Studies on the stimulation of human blood platelets by semi-synthetic platelet-activating factor.

"Saturated" and "unsaturated" platelet-activating factor (PAF) obtained from ratfish liver oil were proved to exert potent stimulation on human blood platelets. Using 0.025 to 1.0 mumol/1 PAF a dose-dependent platelet aggregation in platelet-rich plasma was observed. During PAF-induced irreversible aggregation a 9 to 40% release of platelet bound serotonin occurred. The specific effect of PAF, however, seems to be limited to induce reversible aggregation since second wave of aggregation and serotonin release were suppressed by a combination of acetylsalicylic acid and an ADP scavenging system. Incubation of PAF for 30 min in plasma resulted in a 90% loss of its platelet aggregating power. Subthreshold concentrations of PAF enhanced the platelet aggregation triggered by suboptimal concentrations of ADP, epinephrine, or collagen. Vice versa non-aggregating concentrations of ADP, epinephrine, collagen, Ca-ionophore A 23,187, or arachidonic acid amplified PAF-induced platelet aggregation. The synergistic effect of PAF and other stimuli of blood platelet activation can be partly interpreted as a stimulating effect of PAF on the metabolization of arachidonic acid.

Cooperative effects of 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine (PAF-acether) and exogenous arachidonic acid in stimulation of human blood platelets.

Cooperative effects of PAF-acether and arachidonic acid in blood platelet activation were studied in human platelet-rich plasma. Using a combination of low concentrations of PAF-acether and subthreshold amounts of exogenous arachidonic acid an enhancement of aggregation and an increased formation of malondialdehyde are obtained. Both effects are completely suppressed either by acetylsalicylic acid or by use of PAF-acether desensitized platelets, indicating that cyclooxygenase products and intact receptors for PAF-acether are of primary importance in the observed synergism. Stimulating activity of PAF-acether on the formation of cyclooxygenase products is also seen with use of higher concentrations of arachidonic acid during mild stimulation of human platelets. This effect of PAF-acether is dependent on the concentration used and is obviously due to changes in the platelet membrane as derived from experiments with lysed platelets. Enhanced capacity of platelets to synthesize endoperoxides and thromboxane in consequence of an in vivo release of PAF-acether into the circulation might be of pathobiochemical relevance in some circumstances.

Use of the microanalytical system KAPA for serial determinations of triglycerides, total cholesterol and HDL cholesterol in capillary plasma.

Enzymatic methods adapted to the KAPA system can be carried out in a final test volume of less than 10 mu 1. The procedure is mechanized, including the pre-analytical steps. Day-to-day precision is 2-6% C.V. for single determinations. Advantages of the technique are: (1) capillary plasma can be used; (2) reagent waste is reduced by about two orders of magnitude compared to common procedures, and (3) 400 specimens can be assayed by one technician per day. These conditions facilitate serial determinations, large scale screening, or repetitive monitoring of values in a group of individuals.

Enhanced net mass transfer of HDL cholesteryl esters to Apo B-containing lipoproteins in patients with peripheral vascular disease.

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.

[Some remarks on recent trends in pathology (author's transl)]. / Gedanken zur Entwicklung der Pathologie

The present situation of pathology is analysed. Concerning the German Democratic Republic the situation is characterized by a consequent separation of the pathology into the special branches of pathomorphology, pathophysiology, and pathobiochemistry.

Intraindividual variability of plasma cholesterol and triglycerides and the effect of propranolol treatment.

In 114 male subjects cholesterol and triglyceride concentrations in plasma after overnight fasting were followed up during 12 weeks. About one third of the individuals showed pronounced day to day variations of their cholesterol or/and triglyceride levels. Considerable intraindividual variability was moreover found for HDL cholesterol. Plasma lipid instability was more frequent in hyperlipemics and in subjects with low HDL cholesterol than in normolipemics. It was tried to level out the fluctuations by a beta blocking agent. Propranolol was used and proved to reduce the fluctuations of total cholesterol. However, at the same time the concentrations of triglycerides were increased and those of HDL cholesterol decreased. Practical consequences of both the fluctuating plasma lipid concentrations and the propranolol effects are stressed.

[The binding of blood plasma proteins and salivary proteins to hydroxyapatite columns and its effect on demineralization in an in vitro system]. / Die Bindung von Blutplasma- und Speichelproteinen an Hydroxylapatitsäulen und deren Einfluss auf die Demineralisation in einem In-vitro-System.

Demineralization of hydroxyapatite (HAP) columns by acidic acetate buffer proceeds in two phases. In an initial phase which is characterized by the formation of pH gradient within the HAP sediment, calcium but no phosphate ions are eluted from the column. In the second phase when a low pH value has been established in the pellet calcium and phosphate are eluted in a ratio identical to that of the original HAP. The binding of human blood plasma proteins and saliva proteins to the HAP as well as the effect of the protein binding on HAP demineralization was investigated. The maximum binding capacity of HAP for the proteins of unstimulated mixed saliva is about four times lower than the binding of human serum albumin or blood plasma proteins. However, saliva proteins affect HAP demineralization more markedly than the other proteins. Whereas the pretreatment of HAP with human serum albumin does not alter the the demineralization process. binding of blood plasma proteins inhibits the initial phase of demineralization. Both phases of HAP demineralization are strongly inhibited when saliva proteins are bound to HAP. It is also shown that demineralization modifies the protein binding to HAP.

Effect of diamide (azodicarboxylic acid-bis-dimethylamide) on arachidonic acid release from human blood platelet phospholipids.

It was found that the thiol-oxidizing agent diamide inhibits the formation of arachidonic acid metabolites in thrombin-stimulated platelets. An inhibition of arachidonic acid release from membrane phospholipids in diamide-treated platelets is concluded since diamide does not diminish the formation of stable metabolites from exogenous arachidonic acid. The role of the cellular thiol-disulfide status for the activities of phospholipases involved in arachidonic acid release is discussed.

Effect of talinolol and the optical isomers of propranolol on LCAT activity in vitro.

In vitro effects of DL-propranolol (a therapeutically used beta-blocker with local anesthetic properties), L-propranolol (having both beta-blocking and unspecific local anesthetic properties), D-propranolol (having local anesthetic, but much less beta-blocking properties), and talinolol (a therapeutically used beta-1-blocker with much less local anesthetic properties) on lecithin: cholesterol acyl transferase (LCAT) activity in human plasma were compared. Both D-propranolol and L-propranolol inhibited LCAT activity, the former being a little more effective. The racemic mixture showed intermediate dose-effect curves. In no case could a complete inhibition be achieved. Talinolol exhibited no effect on LCAT activity. Therefore, the inhibition of LCAT activity by beta-blocking drugs may be connected with their local anesthetic properties. Because LCAT inhibition is followed by a decrease of HDL cholesterol and an increase of triglycerides in plasma, it is recommended to use only beta-blockers without such side effects on lipoprotein metabolism for therapeutical purposes.

Stimulation of lecithin:cholesterol acyl transfer by intravenous injection of heparin.

Intravenous injection of 100 IU heparin per kg body weight caused elevation of the lecithin:cholesterol acyl transfer rate in each one of 11 men, in whom before as well as 5 min and 30 min after heparin application the concentration of unesterified fatty acids in plasma did not reach 0.8 mmol/l. The plasma concentration of unesterified cholesterol in high-density lipoproteins (HDL) increased in parallel at the expense of unesterified cholesterol in very low-density lipoproteins (VLDL) and low-density lipoproteins (LDL). For HDL cholesteryl esters an opposite effect was observed. The results support the hypothesis of a participation of the lecithin:cholesterol acyl transferase (LCAT) in VLDL breakdown.

The effect of propranolol treatment on total cholesterol, HDL cholesterol, triglycerides. Postheparin lipolytic activity and lecithin:cholesterol acyltransferase in hypertensive individuals.

Administration of 120 mg propranolol per day to hypertensive patients is followed by decreased plasma concentrations of total cholesterol and HDL cholesterol and increased concentrations of triglycerides. Postheparin lipolytic activity was reduced when measured 5 min after heparin administration. The activity of lecithin:cholesterol acyltransferase is diminished. Reduced activity of this enzyme by propranolol could be demonstrated in vitro as well.

Principle and application of a multicuvette for determination of enzyme activities and substrate concentrations in microsamples.

A method of photometric analysis is described that allows to determine enzyme activities or substrate concentrations of 50 samples simultaneously in a multicuvette. Test volume is in the range of 30-85 micronl, light path between 0.5 and 2.0 mm. The testing principle can be used for colorimetric measurements and for NADH dependent reactions as well. The procedure is particularly suited for kinetic determinations. Variation coefficients for determinations of substrate concentrations and enzyme activities are in the range of conventional enzymatic tests.

Inhibition of lecithin: cholesterol acyl transfer by propranolol treatment in man.

The changes in some parameters of plasma lipoprotein metabolism in 25 hypertensive men, caused by a two months lasting oral treatment with 120 mg propranolol/day, were compared to the changes that occur during the same time in the subjects (n = 9) of an untreated control group. Propranolol led to an increase of triglycerides and a decrease of HDL cholesterol in plasma, the latter consisting in a small increase of unesterified cholesterol and a larger decrease of cholesteryl esters. In parallel a decrease in the lecithin:cholesterol acyl transfer, measured as initial rate of cholesterol esterification, was observed. The extent of the plasma lipid and enzyme alterations was shown to be dependent on the patient's pretreatment situation. Changes in lecithin:cholesterol acyl transfer rate after oral treatment correlated significantly with similar changes after propranolol addition in comparable amounts to plasma in vitro. A similarity between propranolol induced alterations in the metabolism of plasma lipoproteins and the situation in hereditary LCAT deficiency is obvious and may be interpreted as a harmful component of propranolol action.

Hydroxyapatite columns as an in vitro system to study demineralization processes.

Acetate buffers were passed through small hydroxyapatite columns and the column effluents were analysed for calcium and phosphate. Changes in the pH of the column effluents were also measured. As long as the pH of the column effluent was higher than about 5.5, only calcium was eluted from the column. However, when the pH fell below 5.5, the rate of calcium elution decreased and phosphate elution increased until calcium and phosphate elution reached constant values. Pretreating the hydroxyapatite with sodium fluoride or 1-hydroxyethylidene-1,1-bisphosphonate accelerated the drop of the pH and reduced the initial Ca/P ratio in the column effluent. We suggest that these results are due to the formation of a pH gradient along the progressing buffer fron with calcium and phosphate liberated from the hydroxyapatite reprecipitating to form calcium-poor minerals.

[Proteins and phospholipids of thrombocytes and erythrocytes in glucose-6-phosphate dehydrogenase deficient patients]. / Untersuchungen der Protein und Phospholipide von Thrombozyten und Erythrozyten bei Glukose-6-phosphat-dehydrogenase-defizienten Patienten.

Proteins and phospholipids of platelets and red cell membranes of glucose-6-phosphate-dehydrogenase-deficient patients with chronic hemolytic disease were investigated. The enzyme deficiency is not connected with alteration in the protein and phospholipid pattern of platelets. However, marked differences in the diamide-induced protein polymerization between normal and deficient platelets were found. Whereas normal platelets show only a weak polymerization which is reversible during diamide incubation of the cells, the deficient platelets demonstrate a considerable and long lasting protein polymerization. The results point to a direct connection between the thiol-disulfide status of platelets and intracellular level of glutathione, which is able to repair oxidative damages. Two of the six patients showed already protein polymers in their erythrocyte membrane without addition of any SH-oxidizing agent. The phospholipid pattern of the deficient red-cell membranes were comparable to the controls.

[Suitability and recovery of capillary blood plasma for lipid determinations]. / Eignung und Gewinnung von Kapillarblutplasma für Lipidbestimmungen.

Plasma lipid analysis, particularly in population screening, can be facilitated by the use of capillary blood. A system for serial collection of capillary plasma is described. The concentrations of total cholesterol, HDL-cholesterol and triglycerides in heparinized capillary plasma and venous blood, respectively, are compared. No significant differences were found. Using the KAPA-method hemolytic samples are a source of error. They occurred with a frequency of about 3% in mass screening.

Anticoagulant-free prepared blood platelets and the effect of calcium on their aggregation behaviour.

Human blood platelets were prepared by gel filtration of native blood and subsequent centrifugation in a Ficoll density gradient avoiding any anticoagulant during the preparation. In ADP, ionophore A 23 187 and thrombin induced aggregation an increase in extracellular Ca++ enhanced the velocity of aggregation. From a defined Ca++ concentration threshold on there was a shift from monophasic to biphasic aggregation.