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Biomedical subjects

R Machovich

Publications and source records attributed to R Machovich.

At least 55 records · Page 3Linked to original sources
PubMed1987

Conditions of formation of the heparin-fibronectin-collagen complex and the effect of plasmin.

The formation and composition of the insoluble heparin-fibronectin-collagen complex and its degradation by proteolysis was investigated. At fixed concentrations of the other molecular components of the complex, the maximal rate of complex formation, measured turbidimetrically, was reached at a concentration of 4 microM heparin and 0.9 microM collagen, while the rate of complex formation was linearly related to concentrations of fibronectin as high as 3 microM. Heparin was incorporated into the complex in a saturable manner, and was released in active anticoagulant form by plasmin but not by urokinase. The complex formation was inhibited by 5 mM calcium or 250 mM NaCl as well as by polybrene or spermin. It is suggested that fibronectin binds both heparin and collagen cooperatively to form an insoluble ternary complex of the extracellular matrix.

Binding, Competitive↗
PubMed1987

Interaction of antithrombin III and thrombin-antithrombin III complex with cultured aortic endothelial cells.

The binding of antithrombin III, thrombin, thrombin-antithrombin III complex to endothelial cells was investigated. While the rate of the binding of thrombin to these cells was very rapid, that of antithrombin III was relatively slow and the thrombin-antithrombin III complex was intermediate. Binding kinetics indicated that antithrombin III, like thrombin, showed high affinity to endothelial cells; with a Kd of 3 X 10(-8) M and with 5 X 10(4) binding sites per cell. The dissociation of the inhibitor molecule was also rapid, i.e., approximately 70% bound antithrombin III was released in 2 minutes. Heparin, in a 100-fold molar excess to antithrombin III, or the modification of lysine residues of the inhibitor involved in the interaction with heparin, did not influence the association of antithrombin III with endothelial cells. In addition, antithrombin III did not compete with thrombin blocked in its active center for binding to endothelial cells. It is suggested that the binding sites of endothelial cells are different for thrombin and antithrombin III, and antithrombin III does not bind to these cells through its heparin binding domain.

Animals↗
PubMed1987

Prostanoid synthesis in isolated parenchymal and nonparenchymal mouse liver cells in the presence of arachidonic acid.

Prostanoid synthesis was investigated in suspensions of isolated mouse hepatocytes and nonparenchymal liver cells. A stable metabolite of thromboxane A2 (TXB2) of prostacyclin (6-keto PGF1 alpha) and one of the prostaglandins (PGF2 alpha) was detected by radio-immuno-assay (RIA). Hepatocytes synthesized mainly TXB2, while smaller amounts of 6-keto PGF1 alpha and PGF2 alpha were detected during 60 min incubation. Homogenization of hepatocytes caused a slight increase of TXB2 production and provoked the synthesis of PGF2 alpha and 6-keto PGF1 alpha. The addition of arachidonate to hepatocytes did not influence prostanoid production at concentrations below 10-5M. Higher concentrations further increased TXB2 production and also increased the synthesis of 6-keto PGF1 alpha and PGF2 alpha. Nonparenchymal cells synthesized all the three types of prostanoids and homogenization of these cells did not result in a marked change. The addition of 10(-7)-10(-5)M arachidonate increased the TXB2, 6-keto PGF1 alpha and PGF2 alpha synthesis in nonparenchymal cells. No further increase was found at higher concentrations.

Animals↗
PubMed1986

Anticoagulant effect of sulphated poly/vinyl alcohol-acrylic acid/copolymers.

Copolymers of poly/vinyl alcohol-acrylic acid/ with various content of sulphate and carboxyl groups have been synthetized and tested for their in vitro effect on blood coagulation. The results indicate that the sulphated copolymers display an inhibitory effect but there is a requirement in the charged groups of about 20% in the molecule to possess effective anticoagulation. The biochemical mechanism of their actions is complex, i.e. the inhibition of blood clotting is a consequence of both the accelerated inactivation rate of thrombin by antithrombin-III and a direct inhibitory effect on the thrombin-fibrinogen reaction. Moreover, additional effects may occur on other blood coagulation enzymes than thrombin, depending on the chemical composition of the copolymers.

Acrylic Resins↗
PubMed1986

Species specificity of thrombin-induced changes in vascular tone.

We studied the effects of acetylcholine and human thrombin on the tone of rabbit and dog isolated femoral arteries and aortas with intact endothelium. Acetylcholine (10(-9)-10(-6) mol/l) produced relaxation in the vessels from both species whereas thrombin (10(-9)-3 X 10(-8) mol/l) relaxed only canine arterial smooth muscle. Thrombin pretreatment increased significantly the relaxant potency of acetylcholine in femoral arteries of dogs. The results suggest an interspecies difference in the thrombin-induced endothelium-dependent vasorelaxation.

Acetylcholine↗
PubMed1985

Association of alpha 2-macroglobulin-thrombin and alpha 2-macroglobulin-plasmin complexes with isolated hepatocytes.

125I-labelled alpha 2-macroglobulin complexed with thrombin or plasmin bound to hepatocytes in a concentration- and time-dependent manner. The apparent Kd values calculated from displacement experiments were 7.9 X 10(-8) M for alpha 2-macroglobulin-thrombin and 8.5 X 10(-8) M for alpha 2-macroglobulin-plasmin. Association of these complexes was only partially reversible; after a 180 min incubation period, 50-60% of the bound radioactivity was internalized by the cells. alpha 2-Macroglobulin itself bound also to hepatocytes, but the affinity of the alpha 2-macroglobulin complexes was higher than that of the inhibitor alone, and alpha 2-macroglobulin was not internalized, either. 125I-labelled thrombin or plasmin bound to hepatocytes as well. These bindings were also concentration-dependent and could be decreased with an excess of unlabelled ligands. Binding rates and amounts of the bound proteinases were higher than those of their alpha 2-macroglobulin complexes. The alpha 2-macroglobulin-thrombin complex competed with the alpha 2-macroglobulin-plasmin complex in binding to hepatocytes, whereas there was no competition between these complexes and the antithrombin III-thrombin complex. These results suggest that the binding sites of hepatocytes for alpha 2-macroglobulin-proteinase and antithrombin III-proteinase complexes are different.

Animals↗
PubMed1985

Glucocorticoid receptor is activated by heparin and deactivated by plasmin.

Chick thymus glucocorticoid receptor activation was followed in the presence of heparin and/or plasmin. Heparin, at a concentration of 9 microM, accelerated the rate of activation at 25 degrees C without influencing significantly the maximum activated fraction of the 3H-triamcinolone acetonide-receptor complex. On the contrary, 0.7 microM plasmin added prior to incubation at 25 degrees C (activation) of the complex blocked DNA cellulose binding. Thrombin did not influence receptor activation. Added to the activated complex, plasmin resulted in a rapid deactivation, i.e. an irreversible loss of DNA binding capacity. Plasmin and heparin appeared to exert their effects independent of each other in spite of the fact that they are known to interact in the concentration range in question.

Animals↗
PubMed1985

Sensitivity of thrombin and plasmin to heparin and antithrombin III.

Thrombin and plasmin are inactivated by antithrombin III at different rates. Heparin, at a catalytic amount, increases primarily the inactivation rate of thrombin. The two proteinases compete for heparin, i.e. heparin is preferably attached by thrombin. Furthermore, fibrinogen as well as fibrin protect plasmin against inactivation by antithrombin III. However, at high concentration of heparin, plasmin inactivation by antithrombin III is accelerated even in the presence of fibrinogen or fibrin.

Antithrombins↗
PubMed1985

Blood coagulation-fibrinolytic system and endothelial cells.

Endothelial cells, under normal conditions, possess antithrombotic nature, whereas during damage, various components of the cell may initiate blood coagulation. These functions are greatly influenced and controlled by thrombin and plasmin through their direct actions or their formation in the blood coagulation-fibrinolytic system.

Animals↗
PubMed1984

Uptake of arachidonic acid, arachidic acid, oleic acid and their incorporation into phospholipids and triacylglycerols of isolated murine hepatocytes. Effect of thrombin-antithrombin III complex.

Uptake and metabolism of arachidonic acid, arachidic acid and oleic acid were investigated in isolated hepatocytes prepared from mouse liver with the collagenase perfusion method. The rate of uptake of arachidonic acid was time- and concentration- dependent. 94-98% of the arachidonic acid was incorporated into the phospholipid and triacylglycerol fractions following a 60 min incubation period at 37 degrees C. In the presence of thrombin-anti-thrombin III complex a change in the distribution of arachidonic acid incorporated into lipid fractions was found, i.e. increased incorporation into phosphatidyl-serine and phosphatidylethanolamine, whereas the uptake was not altered. There was no change in the uptake and incorporation of arachidic acid and oleic acid.

Animals↗
PubMed1984

Interaction of plasmin with endothelial cells.

Interaction of human plasmin with a monolayer culture of mini-pig aortic endothelial cells was studied by using the 125I-labelled enzyme. The binding of plasmin was time- and concentration-dependent. Equilibrium between bound and free enzyme was obtained within 90s, and Scatchard analysis indicated a high- and a low-affinity population of binding sites of approx. 1.24 X 10(4) sites/cell having a Kd of 1.4 X 10(-9) M and 7.2 X 10(4) sites/cell with a Kd of 2 X 10(-8) M respectively. Plasmin, bound to cell, was spontaneously released within 2 min, suggesting a rapid equilibrium. Chemical modification of the enzyme with phenylmethanesulphonyl fluoride or pyridoxal 5'-phosphate revealed that neither the active centre nor the heparin-binding site of plasmin was involved in the interaction with the endothelial cell. In terms of endothelial-cell receptors, the binding sites of cells for plasmin and thrombin were different: the two enzymes did not compete with each other, and the pretreatment of cells with neuraminidase or chondroitin ABC lyase resulted in a 50% decrease of thrombin or plasmin binding respectively. Arachidonic acid incorporated into phospholipids of the cell was released by plasmin, but a change in the rate of prostacyclin formation was not measurable. The interaction of plasmin with endothelial cells seems to be specific in the fibrinolytic system, since plasminogen did not bind to these cells under similar conditions.

Animals↗
PubMed1984

Prostaglandin and thromboxane synthesizing activity in isolated murine hepatocytes and nonparenchymal liver cells.

Prostanoid synthesis from 3H-arachidonic acid was compared in isolated parenchymal and nonparenchymal murine liver cells. The cells incorporated arachidonic acid into phospholipids but no prostanoid synthesis could be measured during 30 min incubation. Conditions necessary for prostanoid synthesis were different in parenchymal and nonparenchymal cells and the products were also different. Prostanoid synthesis could be induced by in vitro partial hepatectomy: parenchymal cells synthesized thromboxane A2 whereas nonparenchymal cells produced prostaglandin E2 and F2 alpha. Prostaglandin E2 and F2 alpha synthesis could be provoked also by homogenization of the nonparenchymal cells prepared from normal liver, while the homogenates of parenchymal cells prepared from normal liver did not synthesize thromboxane. Imidazole and indomethacin inhibited the production of thromboxane and prostaglandins, respectively. Our results suggest that the various cell types of the liver respond by the synthesis of different and specific prostanoids after the same injury.

Animals↗
PubMed1983

Interaction of heparin with lipoproteins - role of the complex in the inactivation of thrombin and plasmin.

Heparin forms a complex with human low density lipoprotein (LDL) in the presence of Ca2+. The complex is dissociable by 0.5 M NaCl. Thrombin and plasmin causes the dissociation of the LDL-heparin complex, whereas factor Xa does not. Heparin, complexed with LDL, retains its enhancing effect on the rate of thrombin and plasmin inactivation by antithrombin III. LDL isolated from the plasma of persons with different pathological conditions did not alter the rate of thrombin inactivation by antithrombin III either in the absence or in the presence of heparin. Heparin seems to maintain its biological functions when it is in a complex with LDL.

Chromatography, Gel↗
PubMed1983

The interaction of heparin with human plasmin.

1. The interaction of heparin with human plasmin was investigated measuring plasmin activity and enzyme inactivation in the presence of heparin. Hydrolysis of synthetic substrates (H-D-Val-Leu-Lys-pNA, H-D-Val-Phe-Lys-pNA and H-D-Pro-Phe-Lys-pNA) by plasmin was enhanced by heparin through an increase in kcat values. 2. This effect was the consequence of a change of Vmax since Km values were not altered in the presence of heparin. The polysaccharide also enhanced the rate of enzyme inactivation using TLCK as an active site blocking reagent. 3. Furthermore, heparin increased the heat sensitivity of plasmin, when synthetic substrate H-D-Val-Leu-Lys-pNA was used but it did not affect enzyme activity towards N-benzoyl-L-arginine-ethylester substrate. 4. The data show that microenvironmental conformation around the active center of plasmin is influenced by heparin.

Binding Sites↗
PubMed1983

Mechanism of thrombin binding to endothelial cells.

The interaction of human alpha-thrombin with mini-pig aortic endothelial cells was studied using 125I-labeled enzyme. Equilibrium between bound and free thrombin was attained within 1 min, and the Klotz-Hunston equations indicated two populations of binding sites. Approximately 30,000 sites/cell belonged to the high-affinity class with a Kd of about 3 x 10(-8) M. Modification of two lysine residues of thrombin with pyridoxal 5'-phosphate (PLP2-thrombin) destroyed the high-affinity binding and about three-fourths of the low-affinity bindings. When the lysine residue of thrombin involved in heparin binding was protected with heparin against chemical modification (PLP-thrombin), the modified enzyme remained similar to the native one with respect to cellular binding, with some loss of low-affinity binding only. Heparin, in a tenfold molar excess to enzyme, inhibited the binding of the native as well as the PLP-thrombin, whereas it did not influence the interaction between PLP2-thrombin and the cell. Since heparin might interfere with both the enzyme and the cell, the binding of heparin to endothelial cells was also examined. The results revealed that 3H-heparin also bound to cells. This binding was characterized by a Kd of 3 x 10(-7) M, approximately 10(6) sites/cell. Furthermore, thrombin bound to endothelial cells was released by antithrombin III. On the basis of these and other data in the literature, a model is proposed for the mechanism of the binding of thrombin to endothelial cells.

Antithrombin III↗