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

Using radioimmunoassay techniques we studied the formation of the 5-lipoxygenase-derived cysteinyl-leukotrienes (LT) in comparison to the cyclooxygenase product thromboxane (TX) B2 in whole human blood allowed to clot at 37 degrees C in vitro. Spontaneous clotting resulted in a time-dependent release of smaller amounts of cysteinyl-LT as well as release of large amounts of TXB2 into the serum. Cysteinyl-LT were characterized by their immunoreactive behaviour and their biological activity in the guinea pig ileum bioassay, an effect which could be antagonized by the SRS-A antagonist FPL 55712 (0.38 microM). By reversed phase HPLC cysteinyl-LT in the serum were identified as a mixture of LTC4, LTD4 and LTE4. At 90 and 120 min part of the immunoreactive material consisted of the omega-oxidized metabolite 20-OH-LTE4. Almost complete inhibition of cyclooxygenase activity by indomethacin (2.8 microM) did not affect cysteinyl-LT formation by clotting whole human blood in vitro nor did activation of platelets by compounds such as the TX mimetic U 46619 (10 microM), platelet-activating factor (PAF, 1 microM) or thrombin (3 IU/ml). In contrast, the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA, 10 microM), the Ca2+-chelating anticoagulants trisodium citrate (10 microM) and edetate disodium (EDTA, 5.4 mM) as well as the functionally unrelated heparin (20 IU/ml) significantly inhibited the formation of cysteinyl-LT as well as of TXB2. Thus, an event related to the process of clotting of whole human blood appears to be able to induce cysteinyl-LT formation in amounts which might be functionally relevant during thromboembolic events.
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PMID:Clotting of whole human blood induces cysteinyl-leukotriene formation. 254 55

A cooperation between leukocytes and platelets relative to metabolism of arachidonic acid has been observed in animal studies. To determine potential stimulatory effects of leukotrienes (LTs) on human platelets, LTs were incubated with platelet rich plasma followed by addition of subthreshold concentration of aggregatory stimulus. LTs (LTE4 LTD4 LTC4) alone had no direct effect on platelet aggregation, but potentiated the effects of subthreshold concentrations of epinephrine and thrombin and caused complete platelet aggregation. This potentiation was similar in citrated or heparinized blood and was unaffected by exogenous CaCl2. LTs did not induce secondary wave of aggregation in aspirin or selective TXA2-synthetase blocker OKY-046-treated platelets. In addition, LTs stimulated TXA2 biosynthesis by platelets in the presence of subaggregatory concentrations of epinephrine, but not when platelets had been pretreated with OKY-046. These data indicate that LTs potentiate epinephrine-induced platelet aggregation by modulating TXA2 synthetase activity.
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PMID:Leukotrienes potentiate the effects of epinephrine and thrombin on human platelet aggregation. 300 72

Contact activation of the intrinsic coagulation cascade in whole human blood in vitro has previously been demonstrated to trigger release of leukotrienes (LT) into serum samples. In our present study we intended to identify the cellular origin of the activated 5-lipoxygenase pathway leading to LT formation under these experimental conditions. Therefore, whole human blood samples incubated for 60 min in vitro were supplemented with Percoll-isolated, 5-lipoxygenase-carrying, autologous blood cells. Surprisingly, exogenously added polymorphonuclear neutrophils (PMN, 5 x 10(6) or 15 x 10(6)/ml) capable of producing cysteinyl-LT in response to ionophore A23187 (1 microM) stimulation, had no effect neither on immunoreactive cysteinyl-LT nor on thromboxane (TX) B2 formation. However, exogenously added mononuclear cells (MNC, 5 x 10(6) or 15 x 10(6)/ml) led to a cell number-dependent increase in cysteinyl-LT generation as did supplementation with peripheral monocytes (PM, 5 x 10(5) or 15 x 10(5)/ml). While MNC enhanced the TXB2 production, PM had no such effect. Incubation of PM (5 x 10(5) or 15 x 10(5)/ml) in recalcified platelet-rich plasma (PRP) induced a cysteinyl-LT formation comparable to that in whole human blood. In contrast to the TXB2 generation, the cysteinyl-LT formation appears to be largely independent from thrombin, since recombinant hirudin (HBW 023, 2 microM), a specific thrombin inhibitor, had no significant effect on the cysteinyl-LT production but nearly completely abolished the TXB2 formation. By reverse phase HPLC the immunoreactive cysteinyl-LT were shown to consist of a mixture of LTC4, LTD4 and LTE4, with LTC4 being the predominant metabolite in all samples studied.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Leukotriene formation by peripheral monocytes in contact-activated human blood. 821 86

Human platelets are devoid of 5-lipoxygenase activity but convert exogenous leukotriene A4 (LTA4) either by a specific LTC4 synthase to leukotriene C4 or via a 12-lipoxygenase mediated reaction to lipoxins. Unstimulated platelets mainly produced LTC4, whereas only minor amounts of lipoxins were formed. Platelet activation with thrombin, collagen or ionophore A23187 increased the conversion of LTA4 to lipoxins and decreased the leukotriene production. Maximal effects were observed after incubation with ionophore A23187, which induced synthesis of comparable amounts of lipoxins and cysteinyl leukotrienes (LTC4, LTD4 and LTE4). Chelation of intra- and extracellular calcium with quin-2 and EDTA reversed the ionophore A23187-induced stimulation of lipoxin synthesis from LTA4 and inhibited the formation of 12-hydroxyeicosatetraenoic acid (12-HETE) from endogenous substrate. However, calcium did not affect the 12-lipoxygenase activity in the 100,000 x g supernatant of sonicated platelet suspensions. Furthermore, the stimulatory effect on lipoxin formation induced by platelet agonists could be mimicked in intact platelets by the addition of low concentrations of arachidonic acid, 12-hydroperoxyeicosatetraenoic acid (12-HPETE) or 13-hydroperoxyoctadecadienoic acid (13-HPODE). The results indicate that the elevated lipoxin synthesis during platelet activation is due to stimulated 12-lipoxygenase activity induced by endogenously formed 12-HPETE.
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PMID:Stimulation of lipoxin synthesis from leukotriene A4 by endogenously formed 12-hydroperoxyeicosatetraenoic acid in activated human platelets. 830 92

The purpose of this study was to identity the trigger mechanism activating the 5-lipoxygenase pathway during blood-brain cell contact and to estimate the contribution of blood and brain cells to the cysteinyl-leukotriene (LT) biosynthesis observed under these conditions. Incubation of dissociated rat brain cells in Krebs-Henseleit solution for up to 60 min did not stimulate any detectable cysteinyl-LT biosynthesis. Incubation of recalcified rat whole blood in vitro for up to 60 min led to release of only small amounts of cysteinyl-LT into the serum samples. However, coincubation of dissociated rat brain cells with physiologically recalcified autologous whole blood triggered a time-dependent release of large amounts of immunoreactive cysteinyl-LT into the serum samples. By reverse-phase HPLC, immunoreactive cysteinyl-LT was identified as a mixture of LTC4, LTD4, and LTE4. The extent of the 5-lipoxygenase stimulation depended on the amount of autologous blood coincubated with the dissociated brain cells. Activation of the 5-lipoxygenase pathway also occurred with coincubation of dissociated rat brain cells with recalcified autologous plasma. Stimulation of cysteinyl-LT biosynthesis during blood-brain cell contact remained unaffected by aprotinin, but concentration-dependent inhibition by the structurally and functionally unrelated thrombin inhibitors D-Phe-Pro-Arg-CH2Cl and recombinant hirudin was seen. Finally, when dissociated rat brain cells were incubated in Krebs-Henseleit solution in the presence of human alpha-thrombin, a concentration-dependent release of cysteinyl-LT into the buffer samples was observed. These data demonstrate that, in rats, during blood-brain cell contact, stimulation of the 5-lipoxygenase pathway in brain cells proceeds via alpha-thrombin as effector molecule.
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PMID:Thrombin stimulates activation of the cerebral 5-lipoxygenase pathway during blood-brain cell contact. 896 15

The contribution of platelets to arachidonic acid transcellular metabolism may represent an important pathway of leukotriene (LT) production. The aim of this study was to investigate the role of platelets on LT production in an acute inflammatory model in the rabbit. Preliminary experiments showed that rabbit whole blood (5 ml) stimulated in vitro with the calcium ionophore A23187 produced LTB4 (52.7+/-13.9 ng) and the mixed 5,12-DiHETE (7.25+/-0.75 ng). In A23187-stimulated thrombocytopenic blood, LTB4 was significantly reduced to 19.5+/-8.6 ng and 5,12-DiHETE was undetectable. Peptido-LTs were undetectable in both conditions. In experiments using washed cells, addition of thrombin-activated platelets to fMLP-activated PMN resulted in the appearance of 5,12-DiHETE and in more than twofold increase of LTB4 synthesis. When 3H-arachidonic acid-labelled platelets were mixed with unlabelled PMN and challenged with fMLP and thrombin, radioactive LTB4 and 5,12-DiHETE were produced, indicating that platelet-derived arachidonic acid was utilized by PMN 5-lipoxygenase. Intravenous infusion of fMLP (2.5 nmol/kg/min) in the rabbit induced marked granulocytopenia, thrombocytopenia and increased TxB2 plasma concentrations within 3 min. Electron microscopy of lungs showed morphologically activated and aggregated platelets occluding the capillary lumen. Activation and recruitment of circulating cells was accompanied by the production of LTB4 (peak levels at 1 min: 30.0+/-9.5 ng/ml) and LTE4 (peak levels at 10 minutes: 77.8+/-11.6 ng/ml). The areas under the blood concentration-time curve (AUC, ng min/ml) corresponded to 812+/-182 and 3692+/-658 for LTB4 and LTE4, respectively. In immunologically thrombocytopenic rabbits, the AUC for LTB4 (86.0+/-23.0) and LTE4 (1165+/-542) were both significantly different from controls while in rabbits treated with an anti-leukocyte antiserum, both LTB4 and LTE4 were similar to controls. This experimental model provides in vivo evidence that platelets, involved in an acute inflammatory event contribute to the transcellular production of LTs.
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PMID:Platelet contribution to leukotriene production in inflammation: in vivo evidence in the rabbit. 1010 75

Cysteinyl leukotrienes (cysLT), i.e., LTC4, LTD4, and LTE4, are lipid mediators derived from the 5-lipoxygenase pathway, and the cysLT receptors cysLT1-R/cysLT2-R mediate inflammatory tissue reactions. Although endothelial cells (ECs) predominantly express cysLT2-Rs, their role in vascular biology remains to be fully understood. To delineate cysLT2-R actions, we stimulated human umbilical vein EC with LTD4 and determined early induced genes. We also compared LTD4 effects with those induced by thrombin that binds to protease-activated receptor (PAR)-1. Stringent filters yielded 37 cysLT2-R- and 34 PAR-1-up-regulated genes (>2.5-fold stimulation). Most LTD4-regulated genes were also induced by thrombin. Moreover, LTD4 plus thrombin augmented gene expression when compared with each agonist alone. Strongly induced genes were studied in detail: Early growth response (EGR) and nuclear receptor subfamily 4 group A transcription factors; E-selectin; CXC ligand 2; IL-8; a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif 1 (ADAMTS1); Down syndrome critical region gene 1 (DSCR1); tissue factor (TF); and cyclooxygenase 2. Transcripts peaked at approximately 60 min, were unaffected by a cysLT1-R antagonist, and were superinduced by cycloheximide. The EC phenotype was markedly altered: LTD4 induced de novo synthesis of EGR1 protein and EGR1 localized in the nucleus; LTD4 up-regulated IL-8 formation and secretion; and LTD4 raised TF protein and TF-dependent EC procoagulant activity. These data show that cysLT2-R activation results in a proinflammatory EC phenotype. Because LTD4 and thrombin are likely to be formed concomitantly in vivo, cysLT2-R and PAR-1 may cooperate to augment vascular injury.
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PMID:Cysteinyl leukotriene 2 receptor and protease-activated receptor 1 activate strongly correlated early genes in human endothelial cells. 1660 35