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)

Leukotriene (LT) A4 metabolism was studied in human platelets and endothelial cells, since both cells could be involved in transcellular formation of LTC4. Upon addition of exogenous LTA4, both cells produced LTC4 as a major metabolite at various incubation times, and no LTB4, LTD4, or LTE4 was detected. Kinetic studies revealed a higher apparent Km for LTA4 in endothelial cells as compared to platelets (5.8 microM for human umbilical vein endothelial cells (HUVEC) versus 1.3 microM for platelets); platelets were more efficient in this reaction with a higher Vmax (174 pmol/mg protein/min) versus 15 pmol/mg protein/min in HUVEC. The formation of LTC4 and corresponding kinetic parameters were not modified when platelets or endothelial cells were stimulated by thrombin prior to or simultaneously with the addition of LTA4. In both cells LTC4 synthase activity was not modified by repeated addition of LTA4 showing that it is not a suicide-inactivated enzyme. Furthermore, in platelets and endothelial cells, the enzyme activity was localized in the membrane fraction and was distinct from cytosolic glutathione-S-transferases. Platelet membrane fractions showed apparent Km values of 31 microM and 1.2 mM for LTA4 and GSH, respectively. Inhibition of LTC4 formation from platelets and endothelial cells preparations by S-substituted glutathione derivatives was correlated to the length of the S-alkyl chain. The same substances inhibited cytosolic glutathione-S-transferases with significantly lower IC50, confirming the distinct nature of the two enzymes. These results show that platelets and HUVEC possess similar enzymes for the production of LTC4 from LTA4; however, platelets seem to have a higher efficiency than HUVEC in performing this reaction.
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PMID:Comparison of leukotriene A4 metabolism into leukotriene C4 by human platelets and endothelial cells. 132 61

Interactions of human platelets with cadmium in vitro were studied with respect to the platelet activation process as indicated by malondialdehyde (MDA) formation and also to the components of the cellular antioxidant defence system such as catalase, glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S-transferase (GST), glucose-6-phosphate dehydrogenase (G6PDH), and reduced glutathione (GSH). Cadmium treatment stimulated platelet MDA formation after a lag phase of at least 15 min and this effect was completely blocked by either 1 mM aspirin or 1 mM CaCl2. Cadmium pretreated platelets also displayed a much higher (5 fold) MDA formation when stimulated by thrombin. Platelet catalase activity was decreased by almost 50% after incubation with cadmium. There was also a moderate decline in platelet GSH and GR activity along with a stimulation of GST and G6PDH activity. These results suggest: (1) the cadmium effect on platelets as observed by enhanced formation of MDA via the cyclooxygenase pathway involves intraplatelet accumulation of cadmium which is inhibited by calcium, (2) a modest decline in GSH, presumably due to the inadequacy of H2O2 detoxification mechanism, does not adversely affect platelet function because of the adaptive response of G6PDH, and (3) intracellular accumulation of cadmium may result in platelet hyperactivity through higher intraplatelet free calcium levels resulting directly through cadmium action or indirectly through higher H2O2 levels due to catalase inhibition.
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PMID:Effects of cadmium treatment in vitro on the antioxidant protection mechanism and activation of human blood platelets. 313 42

Selenium added to the culture medium of confluent pig aortic endothelial cells caused a time-related elevation in the activity of the hydroperoxide scavenging enzyme: glutathione peroxidase. This increased activity was associated with an enhanced ability to produce prostacyclin irregardless of whether the agonist was arachidonic acid or thrombin. Since prostacyclin synthetase is believed to be irreversibly inhibited by alkyl hydroperoxides, we feel that the greater production of prostacyclin by selenium-treated cells as compared with control cells may reflect a protective effect of GSH.Px towards the synthetase enzyme. The results from this study may explain the observations made on a group of human volunteers ingesting selenium as a dietary supplement. After six weeks treatment with selenium, bleeding time in this group was prolonged suggesting an improved ability to synthesize prostacyclin as a result of selenium-dependent glutathione peroxidase activation in the vessel wall.
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PMID:Selenium enhances prostacyclin production by cultured endothelial cells: possible explanation for increased bleeding times in volunteers taking selenium as a dietary supplement. 637 71

Diabetes is characterized by the existence of a thrombosis-prone condition, possibly related to hyperglycemia. However, the mechanism linking hyperglycemia to the activation of the coagulation cascade is still unclear. It has been recently suggested that diabetes is accompanied by increased oxidative stress. In this work, the possibility that oxidative stress may be involved in the hyperglycemia-induced coagulation activation has been evaluated. Prothrombin fragment 1 + 2 (F1 + 2), which represents a reliable marker of the amount of thrombin released in the circulation, has been chosen for studying thrombin formation in vivo. In nine type II diabetic patients and in seven healthy control subjects, matched for age and body mass index, three different experiments were performed: oral glucose tolerance test (OGTT), intravenous antioxidant glutathione (GSH) administration for 2 h, and OGTT plus intravenous GSH administration. Samples were drawn at -15 min and every 30 min from 0 to 180 min. During the OGTT, F1 + 2 significantly increased in both diabetic and healthy subjects. GSH administration during OGTT normalized this phenomenon. GSH administration alone significantly decreased F1 + 2 in diabetic patients, while no effect was observed in the normal subjects. These data suggest that hyperglycemia may induce thrombin activation, possibly inducing an oxidative stress, and that antioxidant GSH may counterbalance this effect.
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PMID:Hyperglycemia-induced thrombin formation in diabetes. The possible role of oxidative stress. 762 98

Selenium (Se) is an essential component of glutathione peroxidase (GSH-Px), an enzyme that protects cells by reducing intracellular peroxides. Impaired Se status and GSH-Px activity seem associated with increased risk of atherosclerotic vascular diseases. This study reports the effects of Se supplementation on GSH-Px activity, on prostacyclin (PGI2) production, on 12-hydroxy-eicosatetraenoic acid (12-HETE) levels, and on GSH-Px mRNA expression in cultured human umbilical vein endothelial cells (HUVEC). Se-enriched HUVEC showed significant increase of both GSH-Px activity and thrombin-stimulated production of PGI2 in the presence of stable concentrations of 12-HETE. On the other hand, an inverse correlation between Se concentrations in culture media and GSH-Px mRNA levels in Northern blot analysis was shown; this suggests that a major degree of regulation for GSH-Px expression by Se is most likely exerted at the posttranscriptional level. These observations may help to explain the increased incidence of atherosclerosis described in Se-deficient individuals.
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PMID:Selenium enhances glutathione peroxidase activity and prostacyclin release in cultured human endothelial cells. Concurrent effects on mRNA levels. 788 76

1. The aim of this study was to investigate whether two metabolites of glyceryl trinitrate (GTN), 1,2 and 1,3-glyceryl dinitrate (1,2-GDN and 1,3-GDN) could account for the pharmacological effects of GTN. To this end the formation of nitric oxide (NO) from 1,2- and 1,3-GDN in the presence of bovine aortic smooth muscle cells (SMC) or endothelial cells (EC) was studied. The effects of various thiols on NO formation from these dinitrates was also evaluated. 2. 1,2-GDN or 1,3-GDN (10(-10)-10(-5) M) caused a dose-dependent relaxation of rabbit aortic strips denuded of endothelium and precontracted with phenylephrine. The dinitrates were less than one tenth as potent as GTN. 3. Incubation of 1,2-GDN or 1,3-GDN (75-2400 microM) with SMC for 30 min led to a concentration-dependent increase in nitrite (NO2-) formation but this increase was less than that produced from GTN. Likewise incubation of 1,2-GDN or 1,3-GDN with N-acetylcysteine (NAC), glutathione (GSH) or thiosalicylic acid (TSA) (all at 1 mM) for 30 min at 37 degrees C produced a concentration-dependent increase in NO2- formation. 4. Platelet aggregation induced by thrombin (40 mu ml-1) was not modified by high concentrations of 1,2-GDN or 1,3-GDN (175-700 microM). However, aggregation was inhibited when platelets were exposed to 1,2-GDN or 1,3-GDN (700 microM) in the presence of SMC (0.24-1.92 x 10(5) cells) or EC (0.8-3.2 x 10(5) cells). These effects were abrogated by co-incubation with oxyhaemoglobin (OxyHb, 10 microM) indicating that they were due to NO release. The concentrations of the dinitrates required to inhibit platelet aggregation by 50% were about 15 times higher than for GTN in the presence of the same numbers of SMC or EC.5. When NAC or TSA (both at 0.5 mM) were co-incubated with platelets for 3 min in the presence of1,2-GDN or 1,3-GDN, a concentration-dependent inhibition of platelet aggregation was observed. These anti-platelet effects were abolished by co-incubation with OxyHb (10 microM). Glutathione had no potentiating effects.6. Thus the dinitrate metabolites of GTN are metabolized to NO by SMC or EC and are acted upon by thiols to form NO at concentrations about 10 times higher than those of GTN. In vivo, after oral or intravenous GTN, GDN levels are reached which are more than 10 times higher than those of GTN.These data support the notion that part of the effects of GTN are due to the generation of NO from 1,2-GDN and 1,3-GDN by the cells of the vascular wall.
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PMID:Vascular and anti-platelet actions of 1,2- and 1,3-glyceryl dinitrate. 829 17

In 41 patients with coronary heart disease (CHD) the concentrations of total blood platelet malonyldialdehyde (MDA: 2.11 +/- 0.25 nmol/10(9) platelets) and MDA corresponding to thromboxane A2 (TXA2 0.84 +/- 0.13 nmol/10(9) platelets) were increased in comparison with values in blood platelets of healthy subjects (1.19 +/- 0.09 and 0.71 +/- 0.05 nmol/10(9) platelets), respectively. The increased aggregability with ADP and thrombin of patient platelets was also observed. In relation to the blood platelets of healthy subjects, the antioxidant enzymes activities of patient blood platelets were significantly (P < 0.001) decreased. Platelet glutathione peroxidase (GSH-Px) activity of the patients (11.3 +/- 0.85 U/g protein) was significantly lower than controls (18.3 +/- 1.12 U/g protein). In patients with CHD the activities of the other antioxidative platelet enzymes: catalase (Cat, 7.37 +/- 1.38 U/g protein) and superoxide dismutase (SOD, 1529.4 +/- 167 U/g protein) were also significantly decreased in comparison with values for healthy subjects (Cat: 9.06 +/- 1.30 U/g protein and SOD: 1987 +/- 230 U/g protein, respectively). It is suggested that antioxidative defense in blood platelets may affect the haemostatic processes and lipid peroxidation in patients with CHD.
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PMID:Changes in antioxidant enzymes activities, aggregability and malonyldialdehyde concentration in blood platelets from patients with coronary heart disease. 835 54

The expression vector pGEX-2T under the control of the IPTG-inducible tac promotor is effective for the production of a fusion protein of glutathione transferase (GST, 26 kDa) and promatrilysin (28 kDa) separated from the C-terminus of GST by a thrombin cleavage site. Zwittergen (palmityl sulfobetaine), 2%, solubilizes the fusion protein that is found associated with inclusion bodies. The solubilized fusion protein is purified by affinity chromatography on GSH agarose. Promatrilysin is obtained by thrombin cleavage either on the column or after GSH elution of the fusion protein. Mono S chromatography of the recovered protein yields homogeneous promatrilysin. The zinc content of promatrilysin and its activated enzyme product is slightly greater than 2 mol of zinc per mole of protein. The results indicate that the matrix metalloproteinases (MMPs) contain two metal-binding sites at which zinc is firmly bound and possibly a third site at which it is weakly bound. Primary sequence alignments for all the MMPs have a sequence homologous to the zinc-binding site of astacin, HExxHxxGxxH, suggesting one of the zinc sites is a catalytic one, in agreement with the known inhibition of these enzymes by chelators. However, the other zinc-binding site(s) likely reflect the different ways that astacin and the MMP subfamilies are stabilized, i.e., disulfides in astacin and metal ions in the MMPs.
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PMID:Matrilysin: expression, purification, and characterization. 856 47

Highly reactive oxygen species rapidly inactivate nitric oxide (NO), and endothelial product which inhibits platelet activation. We studied platelet inhibition by NO in two brothers with a cerebral thrombotic disorder. Both children had hyperreactive platelets, as determined by whole blood platelet aggregometry and flow cytometric analysis of the platelet surface expression of P-selectin. Mixing experiments showed that the patients'platelets behaved normally in control plasma; however, control platelets suspended in patient plasma were not inhibited by NO. As determined by flow cytometry, in the presence of plasma from either patient there was normal inhibition of the thrombin-induced expression of platelet surface P-selectin by prostacyclin, but not NO. Using a scopoletin assay, we measured a 2.7-fold increase in plasma H2O2 generation in one patient and a 3.4-fold increase in the second patient, both compared woth control plasma. Glutathione peroxidase (GSH-Px) activity was decreased in the patients' plasmas compared with control plasma. The addition of exogenous GSH-Px led to restoration of platelet inhibition by NO. These data show that, in these patients' plasmas, impaired metabolism of reactive oxygen species reduces the bioavailability of NO and impairs normal platelet inhibitory mechanisms. These findings suggest that attenuated NO-mediated platelet inhibition produced by increased reactive oxygen species or impaired antioxidant defense may cause a thrombotic disorder in humans.
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PMID:Decreased platelet inhibition by nitric oxide in two brothers with a history of arterial thrombosis. 861 52

Methylglyoxal generates a slight increase in the basal level of hydrogen peroxide in platelets. The oxidation effect of methylglyoxal significantly potentiated by thrombin, depends on both the ketoaldehyde and the agonist concentrations. A further significant increase in hydrogen peroxide accumulation was obtained in platelets pretreated with the alkylating agent N-ethylmaleimide which depletes GSH and blocks glutathione peroxidase. Resting platelets completely transform the ketoaldehyde into D(-)lactate, whereas stimulated platelets transform about 10-15 per cent of the metabolized methylglyoxal into D(-)lactate. The metabolic modifications generated by methylglyoxal such as the GSH depletion and hydrogen peroxide accumulation induce modifications in platelet function. Methylglyoxal inhibits platelet aggregation induced by several agonists and ATP release induced by thrombin.
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PMID:Effects of methylglyoxal on platelet hydrogen peroxide accumulation, aggregation and release reaction. 864 Sep 57


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