Biomedical subjects
Publications and source records attributed to U Till.
Human hypercholesterolemic serum increases proliferation of human umbilical endothelial cells in vitro. This might be due to an increased content of low molecular weight growth factors, which are released by blood cells, mainly thrombocytes. Hypercholesterolemia could increase endothelial cell turnover and expression of dysfunctional cells which may support atherogenic processes.
This study investigates the effect on human platelet function of two sesquiterpene lactones from Arnica montana L., helenalin (H) and 11 alpha,13-dihydrohelenalin (DH). Both compounds inhibited collagen-induced platelet aggregation, thromboxane formation and 5-hydroxytryptamine secretion in a concentration-dependent manner at 3-300 microM. When arachidonic acid was used as stimulus, thromboxane formation remained unaffected despite of inhibition of platelet aggregation. Both H and DH reduced the number of acid-soluble sulfhydryl groups in platelets, by up to 78% at anti-aggregatory concentrations. Moreover, H- and DH-induced platelet inhibition could be prevented by the thiol containing amino acid cysteine. It is concluded that H and DH inhibit platelet function via interaction with platelet sulfhydryl groups, probably associated with reduced phospholipase A2 activity.
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An inbred strain of fawn hooded rats with a congenital platelet defect shows a marked bleeding tendency with prolonged bleeding time. This haemorrhagic disorder has been exclusively related to a deficiency of nucleotides in platelet dense granules. When tested in cell electrophoresis platelets from fawn hooded bleeder rats showed a significantly lower electrophoretic mobility than normal rat platelets. Subsequent studies on the platelet membrane protein pattern by high resolution two-dimensional gel electrophoresis revealed the deficiency of a membrane glycoprotein (apparent molecular mass 90.000, isoelectric point 5.6), which is detectable in normal rat platelets after surface labeling by periodate-tritiated sodium borohydride. It seems likely, that this glycoprotein defect contributes at least partially to the disorder of platelet function in fawn hooded bleeder rats.
A newly synthesised structural analogue of PAF, coded KO-286011 (1-O-hexadecyl-2-O-ethyl-rac-glycero-3-phosphoric acid 4-(N,N-dimethylamino)pyridinium butylester), was proved for its ability to inhibit PAF-mediated platelet responses in vitro and in vivo. The compound inhibited effectively the PAF-induced aggregation and secretion of human and rabbit platelets. In contrast, there was little influence on ADP-, collagen-, and arachidonic acid-triggered platelet responses. Schild-analysis of aggregation data ascertained in human platelet-rich plasma was consistent with a simple competitive antagonism and yielded a pA2 of 6.44. Proaggregatory activity of KO-286011 was excluded turbidimetrically as well as by means of a single cell counting technique. [3H]PAF binding studies provided evidence that KO-286011 exerts its inhibitory action at the PAF-receptor level. A significant inhibition of the ex vivo PAF-induced platelet aggregation was found after i.v. administration of 0.5 mg/kg KO-286011 to rabbits. The effect was most pronounced 5 min after dosing the inhibitor and detectable over a period of 30 min. Intravenous administration of 10 and 25 micrograms/kg KO-286011 to guinea pigs prevented dose-dependently the PAF-induced formation of thromboxane A2. The PAF-inhibitory action of KO-286011 was more potent than that of the ginkgolide BN 52021.
Phorbolester (PMA) and forskolin (FSK) cause a dramatic reorganization of microfilaments in cultured cells. We have incubated human blood platelets with PMA and FSK and we investigated the G-/F-actin equilibrium by the DNase I inhibition assay. PMA incubation (0.8 microM, 5 min, 37 degrees C) leads to an increase of filamentous actin (14.4 +/- 4.0%) compared to control platelets. The effect is rapid, dose-dependent and specific, since the biologically inactive derivative phorbol 12,13-didecanoate has no effect. FSK incubation (4 microM, 5 min, 37 degrees C) causes a decrease of F-actin (12.8 +/- 9.0%), the effect is rapid and dose-dependent too. Since 8-bromoadenosine 3':5' cyclic monophosphate also decreases F-actin in human blood platelets, the FSK effect seems to be mediated by cyclic AMP due to affecting the adenylate cyclase.
In vitro net mass transfer of HDL cholesteryl ester to apolipoprotein (Apo) B-containing lipoproteins (HDL-CET) was found to be nearly twofold higher in plasma from 35 male patients with peripheral vascular disease compared to the values of 27 age- and sex-matched healthy controls (P less than 0.001). Differences in HDL-CET were predominantly observed between normolipidemic patients and controls, and were also demonstrable in pairs of patients and controls with similar HDL cholesterol, VLDL + LDL cholesterol, and triglyceride concentrations. Within the control group, higher HDL-CET was found in individuals with enhanced triglyceride or VLDL + LDL cholesterol levels. This dependence was not observed in the patient group. Consequently, enhanced HDL-CET in the patients seems to be independent of plasma lipid levels.
Blood platelets have the capacity to participate in a number of physiological as well as pathological processes within the circulation. In order to evaluate their cellular reactivity a number of platelet function tests have been developed. The main in vitro function tests are assessment of aggregation and adhesion, secretion, arachidonate metabolism, coagulant activities and the characterization of surface membrane glycoproteins (Day and Rao, 1986). Here we measure alterations of the G-/F-actin equilibrium of platelets. High F-actin values of unstimulated platelets indicate a hyperreactivity of the cell as examined in platelets from diabetics. Determination of the actin filament content in platelets can be considered as a new sensitive function test.
Platelets appear to be involved in the development of vascular diseases in diabetic patients. In a number of studies, platelet adhesion and aggregation has been found to be enhanced in diabetic patients indicating a platelet hyperreactivity. However, contradictory results on hyperreactivity of diabetic platelets towards agonists published in the literature leading to the problem of reliability of agonist-induced aggregation as a parameter which is able to reflect an altered platelet reactivity. Therefore, we introduced the quantification of actin filaments of platelets as an early indicator of platelet hyperreactivity. In 20 patients with type I diabetes mellitus agonist-induced aggregation, spontaneous aggregation, malondialdehyde (MDA)-formation, plasma lipid status and the ability of plasma to degrade platelet activating factor (PAF) as well as the G- to F-actin equilibrium of platelets were assayed. Compared to an age- and sex-matched control group F-actin values, spontaneous aggregation and PAF-hydrolase were significantly increased in diabetic patients.
Platelet-activating factor (PAF) is rapidly degraded in vivo by the action of a specific acetylhydrolase. Measuring the PAF-acetylhydrolase activity in serum from 12 healthy volunteers a maximum velocity of 67.2 +/- 11.8 nmol/min X ml and a Km value of 15.8 +/- 2.9 mumol/l were ascertained. More than 90% of the activity was found to be associated with lipoproteins. It was detected in isolated VLDL, LDL, Lp(a) as well as HDL2 but not in HDL3. The PAF-acetylhydrolase activities associated with the various lipoproteins were shown to behave like a unique enzyme with distinct kinetic properties. Because VLDL and LDL were found to take up about 5 and 2.5 times more PAF with respect to their mass than HDL, it is concluded that lipoproteins affect the PAF-acetylhydrolase activity at the level of substrate presentation. As a consequence of the distinct kinetic properties, the lipoprotein-associated PAF-acetylhydrolase activities do not contribute proportionately to the degradation of PAF in serum. The latter is shown to depend primarily on the amount of PAF-acetylhydrolase bound to the apoprotein B-containing lipoproteins.
Both, the phagocytic process and the activation of phagocytes with soluble stimuli are accompanied by increased production of reactive oxygen species (ROS). Chemiluminescence (CL) measurement is a simple and sensitive method for the detection of ROS generation. Phagocytes (mainly polymorphonuclear leukocytes, PMNL) were stimulated with soluble stimulus or via phagocytosis in diluted whole blood, and the generation of Luminol-enhanced CL was registered. The time dependence of CL, determined in whole blood, corresponds to the CL from isolated leukocytes. A relationship between peak CL and the number of leukocytes as well as of PMNL was observed. The specific CL, i.e. the CL response related to a defined PMNL number, increases with the age of investigated healthy individuals. No correlations were found between CL and the capacity of PMNL to ingest zymosan particles. Relations between CL and spontaneous platelet aggregation suggest, that reactivity of blood platelets may be a contributing factor to the kinetics of the CL signal in our test system. The inhibition of CL by the sulphydryl reagents diamide and fever few extract indicate the role of cellular sulphydryl groups for phagocyte function. Measurement of CL in whole blood is proved to be a simple assay for assessment of PMNL function and allows measurements in very small blood samples (greater than or equal to 10 ul).
Treatment of polymorphonuclear leukocytes with the soluble and cell penetrating thiol N-(2-mercaptopropionyl)-glycine (2-MPG) inhibits luminol-enhanced chemiluminescence induced by opsonized zymosan, which is believed to be a measure for the generation of reactive oxygen species, and accelerates the rate of phagocytosis measured as ingestion of zymosan particles. It is discussed that 2-MPG protects the phagocyting cells from oxidative damage by reactive oxygen species that are formed during particle ingestion.
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Incubation of citrated or heparinized blood samples with 25 or 50 mmoles/l glucose for 1 hour at 37 degrees C results in a decrease in the osmotic and mechanical resistance of red blood cells as well as in an increase in the red blood cell volume measured as increase in haematocrit. Fructose, but not sorbitol or saccharose has effects on red blood cells that are similar to those of glucose. It is discussed that an uptake of glucose into red blood cells with subsequent water influx results in swelling and decreased stability of the cells.
The general anaesthetic halothane decreases both the metabolic ATP level and the aggregability of blood platelets. Evaluation of the dose dependence of these halothane effects reveals that the loss of aggregability is not the direct result of an impaired energy metabolism.
Blood platelets from malignant hyperthermia patients share the same defect as skeletal muscle. This could be verified by the effect of halothane on the energy metabolism of blood platelets under optimal experimental conditions. The sensitivity of changes in adenine nucleotide metabolization to halothane was significantly higher in platelets from malignant hyperthermia patients than in controls. The enhanced sensitivity is not caused by an adenylate kinase deficiency.
Platelet-activating factor (PAF) is transformed in vivo rapidly into the biologically inactive lyso-PAF. This reaction as well as lipid parameters were quantified in serum from 40 survivors of myocardial infarction and 36 healthy controls matched for age and body weight. The PAF-degrading capacity was 23% (p less than 0.001) higher in patients compared with the control group. Using the degradation of PAF as an univariate discriminator more than 70% of subjects were classified correctly. This is comparable with the discriminatory value of the best lipid variables, apolipoprotein B and HDL-cholesterol. Statistically significant differences in the degradation of PAF were found also by comparing subgroups which were matched for plasma levels of total cholesterol, VLDL/LDL-cholesterol or apolipoprotein B. The ratio by 48% (p less than 0.0001) in the case group was identified as an additional good discriminator between both groups. In contrast, platelet aggregation tests which were performed in acetylsalicyclic acid treated platelet-rich plasma discriminated poorly between patients and controls.
Electrorotation of single platelets was compared with [14C]serotonin release, aggregation and electron microscopy. Activation of washed and degranulated platelets was induced by thrombin, arachidonic acid, collagen, adrenaline, platelet activation factor (PAF), ADP and A23187. A strong correlation between electrorotation decrease and serotonin release was found. Electrorotation did not correlate with aggregation. It was concluded that an increase of the specific conductivity of the platelet membrane by three orders of magnitude (approx. 1.0.10(-7) S.m-1 to 1.0.10(-4) S.m-1) upon activation was responsible for the observed decrease of anti-field rotation and the shift of the first characteristic frequency towards higher values. Electrorotation allowed for time-dependent measurements of activation. Characteristic activation times in the order of minutes were found. There was the following sequence of activators classified by increasing activation time constants: A23187 was the fastest followed by thrombin, collagen, PAF, arachidonic acid, adrenaline, and ADP.