Decrease of platelet intracellular pH and adhesion by ticlopidine in patients with vascular disease.

BACKGROUND: Ticlopidine inhibits platelet aggregation by preventing the binding of fibrinogen to its platelet receptor. We examined whether this inhibition involved platelet transduction system such as Na+/H+ pump and platelet intracellular calcium. METHODS: Platelet adhesion in 13 patients with peripheral vascular disease treated with ticlopidine, 250 mg b.i.d for 30 days, was measured in culture microplates before and after therapy. The microplate wells were coated with human plasma, fibrinogen or collagen, and platelet adhesion was studied in the resting condition and after stimulation with 1 and 10 microM ADP. At the same time, platelet intracellular calcium and ADP-induced calcium increases were measured with the fluorescent indicator Fura 2. In addition, intracellular pH and thrombin-induced pH variations were measured with the fluorescent probe BCECF. RESULTS: Platelet adhesion to plasma and fibrinogen was significantly reduced (about 50%) after treatment with ticlopidine, while adhesion to collagen was not modified. Basal calcium and ADP-induced calcium increase were not significantly different before and after ticlopidine. Platelet basal intracellular pH was reduced (from 7.44+/-0.009 to 7.41+/-0.017, p<0.05), but agonist-induced alkalinisation was not significantly different. Early acidification, not dependent on Na+/H+ exchange, was also reduced (p<0.05). CONCLUSIONS: These data do not seem to support the hypothesis that ticlopidine-induced reduction of platelet adhesion depends on alteration of the mechanisms determining signal transduction, at least as far as basal and post-stimulation intracellular calcium is concerned. On the contrary, the possibility that ticlopidine inhibits the Na+/H+ antiport remains open to consideration.

Oxidized LDL and reduction of the antiaggregating activity of nitric oxide derived from endothelial cells.

Oxidized LDL has been observed to induce abnormalities in endothelial function which may be relevant for the progression of atherosclerotic lesions. We studied in vitro the possible effects of oxidized LDL on the antiaggregating activity of endothelial cells, which is dependent on release of prostacyclin and nitric oxide. We used an experimental model in which cultured human endothelial cells were placed in the aggregometer in contact with human platelets, after blockade of cyclo-oxygenase by adding acetylsalicylic acid. In this way the antiaggregant effect of endothelial cells was dependent on the release of nitric oxide alone; prevention of antiaggregant activity by preincubation of endothelial cells with 300 microM L-NG-mono-methylarginine confirmed this. When this system was used, endothelial cells (2-7.5 x 10(5)/ml) almost completely inhibited thrombin-induced (0.02-0.08 U/ml) platelet aggregation (2 x 10(8) platelets/ml), measured according to Born (11.1% +/- 8.5 vs 68.6% +/- 12.6, M +/- SD). This antiaggregating activity was reduced when slightly oxidized LDL 100 micrograms/ml (35.2% +/- 14.9, p < 0.001), but not native LDL 100 micrograms/ml (7.5% +/- 7.6), was added immediately before aggregation was induced. Incubation of endothelial cells with oxidized LDL 100 micrograms/ml for 1 h did not affect the antiaggregating capacity, unless oxidized LDL was present during aggregation (18.3% +/- 10.2 vs 35.8% +/- 9.6, p < 0.02). No significant direct effect of either oxidized or native LDL on stimulated platelet aggregation was observed. Our results indicate that slightly oxidized LDL can reduce the antiaggregating properties of the endothelium, probably by interaction with NO rather than through inhibition of its synthesis.

Computer assisted laboratory diagnosis: a ten-year experience.

An automated procedure to help general practitioners in clinical diagnosis and decision making is presented. The computer-based program is conceived to process results from laboratory tests performed on outpatients, providing general practitioners with possible causes of abnormal results. When only one or two abnormal tests are observed, a series of suggestions pertinent to each abnormality is printed. When there are more abnormal test results, the program performs a more complex procedure ending with the output of some diagnostic hypotheses. Messages are also printed to focus the physician's attention to particular aspects of patient pathology that were sometimes missed or disregarded and to suggest new investigations the laboratory can perform to improve diagnostic efficiency. Moreover some advice is supplied to allow a better evaluation of particular risk conditions, as those associated with the development of coronary heart disease. The program has been recently extended with the calculation of intraindividual reference intervals. The system described has been working since 1976 and appears particularly useful when the general practitioner is faced with a number of pathological results of difficult interpretation.

Spectrin extractability from erythrocytes in Duchenne muscular dystrophy patients and carriers and in other myopathies.

Spectrin extractability was measured in the erythrocyte membranes from patients with Duchenne Muscular Dystrophy (DMD), from DMD definite carriers (in whom serum creatine kinase (CK) was also measured) and patients affected by other myopathies. After the extraction of spectrin from ghosts with EDTA, membrane proteins were examined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Spectrin extractability was also investigated in the presence of an excess of calcium. Spectrin extraction from erythrocyte ghosts was significantly reduced with respect to controls in DMD patients, in DMD definite carriers and in patients affected by limb-girdle dystrophy, but not in patients suffering from other non-dystrophic myopathies. Fifty percent of DMD definite carriers showed a reduced extraction of spectrin and some of them had normal serum CK. Reduced extractability was also observed in red blood cells incubated in media containing excess calcium. Our results could suggest that reduced spectrin extractability is connected with a modification of intracellular calcium levels.

Increased leukocyte aggregation in patients with hypercholesterolaemia.

Leukocyte aggregation induced by N-formylmethionyl-leucyl-phenylalanine (FMLP) has been measured in a group of patients with hypercholesterolaemia (n = 22) and in a group of control subjects (n = 26), age- and sex-matched. When hypercholesterolaemic patients were divided into two groups, with a discriminatory level of 7.7 mmol/l of plasma cholesterol, FMLP-induced leukocyte aggregation of patients with greater than 7.7 mmol/l of plasma cholesterol was significantly higher compared both with control subjects and patients with plasma cholesterol levels less than 7.7 mmol/l. A positive significant correlation was found between leukocyte aggregation and plasma cholesterol, whereas no correlation was observed between leukocyte aggregation and plasma triglycerides or APO-B levels. These findings provide further support for the hypothesis that elevated plasma cholesterol levels may induce a cellular membrane abnormality responsible for the increased leukocyte aggregation and, subsequently, endothelial damage.

Selenium enhances prostacyclin production by cultured endothelial cells: possible explanation for increased bleeding times in volunteers taking selenium as a dietary supplement.

Selenium added to the culture medium of confluent pig aortic endothelial cells caused a time-related elevation in the activity of the hydroperoxide scavenging enzyme: glutathione peroxidase. This increased activity was associated with an enhanced ability to produce prostacyclin irregardless of whether the agonist was arachidonic acid or thrombin. Since prostacyclin synthetase is believed to be irreversibly inhibited by alkyl hydroperoxides, we feel that the greater production of prostacyclin by selenium-treated cells as compared with control cells may reflect a protective effect of GSH.Px towards the synthetase enzyme. The results from this study may explain the observations made on a group of human volunteers ingesting selenium as a dietary supplement. After six weeks treatment with selenium, bleeding time in this group was prolonged suggesting an improved ability to synthesize prostacyclin as a result of selenium-dependent glutathione peroxidase activation in the vessel wall.

Kidney 15-hydroxy-prostaglandin-dehydrogenase activity during the development of experimental hypertension in the rat.

15-hydroxy-prostaglandin-dehydrogenase (PGDH) activity was studied in rat kidney homogenates during the development of hypertension, within 20 days after left renal artery constriction by a solid silver clip. In the ischemic kidney PGDH activity increased at day 6, reached maximum at day 10, then progressively at day 15 and returned to normal levels at day 20. No difference was found between contralateral kidneys and kidneys of normotensive control rats. Variations of PGDH activity did not seem to be related to either renal perfusion pressure or renin production. Increased PGDH activity may be a consequence of an enzyme induction following increased PG-synthetase activity, or it could be viewed as a defence mechanism, according to the hypothesis of a prohypertensive effect of PG in the rat.

125I-iothalamate and creatinine clearances in patients with chronic renal diseases.

In 196 adult patients with chronic renal disease or primary hypertension, the evaluation of glomerular filtration rate (GFR) by means of creatinine clearance, 'predicted' creatinine clearance and [125I]-iothalamate clearance was performed. Iothalamate clearance was evaluated after subcutaneous injection of the substance . In patients with normal or upper borderline plasma creatinine values, the iothalamate clearance ranged from 44 to 117 ml/min/1.73 m2 and the overestimation of GFR from creatinine clearance was negligible. In patients with mild or advanced renal failure, the overestimation of GFR from creatinine clearance increased up to 18 and 32%, respectively. The clinical usefulness of iothalamate clearance is evident especially in patients with mild renal failure, in whom an accurate evaluation of GFR is often important for a correct dietary and therapeutic approach.

In vivo and in vitro variations of human erythrocyte glutathione peroxidase activity as result of cells ageing, selenium availability and peroxide activation.

Cases showing erythrocyte glutathione peroxidase (GSH-Px) defects have been previously described. Our experiments demonstrate that a number of non genetic factors may influence the GSH-Px activity in human erythrocytes. Selenium administration in vivo was followed in four subjects by elevation in erythrocyte GSH-Px activity ranging from 30% to 1400%. Selenium operates mainly in the bone marrow erythroblasts by facilitating the synthesis of active GSH-Px molecules; experiments in vivo demonstrate that, in the youngest erythrocytes, selenium can raise the enzyme activity, but by a different mechanism. The reticulocyte GSH-Px activity appears to depend on selenium availability and may vary over a wide range. In some normal and iron deficient subjects the GSH-Px activity in the youngest erythrocyte fraction was equal or lower than that previously found in whole erythrocytes of patients affected by haemolytic anaemia. During erythrocyte life, GSH-Px activity may either diminish or increase, and these variations are inversely related to the initial GSH-Px activity in youngest cells. In vitro experiments with the addition of acetyl-phynyl-hydrazine strongly suggest that elevation of GSH-Px activity may be due to allosteric enzyme activation by activated oxygen.

Urinary kallikrein excretion and plasma renin activity in patients with essential hypertension and primary aldosteronism.

1. The 24 h urinary excretion of kallikrein has been studied in 40 normotensive control subjects and in 74 age-matched patients with essential hypertension under similar conditions. By use of the renin-sodium index, hypertensive patients were divided into two subgroup: low-renin hypertension and normal-renin hypertension patients. Urinary kallikrein determinations were also obtained from six hypertensive patients with primary aldosteronism. 2. Urinary kallikrein was significantly lower both in patients with normal-renin and low-renin essential hypertension. Urinary kallikrein excretion was very high in the patients with primary aldosteronism. 3. In nine hypertensive patients beta-adreno-receptor-blocking therapy caused a significant decrease of plasma renin activity, but had no significant effect on urinary kallikrein excretion. 4. The results support the concept that low urinary kallikrein is likely to be a marker of essential hypertension. Under certain conditions its excretion is positively related to mineralocorticoid hormone concentrations but it is not primarily related to the renin-angiotensin system.

Unilateral cryptorchidism with compensatory hypertrophy of descended testicle in prepubertal boys.

5 prepubertal boys with unilateral cryptorchidism and compensatory hypertrophy of the descended testicle, 22 prepubertal boys with unilateral cryptorchidism and without CTH, and 14 prepubertal normal boys were submitted to LH-RH and to HCG tests in order to study the hormonal behaviour in CTH phenomenon before puberty. High but normal peaks of plasma LH and FSH were observed after LH-RH in CTH boys who showed a significant increase of testosterone after HCG stimulation. On the contrary the LH response to LH-RH and the testosterone response to HCG of the boys with unilateral cryptorchidism and without CTH were, as expected, significantly lower than in the control ones.