelets.
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PMID:Secreted ADP plays a central role in thrombin-induced phospholipase D activation in human platelets. 986 70

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

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

Mitogen-induced activation of a nuclear-acting PC-phospholipase D (PLD) is mediated, at least in part, by the translocation of RhoA to the nucleus. A remaining question is whether PLD in all subcellular compartments is regulated in the same manner. To address this question, we identified PLD in another subcellular compartment and determined whether its activity was influenced by alpha-thrombin in a RhoA-dependent manner. The data in this manuscript show that nuclear PLD is selectively regulated. alpha-Thrombin stimulates an increase in PLD activity in IIC9 fibroblast nuclei while Golgi PLD activity is unaffected. We cloned PLD1 from IIC9s (hamPLD1b) to show that it is present in both nuclei and Golgi. Interestingly, only nuclear PLD1 is modulated by alpha-thrombin, demonstrating that this activity is selectively regulated. These data provide support for the physiological importance of agonist-induced nuclear signalling enzymes.
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PMID:PLD1b in IIC9 fibroblasts is selectively activated in the nucleus and not in the Golgi apparatus. 1174 13

Heterotrimeric GTP-binding (G) proteins transduce hormone-induced signals to their effector enzymes, which include several phospholipases. In particular, the G(o)/G(i) and G(q) protein families have been shown to couple signaling to phospholipase A(2) (PLA(2)), phospholipase C, and phospholipase D, while the G(12)/G(13) family has been linked to the activation of small GTPases of the Rho family, and hence, to phospholipase D activation. Here, we demonstrate that in CHO cells, the G(12)/G(13) family is also able to activate cPLA(2)alpha, through the activation of RhoA and, subsequently, ERK1/2. Hormone-induced arachidonic acid release increased as a consequence of Galpha(13) overexpression, and was inhibited through inhibition of Galpha(13) signaling. The Galpha(13)-mediated cPLA(2)alpha activation was inhibited by pharmacological blockade of ERK1/2 with either U0126 or PD98059, and by RhoA inactivation with C3 toxin or a dominant-negative RhoA (N19RhoA), and was stimulated by the serine-threonine phosphatase inhibitor calyculin A. Our data thus identify a pathway of cPLA(2)alpha regulation that is initiated by thrombin and purinergic receptor activation, and that signals through Galpha(13), RhoA and ERK1/2, with the involvement of a calyculin-sensitive phosphatase.
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PMID:Galpha13 mediates activation of the cytosolic phospholipase A2alpha through fine regulation of ERK phosphorylation. 1680 23

Pathological conditions such as coronary artery disease are clinically controlled via therapeutic regulation of platelet activity. Thrombin, through protease-activated receptor (PAR) 1 and PAR4, plays a central role in regulation of human platelet function in that it is known to be the most potent activator of human platelets. Currently, direct thrombin inhibitors used to block platelet activation result in unwanted side effects of excessive bleeding. An alternative therapeutic strategy would be to inhibit PAR-mediated intracellular platelet signaling pathways. To elucidate the best target, we are studying differences between the two platelet thrombin receptors, PAR1 and PAR4, in mediating thrombin's action. In this study, we show that platelet activation by PAR1-activating peptide (PAR1-AP) requires a phospholipase D (PLD)-mediated phosphatidic acid (PA) signaling pathway. We show that this PAR1-specific PA-mediated effect is not regulated through differential granule secretion after PAR-induced platelet activation. Perturbation of this signaling pathway via inhibition of lipid phosphate phosphatase-1 (LPP-1) by propranolol or inhibition of the phosphatidylcholine-derived phosphatidic acid (PA) formation by PLD with a primary alcohol significantly attenuated platelet activation by PAR1-AP. Platelet activation by thrombin or PAR4-AP was insensitive to these inhibitors. Furthermore, these inhibitors significantly attenuated activation of Rap1 after stimulation by PAR1-AP but not thrombin or PAR4-AP. Because PA metabolites such as diacylglycerol play an important role in intracellular signaling, identifying crucial differences in PA regulation of PAR-induced platelet activation may lead to a greater understanding of the role of PAR1 versus PAR4 in progression of thrombosis.
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PMID:Protease-activated receptors differentially regulate human platelet activation through a phosphatidic acid-dependent pathway. 1715 Dec 88

Increased phosphatidic acid (PA) and phospholipase D (PLD) activity are frequently observed in various disease states including cancers, diabetes, sepsis, and thrombosis. Previously, PA has been regarded as just a precursor for lysophosphatidic acid (LPA) and diacylglycerol (DAG). However, increasing evidence has suggested independent biological activities of PA itself. In the present study, we demonstrated that PA can enhance thrombogenic activities in human erythrocytes through phosphatidylserine (PS) exposure in a Ca(2+)-dependent manner. In freshly isolated human erythrocytes, treatment of PA or PLD induced PS exposure. PA-induced PS exposure was not attenuated by inhibitors of phospholipase A(2) or phosphatidate phosphatase, which converts PA to LPA or DAG. An intracellular Ca(2+) increase and the resultant activation of Ca(2+)-dependent PKC-alpha appeared to underlie the PA-induced PS exposure through the activation of scramblase. A marginal decrease in flippase activity was also noted, contributing further to the maintenance of exposed PS on the outer membrane. PA-treated erythrocytes showed strong thrombogenic activities, as demonstrated by increased thrombin generation, endothelial cell adhesion, and erythrocyte aggregation. Importantly, these procoagulant activations by PA were confirmed in a rat in vivo venous thrombosis model, where PA significantly enhanced thrombus formation. In conclusion, these results suggest that PA can induce thrombogenic activities in erythrocytes through PS exposure, which can increase thrombus formation and ultimately contribute to the development of cardiovascular diseases.
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PMID:Procoagulant and prothrombotic activation of human erythrocytes by phosphatidic acid. 2049 45

In aspirin-treated platelets labelled by preincubation with [(3)H]-choline, enhanced release of both [(3)H]-choline and [(3)H]-choline phosphate resulted from stimulation by collagen or thrombin. No such release accompanied stimulation by ADP, platelet-activating factor or adrenaline. Release of [(3)H]-choline phosphate was entirely dependent on aggregate formation whereas release of [(3)H]-choline was reduced but not eliminated, if aggregation was prevented. The properties of [(3)H]-choline and [(3)H]-choline phosphate release indicated that both collagen and thrombin induced activation of phospholipase D in the absence of aggregate formation. Such activation was augmented if aggregate formation occurred. Aggregation induced by these two agonists also caused activation of phosphatidylcholine-specific phospholipase C. These effects were prevented in the presence of staurosporine and could also be induced by addition of a synthetic 1,2-diacylglycerol indicating a role for protein kinase C.
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PMID:Release of Choline Metabolites from Human Platelets: Evidence for Activation of Phospholipase D and of Phosphatidylcholine-specific Phospholipase C. 2104 50


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