Search PubMed⌕ Search

Biomedical subjects

R Machovich

Publications and source records attributed to R Machovich.

At least 73 records · Page 4Linked to original sources
PubMed1982

Specific binding of thrombin-antithrombin III complex to hepatocytes.

Thrombin-antithrombin III complex binds selectively to isolated hepatocytes, whereas antithrombin III alone does not. The binding is time and concentration dependent at 37 degrees C: the apparent Km value is 0.8/microM. The rate of binding is approximately 1.6 X 10(5) molecules h-1 cell-1 at this concentration. At 4 degrees C there is no measurable interaction between the complex and the hepatocytes. The binding is also prevented by pretreatment of cells with trypsin. On the other hand, about 80% of the thrombin-antithrombin III complex bound to hepatocytes is releasable by trypsin digestion. NaF or carboxyatractyloside does not inhibit the process. The interaction of thrombin-antithrombin III complex with hepatocytes seems to be specific, since the complexes of antithrombin III with other proteinases, like trypsin or plasmin, are not bound at the concentrations used. Based on these data, a mechanism for the binding of the inactive complexed form of thrombin to hepatocytes is suggested.

Animals↗
PubMed1981

Kinetic analysis of the heparin-enhanced plasmin--antithrombin III reaction. Apparent catalytic role of heparin.

Inactivation of plasmin by a 3-4-fold molar excess of antithrombin III follows pseudo-first-order kinetics and the apparent rate constants are proportional to the concentration of the inhibitor. Heparin accelerates the inactivation reaction without changing its pseudo-first-order character, and the apparent rate constants are also proportional to the concentration of the polysaccharide. Heparin results in a minimum 20-fold rate enhancement of the reaction between plasmin and antithrombin III when the concentrations of heparin and plasmin are approx. 0.5mum and 1mum respectively. Heparin at a molar concentration well below that of plasmin still accelerates the reaction: one molecule of the polysaccharide is able to facilitate the inactivation of about 100 molecules of plasmin. Heparin must bind to plasmin to accelerate the plasmin-antithrombin III reaction, since the modification of four to five lysine residues of the enzyme inhibits the rate-enhancement effect of heparin and the dissociation of heparin-plasmin complex decreases the inactivation rate of plasmin. Increasing the concentration of antithrombin III, at a constant amount of heparin, results in increase of the inactivation rate. By contrast, the effect of increasing the amount of plasmin in the presence of constant amount of heparin and antithrombin III is such that higher plasmin-to-heparin ratios are associated with lower rates of inactivation. It seems, therefore, that to obtain ;optimal' conditions for fast enzyme inactivation, the amount of heparin should be matched to plasmin rather than to antithrombin III. Arrhenius plots of the plasmin-antithrombin III reaction are linear both in the absence and presence of heparin, at concentrations of 1 or 2mug/ml, over a range of 26K. Under these experimental conditions, heparin increases activation entropy. The findings show that heparin seems to fulfil some criteria that are characteristic for biological catalysis: binding, reaction-rate enhancement (increasing activation entropy), recycling of heparin (effectiveness of non-stoichiometric amounts of the polysaccharide) and specificity.

Antithrombin III↗
PubMed1981

Thrombin and haemostasis: regulation of the biological functions of thrombin. (Facts and perspectives).

Thrombin plays an essential role in the maintenance of haemostatic balance through several biochemical reactions. Its functions as well as the regulation of its activities seem to be decisive in controlling thrombus formation. The present paper endeavours to give a brief summary of the control of thrombin activity and the regulation of biological actions of thrombin. As to the control of the amount of the active enzyme, three possible ways are discussed, i.e. prothrombin synthesis, activation of prothrombin, and inactivation of thrombin by plasma proteinase inhibitors (antithrombin III, alpha-2-macroglobulin, alpha-1-proteinase inhibitor). Regarding the regulation of the various actions of thrombin, the most important biological targets of this enzyme are summarized, namely interaction with fibrinogen, activation of Factor XIII, Factor IX, Factor VIII, Factor V and prothrombin, and the binding to platelets, fibroblasts and endothelial cells. The limits of our present knowledge about the determination of the possible reaction routes catalyzed by thrombin are also considered. Finally, a tentative hypothesis is put forward to explain how the endogenous modulators like heparin determine the biological functions of thrombin.

Antithrombin III↗
PubMed1979

Decreased heparin sensitivity of cyclohexanedione-modified factor Xa.

Modification of 12 arginine residues of bovine Factor Xa with 1,2-cyclohexanedione has resulted in a decrease in heparin sensitivity of the reaction between enzyme and antithrombin-III, whereas the antithrombin-III sensitivity of modified Factor Xa was only slightly affected. It is suggested that heparin accelerates the Factor Xa-antithrombin-III reaction by interacting with Factor Xa.

Animals↗
PubMed1979

Inhibition of esterase and amidase activities of alpha- and beta-thrombin in the presence of antithrombin III and heparin.

Inhibition of the esterase and amidase activities of bovine alpha- and beta-thrombin in the presence of antithrombin III and heparin has been studied. It was found that both the esterase and amidase activities of alpha-thrombin were inhibited by antithrombin III and the reactions were accelerated by heparin. The inhibition of amidase and esterase activities of beta-thrombin by antithrombin III has also been demonstrated. Heparin however did not increase the rate of inactivation of the enzyme.

Amidohydrolases↗
PubMed1978

Effect of heparin on thrombin inactivation by antithrombin-III.

The inactivation of thrombin by heat and by its physiological inhibitor, antithrombin-III, shows quite different dependence on heparin concentration. Heparin at 250 microgram/ml protects thrombin against heat inactivation, and thrombin behaves heterogeneously in this reaction. In the absence of heparin, the thermodynamic activation parameters change with temperature (deltaH+ = 733 kJ/mol and 210 kJ/mol at 50 and 58 degrees C respectively). When heparin is present, heat inactivation of the protected thrombin species proceeds with deltaH+ = 88 kJ/mol and is independent of temperature in the same range. On the other hand, heparin at 0.125-2.5 microgram/ml accelerates the thrombin-antithrombin-III reaction. Thrombin does not show heterogeneity in this reaction and the time courses at any heparin concentration and any temperature between 0 and 37 degrees C appear to follow first-order kinetics. Activation enthalpy is independent of heparin concentration or temperature, deltaH+ = 82-101 kJ/mol, varying slightly with antithrombin-III concentration and thrombin specific activity. Heparin seems to exert its effect by increasing activation entropy. On the basis of these data we suggest a mechanism of action of heparin in the thrombin-antithrombin-III reaction which accounts for all the important features of the latter and seems to unify the different hypotheses that have been advanced.

Antithrombin III↗
PubMed1978

Decreased heparin sensitivity of cycholhexanedione-modified thrombin.

Modification of 5--6 arginine residues of thrombin with 1,2-cyclohexanedione has resulted in the selective abolition of the heparin sensitivity of the enzyme's reaction with antithrombin-III, whereas the antithrombin-III sensitivity of native and modified thrombin was indistinguishable. It is suggested that heparin accelerates the thrombin antithrombin-III reaction by interacting with thrombin.

Animals↗
PubMed1978

Air reoxidation and reactivation of reduced thrombin.

About 80% of thrombin was inactivated after 50 minutes chemical reduction at 22 degrees C in a reaction mixture containing o.1 M mercaptoethanol and 2.6 M urea. The reduced protein was spontaneously reoxidated in air at 22 degrees C in 30--60 minutes. The reoxidation of disulphide bonds in thrombin led to partial reactivation of the enzyme. Recovery of thrombin activity after oxidation ranged from 0 to 60% according to the conditions of reoxidation in air. Heparin and copper ion increased the rate of reactivation, whereas in the presence of 10 mM iodoacetamide there was no reactivation.

Air↗
PubMed1977

Inactivation of alpha- and beta-thrombin by antithrombin-III, alpha 2-macroglobulin and alpha 1-proteinase inhibitor.

Inactivation of alpha- and beta-thrombin by alpha 2-macroglobulin, by alpha 1-proteinase inhibitor and by antithrombin-III and heparin was studied. The amount of alpha- and beta-thrombin inactivated by antithrombin-III was proportional to the concentration of the inhibitor, but the inactivation rates of the two forms of thrombin were different. Heparin facilitated complex-formation between alpha-thrombin and antithrombin-III, whereas inactivation of beta-thrombin by antithrombin was only slightly influenced, even at a heparin concentration two orders of magnitude higher. alpha 2-Macroglobulin inhibited both alpha- and beta-thrombin activity similarly, i.e. the amount of alpha- and beta-thrombin inactivated as well as the rates of their inhibition were the same. alpha 1-Proteinase inhibitor also formed a complex with alpha- and beta-thrombin, similarly to antithrombin-III, although the inactivation of the enzyme needed high inhibitor concentration and long incubation time. These results suggest that the inactivation of beta-thrombin, if it occurs in the plasma, is also controlled by plasma inhibitors.

Antithrombins↗
PubMed1977

Effect of calcium ion on the interaction between thrombin and heparin. Thermal denaturation.

Heat inactivation of thrombin at 54 degrees C followed first order kinetics with a rate constant of 1.0 min-1 approximately. Addition of heparin resulted in protection against thermal denaturation and, at the same time, rendered denaturation kinetics more complex. Analysis of the biphasic curve of heat inactivation in the presence of heparin revealed that the rate constants of the second phase changed systematically with heparin concentrations. Namely, at 4.5 x 10(-6)M, 9 x 10(-6)M, 1.8 x 10(-5)M and 3.6 x 10(-5)M heparin concentrations, the rate constants were 0.27 min -1, 0.17 min-1, 0.11 min-1 and 0.06 min-1, respectively. Sulfate as well as phosphate ions displayed also enzyme protection against heat inactivation, however, the same effect was obtained already at a heparin concentration, lower by three orders of magnitude. The kinetics of enzyme denaturation was not affected by calcium ions, whereas in the presence of heparin the inactivation rate of thrombin changed, i.e. calcium ions abolished the biphasic character of time course of thermal denaturation. Thus, the data suggest that calcium ions contribute to the effect of heparin on thrombin.

Animals↗
PubMed1977

Effect of collagen on thrombin inactivation by antithrombin-III and heparin.

Thrombin inactivation by antithrombin-III and heparin has been found to decrease in the presence of collagen, whereas thrombin activity and the rate of thrombin inactivation by antithrombin-III alone are not affected. Albumin, at the same concentration as collagen, does not influence either thrombin activity or thrombin inactivation by antithrombin-III or by antithrombin-III plus heparin.

Animals↗
PubMed1977

Some properties of human progressive antithrombin.

The effect of some mono- and divalent cations was examined on thrombin--antithrombin reaction in vitro. It was found that 0--0.1 M sodium- or potassium chloride did not affect either thrombin or antithrombin activity; at higher concentrations thrombin activity decreased. Calcium chloride as well as magnesium chloride at concentrations from 0 to 0.5 M increased enzyme activity, whereas at higher concentrations the activity decreased. Thrombin inactivation by antithrombin was also accelerated at calcium or magnesium chloride concentrations above 0.04 M. Antithrombin was inactivated at pH 7.3 at 65 degrees C in some minutes and heparin failed to protect it against heat denaturation. Thrombin inactivation by antithrombin did not proceed at 0 degrees C in 60 min, but the interaction between thrombin and antithrombin was facilitated in the presence of heparin.

Antithrombin III↗
PubMed1976

Inactivation of alpha- and beta- thrombin by antithrombin-III and heparin.

Inactivation of alpha- and beta-thrombin by antithrombin-III and heparin was studied, since it had been suggested that two forms of thrombin exist with respect to heparin sensitivity (Machovich 1975b). It was found that the inactivation rates of alpha- and beta-thrombin by antithrombin were different, namely alpha-thrombin was more sensitive to antithrombin than beta-thrombin. Heparin facilitated the complex formation between alpha-thrombin and antithrombin-III, whereas beta-thrombin inactivation was only slightly affected. Furthermore, heparin protected alpha-thrombin against the inactivating effect of heat, while beta-thrombin lost its activity during the heat treatment. These findings suggest that the formation of beta-thrombin in blood circulation may have an important role in thrombosis predisposition.

Alpha-Globulins↗