Development of research into the physiology and pathophysiology of the fibrinolysis system

A modern data review on the importance of fibrinolysis system is given. A considerable success has been scored during the study of molecular parameters of fibrinolysis system: the plasminogen, plasmin, its inhibitors, plasminogen activators and the mechanism of activation system have been characterized. The entrance of A, K, C, P and PP vitamins has been established to be necessary for the normal functioning of the fibrinolysis system; the dependence of the blood fibrinolytic activity upon the initial plasminogen content and concentration of its activators in blood has been revealed. The plasminogen activator depletion in tissues has been shown to be one of the reasons of some pathological states development, especially at cardiovascular diseases. The increase of fibrinolysis level by the active fibrinolytic ferment injection in blood has a medical effect at thrombosis. The ferment fibrinolysin received in the laboratory is successfully used in clinical practice. Some other activators of fibrinolytic system: tricholysine and longolytin from the culture of saprophyte fungi, plasminogen activator from the pig heart and the cells culture of the calf kidney have been received and are being studied.     

Plasmin converts factor X from coagulation zymogen to fibrinolysis cofactor

Known anticoagulant pathways have been shown to exclusively inhibit blood coagulation cofactors and enzymes. In the current work, we first investigated the possibility of a novel anticoagulant mechanism that functions at the level of zymogen inactivation. Utilizing both clotting and chromogenic assays, the fibrinolysis protease plasmin was found to irreversibly inhibit the pivotal function of factor X (FX) in coagulation. This was due to cleavage at several sites, the location of which were altered by association of FX with procoagulant phospholipid (proPL). The final products were approximately 28 and approximately 47 kDa for proPL-bound and unbound FX, respectively, which did not have analogues when activated FX (FXa) was cleaved instead. We next investigated whether the FX derivatives could interact with the plasmin precursor plasminogen, and we found that plasmin exposed a binding site only on proPL-bound FX. The highest apparent affinity was for the 28-kDa fragment, which was identified as the light subunit disulfide linked to a small fragment of the heavy subunit (Met-296 to approximately Lys-330). After cleavage by plasmin, proPL-bound FX furthermore was observed to accelerate plasmin generation by tissue plasminogen activator. Thus, a feedback mechanism localized by proPL is suggested in which plasmin simultaneously inhibits FX clotting function and converts proPL-bound FX into a fibrinolysis cofactor. These data also provide the first evidence for an anticoagulant mechanism aimed directly at the zymogen FX.     

Genetic Mechanisms of Hereditary Hemostasis Disorders

This review summarizes known human genes whose mutations are associated with inherited hemostasis defects. These genes are divided into three groups. The genes of the first group are responsible for platelet adhesion, activation, and aggregation. The genes of the second group control the biosynthesis of blood-clotting factors and cofactors. The genes of the third group are required for the functioning of proteins involved in the anticoagulant system and fibrinolysis.     

Grape intake reduces thrombin generation and enhances plasma fibrinolysis. Potential role of circulating procoagulant microparticles

Phytochemicals contained in grapes down-regulate several prothrombotic pathways in vitro. We evaluated the effect of grape consumption on coagulation and fibrinolysis in healthy volunteers. Thirty subjects were enrolled: 20 were given grape (5 g/kg body weight/day for 3 weeks), while 10 served as controls. Blood samples were taken at baseline (T0), at the end of the grape diet (T1) and after 4-week wash-out (T2). Grape intake caused a significant decrease of the procoagulant and inflammatory responses of whole blood and/or mononuclear cells to bacterial lipopolysaccharide at both T1 and T2. At plasma level, grape diet decreased thrombin generation at T1 and T2, largely through a reduction in the number and/or activity of procoagulant microparticles. This anticoagulant effect resulted in the formation of clots that were more susceptible to fibrinolysis, mainly because of a lesser activation of thrombin activatable fibrinolysis inhibitor. No difference in any variables was detected in controls at the time points considered. In conclusion, chronic grape consumption induces sustained anticoagulant and profibrinolytic effects with potential benefits for human health.     

Studies on the different modes of action of the anticoagulant protease inhibitors DX-9065a and Argatroban. II. Effects on fibrinolysis

The accompanying paper (Nagashima, H. (2002) J. Biol. Chem. 277, 50439-50444) has demonstrated that argatroban can yield a stronger inhibitory effect on thrombin generation than DX-9065a during extrinsic pathway-stimulated human plasma coagulation, while these anticoagulant compounds have comparable abilities to prolong clot time. Since thrombin generation is known to be an important determinant for fibrinolytic resistance of clots formed during coagulation, the two compounds are compared by tissue plasminogen activator-induced clot lysis assays. The results demonstrated that, in the presence of thrombomodulin, argatroban dose dependently accelerated fibrinolysis of the clots, whereas DX-9065a did not. The activation of thrombin activatable fibrinolysis inhibitor (TAFI) determined in separate assays reflected the differential influence on thrombin generation by these compounds. Moreover, TAFI activation correlated closely with the fibrinolytic resistance observed during tissue plasminogen activator-induced clot lysis. This study demonstrates the differential effects of DX-9065a and argatroban on thrombin generation, which in turn results in a differential acceleration of fibrinolysis as well as TAFI activation in the clots formed under the influence of these compounds. The data implicate a possible difference in the antifibrinolytic properties of clots formed during treatment with these compounds.     

Platelet factor 4 inhibits thrombomodulin-dependent activation of thrombin-activatable fibrinolysis inhibitor (TAFI) by thrombin

Thrombomodulin (TM) is a cofactor for thrombin-mediated activation of protein C and thrombin-activatable fibrinolysis inhibitor (TAFI) and thereby helps coordinate coagulation, anticoagulation, fibrinolysis, and inflammation. Platelet factor 4 (PF4), a platelet α-granule protein and a soluble cofactor for TM-dependent protein C activation, stimulates protein C activation in vitro and in vivo. In contrast to stimulation of protein C activation, PF4 is shown here to inhibit activation of TAFI by thrombin-TM. Consequences of inhibition of TAFI activation by PF4 included loss of TM-dependent prolongation of clot lysis times in hemophilia A plasma and loss of TM-stimulated conversion of bradykinin (BK) to des-Arg(9)-BK by TAFIa in normal plasma. Thus, PF4 modulates the substrate specificity of the thrombin-TM complex by selectively enhancing protein C activation while inhibiting TAFI activation, thereby preventing the generation of the antifibrinolytic and anti-inflammatory activities of TAFIa. To block the inhibitory effects of PF4 on TAFI activation, heparin derivatives were tested for their ability to retain high affinity binding to PF4 despite having greatly diminished anticoagulant activity. N-acetylated heparin (NAc-Hep) lacked detectable anticoagulant activity in activated partial thromboplastin time clotting assays but retained high affinity binding to PF4 and effectively reversed PF4 binding to immobilized TM. NAc-Hep permitted BK conversion to des-Arg(9)-BK by TAFIa in the presence of PF4. In a clot lysis assay on TM-expressing cells using hemophilia A plasma, NAc-Hep prevented PF4-mediated inhibition of TAFI activation and the antifibrinolytic functions of TAFIa. Accordingly, NAc-Hep or similar heparin derivatives might provide therapeutic benefits by diminishing bleeding complications in hemophilia A via restoration of TAFIa-mediated protection of clots against premature lysis.     

Purification and properties of the anticoagulant principle of Trimeresurus gramineus venom.

By means of DEAE-Sephadex A-50 column chromatography, Trimeresurus gramineus venom was separated into 12 fractions. Fraction 8 had marked anticoagulant action in the tests of whole blood clotting time, calcium clotting time and plasma prothrombin time. Fraction 8 was rechromatographed on Sephadex G-100, then on DEAE-Sephadex A-50 again, and finally on Sephadex G-100, and a single peak was obtained. The patterns of microzone and disc electrophoresis also showed a single band. A single symmetrical boundary with 1.70 Svedberg units was obtained by ultracentrifugation. The estimated molecular weight was 19 500. The isoelectric point was pH 4.5. Chemical analysis showed that the anticoagulant principle was a glycoprotein and that it was thermolabile. The anticoagulant activity of this purified principle was 3.5 times higher than that of the crude venom. Fraction 5 potentiated its anticoagulant activity to 10 times higher than that of the crude venom. This principle did not possess caseinolytic, tosyl-L-arginine methyl ester esterase, phospholipase A, phosphodiesterase, alkaline phosphomonoesterase, fibrinolytic, hemorrhagic or local irritating activities. The purified anticoagulant principle did not destroy fibrinogen, induce fibrinolysis, inactivate thrombin nor interfere with the interaction between thrombin and fibrinogen. However, a marked inhibition of prothrombin activation was caused by the anticoagulant principle. The inhibition of prothrombin activation was not due to the destruction of prothrombin or its activation factors, but due to an interference in the interaction between prothrombin and its activation factors because of the reversible binding of these factors with the anticoagulant principle of the venom.     

Protein C system and thrombosis diseases

The mechanism of protein C system functioning and role of its components in the regulation of the coagulation and the fibrinolysis was considered in the review. There are also new data about the anticoagulation, profibrinolysis and antiinflammatory functions of protein C system. Activated protein C is inhibited by the protein C inhibitor, alpha 1-antitripsin, and inhibitor of tissue activator plasminogen-1. Hereditary or acquired deficiency of protein C, protein S and thrombomodulin lead to thrombotic diseases. Injection of exogenous recombinant protein C or activated protein C into the blood increases the anticoagulant activity of the blood and produced the antithrombotic effect.     

Pathophysiology of thrombophilic states

The coagulation system can be considered as a balance in which clotting and fibrinolysis have to be in a state of equilibrium. Increased fibrin formation or decreased fibrinolysis can predispose to thromboembolic diseases. Derailments in the clotting system leading to thrombosis center around the regulatory mechanisms, antithrombin III, protein C, protein S and possibly heparin cofactor II. Many cases of congenital or acquired deficiencies or abnormalities or antithrombin III, protein C and S have been described, all predisposing to thrombotic events. Alterations of the fibrinolytic system can also be associated with thromboembolisms. In particular, abnormalities of plasminogen, tissue plasminogen activator release and elevated tissue plasminogen activator inhibitor levels seem to be associated with thromboses. Conceivably also factor XIIa (Hageman factor) and prekallikrein deficiencies, when associated with thrombosis, exert their mechanism through the fibrinolytic system. Finally, about 50% of patients with lupus anticoagulant seem to suffer from thromboembolic disorders. The pathophysiology of this particular association is not known with certainty. Undoubtedly, there will be more disturbances discovered in the hemostasis system that are associated with increased intravascular fibrin formation. The understanding of these derailments is at this time only in its earliest stages of development.     

Status of the antioxidant system and lipid peroxidation in rat liver after poisoning animals with aminobiphenyl]

It was found that intoxication of animals with aminobiphenyls leads to the activation of such glutathione-dependent enzymes as glutathione-S-transferase and glutathione reductase. This is accompanied by the induction of activities of individual isoforms of the multifunctional family of glutathione-S-transferases. There was a decrease in the glutathione peroxidase activity after intoxication with benzidine derivatives. It was found that the GSH content in rat liver decreased after benzidine intoxication and sharply increased after effects of 3,3'-dimethylbenzidine and 3,3'-dimethoxybenzidine. In all cases studied there was a diminution in the level of diene conjugates. It was supposed that the specificity of the catalytic glutathione redox system reaction is due to structural peculiarities of the aminobiphenyls being injected. Analysis of functional pairs of glutathione-dependent enzymes revealed a certain imbalance in the antioxidant system function after aminobiphenyl poisoning.     

Thrombin-activable fibrinolysis inhibitor zymogen does not play a significant role in the attenuation of fibrinolysis

Activated thrombin-activable fibrinolysis inhibitor (TAFIa) plays a significant role in the prolongation of fibrinolysis. During fibrinolysis, plasminogen is activated to plasmin, which lyses a clot by cleaving fibrin after selected arginine and lysine residues. TAFIa attenuates fibrinolysis by removing the exposed C-terminal lysine residues. It was recently reported that TAFI zymogen possesses sufficient carboxypeptidase activity to attenuate fibrinolysis through a mechanism similar to TAFIa. Here, we show with a recently developed TAFIa assay that when thrombin is used to clot TAFI-deficient plasma supplemented with TAFI, there is some TAFI activation. The extent of activation was dependent upon the concentration of zymogen present in the plasma, and lysis times were prolonged by TAFIa in a concentration-dependent manner. Potato tuber carboxypeptidase inhibitor, an inhibitor of TAFIa but not TAFI, abolished the prolongation of lysis in TAFI-deficient plasma supplemented with TAFI zymogen. In addition, TAFIa but not TAFI catalyzed release of plasminogen bound to soluble fibrin degradation products. The data presented confirm that TAFI zymogen is effective in cleaving a small substrate but does not play a role in the attenuation of fibrinolysis because of its inability to cleave plasmin-modified fibrin degradation products.     

Lys-plasminogen is a significant intermediate in the activation of Glu-plasminogen during fibrinolysis in vitro.

Plasminogen, the zymogen form of the fibrinolytic enzyme plasmin, is known to undergo plasmin-mediated modification in vitro. The modified form, Lys-plasminogen, is superior to the native Glu-plasminogen in fibrin binding and as a substrate for activation by tissue-type plasminogen activator (t-PA). The present study was undertaken to determine the existence and significance of the Glu- to Lys-plasminogen conversion during t-PA-mediated lysis of plasma clots in vitro. When human plasma was supplemented with exogenous Lys-plasminogen and clotted, a dose-dependent shortening of lysis time was observed. Formation of Lys-plasminogen in situ during fibrinolysis was determined using 131I-Glu-plasminogen-supplemented plasma. By the time of lysis, Lys-plasminogen had accumulated to about 20% of the initial concentration of Glu-plasminogen. Quantitation of activation of both Glu- and Lys-plasminogen as well as the conversion of Glu- to Lys-plasminogen in plasma supplemented with both 131I-Glu-plasminogen and 125I-Lys-plasminogen was accomplished by determining the flux of the isotopically labeled species along three pathways: Glu-plasminogen-->Glu-plasmin, Glu-plasminogen-->Lys-plasminogen, and Lys-plasminogen-->Lys-plasmin. After a brief lag, the Glu-plasminogen activation rate was constant until lysis was achieved, at which point activation ceased. The Lys-plasminogen activation rate also was essentially constant until lysis but was not characterized by a lag phase. The rate of conversion of Glu- to Lys-plasminogen was nonlinear and correlated directly with the rate of fibrinolysis. By the time lysis had occurred, Glu-plasminogen consumption had been distributed equally between direct activation to plasmin and conversion to Lys-plasminogen, and 45% of the plasmin which had been formed was derived from Lys-plasminogen. These results demonstrate both the formation and the subsequent activation of Lys-plasminogen during fibrinolysis. As a result of improved fibrin binding and activation of Lys-plasminogen compared to Glu-plasminogen, the formation of Lys-plasminogen within a clot constitutes a positive feedback mechanism that can further stimulate the activation of plasminogen by t-PA as fibrinolysis progresses.     

Effect of Mild Hypothermia on the Coagulation-Fibrinolysis System and Physiological Anticoagulants after Cardiopulmonary Resuscitation in a Porcine Model

The aim of this study was to evaluate the effect of mild hypothermia on the coagulation-fibrinolysis system and physiological anticoagulants after cardiopulmonary resuscitation (CPR). A total of 20 male Wuzhishan miniature pigs underwent 8 min of untreated ventricular fibrillation and CPR. Of these, 16 were successfully resuscitated and were randomized into the mild hypothermia group (MH, n = 8) or the control normothermia group (CN, n = 8). Mild hypothermia (33°C) was induced intravascularly, and this temperature was maintained for 12 h before pigs were actively rewarmed. The CN group received normothermic post-cardiac arrest (CA) care for 72 h. Four animals were in the sham operation group (SO). Blood samples were taken at baseline, and 0.5, 6, 12, 24, and 72 h after ROSC. Whole-body mild hypothermia impaired blood coagulation during cooling, but attenuated blood coagulation impairment at 72 h after ROSC. Mild hypothermia also increased serum levels of physiological anticoagulants, such as PRO C and AT-III during cooling and after rewarming, decreased EPCR and TFPI levels during cooling but not after rewarming, and inhibited fibrinolysis and platelet activation during cooling and after rewarming. Finally, mild hypothermia did not affect coagulation-fibrinolysis, physiological anticoagulants, or platelet activation during rewarming. Thus, our findings indicate that mild hypothermia exerted an anticoagulant effect during cooling, which may have inhibitory effects on microthrombus formation. Furthermore, mild hypothermia inhibited fibrinolysis and platelet activation during cooling and attenuated blood coagulation impairment after rewarming. Slow rewarming had no obvious adverse effects on blood coagulation.     

Anticoagulant and anticomplement effects of an endogenous inhibitor from hepatopancreas of red king crab (<Emphasis Type="Italic">Paralithosed camtschaticus</Emphasis>) on human blood

Serpins are the superfamily of serine and cysteine protease inhibitors (SERine Protease Inhibitors) acting by an irreversible suicide mechanism. A novel serpin from hepatopancreas of red king crab (Paralithosed camtschaticus) was isolated and its effect on the process of human blood plasma coagulation was investigated. The investigated serpin exhibited a significant anticoagulant effect, which dramatically increased in the combination with heparin. The study of the crab serpin on C1s (C1 esterase) revealed its competition with the C1 inhibitor from blood plasma. Although the inhibitor weakly influenced thrombin activity, inhibition constant for C1s was (2.02 ± 0.71) 10⁻⁷ M. Unlike the C1 inhibitor the novel red king crab serpin does not inhibit fibrinolysis but inhibits blood coagulation. This creates certain clinical perspectives.     

Venous Thrombogenesis and Functional State of the Hemorheology and Hemostasis System in Stroke Patients

Investigation of the hemorheology and hemostasis system in patients with stroke of various types, location, and severity as well as with and without venous thromboembolic complications (VTECs), who were treated at critical and intensive care units, demonstrated the significance of impairments in the hemorheology and hemostasis system for VTEC pathogenesis in stroke patients. Despite ongoing anticoagulant therapy, aggravation of the prothrombogenic state was observed in VTEC patients. Ischemic stroke was associated with more severe changes compared to hemorrhagic stroke. Hemostasiological predictors of VTEC were identified. In patients without VTEC, both the coagulation and anticoagulation systems as well as the fibrinolysis system were preserved. The D-dimer and thrombophilia markers, such as hyperhomocysteinemia and antiphospholipid syndrome, were shown not to contribute to the development of VTEC in stroke patients.     

Effects of extracellular DNA on plasminogen activation and fibrinolysis

The increased levels of extracellular DNA found in a number of disorders involving dysregulation of the fibrinolytic system may affect interactions between fibrinolytic enzymes and inhibitors. Double-stranded (ds) DNA and oligonucleotides bind tissue-(tPA) and urokinase (uPA)-type plasminogen activators, plasmin, and plasminogen with submicromolar affinity. The binding of enzymes to DNA was detected by EMSA, steady-state, and stopped-flow fluorimetry. The interaction of dsDNA/oligonucleotides with tPA and uPA includes a fast bimolecular step, followed by two monomolecular steps, likely indicating slow conformational changes in the enzyme. DNA (0.1-5.0 μg/ml), but not RNA, potentiates the activation of Glu- and Lys-plasminogen by tPA and uPA by 480- and 70-fold and 10.7- and 17-fold, respectively, via a template mechanism similar to that known for fibrin. However, unlike fibrin, dsDNA/oligonucleotides moderately affect the reaction between plasmin and α(2)-antiplasmin and accelerate the inactivation of tPA and two chain uPA by plasminogen activator inhibitor-1 (PAI-1), which is potentiated by vitronectin. dsDNA (0.1-1.0 μg/ml) does not affect the rate of fibrinolysis by plasmin but increases by 4-5-fold the rate of fibrinolysis by Glu-plasminogen/plasminogen activator. The presence of α(2)-antiplasmin abolishes the potentiation of fibrinolysis by dsDNA. At higher concentrations (1.0-20 μg/ml), dsDNA competes for plasmin with fibrin and decreases the rate of fibrinolysis. dsDNA/oligonucleotides incorporated into a fibrin film also inhibit fibrinolysis. Thus, extracellular DNA at physiological concentrations may potentiate fibrinolysis by stimulating fibrin-independent plasminogen activation. Conversely, DNA could inhibit fibrinolysis by increasing the susceptibility of fibrinolytic enzymes to serpins.     

Mechanism of action of θ-amino acids on plasminogen activation and fibrinolysis induced by staphylokinase

Stimulation of Lys-plasminogen (Lys-Pg) and Glu-plasminogen (Glu-Pg) activation under the action of staphylokinase and Glu-Pg activation under the action of preformed plasmin-staphylokinase activator complex (Pm-STA) by low concentrations and inhibition by high concentrations of omega-amino acids (>90-140 mM) were found. Maximal stimulation of the activation was observed at concentrations of L-lysine, 6-aminohexanoic acid (6-AHA), and trans-(4-aminomethyl)cyclohexanecarboxylic acid 8.0, 2.0, and 0.8 mM, respectively. In contrast, the Lys-Pg activation rate by Pm-STA complex sharply decreased when concentrations of omega-amino acids exceeded the above-mentioned values. It was found that formation of Pm-STA complex from a mixture of equimolar concentrations of staphylokinase and Glu-Pg or Lys-Pg is stimulated by low concentrations (maximal at 10 mM) of 6-AHA. Negligible increase in the specific activities of plasmin and Pm-STA complex was detected at higher concentrations of 6-AHA (to maximal at 70 and 50 mM, respectively). Inhibitory effects of omega-amino acids on the rate of fibrinolysis induced by staphylokinase, Pm-STA complex, and plasmin were compared. It was found that inhibition of staphylokinase-induced fibrinolysis by omega-amino acids includes blocking of the reactions of Pm-STA complex formation, plasminogen activation by this complex, and lysis of fibrin by forming plasmin as a result of displacement of plasminogen and plasmin from the fibrin surface. Thus, the slow stage of Pm-STA complex formation plays an important role in the mechanism of action of omega-amino acids on Glu-Pg activation and fibrinolysis induced by staphylokinase. In addition to alpha-->beta change of Glu-Pg conformation, stimulation of Pm-STA complex formation leads to increase in Glu-Pg activation rate in the presence of low concentrations of omega-amino acids. Inhibition of Pm-STA complex formation on fibrin surface by omega-amino acids is responsible for appearance of long lag phases on curves of fibrinolysis induced by staphylokinase.     

Influencing the fibrinolytic activity by antithrombotics in patients with atherosclerosis

In 106 atherosclerotic patients receiving an anticoagulant therapy and 91 patients receiving acetylsalicylic acid, fibrinogen and fibrin degradation products were determined as well as euglobulin lysis before and after venous occlusion. Platelet function data and thromboxane (TXB2) were also determined. Since "moderate" anticoagulant therapy with thromboplastin time values 26-40% results in deteriorated fibrinolysis data, anticoagulant therapy is strictly to remain within the therapeutic range of 15-20% up to 25% thromboplastin time at maximum. Treatment with acetylsalicylic acid proved useful on condition that the required dose was determined individually. This type of treatment will then be able to reduce the thromboxane level and positively influence the fibrinolytic potential.     

Purification and characterization of the anticoagulant principle of Trimeresurus mucrosqualatus venom

By means of CM-Sephadex column chromatography, Trimeresurus mucrosquamatus venom was separated into 20 fractions. Fraction XX had the marked anticoagulant action. This fraction was refractionated three times on Sephadex G-75, and a single peak was obtained. The patterns of microzone and disc electrophoresis also showed a single band. A single, symmetrical boundary with a value of 1.61 S was obtained by ultracentrifugation. It was a single peptide chain with a molecular weight of 11 700. The isoelectric point was higher than pH 10.The anticoagulant principle possesses phospholipase A activity and was calcium ion dependent. It did not possess proteolytic, tosyl-L-arginine methyl ester esterase, phosphodiesterase and alkaline phosphomonoesterase activities of the crude venom. The phospholipase A activity was heat-labile at pH 7.4, but was heat-stable at pH 5.6. The anticoagulant activity was more resistant to heat treatment as compared with phospholipase A activity.The anticoagulant action of the purified principle was competitively inhibited by platelet phospholipid, tissue thromboplastin and cephalin, and was neutralized by antiserum. The anticoaugulant principle inhibited platelet aggregation induced by ADP. It did not destroy fibrinogen, Factor X, prothrombin and thrombin; nor did it induce fibrinolysis nor interfere with the interaction between thrombin and fibrinogen. It is concluded that the anticoagulant action of this phospholipase A was due to the inhibition of the activations of Factors X and II through the activation of the procoagulant activity of phospholipids mediated partly by phospholipid-binding activity of this venom enzyme and partly by its enzymatic hydrolysis of phospholipids.     

Blocking NMDA receptors prevents the oxidative stress induced by acute ammonia intoxication

Acute ammonia intoxication diminishes the activities of antioxidant enzymes and increases superoxide formation in brain. These effects could play a role in the mechanism of ammonia toxicity. It has been shown that ammonia toxicity is mediated by activation of NMDA receptors. The aim of this work was to assess whether ammonia-induced changes in antioxidant enzymes and in superoxide formation are mediated by activation of NMDA receptors. It is shown that MK-801, an antagonist of NMDA receptors prevents ammonia-induced changes in superoxide dismutase, glutathione peroxidase and catalase. Ammonia intoxication also induces a depletion of glutathione and an increase in lipid peroxidation. Both effects, as well as ammonia-induced increase in superoxide formation are prevented by MK-801. These results indicate that ammonia-induced oxidative stress in brain is mediated by excessive activation of NMDA receptors and support the idea that oxidative stress can play a role in the mechanism of ammonia toxicity.