LDL are oxidatively modified by platelets via GP91(phox) and accumulate in human monocytes.

Oxidative stress-mediated LDL modification has a key role in initiation of the atherosclerotic process. Platelets produce reactive oxidant species (ROS) upon stimulation with agonist, but it is uncertain whether they are able to oxidatively modify LDL. Human platelets taken from healthy subjects were incubated with LDL, then stimulated with collagen. Compared with unstimulated platelets, collagen-stimulated platelets induced LDL modification as shown by enhanced conjugated dienes and lysophosphatidylcholine formation, electrophoretic mobility, Apo B-100 degradation, and monocyte LDL uptake. Activated platelets also induced a marked reduction of vitamin E contained in LDL. A significant inhibition of LDL oxidation was observed in platelets treated with arachidonyl trifluomethyl ketone (AACOCF3), an inhibitor of phospholipase A2. The experiments reported above were also conducted in patients with hereditary deficiency of gp91phox, the central core of NADPH oxidase, and in patients with hypercholesterolemia. Platelets from gp91 phox-deficient patients produced a small amount of ROS and weakly modified LDL. Conversely, platelets from hypercholesterolemic patients showed enhanced ROS formation and oxidized LDL more than platelets from healthy subjects. This study provides evidence that platelets modify LDL via NADPH oxidase-mediated oxidative stress, a phenomenon that could be dependent on arachidonic acid activation. This finding suggests a role for platelets in favoring LDL accumulation within atherosclerotic plaque.

Oxidative stress-mediated platelet CD40 ligand upregulation in patients with hypercholesterolemia: effect of atorvastatin.

OBJECTIVES: We speculated that in patients with hypercholesterolemia CD40L overexpression could depend on low-density lipoprotein (LDL)-induced enhanced intraplatelet formation of O(2)*(-) and statin could reduce platelet CD40L via interference with platelet O(2)*(-) production. BACKGROUND: CD40L is a protein with inflammatory and thrombotic properties. CD40L is upregulated in platelets from hypercholesterolemic (HC) patients but the underlying mechanism is unclear. METHODS: Collagen-induced platelet CD40L and platelet O(2)*(-) expression were investigated in 40 HC patients and 40 healthy subjects. HC patients were then randomized to either a diet (n = 20) (group A) or atorvastatin 10 mg day (n = 20) (group B); the above variables were measured at baseline and after 3 and 30 days of treatment. O(2)*(-) and CD40L were also measured in vitro in LDL-treated platelets with or without nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor or atorvastatin added. RESULTS: Compared with controls, HC patients showed higher values of platelet CD40L (P < 0.001) and O(2)*(-) (P < 0.001). Platelet CD40L was significantly correlated with O(2)*(-) (P < 0.001). The interventional trial showed no changes in group A and a significant and parallel decrease in platelet CD40L (P < 0.001) and O(2)*(-) (P < 0.001) in group B. In vitro studies demonstrated that LDL-induced platelet CD40L and GP IIb/IIIa (PAC1 binding) activation via the NADPH oxidase pathway. CD40L upregulation was counteracted by atorvastatin in a dose-dependent fashion. CONCLUSIONS: This study suggests that in patients with hypercholesterolemia platelet CD40L is upregulated via NADPH oxidase-dependent O(2)*(-) generation. Atorvastatin downregulated CD40L with an oxidative stress-mediated mechanism likely involving platelet NADPH oxidase, an effect that seemed to be independent of its cholesterol-lowering action.

Polyphenols enhance platelet nitric oxide by inhibiting protein kinase C-dependent NADPH oxidase activation: effect on platelet recruitment.

Several studies demonstrated an inverse association between polyphenol intake and cardiovascular events. Platelet recruitment is an important phase of platelet activation at the site of vascular injury, but it has never been investigated whether polyphenols influence platelet recruitment. The aim of the study was to analyze in vitro whether two polyphenols, quercetin and catechin, were able to affect platelet recruitment. Platelet recruitment was reduced by NO donors and by NADPH oxidase inhibitors and was enhanced by L-NAME, an inhibitor of NO synthase. Quercetin and catechin, but not single polyphenol, significantly inhibited platelet recruitment in a concentration-dependent fashion. The formation of superoxide anion was significantly inhibited in platelets incubated with quercetin and catechin but was unaffected by a single polyphenol. Incubation of platelets with quercetin and catechin resulted in inhibition of PKC and NADPH oxidase activation. Treatment of platelets with quercetin and catechin resulted in an increase of NO and also down-regulated the expression of GpIIb/IIIa glycoprotein. This study shows that the polyphenols quercetin and catechin synergistically act in reducing platelet recruitment via inhibition of PKC-dependent NADPH oxidase activation. This effect, resulting in NO-mediated platelet glycoprotein GpIIb/IIIa down-regulation, could provide a novel mechanism through which polyphenols reduce cardiovascular disease.

gp91phox-dependent expression of platelet CD40 ligand.

BACKGROUND: CD40 ligand (CD40L) expression on platelets is mediated by agonists, but the underlying mechanism is still unclear. METHODS AND RESULTS: CD40L expression was measured in platelets from healthy subjects both with and without the addition of antioxidants or a phospholipase A2 (PLA2) inhibitor and in platelets from 2 patients with an inherited deficiency of gp91phox. Immunoprecipitation analysis was also performed to determine whether normal platelets showed gp91phox expression. Unlike catalase and mannitol, superoxide dismutase inhibited agonist-induced platelet CD40L expression in healthy subjects. Immunoprecipitation analysis also showed that platelets from healthy subjects expressed gp91phox. In 2 male patients with inherited gp91phox deficiency, collagen-, thrombin-, and arachidonic acid-stimulated platelets showed an almost complete absence of superoxide anion (O(2)(-)) and CD40L expression. Incubation of platelets from healthy subjects with a PLA2 inhibitor almost completely prevented agonist-induced O(2)(-) and CD40L expression. CONCLUSIONS: These data provide the first evidence that platelet CD40L expression occurs via arachidonic acid-mediated gp91phox activation.

Superoxide anion and hydroxyl radical release by collagen-induced platelet aggregation--role of arachidonic acid metabolism.

Previous study demonstrated that platelets undergoing anoxia-reoxygenation generate superoxide anion (O2-) and hydroxyl radical (OH ) which in turn contribute to activate arachidonic acid (AA) metabolism. However it has not been clarified if oxygen free radicals (OFRs) are also generated when platelets are aggregated by common agonists. We used two probes, i.e. lucigenin and salicylic acid (SA), to measure platelet release of O2- and OH(0), respectively. Among the agonists used, such as ADP, thrombin and collagen, the release of O2- and OH was observed mainly when platelets were stimulated with collagen. Such release was inhibited in platelets pre-treated by aspirin suggesting that AA metabolism was the main source of O2- and OH(0) formation. To further analyze this relationship, O2- and OH(0) formation was measured during AA-stimulated platelet aggregation (PA); we observed that O2- and OH(0) release were dependent upon AA concentration. Furthermore, we found that the incubation of platelets with AACOCF3, a potent inhibitor of cytosolic phospholipase A2, inhibited collagen-induced platelet O2- and OH(0) release. The incubation of platelets with salicylic acid or ascorbic acid, which blunt OH and O2- respectively, inhibited both collagen-induced platelet aggregation and AA-release. This study demonstrated that collagen-induced platelet aggregation is associated with O2- and OH formation, which is dependent upon AA release and metabolism.

Effect of amlodipine on exercise-induced platelet activation in patients affected by chronic stable angina.

BACKGROUND: Literature concerning exercise-induced platelet activation in chronic stable angina is somewhat confusing. The reason lies in the type of exercise as well as in methodological problems. A powerful, recently introduced procedure to detect platelet activation is flow cytometry. Platelet response to activating factors is mediated by calcium uptake; however, calcium antagonist effect on platelet activity is still unclear. HYPOTHESIS: The study was undertaken to investigate exercise-induced platelet activation before and after treatment with amlodipine in chronic stable angina. METHODS: Twenty patients with chronic stable angina were entered into the study. Each subject underwent a symptom-limited cycloergometer stress test following a washout period of 2 weeks. Blood samples were collected before and immediately after exercise. All subjects were then randomized into two groups of 10 patients each, with Group 1 and Group 2 taking amlodipine 10 mg/day, and placebo for 4 weeks, respectively. They subsequently underwent a second exercise stress test, and blood samples were obtained before and immediately after exercise. Flow-cytometric evaluation of platelet activity was performed in order to recognize GMP-140 expression on platelet membrane. RESULTS: Strenuous exercise induced a significant increase in platelet activation in all subjects prior to therapy. No significant differences were observed in platelet activity at rest between Groups 1 and 2, whereas a significant decrease in exercise-induced platelet activation was demonstrated in Group 1 compared with Group 2. CONCLUSION: Our data provide evidence of the favorable effect of amlodipine on exercise-induced platelet activation in patients affected by chronic stable angina.

Cholesterol and fibrinogen as predictive factors of progressive carotid atherosclerosis.

In order to detect the presence of determining factors as predictors of progressive carotid atherosclerosis, the incidence of total serum cholesterol and fibrinogen elevation was evaluated in patients affected by coronary artery disease (CAD). 61 subjects with CAD (mean age 62 years) and significative lesions (> 50%) underwent periodic Echo-Doppler (Duplex scanning) of the supra aortic branches. Total serum cholesterol, HDL, LDL and fibrinogen were monitored, as well. A 24 month follow-up period was performed. Indicative of the progression of carotid atherosclerosis has been considered the presence of a stenosis degree over 20% than the initial one. In 14 of the 61 subjects who entered the study, there was evidence of progressive carotid atherosclerosis. The same patients showed higher levels of LDL cholesterol (130 +/- 36.3 vs 96.5 +/- 33.2) and Fibrinogen (398.3 +/- 59.4 vs 328 +/- 36.8) and lower levels of HDL cholesterol (27.2 +/- 4.2 vs 34.4 +/- 10.1). Our results confirm the importance of cholesterol and fibrinogen as determining risk factors, especially in patients with multiple vascular disease (coronary and carotid).

Incidence of carotid artery atherosclerosis in patients with coronary artery disease.

The natural history of coronary artery disease (CAD) is often complicated by cerebrovascular accidents. The real incidence of atherosclerotic lesions of carotid arteries in coronary patients is not well established. In order to detect a high-risk group for stroke development, 184 patients affected by CAD, either partially symptomatic or asymptomatic for carotid artery stenosis, underwent Echo-Doppler ultrasonography (duplex scanning) of supra-aortic branches. Significant carotid stenosis (> 50%) was demonstrated in 51 subjects (27.7%); 21 subjects (41.2%) were partially symptomatic (dizziness, vertigo, lipothymia, etc), and 30 subjects (58.8%) were completely asymptomatic. The authors' data suggest that carotid disease can develop concurrently with coronary disease in a significant proportion of patients, even though completely asymptomatic. In order to obtain optimal long-term results, both coronary and carotid artery disease require appropriate evaluation and either medical or surgical management. For these reasons they recommend duplex scanning as a routine screening procedure in patients affected by CAD.

[Therapy and prevention of infective endocarditis]. / Terapia e profilassi delle endocarditi infettive.

Antibiotic therapy has improved infective endocarditis prognosis. The observance of general rules to choose the more suitable antibiotic drugs, as regard to their effectiveness, pharmacodynamic peculiarities and use, is mandatory. If the infection is due to antibiotic resistant microorganisms, microbiological analyses to estimate the bactericidal effect of the antibiotics, must be carried out. Resistance to penicillins, oligopeptides and aminoglycosides makes endocarditis produced by Enterococcus spp difficult to treat. The identification of patients at risk for infective endocarditis after surgical and invasive instrumental procedures, allows to introduce antibiotic prophylaxis regimens which can reduce the probability of acquiring the disease.

Evaluation of total anomalous pulmonary venous drainage with cross-sectional colour-flow Doppler echocardiography.

Three patients with total anomalous pulmonary venous drainage (TAPVD) were studied by real-time cross-sectional colour-flow Doppler echocardiography. Serial suprasternal, parasternal and subcostal scans were obtained. In all cases surgical or angiocardiographic confirmation was available. Two patients had supracardiac drainage (to the left vertical vein or to the right superior vena cava) and one patient had infracardiac drainage. An abnormal forward flow in the left innominate vein and vertical vein was visualized in those patients with supracardiac TAPVD. Abnormal venous flow was also imaged in one patient with mixed drainage. In the patient with infradiaphragmatic TAPVD characteristic flow signals were identified in the inferior vena cava (retrograde flow) and in the descending aorta and anomalous pulmonary venous channel (forward flow). In all patients the patterns of pulmonary venous flow allowed us to distinguish TAPVD from contiguous structures and to validate two-dimensional cross-sectional imaging.

Effects of coffee on serum cholesterol and lipoproteins: the Italian brewing method. Italian Group for the Study of Atherosclerosis and Dismetabolic Diseases, Rome II Center.

In order to evaluate the effects of Italian brewed coffee (moka) on cholesterol and serum lipoproteins, a randomized double-blind 14-week clinical trial was performed. After a coffee-free period of four weeks, 49 subjects drank coffee, caffeinated and decaffeinated, for ten weeks. There was no evidence that the Italian method of brewing coffee affects serum lipoproteins since no statistically significant differences were found.

[Silent myocardial ischemia: prevalence, prognostic significance, diagnosis]. / Ischemia miocardica silente: prevalenza, significato prognostico, diagnostica.

Silent myocardial ischemia (SMI) has been demonstrated in 2 to 5% of subjects in totally asymptomatic population, in 30% of patients with history of previous myocardial infarction and in 60 to 100% of patients with stable or unstable angina pectoris. In these patients, 60 to 80% of transient episodes of ischemia are silent and SMI is induced by daily activities and so can be registered during continuous ECG monitoring. The finding of SMI during an exercise testing or during ambulatory monitoring has an unfavourable prognostic significance both in apparently asymptomatic subjects and in patients suffering from stable or unstable angina pectoris or survivors to a myocardial infarction. Stress testing and Holter monitoring are the most used non invasive tests to detect SMI. The sensitivity and specificity of ergometer test can be improved by 201-Tl myocardial scintigraphy. Moreover, the ergometer test can be used as a provocative test to induce changes in regional wall kinesis and so these alterations can be evaluated by using echocardiogram and radioisotopic or contrast ventriculography. The echocardiogram allows to evaluate the presence of kinesis changes induced by stress test or by pharmacological stimulation with dipyridamole or dobutamine. SMI can be also detected through the study of metabolic alterations during cardiac catheterism.

Carnitine inhibits arachidonic acid turnover, platelet function, and oxidative stress.

Carnitine is a physiological cellular constituent that favors intracellular fatty acid transport, whose role on platelet function and O(2) free radicals has not been fully investigated. The aim of this study was to seek whether carnitine interferes with arachidonic acid metabolism and platelet function. Carnitine (10-50 microM) was able to dose dependently inhibit arachidonic acid incorporation into platelet phospholipids and agonist-induced arachidonic acid release. Incubation of platelets with carnitine dose dependently inhibited collagen-induced platelet aggregation, thromboxane A(2) formation, and Ca(2+) mobilization, without affecting phospholipase A(2) activation. Furthermore, carnitine inhibited platelet superoxide anion (O(2)(-)) formation elicited by arachidonic acid and collagen. To explore the underlying mechanism, arachidonic acid-stimulated platelets were incubated with NADPH. This study showed an enhanced platelet O(2)(-) formation, suggesting a role for NADPH oxidase in arachidonic acid-mediated platelet O(2)(-) production. Incubation of platelets with carnitine significantly reduced arachidonic acid-mediated NADPH oxidase activation. Moreover, the activation of protein kinase C was inhibited by 50 microM carnitine. This study shows that carnitine inhibits arachidonic acid accumulation into platelet phospholipids and in turn platelet function and arachidonic acid release elicited by platelet agonists.

[Effects of physical activity on lipids and coagulation]. / Effetto dell'attività fisica sull'assetto lipidico e sulla coagulazione.

In order to evaluate the effects of physical activity on seric lipoproteins and coagulation parameters, an 8-week clinical trial was performed. Fifteen healthy young subjects (average age 23 years) with no history of previous agonistic physical activity, entered the study. Each subject underwent a physical programme consisting of three times a week bicycle ergometer exercise with progressive increases in work rate by using stages of 3 min duration until the 85% of the predictive heart rate was reached. Each individual was subjected to four blood drawings according to the following schedule: at the beginning of the study, after 4 weeks, after 8 weeks (at the end of the programme) and 4 weeks after the interruption of training. As far as the seric lipoproteins are concerned, the following parameters were monitored: total cholesterol, HDL-C, LDL-C, VLDL-C, triglycerides, Apo-A1, Apo-B100, NEFA and phospholipids. On the other hand the following coagulation parameters were monitored: fibrinogen PT, aPtt, coagulation factors (II-XII), red cells, leucocytes, platelets, hemoglobin and hematocrit. From the analysis of the data, the following statistically significant results were observed: HDL-C increased by 14%, LDL-C decreased by 13%, Apo-A1 increased by 6%, fibrinogen increased by 31.7%, Ptt decreased by 3.7% and leucocytes increased by 15%. Four weeks after exercise was terminated, all monitored parameters turned into the basal range. Our data seem to demonstrate a positive effect of physical exercise on seric lipoproteins in the short period. Nevertheless they provide evidence of an hypercoagulability condition demonstrated by the important fibrinogen increase and the Ptt decrease.

Metformin decreases platelet superoxide anion production in diabetic patients.

BACKGROUND: Patients with type 2 diabetes mellitus are usually treated with oral antidiabetic agents but it is still not known whether these drugs have antioxidant effects in humans. METHODS: We studied 60 patients with type 2 diabetes mellitus, divided into three groups on the basis of hypoglycaemic treatment (Group A: metformin, Group B: glibenclamide, Group C: diet). All patients were followed for at least 1 year. The three subgroups had similar clinical characteristics. Twenty healthy subjects, of comparable sex and age, were enrolled as controls. In each subject, platelet production of superoxide anion (O(2)(-)) elicited by collagen, was determined by lucigenin assay. RESULTS: Patients with diabetes had higher platelet O(2)(-) production than controls; no correlation was observed between blood glucose and platelet O(2)(-) production. Group A patients had platelet O(2)(-) production similar to that of healthy subjects but lower than Group B and Group C patients. CONCLUSION: The present findings suggest an in vivo antioxidant activity of metformin and warrant prospective studies to further explore this hypothesis.