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)

Increased adherence of leucocytes to the vessel wall is thought to be a key event in potentially deleterious leucocyte-vessel wall interactions in a variety of diseases associated with microvasculatory dysfunction. As measured by videomicroscopy in mesenteric venules (phi 40 +/- 8 microns) of anaesthetized rats, an injury caused by one short electrical stimulus to the venule wall led to a prolonged increase of marginated (sticking and rolling) leucocytes from 228/mm2 to 797/mm2 without affecting red cell velocity and shear rate. Iloprost (0.1 micrograms/kg/min i.v.), PGE1 (2.0 micrograms/kg/min i.a.), BW 755 C (40 mg/kg s.c.), forskolin (0.3 mg/kg i.v.), and hirudin (500 IU/kg + 200 IU/kg/h i.v.) all significantly attenuated injury-induced leucocyte margination. Indomethacin (10 mg/kg s.c.) had no effect and sulotroban (0.5 mg/kg/min) showed borderline efficacy. Platelet depletion by rat antiplatelet serum completely inhibited increased leucocyte margination. PGI2 mimetics, drugs interfering with lipoxygenase metabolism, and thrombin inhibitors may therefore be useful to prevent exaggerated leucocyte-vessel wall interactions.
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PMID:Adherence of leucocytes to electrically damaged venules in vivo. Effects of iloprost, PGE1, indomethacin, forskolin, BW 755 C, sulotroban, hirudin, and thrombocytopenia. 248 44

We examined the mechanism of the neutrophil (PMN)-dependent increase in pulmonary vascular permeability to protein after thrombin-induced pulmonary microembolism. Humoral factors that activate PMNs after thrombin-induced pulmonary microembolism were characterized in pulmonary lymph obtained from unanesthetized sheep challenged with intravenous infusion of alpha-thrombin. Time-dependent increases in PMN migration, aggregation, and superoxide anion (O2-) generation were induced by the pulmonary lymph obtained within 20 minutes after thrombin infusion. The pulmonary lymph neutrophil activating factors present in ether extracts of lymph had retention times of leukotriene B4 (LTB4) and monohydroxyeicosatetraenoic acids (HETEs) by high-performance liquid chromatography. The postthrombin lymph samples containing the LTB4 and HETEs increased PMN O2- generation and endothelial monolayer permeability to 125I-albumin in the presence of PMNs layered on the endothelial monolayers. Control lymph samples replete with LTB4, 5-HETE, and 15-HETE induced increases in PMN O2- generation and endothelial monolayer permeability to 125I-albumin in the presence of PMNs layered on the endothelial monolayers. Maximal increases in PMN O2- production and endothelial permeability occurred when LTB4, 5-HETE, and 15-HETE were coincubated with PMNs, indicating a synergistic action of these mediators in inducing PMN activation. Endothelial monolayer permeability to 125I-albumin did not increase with postthrombin lymph samples obtained after pretreatment with the 5-lipoxygenase inhibitor, L-651,392. The results indicate that lipoxygenase products generated in the lungs after thrombin-induced microembolism contribute to increased endothelial permeability secondary to PMN activation.
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PMID:Lipoxygenase products induce neutrophil activation and increase endothelial permeability after thrombin-induced pulmonary microembolism. 249 95

The origin of the arachidonate released from platelets on stimulation with thrombin was investigated by comparing the specific activities of released arachidonate and of arachidonoyl-containing phospholipids using rat platelets prelabelled with arachidonate. Quantification of the released arachidonate was determined in the presence of BW 755 C, a dual cyclo-oxygenase/lipoxygenase inhibitor, which was found not to modify the arachidonate mobilization between the platelet phospholipids. The phospholipid molecular species were analysed by h.p.l.c. of diradylglycerol benzoate derivatives of diacyl, alkylacyl and alkenylacyl classes. The labelled/unlabelled arachidonate ratio varied greatly in the phospholipids depending on whether an ether or acyl bond was present in sn-1 position of the glycerol, on the length and degree of unsaturation of this fatty chain and on the polar head group. Between 15 s and 5 min of stimulation by thrombin, the released arachidonate kept a constant specific activity which was considerably lower than the specific activity of diacyl-GPC. The specific activity of the released arachidonate was intermediate between the specific activities of the 16:0-20:4 and 18:0-20:4 species of diacyl-GPI and diacyl-GPE, and corresponded to the mean specific activity of alkylacyl-GPC. The data indicate that the released arachidonate cannot come directly from diacyl-GPC, and that two phospholipids in particular can act as direct precursors of the released arachidonate. These are (1) the alkylacyl-GPC and (2) the diacyl-GPE whose hydrolysis would induce an arachidonate transfer from diacyl-GPC.
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PMID:Arachidonate cannot be released directly from diacyl-sn-glycero-3-phosphocholine in thrombin-stimulated platelets. 249 34

The effects of Ca-antagonists on the thrombin-induced mobilization of radiolabeled arachidonate preincorporated into rat platelets as well as the subsequent formation of labeled cyclooxygenase and lipoxygenase products were analyzed in the presence of either Ca2+ or Ca2+-substitutes, Sr2+ and Ba2+. Results indicate that following thrombin stimulation (0.2 U/ml) in the presence of Ca2+, nitrendipine (Nit), Cd2+ or Mn2+ reduced the release of arachidonate and the biosynthesis of thromboxane B2. Inhibition of arachidonic acid release and metabolism were also obtained by both Nit and Cd2+ in the presence of Sr2+ and Ba2+. Results from studies with a semi-purified phospholipase A2 fraction prepared from rat platelets indicated that the activity was almost unaffected by Nit or Cd2+. From these findings, we concluded that inhibition of platelet-induced release and metabolism of arachidonic acid by the Ca-antagonists tested require intact platelets. These data support the hypothesis of an interaction of these agents at an unknown surface membrane level.
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PMID:Effects of organic and inorganic Ca antagonists on rat platelet arachidonic acid metabolism in the presence of Ca2+, Sr2+ and Ba2+. 249 42

The addition of arachidonic acid induced a rapid release of 45Ca2+ from human platelet membrane vesicles which accumulated 45Ca2+ in the presence of ATP. Docosahexaenoic acid, eicosapentaenoic acid, linolenic acid and linoleic acid were less active than arachidonic acid. In contrast, oleic acid, myristic acid and palmitic acid were without effect. The thromboxane A2 analogue induced no 45Ca2+ release. The cyclooxygenase/lipoxygenase inhibitor failed to suppress arachidonic acid-induced 45Ca2+ release at the concentration which inhibited the production of lipid peroxides. These data indicate that the activity of arachidonic acid may be due to fatty acid itself and not to its metabolites. The combination of arachidonic acid and inositol 1,4,5-trisphosphate (IP3) resulted in a greater 45Ca2+ release from platelet membrane vesicles than either compound alone. When the intracellular free Ca2+ concentration ([Ca2+]i) was measured using fura-2, the thrombin-induced [Ca2+]i increase was reduced in platelets which had been treated with a phospholipase A2 inhibitor, ONO-RS-082 (2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid). These results provide evidence that arachidonic acid alone may cause Ca2+ increase and also may induce an additional Ca2+ mobilization to IP3-induced Ca2+ release in human platelets.
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PMID:Evidence of Ca2+ mobilizing action of arachidonic acid in human platelets. 249 58

The techniques we developed to propagate HTM cells in serial cell culture have provided an opportunity to investigate the spectrum of endogenous PGs and other eicosanoids that are produced by these cells. PGE2 and PGF2 alpha were the major cyclooxygenase products detected by both radioimmunoassay and thin-layer chromatography. A small amount of 6-keto PGF1 alpha was also detected, indicating that these cells are able to produce prostacyclin. The observation of a substantial increase in the proportion of PGF2 alpha relative to PGE2 at later time periods after a media change suggests a metabolic conversion of PGE2 to PGF2 alpha by these cells. Bradykinin, thrombin, platelet activating factor, and serum were found to be effective stimulators of PG production by HTM cells, whereas calcium ionophore produced only a minor effect. Using high pressure liquid chromatography, elution profiles of radiolabeled metabolites of AA suggested the presence of certain lipoxygenase products, including LTB4, 12-HETE, 15-HETE, and a small amount of 5-HETE in HTM cells. The formation of these products was inhibited by both DEX and BW 755c, reinforcing the view that metabolic conversions of AA through the lipoxygenase pathway were possible in the trabecular meshwork. We also examined the effects of glucocorticoids on specific protein synthesis in the HTM cells, using 35S-methionine labeling and SDS-PAGE techniques. Short-term (1 day) DEX treatment revealed a major induction of a protein band at approximately 30 kDa. Longer treatments (1 to 3 weeks) resulted in major inductions at approximately 55 kDa inside the cells, with the presence of secreted forms (probably glycoproteins) between 55 and 72 kDa. The short-term DEX effect on protein synthesis a phospholipase inhibitor regulating eicosanoid production within the HTM. The longer-term induction may, on the other hand, be related more directly to the development of steroid glaucoma, based on our findings that the inductions of these proteins correlate with the observed time course and dose-dependence topical glucocorticoid effects on IOP. Continued in vitro and in vivo evaluations of the eicosanoid pathways in cultured HTM cells obtained from normal and glaucomatous human eyes may help to delineate their relationship to IOP regulation and the pathogenesis and treatment of glaucoma. Glucocorticoid-induced proteins may be key participants in the regulation of phospholipase activity and hence may represent a major control mechanism of the AA cascade.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Eicosanoid production and glucocorticoid regulatory mechanisms in cultured human trabecular meshwork cells. 250 21

Human platelets were incubated alone or in whole blood with specific agonists such as thrombin or collagen, and 12-hydroxy-heptadecatrienoic (HHT) and 12-hydroxy-eicosatetraenoic (12-HETE) acids were measured by HPLC as indices of platelet cyclooxygenase and lipoxygenase activities, respectively. We found that both arachidonic acid metabolites are significantly formed at concentrations of thrombin insufficient to provoke platelet aggregation. The ratio HHT/12-HETE varied with increasing concentrations of thrombin, with an increase in the absence and a decrease in the presence of albumin in the incubation. When platelets were stimulated in whole blood, this ratio favoured HHT and the addition of albumin to isolated platelets had the same effect. The formation of oxygenated products of 12-HETE by leukocyte LTB w-hydroxylase and 5-lipoxygenase in unstimulated and stimulated leukocytes, respectively, was also investigated. We failed to detect any significant amounts of these products in whole blood incubated with relatively high concentrations of collagen in the presence or absence of the chemotactic peptide FMLP. We conclude that, although 12-HETE is a good substrate for leukocyte oxygenases when incubated at high concentration with the cells alone, its oxygenation is unlikely to occur in whole blood, making 12-HETE and/or HHT potential markers of platelet activation in vivo, provided they are not substantially degraded during passage of the blood through various organs.
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PMID:Metabolism of endogenous arachidonic acid in weakly activated platelets. Absence of leukocyte cooperative products in whole blood. 251 43

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

Albumin is a major determinant of eicosanoid formation, affecting autacoids important in cell-cell interactions. We delineated three mechanisms by which albumin controlled platelet eicosanoid formation: 1) Albumin diverted free arachidonate toward 12-lipoxygenation. 2) Albumin enhanced release of arachidonate from phospholipids. 3) Albumin inhibited incorporation of arachidonate from the medium into platelet phospholipids. 12(S)-Hydroxyheptadecatrienoic acid (12-HHTrE) formation was reduced 70% by albumin as compared to that formed in albumin-free medium. In sharp contrast, formation of 12(S)-hydroxyeicosatetraenoic acid (12-HETE), the platelet lipoxygenase product, was much less influenced by albumin. Moreover, 12-HETE production in the presence of albumin was markedly increased and prolonged after aspirin treatment. These data suggested that albumin redirected released endogenous arachidonate from cyclooxygenase to lipoxygenase. Therefore, the metabolic fate of arachidonate present in the medium of stimulated platelets was studied by adding tracer [3H]arachidonate 30 sec before thrombin. Albumin increased arachidonate metabolism by lipoxygenase 7-fold as compared to albumin-free controls, while cyclooxygenation increased 2.7-fold. Redirection of eicosanoid metabolism by albumin toward lipoxygenase products constitutes a heretofore undescribed and potentially important physiological role for albumin. In vitro utilization of albumin may reflect in vivo events in thrombosis and hemostasis more accurately than previous studies without albumin could appreciate.
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PMID:Albumin redirects platelet eicosanoid metabolism toward 12(S)-hydroxyeicosatetraenoic acid. 262 19

Regulation of normal haemostasis and blood flow involves complex interactions between plasma proteins and blood cells, including platelets, leukocytes and the endothelial lining of blood vessels. Thrombin acts as a pivot in the maintenance of the haemostatic balance; the vascular endothelial cell in particular limits the generation of thrombin by localisation of anticoagulant processes on its luminal membrane. The endothelial cell synthesises key molecules in this process and also binds exogenously derived molecules, as well as releasing proteins of the fibrinolysis cascade. The thromboresistance of the luminal surface is further regulated by lipoxygenase and cyclo-oxygenase metabolites of unsaturated fatty acids synthesised by the endothelial cell. In response to trauma, inflammatory reactions, normal wound healing and in association with a variety of disease states, the anticoagulant and fibrinolytic mechanisms are downregulated and the procoagulant and thrombotic mechanisms predominate with resultant generation of thrombin, fibrin clot formation and subsequent platelet adhesion and aggregation. Pro-inflammatory and prothrombotic cytokines downregulate the fibrinolytic and activated protein C pathways as well as inducing synthesis of specific procoagulant and prothrombotic mediators by platelets and leukocytes as well as endothelium.
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PMID:Normal haemostasis and its regulation. 269 45


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