The simultaneous effect of albumin bound fatty acids on platelets and endothelial cells.
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Biomedical subjects
Publications and source records attributed to B Svensson.
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This study was carried out to evaluate the effects of albumin-bound fatty acids on the anti-platelet effects of endothelial cells. Primary cultures of human endothelial cells (ECM), grown in confluent monolayers, were incubated with plasma or growth medium enriched with albumin-bound fatty acids (FA) for 2-20 h. The effects of ECM on ADP-induced platelet aggregation (PA) and collagen-induced PA and prostaglandin synthesis in platelet-rich plasma were tested. ECM released an inhibitor of platelet reactions which resembled the activity of PGI2 (prostacyclin). The inhibitory activity was increased by preincubation of ECM with arachidonic acid (AA). A moderate decrease of the activity was obtained by incubation with long-chain saturated, monoenoic and dienoic unsaturated fatty acids. A pronounced decrease of the inhibitor was obtained by incubation with di-homo-gamma-linolenic acid (DHLA). Paired combinations of AA with the other fatty acids in the incubation medium partially restored the inhibitory activity obtained by the separate FA. The stimulation of the inhibitor by AA was dose dependent and high concentrations of AA reduced this activity. The present study indicates that the quantity and quality of the plasma free fatty acids can affect the endothelial cells' ability to act as a non-thrombogenic surface.
In order to avoid the potential risk of a short refractory period and the inconvenience of the electronic blocking mechanism at the highest synchronous rate with the conventional atrial triggered pulse generator, two more functions have been added to the normal atrial triggered (VAT) pulse generator. First, it has been designed to be ventricular inhibited; second, a highest synchronous rate detector has been added. When the highest synchronous rate of about 140 is exceeded, the atrial signals are blocked and the frequency of the basic rate generator is increased to about 130. Two patients could hardly feel the change from atrial triggered pacing to ventricular inhibited stimulation, and both were able to work at higher loads with this type of pacing compared with the conventional atrial triggered pulse generator.
Intestinal dipeptidyl peptidase IV and gamma-glutamyltransferase were compared to the corresponding kidney enzymes with respect to immunological and electrophoretic properties. The influences of selected effectors on the two enzymes were also studied. The two kidney peptidases exhibited the reaction of total identity with the corresponding intestinal enzymes in immunodiffusion. Furthermore, the intestinal dipeptidyl peptidase IV and gamma-glutamyl transferase showed the same inhibition patterns as the corresponding kidney enzymes and the acceptor specificity of the intestinal gamma-glutamyl-transferase was found to be identical to that of the kidney enzyme. The electrophoretic mobilities of dipeptidyl peptidase IV from the two organs differed greatly. The difference was almost abolished by treatment with neuraminidase, suggesting that the variation in mobility was due to different contents of sialic acid. It is suggested that the intestinal brush border peptidases, dipeptidyl peptidase IV and gamma-glutamyltransferase, are closely related to the corresponding enzymes obtained from the kidney.
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Human endothelial cell monolayers prepared from umbilical veins have been incubated with aspirin (1--2 mM) dissolved in Hepes modified solution and in platelet-rich plasma. They have also been incubated with plasma prepared from subjects before and after intake of aspirin giving a mean plasma concentration of 0.5 mM. The effects of the endothelial cells on ADP and collagen-induced platelet aggregation and malondialdehyde production in platelet-rich plasma have been tested. The endothelial cells had a spontaneous inhibitory effect on all three parameters. This effect was abolished when the cells were incubated with aspirin dissolved in MHS for 20 min and the increase in effect observed when platelet-rich plasma was incubated with endothelial cells for a period of 30 min was similarly inhibited when aspirin was dissolved in plasma or when plasma prepared from subjects who had taken aspirin were used. Aspirin had no inhibitory effect on prostacyclin (PGI2) with regard to the effect of PGI2 on platelets. On the contrary, the two compounds had an additive inhibitory effect on platelet aggregation induced by ADP and collagen. These findings should be considered with regard to the use of aspirin as an antithrombotic agent.
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The inhibitory effects of human endothelial cells (ECM) and PGI2 (prostacyclin) on platelet function have been examined in 60 healthy subjects. 5 groups were studied. I. 10 women (19-33 yrs.); II. 10 women (21-31 yrs.) using combined oral contraceptives; III. 10 postmenopausal women (45-66 yrs.); IV. 20 men (24-48 yrs.) and V. 10 men (48-71 yrs.). Spontaneous platelet aggregation in platelet-rich plasma (PRP) was commonly observed in groups II, III, and V. This was prevented when PRP was incubated with ECM, aspirin (ASA), or PGI2. ADP-induced platelet aggregation (PA) was always reduced by ECM, but significantly less in groups II and III. Collagen-induced PA was inhibited by ECM, but less in groups I, II, III and V than in young men (IV). ECM significantly reduced collagen-induced prostaglandin (MDA) production in platelets. Again, the inhibitory effect was significantly reduced in groups II and III. Platelet factor 3 activity, measured after exposure of PRP to ADP, was significantly reduced by ECM, but was significantly less in groups II, III, and V than in group I. Less of an inhibitory, effect of PGI2 upon ADP-induced PA was shown by all groups of females than by the two groups of males.
We investigated the effect of plasma low density lipoproteins (LDL), very low density lipoproteins (VLDL), and high density lipoproteins (HDL) on the platelet inhibitory effect of primary cultures of human endothelial cell monolayers (ECM). ECM incubated with lipoprotein-deficient plasma (LDP) for 2 hours at 37 degrees C had an inhibitory effect on ADP- and collagen-induced platelet aggregation and prostaglandin production in platelet-rich plasma similar to that observed when ECM were preincubated with growth medium or plasma. Concentrations of LDL in LDP up to protein concentration of 1600 microgram/ml had an inhibitory effect on the endothelial cells' ability to modulate these platelet reactions. VLDL at the highest concentration (1600 microgram/ml) had a slightly inhibitory effect, whereas HDL showed no such effect. The inhibitory effect of LDL was not observed during the first hour of incubation. When HDL in concentrations similar to or higher than LDL were combined with LDL, the inhibitory effect of LDL was partially reduced. VLDL combined with LDL or HDL did not interfere with the effects of the later fractions. The inhibitory effect of LDL was significantly reduced when LDL were diluted in whole plasma. Prostacyclin which is synthesized and released from the endothelial cells and contributes to the inhibitory effect upon platelets did not change its effect on platelet reactivity by preincubation with the various lipoprotein fractions. The current studies may indicate that LDL have a direct effect on the endothelial cells and that this effect may be partially counteracted by HDL. This effect of LDL on the endothelial cells reduces the endothelium's ability to inhibit platelet aggregation and thus could favor the tendency to thrombus formation.
A brush-border-specific antiserum was raised in rabbits, with Triton X-100-solubilized brush border proteins from pig intestine being used as antigens. The antiserum was used in immunoelectrophoretic studies of brush border proteins solubilized with Triton X-100. Five immunoprecipitates were obtained which corresponded to microsomal aminopeptidase (EC 3.4.11.2), asparate aminopeptidase (EC 3.4.11.7), lactase (beta-galactosidase, EC 3.2.1.23), maltase (exo-1,4-alpha-glucosidase, EC 3.2.1.3) and sucrase-isomaltase (sucrose alpha-glucohydrolase, EC 3.2.1.48). A faint immunoprecipitate was also found for the glycylprolyl dipeptidyl peptidase (EC 3.4.14.-). The brush border proteins were solubilized on a large scale from a brush border membrane preparation by the use of Triton X-100; the peptidases obtained were homogeneous in size and had hydrophobic properties. By chromatography on columns of concanavalin A-Sepharose, hydroxyapatite, Ultrogel AcA 34, DEAE-cellulose and immunosorbent, gamma-glutamyl transpeptidase (gamma-glutamyl transferase, EC 2.3.2.2) and microsomal aminopeptidase were each isolated in separate fractions. Glycylprolyl dipeptidyl peptidase and asparate aminopeptidase were obtained in another fraction. Immunoelectrophoretic, inhibitor and chromatographic studies showed that the intestinal brush border peptidases are similar to the corresponding particulate peptidases obtained from other organs.
Analogous soluble and insoluble derivatives of subtilisin Novo (EC 3.4.21.14) were prepared by coupling the enzyme to CNBr-activated DEAE-dextran and DEAE-Sephadex, respectively. The DEAE-dextran-subtilisin displayed pH optima and Km values for ester hydrolysis similar to subtilisin, whereas the pH versus activity profiles obtained with DEAE-Sephadex-subtilisin were shifter towards the alkaline pH region and the Km values were increased. Compared with subtilisin, DEAE-dextran-subtilisin showed a 40-65% reduction of kcat for hydrolysis of N-acetyl-L-tyrosine ethyl ester, p-tosyl-L-arginine methyl ester and benzyloxycarbonyl-glycyl-L-tyrosinamide and its maximum velocities for digestion of casein and clupein also amounted to 40-60% of the subtilisin values. With Deae-sephadex-subtilisin, in contrast, the maximum velocity of hydrolysis decreased to a greater extent for polypeptide substrates compared to ester substrates. The present results indicate that the chemical nature of a support can effect intrinsic properties of a matrix-bound enzyme in addition to the steric and diffusional effects usually observed with polymer-attached enzymes.
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In vitro studies have been done on haematopoietic cells from a patient with cyclic neutropenia characterized by severe depression of blood neutrophil levels every 21 days. Serial blood counts reveal periodic fluctuations in neutrophils, monocytes and reticulocytes. Agar culture of marrow cells shows normal concentration of colony forming cells. The percentage of colony forming cells in S phase is highly increased during profound neutropenia and normal during the recovery phase relating the granulocyte production to the peripheral neutrophil level. Studies of ingestion rate, bactericidal activity, lactate production and glucose oxidation during phagocytosis in isolated granulocytes show normal results. Also the ingestion rate in isolated monocytes is normal. Serial karyotype analyses of marrow cells during the neutrophil cycle display a normal pattern. Serum myeloperoxidase levels vary inversely with the peripheral neutrophil count indicating increased granulopoietic activity during profound neutropenia, which might be associated with non effective granulopoiesis during profound neutropenia, leading to a lack of granulocyte reserves in the marrow.