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)

The inhibitory effects of a newly synthesized protease inhibitor, Gabexate mesilate (FOY), on experimental disseminated intravascular coagulation were studied as compared with those of aprotinin or heparin. Thrombin, tissue thromboplastin, and endotoxin were used as DIC trigger substances. As parameters on DIC, platelet counts, white blood cell counts, neutrophilic leukocyte counts, fibrinogen, fibrin degradation products, platelet retention, platelet aggregation, prothrombin time, partial thromboplastin time were served. The drug efficacy in each parameter were expressed by the score system and analyzed statistically. The results were summarized as follows; (1) In thrombin-induced DIC, FOY was apparently superior to the other drugs (p less than 0.05). (2) In thromboplastin-induced DIC, heparin was slightly more effective than FOY or aprotinin. (3) In endotoxin infusion, there were no significant differences among them. In conclusion, the results of the present study suggest that FOY was more effective than heparin or aprotinin on experimental DIC.
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PMID:Inhibitory effects of gabexate mesilate (FOY) on experimental DIC. 22 8

Preparations of low molecular weight porcine heparin with an average specific anticoagulant activity of 94 units/mg were fractionated into "active" and "relatively inactive" forms of the mucopolysaccharide of approximately 6000 daltons each. The active fraction was further subdivided into various species with descending but significant affinities for the protease inhibitor as well as decreasing but substantial anticoagulatn potencies. "Highly active" heparin (approximately 8% of the low molecular weight pool) possesses a specific anticoagulant activity of 350 +/- 10 units/mg. The relatively inactive fraction (67% of the low molecular weight pool) exhibits a specific anticoagulant activity of 4 +/- 1 units/mg. The binding of highly active heparin to antithrombin is accurately described by a single-site binding model with a KHep-ATDISS of approximately 1 X 10(-7) M. Variations in this binding parameter secondary to changes in environmental variables indicate that charge-charge interactions as well as an increase in entropy are critical to the formation of the highly active heparin-antithrombin complex. The interaction of relatively inactive heparin with the protease inhibitor is characterized by an apparent KHep-ATDISS of 1 X 10(-4) M. In large measure, this is due to small amounts of residual active mucopolysaccharide (0.5%). The ability of the highly active heparin to accelerate the thrombin-antithrombin interaction was also examined. We were able to demonstrate that the mucopolysaccharide acts as a catalyst in this process and is able to initiate multiple rounds of enzyme-inhibitor complex formation. The rate of enzyme neutralization is increased to a maximum of 2300-fold as the concentration of heparin is raised until the inhibitor is saturated with mucopolysaccharide. Further increases in heparin concentration result in a reduction in the speed of enzyme neutralization. This appears to be due to the formation of thrombin-heparin complexes. A mathematical model is given which provides a relationship between the initial velocity of enzyme neutralization and reactant concentrations.
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PMID:Fractionation of low molecular weight heparin species and their interaction with antithrombin. 42 27

Aprotinin has been shown to reduce blood loss and blood requirement when administered prior to surgery and this therapeutic benefit appears to be related to its specificity as a protease inhibitor. The inhibition of plasmin by aprotinin is well characterized, but little is known of its effect on thrombin. In preliminary experiments, we showed that aprotinin can prevent platelet aggregation induced by thrombin. Follow-up studies have now been performed in order to clarify the effect of aprotinin on thrombin. A fluorescence study of the direct binding of aprotinin to human alpha-thrombin was analysed according to the Michaelis-Menten model and a dissociation constant of 30 x 10(-6) mol.l-1 was determined. Aprotinin can displace p-aminobenzamidine, a fluorescent-probe molecule which binds to the active site of serine proteases, showing that the active site of thrombin was involved. Aprotinin also inhibited the ability of thrombin to induce a fibrin clot from purified fibrinogen and to induce the hydrolysis of the chromogenic substrate H-D-phenylalanylpipecolylarginine-p-nitroanalidehydrochloride++ + (S-2238). With S-2238, double-reciprocal plots show that the inhibition is competitive with a Ki of 61 microM and a Km of 1.72 microM. Aprotinin was a potent inhibitor of thrombin-induced aggregation. A Schild plot of the aggregation data yielded a slope of 0.97 +/- 0.12 and an apparent dissociation constant of 57.0 +/- 13.1 microM (mean +/- SEM). Thus, the inhibition of thrombin-induced platelet aggregation by aprotinin fits a model of competitive inhibition. Conclusions are that, in addition to a possible direct effect of aprotinin on platelets, the inhibition of thrombin-induced platelet activation by aprotinin can be also explained, in part, by a direct effect of the inhibitor on the thrombin molecule itself. This supports the concept that a proteolytic step is involved in the platelet response to thrombin. Finally, evidence is in favour of the participation of Trp245 in the fluorescence response of thrombin on binding to aprotinin.
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PMID:Aprotinin can inhibit the proteolytic activity of thrombin. A fluorescence and an enzymatic study. 137 6

Thrombin, a peptide with native protease activity, caused a rapid (less than 1 min) increase in glycogenolysis of about 30%, assessed from rates of production of glucose+lactate+pyruvate, and in oxygen uptake in perfused rat liver. These increases were followed by a rapid return to basal values within 5 min. The effect of thrombin on glycogenolysis was dose-dependent and was maximal at perfusate concentrations around 1 U/ml. Interestingly, the effect of thrombin on glycogenolysis could be elicited only once in any given liver. The activation of glycogenolysis by thrombin was diminished nearly 50% by prior infusion of the protease inhibitor, diisopropyl fluorophosphate (10 microM), and over 90% when thrombin was treated with diisopropyl fluorophosphate prior to infusion. The stimulation of glycogenolysis by thrombin could be detected in isolated hepatocytes or in livers stored for 24 h in cold Euro-Collins solution, a treatment which destroys endothelial cells. Further, thrombin stimulated production of prostaglandin D2 from arachidonic acid in cultured hepatic endothelial but not Kupffer cells. The effect of thrombin on carbohydrate output was also blocked by a phospholipase A2 inhibitor (quinacrine, 50 microM) and by an inhibitor of the cyclooxygenase (indomethacin, 20 microM), suggesting the involvement of cyclooxygenase in the mechanism of action of thrombin. In support of this idea, the transient kinetics of stimulation of glycogenolysis by thrombin and arachidonic acid was nearly identical to release of thromboxane B2 (80-420 pg/ml) and prostaglandin D2 (300-900 pg/ml) from the perfused liver. Further, a second addition of thrombin failed to increase thromboxane and prostaglandin D2 release as well as carbohydrate production, supporting a causal link between these phenomena. Taken together, these data support the hypothesis that thrombin interacts with receptors in the liver, possibly on endothelial cells, leading to activation of phospholipase A2 and subsequent transient production of prostaglandins and thromboxanes. These mediators subsequently interact with receptors on parenchymal cells, leading to a transient stimulation of glycogenolysis.
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PMID:Transient activation of hepatic glycogenolysis by thrombin in perfused rat livers. 139 79

The molecular defect responsible for a dramatic prolongation of all standard clotting tests discovered in a 15-yr-old boy has been identified. Initial investigations revealed the presence of an activated Factor X (Factor Xa) and thrombin inhibitor which copurified with alpha 1-antitrypsin (alpha 1-AT), thereby suggesting the occurrence of an alpha 1-AT variant similar to alpha 1-AT Pittsburgh. This was confirmed by dot-blot analysis and direct sequencing after amplification by the polymerase chain reaction. A G to T transition at nucleotide 10038 results in the substitution of Met to an Arg, converting alpha 1-AT into an Arg-Ser protease inhibitor (serpin) that inhibited thrombin and Factor Xa more effectively than antithrombin III. Surprisingly, there was no bleeding history in the proband. The common mutation Z, which may explain a reduced expression of the allele bearing the Arg 358 Met mutation, was not observed in the propositus' DNA. To exclude the presence of another mutation, the coding regions and intron/exon junctions were sequenced. No other mutation was found. Recently, the patient experienced his first hemorrhagic episode at the age of 17. The level of the abnormal inhibitor had increased twofold 2 mo before. The large decrease in protein C concentration may account for the mild bleeding tendency in this case, despite the presence of the alpha 1-AT Pittsburgh mutation. An abnormal protein C pattern was observed in patient's plasma, suggesting that the circulating deficiency might be due to a deleterious effect of the abnormal inhibitor on both intracellular processing and catabolism of protein C.
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PMID:Met 358 to Arg mutation of alpha 1-antitrypsin associated with protein C deficiency in a patient with mild bleeding tendency. 156 92

The relationship between thrombomodulin-associated O-linked glycosammoglycans (GAGs) and the exogenous GAGs heparin or dermatan sulfate was studied in the inhibition of thrombin by antithrombin III (AT III) or heparin cofactor II (HC II). Both rabbit thrombomodulin (TM) and two glycoforms (a high-Mr form containing GAGs and a low-Mr form lacking the majority of O-linked GAGs) of a recombinant human TM deletion mutant (rec-TM) were used. The rapid inactivation of thrombin by HC II in the presence of dermatan sulfate was prevented by both the high-Mr rec-TM and the rabbit TM. In contrast, both rabbit TM treated with chondroitin ABC lyase to remove O-linked GAGs and the low-Mr form of rec-TM had only weak protecting effects. In the absence of exogeneous dermatan sulfate, thrombin inhibition by a high concentration of HC II was slightly accelerated by the high-Mr form of rec-TM but protected by rabbit TM. When thrombin inhibition by AT III in the presence of heparin was studied, both high-Mr rec-TM and rabbit TM again invoked a similar reduction of inactivation rates, whereas in the absence of exogenous heparin, both high-Mr forms accelerated thrombin inhibition by AT III. The diverse reactivities of various forms of TM towards HC II and AT III were also observed during protein C activation by the thrombin-TM complex. These results suggest that thrombin activity at the vessel wall or in fluid phase may undergo major kinetic modulations depending on the type of protease inhibitor, the presence or absence of exogenous GAGs and the glycosylation phenotype of TM. The dependence of TM anticoagulant function on the presence of an intrinsic GAG moiety suggests that variant glycoforms of this endothelial cell cofactor may be expressed differently in a species-, organ-, or tissue-specific manner as a means to regulate TM function in diverse vasculatures.
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PMID:Different glycoforms of human thrombomodulin. Their glycosaminoglycan-dependent modulatory effects on thrombin inactivation by heparin cofactor II and antithrombin III. 164 16

Tryptase from human mast cells has been shown (in vitro) to catalyze the destruction of fibrinogen and high-molecular-weight kininogen as well as the activation of C3a and collagenase. Although large amounts of tryptase are released in tissues by degranulating mast cells and levels as high as 1000 ng/ml have been measured in the circulation following systemic anaphylaxis, no specific physiologic inhibitor has yet been found for the protease. The current work tests several more inhibitors for their effects on tryptase and examines any effect of tryptase on these inhibitors. First, antileukoprotease and low-molecular-weight elastase inhibitor from human lung and hirudin and antithrombin III had no effect on tryptase activity in vitro. Second, the possibility that tryptase, being insensitive to the effects of inhibitors, might instead destroy them was also considered. Tryptase failed to cleave and inactivate antileukoprotease, low-molecular-weight elastase inhibitor, alpha 1 protease inhibitor, alpha 2 macroglobulin, and antithrombin III. Third, based on the knowledge that tryptase stability is regulated by its interaction with heparin, antithrombin III was used as a model heparin-binding protein to demonstrate that a protein competitor for heparin-binding sites, presumably by displacement of tryptase, destabilizes this enzyme. Conversely, tryptase, in excess, blocked the binding of antithrombin III to heparin, thereby attenuating the heparin-mediated inhibition of thrombin by antithrombin III.
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PMID:Interactions of human mast cell tryptase with biological protease inhibitors. 168 95

To clarify the role of thrombin in fibroblast growth and the development of pulmonary fibrosis in bleomycin-induced interstitial lung disease, we examined the relationship of thrombin activity to fibroblast growth-stimulating activity (FGA) in bronchoalveolar lavage (BAL) fluid from bleomycin-treated rats. Male Wistar rats were given a single intratracheal injection of bleomycin, BAL was performed 2, 6, and 15 days later, and the BAL fluid was assayed for thrombin activity and FGA. Higher FGA than the control value was detected in the BAL fluid from rats on day 6 after bleomycin administration. In bleomycin-treated rats, thrombin activity in the BAL fluid was significantly elevated on day 2 and maximal on day 6. The FGA of the BAL fluid from bleomycin-treated rats on day 6 was significantly decreased by its treatment with various thrombin inhibitors, such as alpha 1-protease inhibitor, antithrombin III, hirudin, and MD-805. In our assay, purified rat thrombin also showed FGA in vitro, and its FGA was inhibited by the same concentrations of these thrombin inhibitors as those inhibiting the activity in the BAL fluid. On ammonium sulfate fractionation, most of the thrombin activity was recovered in the fraction of 35 to 50% saturation in which most of the FGA was detected. These results suggest that the FGA of the BAL fluid from bleomycin-treated rats was at least partly due to thrombin is responsible, at least in part, for fibroblast growth and pulmonary fibrosis in bleomycin-induced interstitial lung disease.
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PMID:Thrombin enhances lung fibroblast proliferation in bleomycin-induced pulmonary fibrosis. 171 76

Plasminogen activator inhibitor 1 (PAI-1) is the fast-acting inhibitor of both tissue-type and urokinase-type plasminogen activators (t-PA, u-PA) and is an essential regulatory protein of the fibrinolytic system. In the presence of either the protein vitronectin or the glycosaminoglycan heparin, PAI-1 is also an efficient inhibitor of thrombin. To assess whether these cofactors turn PAI-1 into a general protease inhibitor or whether their influence is restricted to thrombin, the second-order association rate constants between PAI-1 and the human plasma proteases t-PA, u-PA, plasmin, thrombin, Factor Xa (FXa), and Factor XIIa (FXIIa) in the absence and in the presence of either vitronectin or heparin are determined. In addition, the role of the PAI-1 reactive site P3 to P3' residues for the specificity of inhibition was studied by using PAI-1 reactive site mutants. Our results show that: (1) Heparin exclusively increases the rate of inhibition of thrombin by PAI-1, whereas in the presence of heparin the rate of inhibition of the other proteases is not altered; (2) Vitronectin is an obligatory cofactor for the inhibition of thrombin by PAI-1. In addition, vitronectin moderately increases the rate of inhibition by PAI-1 of u-PA and of plasmin, but does not alter the rate of inhibition of t-PA, FXa, or FXIIa; (3) Apart from the important role of the P1 residue, no consensus can be presented on the nature of other residues within the P3 to P3' region with regard to target protease specificity.
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PMID:On the target specificity of plasminogen activator inhibitor 1: the role of heparin, vitronectin, and the reactive site. 171 20

The changes in relevant haemostatic parameters during the course of ten orthotopic liver transplantation were studied when aprotinin was given intra-operatively. Increases of tissue-type (P = 0.008) and urokinase-type (P = 0.009) plasminogen activators during the anhepatic phase could be correlated with hyperfibrinolysis. Thrombin-antithrombin III complexes (TAT) increased after revascularization of the liver graft (P = 0.003). Parallel studies in the perfusate showed that TAT concentrations were 350% and protease inhibitor activities (antithrombin III, protein C) only 52% of the systemic circulation before reperfusion, suggesting that thrombin activation together with protease inhibitor consumption occurs during graft liver reperfusion. The relatively smaller increases in profibrinolytic parameters and a lower blood loss when compared with other groups may be explained by aprotinin administration in our patients.
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PMID:Coagulation changes and the influence of the early perfusate in the course of orthotopic liver transplantation (OLT) when aprotinin is used intra-operatively. 172 11


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