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)

Garlic has been used in herbal medicine for thousands of years. Some reports have shown that garlic has protective effects against atherosclerosis and inhibits platelet function. In this study, we investigated the mechanism by which diallyl trisulfide (DT), a component of garlic, inhibits platelet function. DT inhibited platelet aggregation and Ca(2+) mobilization in a concentration-dependent manner without increasing intracellular cyclic AMP and cyclic GMP. DT also had no inhibitory effects on thromboxane A(2) (TXA(2)) production in cell-free systems. Collagen-related peptide (CRP)-induced Ca(2+) mobilization is regulated by phospholipase C-gamma2 (PLC-gamma2) activation. We evaluated the effect of DT on tyrosine phosphorylation of PLC-gamma2 and the production of inositol-1,4,5-trisphosphate (IP(3)). DT at concentrations that inhibited platelet aggregation and Ca(2+) mobilization had no effects on tyrosine phosphorylation of PLC-gamma2 or on the formation of IP(3) induced by CRP. Similar results were obtained with thrombin-induced platelet activation. DT inhibited platelet aggregation and Ca(2+) mobilization induced by thrombin without affecting the production of IP(3.) We then evaluated the effect of DT on the binding of IP(3) to its receptor. DT at high concentrations partially blocked the binding of IP(3) to its receptor. Taken together, our findings suggest that the agent suppresses Ca(2+) mobilization at a step distal to IP(3) formation. DT may provide a good tool for investigating Ca(2+) mobilization.
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PMID:Inhibition by diallyl trisulfide, a garlic component, of intracellular Ca(2+) mobilization without affecting inositol-1,4, 5-trisphosphate (IP(3)) formation in activated platelets. 1102 Apr 49

Interaction of von Willebrand Factor with glycoprotein Ib-IX-V induces platelet activation through a still poorly defined mechanism. Previous studies have suggested a possible role for the low affinity receptor for immunoglobulin, Fc gamma RIIA, in GPIb-IX-V signaling. Here we show that binding of vWF to platelets induces the tyrosine phosphorylation of Fc gamma RIIA by a Src kinase. Treatment of platelets with the anti-Fc gamma RIIA monoclonal antibody IV.3 specifically inhibits vWF-induced but not thrombin-induced pleckstrin phosphorylation and serotonin secretion. Moreover, vWF fails to induce pleckstrin phosphorylation in mouse platelets, lacking Fc gamma RIIA, and serotonin secretion is impaired. Pleckstrin phosphorylation and serotonin secretion in human platelets stimulated with vWF are blocked by the cyclooxygenase inhibitor acetylsalicylic acid. However, release of arachidonic acid and synthesis of TxA(2) induced by vWF are not affected by the anti-Fc gamma RIIA monoclonal antibody IV.3. Similarly, vWF-induced tyrosine phosphorylation of Fc gamma RIIA, as well as of Syk and PLC gamma 2, occurs normally in aspirinized platelets. Inhibition of the tyrosine kinase Syk by piceatannol does not affect vWF-induced tyrosine phosphorylation of Fc gamma RIIA but prevents phosphorylation of PLC gamma 2. Pleckstrin phosphorylation and platelet secretion induced by vWF, but not by thrombin, are also inhibited by piceatannol. Pleckstrin phosphorylation is also sensitive to the phosphatidylinositol 3-kinase inhibitor wortmannin. These results indicate that PLC gamma 2 plays a central role in platelet activation by vWF and that the stimulation of this enzyme requires coordinated signals through endogenous TxA(2) and Fc gamma RIIA.
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PMID:Platelet activation by von Willebrand factor requires coordinated signaling through thromboxane A2 and Fc gamma IIA receptor. 1134 69

Platelets were labelled separately with six different, radioactive unsaturated fatty acids. The cells were isolated from the radioactive precursors and treated with and without 2 U/ml of thrombin. The formation of radioactive free fatty acid+oxygenated fatty acids and of radioactive radioactive phosphatidic acid+diacylglycerol was taken as a measure of the PLA(2) and PLC reactions, respectively. We found that that in intact platelets PLA(2) prefers phospholipid molecular species containing unsaturated acyls, most likely in the sn-2 position, in the priority order: 20:4>20:5>18:2 = 18:3 = 22:6>>18:1, while PLC prefers its substrates in the priority order 20:5>20:4>18:2>18:3 = 22:6>18:1.
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PMID:Acyl specificity of phospholipases A2 and C in thrombin-stimulated human platelets. 1191 34

Diabetes mellitus (DM) is accompanied by several cardiovascular complications such as coronary artery disease, atherosclerosis, hypertension, cerebral and myocardial infarction, etc. DM induces the alteration of platelet functions including activation, hyperaggregation, adhesiveness, and formation of thrombi. Release of AA from phospholipids of the PM, synthesis of TxA(2),PGE(2), activity of PLA(2), and PLC are increased in the platelets of the DM patients. Stimulation of PLA(2) activity and accumulation of bioactive metabolites such as AA, its oxygenated derivatives, prostaglandins and PAF can evoke glucose production, also. In this study we explored the effect of the 1,4-dihydropyridine compound cerebrocrast at a low concentration (10(-6)-10(-8)M) on the level of intracellular calcium in unstimulated human platelets and those stimulated with thrombin as well as release of [(3)H] AA from phospholipids of platelet PM. Cerebrocrast at a concentration of 10(-6) M decreased the basal level of intracellular calcium concentration (platelets were loaded with Fura-2) in unstimulated as well as in thrombin stimulated platelets. Cerebrocrast at concentrations of 10(-6), 10(-7), 10(-8) M inhibited release of [(3)H] AA from phospholipids of platelet PM. We conclude that blockade of human platelet activation with cerebrocrast can prevent aggregation, adhesion and formation of thrombi. The inhibition of [(3)H] AA release from phospholipids of platelet PM can prevent formation of eicosanoids such as TxA(2), PGG(2), and PGH(2) plus AA oxygenated derivatives. These effects of cerebrocrast are very significant in the treatment of DM-evoked cardiovascular complications.
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PMID:Effect of cerebrocrast on the function of human platelets and release of the arachidonic acid from plasma membrane. 1197 14

Protease-activated receptors (PARs), newly identified members of G protein-coupled receptors, are widely distributed in the brain. Thrombin evokes multiple cellular responses in a large variety of cells by activating PAR-1, -3, and -4. In cultured rat astrocytes we investigated the signaling pathway of thrombin- and PAR-activating peptide (PAR-AP)-induced cell proliferation. Our results show that PAR activation stimulates proliferation of astrocytes through the ERK pathway. Thrombin stimulates ERK1/2 phosphorylation in a time- and concentration-dependent manner. This effect can be fully mimicked by a specific PAR-1-AP but only to a small degree by PAR-3-AP and PAR-4-AP. PAR-2-AP can induce a moderate ERK1/2 activation as well. Thrombin-stimulated ERK1/2 activation is mainly mediated by PAR-1 via two branches: 1) the PTX-sensitive G protein/(betagamma-subunits)-phosphatidylinositol 3-kinase branch, and 2) the G(q)-PLC-(InsP(3) receptor)/Ca2+ -PKC pathway. Thrombin- or PAR-1-AP-induced ERK activation is partially blocked by a selective EGF receptor inhibitor, AG1478. Nevertheless, transphosphorylation of EGF receptor is unlikely for ERK1/2 activation and is certainly not involved in PAR-1-induced proliferation. The metalloproteinase mechanism involving transactivation of the EGF receptor by released heparin-binding EGF was excluded. EGF receptor activation was detected by the receptor autophosphorylation site, tyrosine 1068. Our data suggest that thrombin-induced mitogenic action in astrocytes occurs independently of EGF receptor transphosphorylation.
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PMID:Thrombin (PAR-1)-induced proliferation in astrocytes via MAPK involves multiple signaling pathways. 1237 96

Thrombin responses in human platelets are mediated by the protease-activated receptors (PAR), PAR1 and PAR4. The signalling pathways mediating PAR activation have not been fully delineated for human platelets. We assessed cytoplasmic Ca2+ mobilization in response to activation with thrombin and PAR1 (SFLLRN) and PAR4 (GYPGKF) peptides in two patients whose platelets were deficient in two major signalling proteins, Galphaq or phospholipase (PLC)-beta2. In normal platelets, thrombin induced a biphasic Ca2+ response with a rapid rise to a peak followed by a sustained elevation in Ca2+. The peak Ca2+ rise was impaired in both patients at lower thrombin concentrations. At higher concentrations, it was decreased in PLC-beta2-deficient platelets; the sustained Ca2+ elevation observed in normal and Galphaq-deficient platelets was reduced in PLC-beta2-deficient platelets. The response to SFLLRN was decreased in both patients at lower concentrations. The peak Ca2+ in response to GYPGKF was reduced in both patients; the sustained Ca2+ increase was markedly decreased in PLC-beta2-deficient platelets. These studies provide evidence that, in human platelets, both Galphaq and PLC-beta2 play a major role in responses to PAR1 and PAR4 activation, and that PLC-beta2 is required for the sustained Ca2+ rise upon thrombin activation.
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PMID:Role of Galphaq and phospholipase C-beta2 in human platelets activation by thrombin receptors PAR1 and PAR4: studies in human platelets deficient in Galphaq and phospholipase C-beta2. 1271 74

1. Docosahexaenoic acid (DHA) and arachidonic acid (AA), polyunsaturated fatty acids (PUFAs), are important for central nervous system function during development and in various pathological states. Astrocytes are involved in the biosynthesis of PUFAs in neuronal tissue. Here, we investigated the mechanism of DHA and AA release in cultured rat brain astrocytes. 2. Primary astrocytes were cultured under standard conditions and prelabeled with [(14)C]DHA or with [(3)H]AA. Adenosine 5'-triphosphate (ATP) (20 micro M applied for 15 min), the P2Y receptor agonist, stimulates release of both DHA (289% of control) and AA (266% of control) from astrocytes. DHA release stimulated by ATP is mediated by Ca(2+)-independent phospholipase A(2) (iPLA(2)), since it is blocked by the selective iPLA(2) inhibitor 4-bromoenol lactone (BEL, 5 micro M) and is not affected either by removal of Ca(2+) from extracellular medium or by suppression of intracellular Ca(2+) release through PLC inhibitor (U73122, 5 micro M). 3. AA release, on the other hand, which is stimulated by ATP, is attributed to Ca(2+)-dependent cytosolic PLA(2) (cPLA(2)). AA release is abolished by U73122 and, by removal of extracellular Ca(2+), is insensitive to BEL and can be selectively suppressed by methyl arachidonyl fluorophosphonate (3 micro M), a general inhibitor of intracellular PLA(2) s. 4. Western blot analysis confirms the presence in rat brain astrocytes of 85 kDa cPLA(2) and 40 kDa protein reactive to iPLA(2) antibodies. 5. The influence of cAMP on regulation of PUFA release was investigated. Release of DHA is strongly amplified by the adenylyl cyclase activator forskolin (10 micro M), and by the protein kinase A (PKA) activator dibutyryl-cAMP (1 mM). In contrast, release of AA is not affected by forskolin or dibutyryl-cAMP, but is almost completely blocked by 2,3-dideoxyadenosine (20 micro M) and inhibited by 34% by H89 (10 micro M), inhibitors of adenylyl cyclase and PKA, respectively. 6. Other neuromediators, such as bradykinin, glutamate and thrombin, stimulate release of DHA and AA, which is comparable to the release stimulated by ATP. 7. Different sensitivities of iPLA(2) and cPLA(2) to Ca(2+) and cAMP reveal new pathways for the regulation of fatty acid release and reflect the significance of astrocytes in control of DHA and AA metabolism under normal and pathological conditions in brain.
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PMID:Docosahexaenoic acid and arachidonic acid release in rat brain astrocytes is mediated by two separate isoforms of phospholipase A2 and is differently regulated by cyclic AMP and Ca2+. 1283 76

PLCepsilon (phospholipase Cepsilon) is a novel PLC that has a CDC25 guanine nucleotide exchange factor domain and two RA (Ras-association) domains of which the second (RA2) is critical for Ras activation of the enzyme. In the present studies, we examined hormonal stimulation to elucidate receptor-mediated pathways that functionally regulate PLCepsilon. We demonstrate that EGF (epidermal growth factor), a receptor tyrosine kinase agonist, and LPA (lysophosphatidic acid), S1P (sphingosine 1-phosphate) and thrombin, GPCR (G-protein-coupled receptor) agonists, stimulate PLCepsilon overexpressed in COS-7 cells. EGF stimulated PLCepsilon in an RA2-dependent manner through Ras and Rap. In contrast, LPA, S1P and thrombin stimulated PLCepsilon by both RA2-independent and -dependent mechanisms. To determine the G-proteins that mediate the effects of these GPCR agonists, we co-expressed constitutively active G-proteins with PLCepsilon and found that G(alpha12), G(alpha13), Rho, Rac and Ral stimulate PLCepsilon in an RA2-independent manner; whereas TC21, Rap1A, Rap2A and Rap2B stimulate PLCepsilon in an RA2-dependent manner similar to H-Ras. Of these G-proteins, we show that G(alpha12)/G(alpha13) and Rap partly mediate the effects of LPA, S1P and thrombin to stimulate PLCepsilon. In addition, the stimulation by LPA and S1P is also partly sensitive to pertussis toxin. These studies demonstrate diverse hormonal regulation of PLCepsilon by distinct and overlapping pathways.
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PMID:Hormonal regulation of phospholipase Cepsilon through distinct and overlapping pathways involving G12 and Ras family G-proteins. 1456 55

Thrombin is a serine protease activated during injury and inflammation. Thrombin and other proteases generated by periodontal pathogens affect the behavior of periodontal cells via activation of protease-activated receptors (PARs). We noted that thrombin and PAR-1 agonist peptide stimulated intracellular calcium levels ([Ca2+]i) of gingival fibroblasts (GF). This increase of [Ca2+]i was inhibited by EGTA and verapamil. U73122 and neomycin inhibited thrombin- and PAR-1-induced [Ca2+]i. Furthermore, 2-APB (75-100 microM, inositol triphosphate [IP3] receptor antagonist), thapsigargin (1 microM), SKF-96365 (200 microM) and W7 (50 and 100 microM) also suppressed the PAR-1- and thrombin-induced [Ca2+]i. However, H7 (100, 200 microM) and ryanodine showed little effects. Blocking Ca2+ efflux from mitochondria by CGP37157 (50, 100 microM) inhibited both thrombin- and PAR-1-induced [Ca2+]i. Thrombin induced the IP3 production of GF within 30-seconds of exposure, which was inhibited by U73122. These results indicate that mitochondrial calcium efflux and calcium-calmodulin pathways are related to thrombin and PAR-1 induced [Ca2+]i in GF. Thrombin-induced [Ca2+]i of GF is mainly due to PAR-1 activation, extracellular calcium influx via L-type calcium channel, PLC activation, then IP3 binding to IP3 receptor in sarcoplasmic reticulum, which leads to intracellular calcium release and subsequently alters cell membrane capacitative calcium entry.
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PMID:Protease-activated receptor-1-induced calcium signaling in gingival fibroblasts is mediated by sarcoplasmic reticulum calcium release and extracellular calcium influx. 1509 14

Geiji-Bokryung-Hwan (GBH) was studied on antiplatelet activity in human platelet suspensions. GBH consists of the 5 herbs Cinnamomi Ramulus, Poria Cocos, Mountan Cortex Radicis, Paeoniae Radix, and Persicae Semen, which have been used in herbal medicine for thousands of years for atherosclerosis. The mechanism involved in the antiplatelet activity of GBH in human platelet suspensions was investigated. GBH inhibited platelet aggregation and Ca2+ mobilization in a concentration-dependent manner without increasing intracellular cyclic AMP and cyclic GMP. GBH had no inhibitory effect on thromboxane B2 (TXB2) production in cell-free systems. Collagen-related peptide (CRP)-induced Ca2+ mobilization is regulated by phospholipase C-2 (PLC-gamma2) activation. We evaluated the effect of GBH on tyrosine phosphorylation of PLC-gamma2 and the production of inositol-1,4,5-trisphosphate (IP3). GBH at concentrations that inhibited platelet aggregation and Ca2+ mobilization had no effects on tyrosine phosphorylation of PLC-gamma2 or on the formation of IP3 induced by CRP. Similar results were obtained with thrombin-induced platelet activation. GBH inhibited platelet aggregation and Ca2+ mobilization induced by thrombin without affecting the production of IP3. We then evaluated the effect of GBH on the binding of IP3 to its receptor. GBH at high concentrations partially blocked the binding of IP3 to its receptor. Therefore, the results suggested that GBH suppresses Ca2+ mobilization at a step distal to IP3 formation. GBH may provide a good tool for investigating Ca2+ mobilization.
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PMID:Inhibitory effect of GBH on platelet aggregation through inhibition of intracellular Ca2+ mobilization in activated human platelets. 1547 58


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