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)

Disseminated intravascular coagulation was induced in rats by injection of a silver colloid suspension or thrombin. Ten min after the injection of colloid, fibrin deposits were observed light microscopically in all major organs. At 30 min, fibrin was no longer present. In rats treated with antifibrinolytics (epsilon-aminocaproic acid or Trasylol) fibrin was still present at 30 and 60 min. Interaction of fibrin with Kupffer cells was studied by electron microscopy. At 3, 10, and 20 min after the colloid injection, all fibrin occurred extracellularly, close to the surface of Kupffer cells. At 30 min, all fibrin had disappeared. In rats pretreated with antifibrinolytics, too, all fibrin was found extracellularly at 10, 30, and 60 min. Comparable results were obtained when thrombin was used to induce coagulation. It is concluded that removal of native fibrin from the circulation by Kupffer-cell phagocytosis is unlikely.
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PMID:Rat liver macrophages will not phagocytose fibrin during disseminated intravascular coagulation. 100 4

Thrombomodulin (TM) is a surface glycoprotein that forms a 1:1 complex with thrombin, thereby interacting to form the basis of a major physiologically relevant natural anticoagulant mechanism. Although initially described as a vascular endothelial cell receptor, TM has been reported to be present in several other cells, including megakaryocytes, platelets, monocytes, and several cultured cells. Other investigators have reported that neutrophils (PMN) may play a role in the hemostatic mechanism by supporting transformation of prothrombin to thrombin. To determine whether PMN might contribute further to the regulation of the coagulation system, we have evaluated these cells for the expression of TM. Large numbers of human leukocytes were isolated by standard techniques, and the PMN fraction was extracted and shown to be free of platelets and monocytes. Membrane preparations were affinity purified on an anti-TM-Affigel-10 matrix and the eluted material was examined by Western blotting, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and silver staining. The purified material was identical in apparent molecular weight to TM from human placenta and human umbilical vein endothelial cells (HUVEC). Using a sensitive and specific immunoassay, we estimated that there are a minimum of 5,220 +/- 1,658 molecules of TM per PMN, as compared with more than 50,000 in HUVEC. Northern analysis of RNA from PMN indicates that specific messenger RNA for TM, as identified by a single 3.8-kb band, is identical to that from HUVEC, and thereby confirms that PMN can also synthesize the receptor. Localization of TM in PMN was attempted by immunofluorescence, and the receptor was visualized only in permeabilized PMN, but was not seen on the surface of nonpermeabilized cells. Flow cytometry was also used, and could detect TM in 10% to 15% of nonpermeabilized PMN, whereas the antigen was present in greater than 80% of permeabilized cells. Biologic function of the PMN-derived TM, as tested by thrombin-dependent activation of protein C, was absent. Our results suggest that TM is synthesized by PMN, but under nonstimulated conditions, the protein is largely excluded from the membrane surface, and lacks the ability to promote activation of protein C by thrombin. TM from PMN may provide a further link between inflammation and thrombosis and may also be a significant source of plasma TM.
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PMID:Human neutrophils synthesize thrombomodulin that does not promote thrombin-dependent protein C activation. 132 11

The localization of tissue plasminogen activator (t-PA) on microthrombi in various organs of disseminated intravascular coagulation rats (DIC rats) was investigated by using microautoradiographic technique. After the injection of [125I]fibrinogen, experimental DIC rats induced by the infusion of thrombin for 1 h were submitted to microautoradiograms (MARGMs) of some major organs. The radioactivity of [125I]fibrin thrombi, which were observed as silver grains, was localized in the glomeruli and parts of small vessels in the kidney. In the liver, microthrombi were seen in sinusoid vessels and on Kupffer cells. In addition, many microthrombi were noted in small vessels in the lung and marginal zones in the spleen. Two min after the intravenous administration of [125I]t-PA to DIC rats, many silver grains were observed on each MARGM of the kidney, lung, liver and spleen showing the formation of microthrombi. From the identical results with the observations of MARGMs after the injection of [125I]fibrinogen, we confirmed that t-PA was highly accumulated to microthrombi formed in small vessels of the organs. The scattered silver grains were widely observed on the hepatocytes. This result suggested that t-PA bound to the parenchymal cell surface might be transported into the hepatocytes by receptor-mediated endocytosis. On the other hand, when [125I]urokinase plasminogen activator [( 125I]u-PA) was administered intravenously to DIC rats, many silver grains were observed on MARGM of the proximal tubules in the kidney but not seen on MARGMs of the glomeruli in the kidney, nor in the lung, liver, and spleen. This observation suggested that u-PA might not have a characteristic to accumulate to thrombi.
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PMID:Localization of tissue plasminogen activator on experimental microthrombi in rats. Microautoradiographic observations. 190 22

The permeability response of endothelial monolayers to some "direct-action" type mediators of vasopermeability were studied in vitro. Endothelial cells, cultured to confluence on denatured collagen-coated dextran microcarriers or gelatin microcarriers, prevented staining of the microcarriers with Evans blue dye. Increases in staining, as determined by the spectrophotometric quantitation of the dye after extraction from the microcarriers with formamide, occurred after treatment of human umbilical vein endothelium with histamine (10(-5) M) or thrombin (0.1 U/ml). These increases in monolayer permeability were reversible. Neither bradykinin nor serotonin had any effect in this system. Endothelial monolayers cultured this way consistently stained with silver nitrate at the cell junction areas. Monolayer response to histamine was characterized morphologically by small openings which occurred randomly along the cell junctions; while with thrombin, the spaces, which had developed at junctions, occurred to a greater extent. Prostaglandin E1 (30 microM) and isoproterenol (10 microM), in the presence of 3-isobutyl-1-methylxanthine (1 mM), partially inhibited histamine- and thrombin-mediated changes in permeability. This model responds to certain vasopermeability-altering agents in a manner similar to that of the microcirculation. These studies support the concept that the vasopermeability enhancing effect of histamine in vivo results, in part, from a direct effect on the endothelium.
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PMID:Increased permeability of microcarrier-cultured endothelial monolayers in response to histamine and thrombin. A model for the in vitro study of increased vasopermeability. 241 88

Glucocorticoids have been shown to decrease prostaglandin I2 synthesis in human endothelial cells, suggesting the possible involvement of lipocortin in the inhibition of arachidonic acid liberation achieved by phospholipase A2 (De Caterina, R., and Weksler, B. B. (1986) Thromb. Haemostasis 55, 369-374). To test this hypothesis, human endothelial cells labeled with [14C]arachidonic acid were stimulated with thrombin (2 units/ml, 10 min), resulting in the secretion of free arachidonic acid together with various 14C-labeled metabolites, mainly 6-keto-prostaglandin F1 alpha, the stable derivative of prostaglandin I2. Under conditions where prior incubation of cells with dexamethasone reduced by 51% 6-keto-prostaglandin F1 alpha production, phospholipid hydrolysis induced by thrombin remained unaffected. Using three rabbit polyclonal antibodies directed against endonexin I, lipocortin I, and lipocortin II, evidence was obtained for the presence in human endothelial cells of equivalent amounts of lipocortin I and an immunologically unrelated 33-kDa protein, together with lower quantities of 67-kDa calelectrin/calcimedin. These Ca2+- and phospholipid-binding proteins were selectively extracted with [ethylene-bis(oxyethylene-nitrilo)]tetraacetic acid (EGTA) from cell membranes precipitated in the presence of Ca2+, and they displayed an inhibitory activity against pig pancreas phospholipase A2. However, the amounts of the three proteins were not changed by cell treatment with 2.5 microM dexamethasone, as detected upon polyacrylamide gel electrophoresis by silver staining, immunoblotting, or autoradiography following [35S]methionine in vivo labeling. Since the antiphospholipase A2 activity of EGTA extracts was hardly modified, it was concluded that an increased synthesis of lipocortin cannot account for the inhibition of prostaglandin synthesis brought about by dexamethasone, suggesting other biological functions for these proteins.
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PMID:Effect of dexamethasone on prostaglandin synthesis and on lipocortin status in human endothelial cells. Inhibition of prostaglandin I2 synthesis occurring without alteration of arachidonic acid liberation and of lipocortin synthesis. 252 36

The aim of this study was to investigate the platelets of a Glanzmann thrombasthenic patient, which in citrated PRP failed to respond to various agonists, but aggregated and secreted to high concentrations of thrombin (0.36, 0.72 and 1 U/ml) and collagen (4, 10 and 20 micrograms/ml) when washed and resuspended in a Tyrode-albumin solution (containing 2 mM Ca2+). Aggregation of the patient platelets was not affected by anti-IIb/IIIa monoclonal antibody (P18) which strongly inhibits thrombin or collagen induced aggregation of normal platelets. Washed platelets of this patient did not aggregate to ADP (10-100 microM) in the presence of added fibrinogen (2 mg/ml) nor bind 125I-labelled fibrinogen (40 to 320 micrograms/ml) when thrombin-stimulated. Different anti-IIb/IIIa monoclonal antibodies (P2, P18) when used in binding or crossed immunoelectrophoretic studies showed a complete absence of the IIb-IIIa glycoprotein complex on the patient platelets. Moreover, glycoproteins IIb or IIIa were absent on silver-stained two-dimensional (non-reduced/reduced) polyacrylamide gel separations of the patient platelets and were not detected by Western blots used in combination with anti-PLA1 (antigen present on IIIa), anti-Leka (antigen present on IIb). This study shows that platelets lacking glycoproteins IIb or IIIa can aggregate in response to high concentrations of collagen or thrombin when resuspended in the presence of physiological concentrations of calcium. Results obtained in this study could indicate the existence of other mechanisms (other than the IIb-IIIa glycoprotein complex) involving glycolipids, heparans, proteoglycans, and/or unknown membrane glycoproteins to mediate platelet aggregation of stimulated thrombasthenic platelets.
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PMID:Aggregation to thrombin and collagen of platelets from a Glanzmann thrombasthenic patient lacking glycoproteins IIb and IIIa. 259 66

The kinetic parameters and some enzymatic characteristics of human platelet and chicken gizzard transglutaminases were determined. Activity of the transglutaminases was regulated by calmodulin. These enzymes co-isolated with alpha-actinin and were dissociated from alpha-actinin by gel filtration and absorption onto a calmodulin affinity column. Silver-stained polyacrylamide gels showed that the protein peak eluted by EGTA from this column contained polypeptides of Mr approximately 58,000 and 63,000. The transglutaminases required Ca2+ for incorporation of monodansylcadaverine into casein and actin substrates. Activity was enhanced 3-fold by calmodulin with a biphasic effect, showing stimulation at 10-200 nM and inhibition at concentrations higher than 300 nM. In the presence of 200 nM calmodulin, half-maximal transglutaminase stimulation was obtained with 2.5 microM free [Ca2+]. Chlorpromazine inhibited calmodulin enhancement of the transglutaminases. Activity of the transglutaminases was independent of proteolytic activation, since inhibitors for Ca2+-dependent proteases failed to inhibit filamin cross-linking. For comparison, factor XIIa, a plasma and platelet transglutaminase, required both Ca2+ and thrombin for activation and was insensitive to calmodulin. The cross-linking pattern of fibrin, fibrin monomers, and fibrinogen by the calmodulin-regulated transglutaminases showed, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, disappearance of fibrinogen alpha-chains with no decrease of beta- and gamma-chains or formation of gamma-gamma dimers. By autoradiography, cross-linked products of 125I-fibrinogen revealed heavily labeled high molecular weight polymers and polypeptides of Mr 98,000, 116,000, and 148,000; the latter appeared to be a transient species. However, when fibrin, fibrin monomers, and fibrinogen were used as factor XIIIa substrates, gamma-gamma dimers and alpha-polymers were formed. Formation of gamma-gamma dimers was slower with fibrinogen than with fibrin. Iodoacetamide blocked activity of factor XIIIa but not of the calmodulin-regulated transglutaminases.
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PMID:Catalytic properties of a calmodulin-regulated transglutaminase from human platelet and chicken gizzard. 286 89

To study association of platelets with factor VIII, the purified protein was 125I-labelled with Bolton-Hunter reagent to a specific activity of 243,000-360,000 cpm/U. Autoradiographs of SDS polyacrylamide gels revealed polypeptides of VIII at Mr about 240 kDa, 90 kDa and intermediate values, as well as some radioactive contaminants, but the light chain (Mr 78/76) seen with silver stain was not labelled. After 2.5-5-fold activation with thrombin, the higher radioactive Mr band disappeared, the band at 90 kDa became more intense, and a band appeared at about 45 kDa. The radioactivity associated with platelets, studied in the presence of haemophilic BaSO4-treated plasma, was maximal after 6-8 min and increased 3-15-fold on activation with thrombin. With activated VIII, autoradiographs of platelet pellets showed only VIIIa but results are expressed as units of unactivated VIII bound. At 0.3-0-0.7 U/ml, 10(8) platelets bound 0.0008-0.004 U VIIIa. The amount bound was not affected by the ratio of unlabelled VIII to VIII labelled in the presence of a 50-fold molar excess of unlabelled Bolton-Hunter reagent. Binding increased to 1.5 U VIIIa/10(8) platelets (about 13,600 molecules per platelet) at 140 U/ml, with no evidence of saturation. Binding was not affected by monoclonal antibodies to platelet glycoproteins IIb/IIIa or Ib, quenching the thrombin before adding platelets, or aggregating the platelets with A23187 in the presence of thrombin. Qualitatively, binding of labelled VIIIa and factor Va studied by others are similar. Binding of 125I-VIIIa to aggregated and unaggregated platelets was normal in patient M.S. whose platelets were shown by others to be deficient in their ability to bind radiolabelled factor Xa and generate coagulant activity. This difference. and the fact that platelet coagulant activity is increased by platelet activation and/or aggregation, suggest that binding of Va or VIIIa alone does not determine the assembly of active proteolytic complexes on the platelet surface.
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PMID:Binding of thrombin-activated human factor VIII to platelets. 309 74

Lysophospholipase released from rat platelets upon activation with thrombin has been purified to near homogeneity by sequential column chromatography on heparin-Sepharose, CM-Sephadex C-50, and TSK gel G2000SW. The final preparation showed a single band with a molecular mass of 32,000 daltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining. The purified enzyme was heat-labile and inactivated after 5 min at 60 degrees C. It showed a broad pH optimum (pH 6-10) and required a divalent cation, such as Ca2+, for the optimal activity. Appreciable activity, however, was observed in the presence of EDTA. Lysophospholipase activity was inhibited by diisopropylfluorophosphate and dithiothreitol. This enzyme activity was retained by a concanavalin A-Sepharose column and eluted with methyl-alpha-D-mannoside. Treatment of lysophospholipase with peptide: N-glycosidase F gave degraded products, suggesting that this protein contain N-linked carbohydrate chains. The purified enzyme was specific to 1-acyl-sn-glycero-3-phospho-L-serine; none of lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylinositol, and 1-acyl-sn-glycero-3-phospho-D-serine was hydrolyzed appreciably.
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PMID:Purification and characterization of lysophospholipase released from rat platelets. 339 99

Human glycoprotein Ib (GPIb) is a major integral membrane protein on human blood platelets responsible for the initial attachment of platelets to the exposed vascular subendothelium. In this report we describe an isolation method for a 'GPIb complex' as well as for its individual components. A three-step procedure involving Triton X-114 phase-partition, affinity chromatography on wheat germ agglutinin and ion-exchange chromatography on DEAE-Sephacel yielded milligram quantities of purified GPIb complex. The single components of the complex were further purified by gel filtration on AcA34 in 0.1% sodium dodecyl sulfate. As well as GPIb, the complex contains GP17, actin-binding protein, actin and a series of unidentified proteins with different molecular masses. In contrast to GPIb alpha, which is very rich in O-linked oligosaccharides, sugar analysis revealed that GPIb beta and GP17 seem to have only N-linked chains of the lactosamine type. The C-terminal alpha-chain remnant, which probably spans the plasma membrane, was identified and isolated for the first time. Western blotting with polyclonal rabbit anti-GPIb antibodies and silver-staining of one- or two-dimensional dodecyl sulfate/polyacrylamide gels revealed that it has an apparent molecular mass of 20 kDa and is linked to GPIb beta by a disulfide bridge close to the membrane. The thrombin-binding site on GPIb is located near the N-terminus on a 40-kDa fragment of GPIb alpha. A disulfide bridge in the N-terminal region is not essential for thrombin binding to GPIb.
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PMID:The glycoprotein Ib complex of human blood platelets. 381 2


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