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)

Normal and mutant forms of human antithrombin-III (AT-III) were synthesized in a cell-free system in order to identify putative functional domains required for heparin binding and complex-formation with alpha-thrombin. Heparin-Sepharose chromatography resulted in the elution of approx. 70% of cell-free-derived normal AT-III-(1-432)-polypeptide as a peak between 0.2 M- and 0.7 M-NaCl. The cell-free-derived normal AT-III also reacted with alpha-thrombin. Approx. 15% of this AT-III formed covalent complexes with alpha-thrombin in 2 min. Unfractionated heparin accelerated the rate of formation of such complexes. Two truncated forms of AT-III (amino acid residues 219-432 and 251-432), containing only the putative thrombin-binding domain, were synthesized independently in this cell-free system. These truncated AT-III polypeptides did not bind heparin and were unable to form stable covalent complexes with alpha-thrombin. However, both of these AT-III polypeptides were cleaved by alpha-thrombin, presumably at the reactive centre Arg-393-Ser-394. The formation of the disulphide bond between Cys-247 and Cys-430 in AT-III-(219-432)-polypeptide had no effect on the results obtained. Mutations in full-length AT-III at Cys-430 had no effect on the ability of AT-III to bind heparin. There was, however, a slight decrease in the formation of stable inhibitory complexes with alpha-thrombin. A cell-free-derived AT-III mutant, devoid of amino acid residues 41-49, which comprise heparin-binding region 1 of AT-III, had slightly decreased heparin binding compared with cell-free-derived normal AT-III-(1-432)-polypeptide. This mutant AT-III polypeptide was unable, however, to form a stable complex with alpha-thrombin. We conclude therefore that the N-terminal domain of AT-III is essential for both heparin binding and complex-formation with alpha-thrombin, but not for the cleavage of AT-III at its reactive centre by alpha-thrombin.
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PMID:The N-terminal domain of antithrombin-III is essential for heparin binding and complex-formation with, but not cleavage by, alpha-thrombin. 154 50

Antithrombin-III-Stockholm is a new structural variant of antithrombin-III (AT-III) with normal heparin affinity but defective serine protease inhibitory activity. The proposita, a white female born in 1966, was diagnosed to have developed a pulmonary embolus while on oral contraceptives at age 19. The proposita, as well as her father, were diagnosed to have a type 2 AT-III deficiency as they had normal levels of immunoreactive AT-III associated with decreased (approximately 60%) functional AT-III when measured with either alpha-thrombin or factor Xa as the substrate, either in the presence or absence of heparin. There was no evidence of abnormal electrophoretic mobility of AT-III from the proposita either in the presence or absence of heparin. Genomic DNA was prepared and all seven AT-III exons were polymerase chain reaction (PCR)-amplified and sequenced in both directions using nested primers. Only exon 7 provided evidence for the presence of a mutation, with the second base of codon 392 having a G----A substitution. Such a mutation would cause the substitution of aspartic acid at the site of the normally appearing glycine in the translated product. Furthermore, this mutation caused the destruction of an Hae III restriction site at this point in the AT-III gene. The absence of this Hae III site was confirmed using restriction fragment length polymorphism analysis of PCR-amplified material from the proposita. Experiments with AT-III from the proposita together with experiments with cell-free translated AT-III-Stockholm provided evidence that the mutant AT-III protein does not efficiently form a stable covalent inhibitory complex with alpha-thrombin, although it exhibits normal heparin affinity. The minimal thrombin-complexing ability of the mutant AT-III protein that was observed was accelerated by heparin, but to subnormal levels.
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PMID:Antithrombin-III-Stockholm: a codon 392 (Gly----Asp) mutation with normal heparin binding and impaired serine protease reactivity. 1190 37

We have reviewed the laboratory methods used to diagnose the prethrombotic state, defined as a procoagulant imbalance between the production and inhibition of enzyme activity in the coagulation pathway short of fibrin deposition. One diagnostic approach is that of measuring the plasma levels of activation peptides which are released from the zymogens of the coagulation cascade when they are activated. Another approach is that of measuring the plasma levels of complexes formed in plasma when enzymes of the coagulation cascade are neutralized by their naturally-occurring inhibitors such as thrombin-antithrombin complex. The clinical usefulness of these methods still needs to be defined with prospective studies.
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PMID:Detection of the prethrombotic state due to procoagulant imbalance. 154 77

Injury to the pleura ultimately results in either repair with fibrosis or repair without fibrosis and a reestablishment of the normal mesothelial monolayer. The role of the mesothelial cell, and of local mediators, in these repair processes remains essentially undefined. In order for repair without fibrosis to occur, mesothelial cells, in response to local mediators, must be capable of migration and/or proliferation to cover the injured and denuded mesothelium. We hypothesized that rat pleural mesothelial cells were capable of both chemotaxis and proliferation in response to thrombin. In an in vitro assay, mesothelial cells demonstrated directed migration in response to a known chemoattractant, formylmethionylleucylphenylalanine. In addition, mesothelial cells demonstrated chemotaxis in a dose-dependent manner in response to thrombin, with a maximal response at a concentration of 10(-8) M. Finally, this chemotaxis was blocked by a specific blocker of thrombin, antithrombin 3. Thrombin also stimulated mesothelial cell proliferation, which was measured both in a [3H]thymidine incorporation assay and by direct cell counts. Again, the response was dose dependent, with the maximal response at 10(-8) M causing the same amount of [3H]thymidine incorporation as 10% fetal bovine serum. As before, this response was completely blocked by antithrombin 3. These results demonstrate that mesothelial cells are capable of both chemotaxis and proliferation in response to thrombin. Thrombin may play an important role in the regulation of pleural repair without fibrosis and the re-establishment of the mesothelial monolayer.
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PMID:Mesothelial cell response to pleural injury: thrombin-induced proliferation and chemotaxis of rat pleural mesothelial cells. 155 Jun 87

There is a controversy about whether or not histidine-rich glycoprotein (HRG), the most abundant plasma protein with glycosaminoglycans-neutralizing capacity, is able to prevent the inhibition of human thrombin by heparin cofactor II (HC II) in the presence of dermatan sulfate (DS). The authors studied the interaction of DS and low molecular weight DS, in a purified system with HRG, platelet factor 4 (PF 4), and with HC II. Their results show that HRG, like PF 4, has an affinity, not only for heparin, but also for DS. However, this affinity seems very weak. In fact, HRG is 10 times less effective than PF 4 in neutralizing the 50% antithrombin activity of HC II in the presence of DS.
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PMID:Interaction between histidine-rich glycoprotein and platelet factor 4 with dermatan sulfate and low-molecular-weight dermatan sulfate. 155 54

Several studies suggest that diabetes is associated with a hypercoagulable state. Therefore determination of thrombin-antithrombin complex (TAT) could represent a sensitive parameter for specific detection of a latent activation of the clotting system. The present study documents increased plasma TAT in a heterogeneous group of non-insulin-dependent diabetic patients. The finding of increased TAT levels both in diabetic patients with vascular complications and in vascular disease patients without diabetes suggests a relationship between existing vascular disease and the hemostatic mechanism that produces augmented thrombin activity. In acute vascular occlusions the presence of diabetes seems to increase activation of the coagulative system.
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PMID:Thrombin-antithrombin III complexes in type II diabetes mellitus. 156 62

We retrospectively evaluated the hemostatic system of 13 patients during implantation (2 to 35 days) of the Jarvik 7-70 total artificial heart (TAH). Although all patients were clinically manageable while on the TAH, 5 had excessive generalized bleeding. After the heart transplant procedure, 2 patients had neurological events and 1 patient, thrombosis of the leg. While the patients were supported by the TAH, the routine coagulation assays (prothrombin time, activated partial thromboplastin time, fibrinogen, factor assays, and platelet count) showed slight abnormalities but no correlation to hemorrhagic or thrombotic events. In contrast, plasma and cellular activation markers, which are highly sensitive and specific for hypercoagulability, fibrinolysis, or platelet activation, revealed activation in all patients. Most striking was the marked activation of the fibrinolytic system (p less than 0.05 to 0.001). Correlations of individual patient data compared with the average TAH group response could be made between excessive enhancement of fibrinolysis (increased D-dimer and tissue plasminogen activator and decreased plasminogen activator inhibitor) and bleeding. A hypercoagulable state (increased fibrinogen and thrombin-antithrombin complex and decreased antithrombin III and protein C), decreased fibrinolysis (decreased tissue plasminogen activator and D-dimer), activated platelets (increased thromboxane B2), or combinations of these were associated with thrombosis. The hemostatic activation returned to normal 1 day after removal of the TAH. These data suggest that the patient with a TAH requires more sophisticated laboratory monitoring and individualized treatment for excessive fibrinolysis, hypercoagulable state, or platelet activation to avoid thrombotic and hemorrhagic complications.
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PMID:Hemostatic abnormalities in total artificial heart patients as detected by specific blood markers. 157 Sep 81

Frequent peroperative sampling of arterial and mixed venous blood was undertaken in eight consecutive patients during cemented hip arthroplasty to obtain a sequential picture of thrombin-antithrombin (TAT) and methylmethacrylate monomer (MMA) concentrations. TAT, detected neo-antigenically, reached its maximum levels after bone preparation, prior to introduction of cement and prosthesis. Furthermore, TAT values were higher in arterial than in mixed venous blood, indicating generation and inactivation of thrombin when blood is passing the lung. MMA concentration, measured by high pressure liquid chromatography, increased very rapidly and declined within 1 min following socket as well as shaft implantation, reaching the highest mean value of 3599 ng/ml only 30 s after femoral impaction of cement. MMA concentration was higher in mixed venous than in arterial blood, indicating elimination of the monomer during lung passage. This study confirms our previous observation that coagulation is activated prior to the implantation of bone cement and prosthesis into the femoral shaft; in addition, it provides further evidence of the thrombin-generating potential of the pulmonary capillary bed. Very frequent sampling was necessary to reveal the sharp rise in MMA levels following impaction of cement. Further investigations are needed to establish whether interaction between activated coagulation and MMA may predispose to cardiovascular complications.
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PMID:Intrapulmonary thrombin generation and its relation to monomethylmethacrylate plasma levels during hip arthroplasty. 159 39

Platelets and coagulation factors were studied during 24-hour heparin-free veno-right ventricular extracorporeal membrane oxygenation (ECMO) in 6 healthy pigs. An endpoint attached and covalently bonded heparin-coated ECMO system was used in these experiments. The veno-right ventricular ECMO supplied the total lung function of the animals, and after 24 hours, all the animals were successfully weaned from ECMO. Lung function and central hemodynamics were not affected by the procedure. Because all the animals showed a significant reduction in plasma volume, the concentration of measured coagulation variables was corrected both for plasma volume changes and for hemodilution. The platelet count and the plasma-free hemoglobin level were not significantly altered by ECMO. Similarly, the prothrombin complex, antithrombin, thrombin-antithrombin complex, factor XII, and the urinary excretion of 2,3-dinor-thromboxane B2 were not significantly altered. Fibrinogen and fibrin monomer increased significantly, whereas von Willebrand factor was significantly decreased after ECMO. In summary, 24-hour heparin-free veno-right ventricular total extracorporeal lung assistance does not affect the platelets and the coagulation system significantly in healthy juvenile pigs.
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PMID:Twenty-four-hour heparin-free veno-right ventricular ECMO: an experimental study. 159 27

The synthetic antithrombin-binding heparin pentasaccharide and a full-length heparin of approximately 26 saccharides containing this specific sequence have been compared with respect to their interactions with antithrombin and their ability to promote inhibition and substrate reactions of antithrombin with thrombin and factor Xa. The aim of these studies was to elucidate the pentasaccharide contribution to heparin's accelerating effect on antithrombin-proteinase reactions. Pentasaccharide and full-length heparins bound antithrombin with comparable high affinities (KD values of 36 +/- 11 and 10 +/- 3 nM, respectively, at I 0.15) and induced highly similar protein fluorescence, ultraviolet and circular dichroism changes in the inhibitor. Stopped-flow fluorescence kinetic studies of the heparin binding interactions at I 0.15 were consistent with a two-step binding process for both heparins, involving an initial weak encounter complex interaction formed with similar affinities (KD 20-30 microM), followed by an inhibitor conformational change with indistinguishable forward rate constants of 520-700 s-1 but dissimilar reverse rate constants of approximately 1 s-1 for the pentasaccharide and approximately 0.2 s-1 for the full-length heparin. Second order rate constants for antithrombin reactions with thrombin and factor Xa were maximally enhanced by the pentasaccharide only 1.7-fold for thrombin, but a substantial 270-fold for factor Xa, in an ionic strength-independent manner at saturating oligosaccharide. In contrast, the full-length heparin produced large ionic strength-dependent enhancements in second order rate constants for both antithrombin reactions of 4,300-fold for thrombin and 580-fold for factor Xa at I 0.15. These enhancements were resolvable into a nonionic component ascribable to the pentasaccharide and an ionic component responsible for the additional rate increase of the larger heparin. Stoichiometric titrations of thrombin and factor Xa inactivation by antithrombin, as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the products of these reactions, indicated that pentasaccharide and full-length heparins similarly promoted the formation of proteolytically modified inhibitor during the inactivation of factor Xa by antithrombin, whereas only the full-length heparin was effective in promoting this substrate reaction of antithrombin during the reaction with thrombin.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Role of the antithrombin-binding pentasaccharide in heparin acceleration of antithrombin-proteinase reactions. Resolution of the antithrombin conformational change contribution to heparin rate enhancement. 161 58


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