Biomedical subjects

P Minuz

Publications and source records attributed to P Minuz.

At least 19 recordsLinked to original sources

PubMed2006

Evaluation of platelet turnover by flow cytometry.

The number of circulating newly produced platelets depends on the thrombopoietic capacity of bone marrow as well as platelet removal from the bloodstream. Flow cytometric analysis with thiazole orange (TO), a fluorescent dye that crosses platelet membranes and binds intracellular RNA, has been used to measure circulating reticulated platelets (RPs) with high RNA content as an index of platelet turnover. We first assessed the specificity of TO flow cytometry and then applied this method in the diagnosis of thrombocytopenia caused by impaired platelet production or increased destruction. We also explored the utility of TO flow cytometry to predict thrombocytopoiesis after chemotherapy-induced bone marrow aplasia. Venous blood, anticoagulated with K(2)EDTA, was incubated with 0.6 microg/ml TO plus an anti-GPIIIa monoclonal antibody. The mean percentage of RPs in control subjects (n = 23) was 6.13 +/- 3.09%. RPs were 10.41 +/- 9.02% in patients (n = 10) with hematological malignancies during aplasia induced by chemotherapy and a significant increase in RPs (35.45 +/- 6.11%) was seen in the recovery phase. In 10 patients with idiopathic thrombocytopenic purpura, the percentage of TO positive platelets was 67.81 +/- 18.79 (P < 0.001 vs. controls). In patients with thrombocytopenia associated with hepatic cirrhosis (n = 21; 21.04 +/- 16.21%, P < 0.001 vs. controls) or systemic lupus erythematosus (n = 6, 29.08 +/- 15.57%; P < 0.001 vs. controls) increases in TO-stained platelets were also observed. Measurement of TO positive platelets may be a reliable tool for the laboratory identification of platelet disorders, with a higher sensitivity than measurement of platelet volume. Measurement of RPs may also prove useful to recognize the underlying pathogenetic mechanisms in thrombocytopenia.

Adolescent↗

PubMed2005

Levels of F2-isoprostanes in systemic sclerosis: correlation with clinical features.

OBJECTIVE: Oxidative stress may be one of the important complex pathogenetic mechanisms that lead to damage in scleroderma; free radicals may provoke endothelial injury, fibroblast proliferation and fragmentation of autoantigens favouring induction of autoantibodies. The present study investigates the oxidant status in scleroderma patients by measuring the urinary concentration of 8-isoprostaglandin-F2alpha, an F2-isoprostane, and a product of free radical-mediated peroxidation of arachidonic acid. METHODS: Forty-three scleroderma patients (42 women and 1 man, mean age 54.1 yr, mean disease duration 9.0 yr) underwent clinical evaluation and instrumental investigations in order to assess skin, vascular, lung and heart involvement. Von Willebrand factor was evaluated as marker of vascular dysfunction in 36 out of the 43 cases. The urinary level of 8-isoprostaglandin-F2alpha was measured in all scleroderma patients and in the 43 age- and sex-matched healthy controls. RESULTS: Urinary levels of 8-isoprostaglandin-F2alpha were higher in scleroderma patients than in the healthy control group (341.7 vs 147.6 pg/mg creatinine; P < 0.001). Values of 8-isoprostaglandin-F2alpha were strongly correlated with the nailfold videocapillaroscopy pattern and lung involvement (P = 0.002 and 0.003, respectively), showing increasing levels with the progression of pulmonary severity. Correlation between 8-isoprostaglandin-F2alpha level and von Willebrand factor narrowly failed to reach statistical significance (P = 0.05). There was no correlation between 8-isoprostaglandin-F2alpha concentration and disease activity, vascular, skin and heart involvement, disease pattern or autoantibody profile. CONCLUSIONS: Our study further supports the role of oxidant stress in the pathogenesis of scleroderma, showing a strong correlation between a marker of free radical damage with both the severity of lung involvement and the videocapillaroscopic patterns.

Adult↗

PubMed2003

Elevated plasma fibrinogen levels in patients with essential hypertension are related to vascular complications.

AIM: We investigated whether or not fibrinogen is related to the cardiovascular risk profile and complications in hypertensive subjects. METHODS: Plasma fibrinogen and laboratory tests including factor VII, homocysteine and microalbuminuria were evaluated in 127 consecutive hypertensive subjects stratified according to cardiovascular risk. Parameters were age, gender, smoking, cholesterol, diabetes, target organ damage: left ventricular hypertrophy (LVH), carotid atherosclerotic complications and retinical vessels. RESULTS: Fibrinogen levels were significantly different between patients according to risk levels (low 290+/-73, n=20, high 342+/-94 mg/dl, n=39, very high risk 350+/-72, n=29, p=0.01), hypertension grade (II-III) and organ damage. Fibrinogen was significantly higher in patients with more severe carotid atherosclerotic lesions and vascular retinal lesions (grades II-III vs 0 and I). Also in patients, matched for age and sex, without and with carotid atherosclerotic lesions, fibrinogen was significantly higher in the latter group. No significant differences were found on the basis of IVS, creatinine and microalbuminuria. In hypertensive patients, fibrinogen directly correlated with age, by multiple linear regression. In hypertensive patients with diabetes, fibrinogen was significantly higher (466+/-176 mg/dL, n=14) than in those hypertensive without diabetes (333+/-87 mg/dL, n=113, p=0.001) and in all patients there was a a significant correlation (r=0.474, p<0.001) between blood glucose and fibrinogen. CONCLUSION: Hyperfibrinogenemia is a marker of vascular damage and could be an important factor contributing to the evolution of the complications.

Blood Coagulation Disorders↗

PubMed2001

Vascular adhesion molecule-1 and markers of platelet function before and after a treatment with iloprost or a supervised physical exercise program in patients with peripheral arterial disease.

Platelet function and levels of vascular adhesion molecule-1 (VCAM-1) were investigated in 24 patients with peripheral arterial disease at Fontaine stage II undergoing a 2 weeks treatment with iloprost (0.5-2 ng/kg/h i.v. infused, 6 h/day) or a 2 weeks supervised physical training, randomly assigned. Patients were studied before (T0) and after (T14) treatments and 10 days later (T24). The adhesion of washed platelets to fibrinogen coated microwells was reduced after treatment both with iloprost (1.9+/-0.4 vs 6.8+/-0.7%; T24 vs T0; M+/-SEM; p<0.05) and physical exercise (3.0+/-1.0 vs 6.7+/-0.7; p<0.05) while adhesion to human plasma coated microwells was reduced only after treatment with iloprost (1.9+/-0.8 vs 5.8+/-0.9; p<0.05). The expression of fibrinogen receptor (glycoprotein IIb/IIIa) on platelets, measured by flow-cytometry was also reduced after iloprost treatment (17.1+/-1.5 vs 31.8+/-4.8 AU; p<0.05) and physical exercise (14.6+/-1.5 vs 34.0+/-3.3; p<0.05). Theurinaryexcretion of platelet thromboxane A2 metabolite 2,3-dinor-thromboxane B2 decreased only in patients treated with iloprost (154.7+/-97.9 vs 256.2+/-106.4 pg mg creatinine(-1); p<0.05). Similarly plasma VCAM-1 was lower in patients who were treated with iloprost (827.7+/-77.4 vs 999.0+/-83.8 ng ml(-1); p<0.05). In conclusion, both iloprost and physical exercise seem to act on reversible phenomena such as the expression of adhesion molecules or ex vivo adhesion, whereas only iloprost reduces thromboxane A2 biosynthesis in vivo. This anti-platelet activity seems to be extended in time and to be associated with an improvement in vascular function.

Aged↗

PubMed2001

NCX4016 (NO-Aspirin) has multiple inhibitory effects in LPS-stimulated human monocytes.

NCX4016 (2 acetoxy-benzoate 2-(2-nitroxymethyl)-phenyl ester, NicOx S.A., France) is an anti-thrombotic agent, chemically related to acetylsalicylic acid (ASA) and able to release NO. We tested the effects of NCX4016 and ASA on the release of the thromboxane (TX) A(2) metabolite TXB(2), tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), expression and activity of tissue factor (TF) in stimulated, adherent human monocytes. Both ASA and NCX4016 1 - 1000 micromol l(-1) dose-dependently reduced TXB(2) concentration, measured by RIA in the supernatant of 10 microg ml(-1) LPS-stimulated cells. NCX4016 activity was comparable to that of equimolar ASA when incubation lasted 6 h (NCX4016 30 micromol l(-1): -86.0+/-10.1%, NCX4016 300 micromol l(-1): -92.2+/-9.0%, ASA 30 micromol l(-1): -92.3+/-7.5%, ASA 300 micromol l(-1): -97.3+/-1.0%, n=6, M+/-s.d.). Most of the activity of NCX4016 up to 100 micromol l(-1) was prevented by 10 micromol l(-1) ODQ, inhibitor of cyclic GMP. NCX4016 100 - 300 micromol l(-1) reduced TNF-alpha (NCX4016 300 micromol l(-1)=-77.2+/-19.9%, n=6) and IL-6 (NCX4016 300 micromol l(-1): -61.9+/-15.2%, n=6) in LPS stimulated monocytes while ASA had no significant effects. TF activity (NCX4016 300 micromol l(-1): 53.7+/-39.9%, n=4) and immunoreactive TF (NCX4016 300 micromol l(-1): -93.9+/-7.9%, n=7), measured in the supernatant of stimulated cells, were also dose-dependently inhibited by NCX4016 but not by ASA. The present results indicate that NCX4016 inhibits TXA(2) generation as well as cytokine release and TF in human monocytes partly via NO-dependent mechanisms. NCX4016 may have a favourable profile of activities in the clinical setting of athero-thrombosis.

Aspirin↗

PubMed2001

8-Iso-PGF2 alpha induces beta 2-integrin-mediated rapid adhesion of human polymorphonuclear neutrophils: a link between oxidative stress and ischemia/reperfusion injury.

F(2)-Isoprostanes are generated from a cyclooxygenase-independent oxidative modification of arachidonic acid. They are present in atherosclerotic plaques and are platelet activators as well as potent vasoconstrictors. Polymorphonuclear neutrophils are major players in ischemia/reperfusion injury and in restenosis after PTCA. The effects of 8-isoprostaglandin (PG) F(2alpha) on very rapid beta(2)-integrin-dependent adhesion was evaluated in human neutrophils in vitro by use of purified integrin as ligand. 8-Iso-PGF(2alpha) (1 nmol/L to 20 micromol/L) triggers a dose-dependent, very rapid neutrophil adhesion to human fibrinogen but not to the endothelial ligand intercellular adhesion molecule-1. Pretreatment with anti-ss(2)-integrin subtypes showed activation of CD11b/CD18 and CD11c/CD18. Adhesion triggering was completely prevented by pertussis toxin. SQ29,548, a specific antagonist of thromboxane A2 receptor, also dose-dependently prevented 8-iso-PGF(2alpha)-triggered neutrophil adhesion. 8-Iso-PGF(2alpha) did not trigger adhesion in human monocytes and lymphocytes and did not induce neutrophil chemotaxis or activation of the oxygen free-radical-forming enzyme NADPH-oxidase. These data highlight the role of 8-iso-PGF(2alpha) as a specific activator of rapid neutrophil adhesion and suggest its involvement in the pathogenesis of ischemia/reperfusion injury and in restenosis after PTCA. The effect is transduced via activation of the receptor for thromboxane A2.

CD18 Antigens↗

PubMed2000

Defective platelet response to arachidonic acid and thromboxane A(2) in subjects with Pl(A2) polymorphism of beta(3) subunit (glycoprotein IIIa).

The membrane complex alpha(IIb)beta(3) is the major receptor for fibrinogen and is involved in platelet adhesion and aggregation. Evidence has been presented that the Pl(A2) allele of the beta(3) Pl(A1/A2) gene polymorphism might be an independent risk factor for coronary thrombosis, but the matter is still controversial. We investigated the relationship between this polymorphism and possible alterations of platelet functions in vitro. The platelet adhesion to fibrinogen-coated microplate wells and the aggregation induced by several different agonists were tested in 63 healthy volunteers, among them, 49 subjects with Pl(A1/A1) polymorphism, 12 subjects with Pl(A1/A2) polymorphism and two subjects with (PlA2/A2) polymorphism. Subjects with PlA1/A2 polymorphism or with Pl(A2/A2) polymorphism showed significantly lower platelet responses as compared with Pl(A1/A1) subjects when either arachidonic acid or the thromboxane A(2) analogue, U46619, were used as agonists. In resting condition and after thrombin or ADP stimulation, platelet function was normal in all the subjects. An increased sensitivity to the anti-aggregatory effect of acetylsalicylic acid was observed in platelets from subjects with the Pl(A2) allele. Finally, using a flow-cytometric evaluation and determining the beta-thromboglobulin plasma levels, we did not find any evidence of a Pl(A2) platelet hyper-reactivity ex vivo. Our findings are not consistent with the hypothesis that the purported increase of cardiovascular risk in these subjects may be as a result of platelet hyperactivation. On the contrary, the Pl(A2) allele is associated with a platelet functional deficiency, specifically linked to the activation of the fibrinogen receptor by thromboxane A(2).

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed1999

Increased endogenous nitric oxide production induced by physical exercise in peripheral arterial occlusive disease patients.

The effects of 14-day physical exercise or iloprost treatment (0.5-2 ng/Kg/min) on endogenous nitric oxide production and neutrophil adhesion were evaluated in 20 patients with peripheral arterial occlusive disease (Fontaine Stage II). Peripheral venous blood samples and 4-h urine samples were collected before, immediately after 14 days of therapy and 7-10 days after therapy in order to evaluate neutrophil adhesion, nitrite/nitrate and cGMP excretion rates. A longer pain free walking distance was observed after exercise, compared to iloprost (>500 m in 3/10 subjects). Urinary nitrite/nitrate, as well as cGMP concentrations, significantly increased after exercise. Nitrite/nitrate excretion rate inversely correlated to neutrophil adhesion. No variations were observed in these parameters in iloprost treated patients. The improvement in claudication and the transient increase in urinary nitrite/nitrate suggest a possible nitric oxide-dependent mechanism for the clinical efficacy of physical exercise. The results from the present and previous observations indicate that, besides pharmacological treatments, a regular aerobic exercise improves peripheral arterial occlusive disease.

Aged↗

PubMed1998

Oxidized low density lipoprotein (LDL) and platelet intracellular calcium: interaction with nitric oxide.

The present study tested the effects of ox-low density lipoprotein (LDL) on nitric oxide (NO)-dependent decrease in agonist-stimulated [Ca2+]i. The effects of ox-LDL on platelet aggregation were also evaluated. Platelets loaded with FURA 2 AM (2 micromol/litre) were incubated with NO-donors for 2-10 min to obtain a 40-50% reduction in \[Ca2+]i and with NO-donors plus ox-LDL (100 microg of protein/ml). Thrombin (0.03 U/ml) was used as an agonist. In some experiments 8-Br-cGMP (0.5-1 mmol/l) was used to investigate the NO-dependent intraplatelet signalling system. Slightly oxidized LDL was obtained by leaving native LDL in the light at room temperature for at least 7 days. Ox-LDL did not cause any increase in thrombin-induced [Ca2+] (control: 215.4 +/- 44.3 nmol/l, ox-LDL 223.4 +/- 35.3 nmol/l, M +/- SEM; n = 8) and platelet aggregation (control: 78.7 +/- 4.9% , ox-LDL: 78.9 +/- 4.2% , n = 12). Ox-LDL antagonized the effects of NO-donors on platelet [Ca2+]i (NO-donor: 137.4 +/- 22.1 nmol/l, NO + ox-LDL: 177.3 +/- 27.6 nmol/l, n = 11; P < 0.001) and platelet aggregation (NO-donor: 15.4 +/- 3.4% , NO + ox-LDL: 28.9 +/- 3.8%, n = 24; P < 0.001). Ox-LDL did not affect the inhibitory activities of 8-Br-cGMP on platelet aggregation (8-Br-cGMP: 22.0 +/- 8.5%, 8-Br-cGMP + ox-LDL: 19.3 +/- 7.8%, n = 5) and platelet [Ca2+]i . In conclusion, slightly oxidized LDL does not directly activate platelets and does not i affect the intracellular NO-dependent signalling system. The present results suggest that LDL reduces the antiplatelet activity of NO mainly by preventing its biological effects.

Journal Article↗

PubMed1998

The F2-isoprostane 8-epiprostaglandin F2alpha increases platelet adhesion and reduces the antiadhesive and antiaggregatory effects of NO.

F2-isoprostanes are prostaglandin (PG) isomers produced in vivo through free radical-catalyzed peroxidation of arachidonic acid, which may affect platelet function. The current study investigated the effects of 8-epiprostaglandin F2alpha (8-epi-PGF2alpha) on critical events of platelet activation. A dose-dependent increase in platelet adhesion to fibrinogen- and plasma-coated microwells by 8-epi-PGF2alpha (1 to 1000 nmol/L) was observed when resting platelets (plasma from 1.3+/-0.2% to 5.5+/-0.2%, EC50 of 48 nmol/L; fibrinogen from 3.3+/-0.3% to 6.4+/-0.2%, EC50 of 35 nmol/L; mean+/-SEM, n=8, P<0.001) and thrombin-stimulated human platelets were used. The expression of the adhesion molecule glycoprotein IIb/IIIa was increased by 10 to 1000 nmol/L 8-epi-PGF2alpha in resting platelets (from 64.8+/-2.1% to 83.9+/-1.3%; n=5, P<0.01) and in stimulated platelets. The secretion of the glycoprotein GMP-140 increased only in the presence of both thrombin and 10 to 1000 nmol/L 8-epi-PGF2alpha (from 48.5+/-3.1% to 63.1+/-2.0%, P<0.05). The antiaggregatory effects of both the NO donor NOR-3 (basal, 21.4+/-4.6%; with 8-epi-PGF2alpha, 30.8+/-6.9%; n=14, P<0.05) and endothelial cells that release NO (basal, 18.5+/-4.6%; with 8-epi-PGF2alpha, 30.7+/-5.3%; n=15, P<0.001) were also reduced. All of these effects were prevented by the thromboxane receptor antagonist GR32191 but not affected by acetylsalicylic acid. An increase in free intracellular calcium concentration, measured with the use of fura 2, was observed with 8-epi-PGF2alpha. In conclusion, F2-isoprostanes may participate in oxidative injury by inducing platelet activation and by reducing the antiplatelet activity of NO: increased platelet adhesiveness and expression of the fibrinogen receptor are induced by nanomolar amounts of 8-epi-PG-F2alpha. Platelet secretion and aggregation can also be induced in the presence of platelet agonists.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed1998

[Patients with vascular diseases undergoing training physical exercise: urinary nitrites/nitrates and platelet adhesiveness].

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Aged↗

PubMed1997

Postischemic hyperemia in subjects with lower limbs obstructive arteriopathy: role of PGI2 and endothelin.

The physiological basis of postischemic hyperemia is not yet fully understood. The present study investigated the effects of pharmacologic manipulation of the prostaglandin system on local hemodynamics. Strain-gauge plethysmography was used to study 8 normal subjects and 9 patients with obliterating arterial disease of the lower limbs. Hemodynamic evaluations were performed before treatment, after seven days of low-dose acetylsalicylic acid (100 mg/day) to inhibit platelet thromboxane synthesis, and after acute infusion of 1 g of acetylsalicylic acid to inhibit endothelial prostacyclin synthesis. In patients with arterial disease, the hemodynamic study was also carried out after infusion of iloprost, a synthetic prostacyclin analogue. Acute infusion of acetylsalicylic acid significantly reduced basal blood flow in normal subjects, but not in patients with arterial disease. In the latter group, iloprost affected neither basal nor maximal postischemic flow. The study also evaluated the role of endothelin in musculocutaneous hemodynamic regulation, both in physiological conditions and in atherosclerosis. This part of the study addressed the possibility that the hemodynamic effects of vasodilator prostanoids like prostacyclin might affect endothelin release in vivo. During reactive hyperemia, plasma endothelin was reduced in normal subjects (-1.02 pg/mL, 95% CI: -2.23, 0.08), but not in patients with atherosclerosis (-0.35 pg/mL, 95% CI: -1.45, 0.75). In both groups, plasma endothelin was not affected by aspirin. These findings confirm the role of prostacyclin in local hemodynamic regulation. In the normal subject, musculocutaneous blood flow seems to depend at least in part on the action of vasodilator prostanoids and endothelin. This is not the case in patients with arterial disease, in whom plasma endothelin does not seem to be affected by postischemic changes in blood flow. A possible explanation for this difference could be alteration of the endothelial function in patients with arterial disease, related to the functional and structural characteristics of the artery wall in atherosclerosis.

Aged↗

PubMed1996

Effect of single and repeated doses of a new nitroderivative of acetylsalicylic acid on platelet TXA2 production in rats.

The effects of a new ASA-nitroderivative compound, NCX 4016 (ASA-NO2), on platelet TXA2 synthesis after single and repeated doses in the rat were investigated. Compared to ASA, cumulative doses of ASA-NO2 showed similar inhibitory effects on platelet TXA2 synthesis and significant increases in nitrite/nitrate plasma concentrations 1 h after the last drug administration: 24 h later nitrite/nitrate plasma levels returned to the control values, while serum TXA2 concentrations did not change. A time-course study after a single dose of ASA-NO2 showed a significant inhibition of platelet TXA2 production also 24 h after drug administration and a significant increase in nitrite/nitrate plasma levels until 10 h.

Animals↗

PubMed1996

Changes in peripheral hemodynamics and vasodilating prostaglandins after high-dose short-term ibuprofen in chronically treated hypertensive patients.

The use of cyclooxygenase inhibitors has been seen to reduce the efficacy of many antihypertensive drugs. However, cyclooxygenase inhibitors are normally non-selective because they affect both vascular tissue, where the endothelial prostanoids exert principally a vasodilatory action, and the kidneys, where they also play an important role in regulating hydroelectrolytic metabolism by redistribution of intraparenchymal flow. To evaluate the relative importance of vascular district in the hypertensive patient, we administered ibuprofen - a drug acting with only a minimal antagonist activity. A group of 20 male hypertensives were randomly allocated, according to a single-blind protocol, to treatment with amlodipine (A, 10 mg/day) or lisinopril (L, 20 mg/day). Blood pressure was significantly reduced after 30 days, with a mean difference of -21.75 mmHg for systolic blood pressure (SBP) (95% confidence interval (Cl): -27.46 to -16.04; P< 0.0001) and -14.15 mmHg for diastolic blood pressure (DBP) (95% Cl: -17.13 to -11.17; P< 0.0001). Brachial artery compliance showed a mean increase of 1.657 x 10(-7) dyn-1 cm(4) (95% Cl: 1.188 to 2.126; P<0.001), and forearm resistances showed a mean decrease of -41.973 mmHg ml(-1)s (95% Cl: -75.479 to -8.467; P = 0.017). Changes in compliance were significantly related to those in SBP (r= -0.546; P= 0.013). The administration of ibuprofen (400 mg, three times a day for 3 days) was accompanied by a slight but significant increase in SBP, but not in brachial artery compliance or forearm resistances. Only SBP was affected, showing a mean increase of 4.25 mmHg (95% Cl: 1.26 to 7.24; P = 0.008). There was also reduced urinary excretion of PGI(2) and TXA(2) metabolites. The mean change in 6-keto-PGF(1 alpha) and 2,3-dinor-6-keto-PGF(1 alpha) was 45.71 ng per g urinary creatinine (uCr) (95% Cl: -0.16 to-91.25; P= 0.049) and -73.17 ng (g uCr)(-1) (95% Cl: -38.81 to -107.53; P<0.001), respectively. The mean decrease in TXA(2) catabolites was highly significant: -39.2 ng (g uCr)(-1) (95% Cl: -18.17 to-60.22; P< 0.001) and -102.87 ng (g uCr)(-1) (95% Cl: -61.86 to -143.88; P< 0.001) for TXB(2) and 2,3-dinor-TXB(2), respectively. Our study highlighted an inverse correlation between changes in blood pressure and those in urinary 2,3-dinor-6-keto-PGF(1alpha) excretion, irrespective of antihypertensive regimen. This suggests that, in the hypertensive patient treated with NSAIDs, inhibition of vascular prostanoid synthesis may play an important role in countering the efficacy of an important vascular tone regulatory mechanism.

6-Ketoprostaglandin F1 alpha↗

PubMed1996

Dual effects of diclofenac on human platelet adhesion in vitro.

The effect of two non-steroidal anti-inflammatory drugs on the adhesion function of human platelets was evaluated. Platelets isolated from healthy human subjects were treated for 10 min with the indicated drugs and then incubated in fibrinogen-coated microwell plates in the absence or in the presence of ADP (10 microM) and thrombin (0.05 U/ml). After 1 h of incubation, adherent platelets were measured using an enzymatic assay. ADP- and thrombin-stimulated adhesion was significantly inhibited by high doses ( > 500 microM) of diclofenac, while doses ranging from 50 to 300 microM stimulated adhesion in the absence of agonists (resting platelets). A similar stimulatory effect on platelet adhesion was observed also with 200-500 microM flurbiprofen. Moreover, immunocytofluorimetry demonstrated that diclofenac dose-dependently (100-500 microM) induced the expression of GMP-140 and increased the expression of GPIIb/IIIa on the membrane of unstimulated platelets. High doses ( > 500 microM) of this drug inhibited thrombin-stimulated expression of GPIIb/IIIa and GMP-140.

Adenosine Diphosphate↗

PubMed1996

In vitro study of the anti-aggregating activity of two nitroderivatives of acetylsalicylic acid.

The antiplatelet activity of two new nitrocompounds, chemically related to acetylsalicylic acid (NCX 4215 and NCX 4016), was studied in vitro to verify the hypothetical dual action of these drugs. Both drugs, in a dose-dependent way, inhibited arachidonic acid-induced platelet aggregation and thromboxane A2 production, measured as thromboxane B2 concentration in whole blood. These effects are likely to be related to cyclo-oxygenase inhibition. NCX 4215 and NCX 4016 in a dose-dependent way inhibited also thrombin-induced aggregation of platelets pretreated with acetylsalicylic acid. These inhibitory effects are related to nitric oxide release and cGMP increase and significantly reversed by oxyhaemoglobin and methylene blue. Either as a cyclo-oxygenase inhibitor or as a nitric oxide donor, NCX 4016 proved to be significantly more potent than NCX 4215.

Arachidonic Acid↗

PubMed1996

Study of platelet adhesion in patients with uncomplicated hypertension.

OBJECTIVE: To evaluate platelet function in patients with essential hypertension by sensitive methods investigating platelet adhesion and expression of some platelet glycoproteins (GP), namely GPIIb/IIIa (CD41/alpha 2 beta 3) and GMP-140 (CD62/P-selectin/PADGEM). Other markers of platelet (beta-thromboglobulin) and endothelium activation (von Willebrand factor) were also measured. METHODS: We studied 21 uncomplicated essential hypertensive patients and 20 healthy normotensive control subjects, non-smokers, matched for age and sex. Resting and stimulated platelet adhesion was performed with a colorimetric method using the activity of platelet acid phosphatase for the determination of the number of platelets adhering to human plasma- or fibrinogen-coated microwells. Platelet activation was characterized by flow cytometric measurement of GPIIb/IIIa and GMP-140 in whole blood and washed platelets suspensions, with antihuman fluorescent monoclonal antibodies. RESULTS: Thrombin-stimulated platelet adhesion to human plasma-coated microwells was significantly higher in hypertensive patients than in control subjects (0.05 U/ml thrombin: 13.4 +/- 1.0 versus 7.7 +/- 0.6% adhesion; 0.1 U/ml thrombin: 19.4 +/- 2.3 versus 12.6 +/- 1.8%; means +/- SEM), whereas platelet adhesion to fibrinogen-coated wells did not differ in the two groups. Flow-cytometry analysis of whole blood demonstrated a significantly increased expression of GMP-140 in hypertensive patients compared with normal subjects (percentage of CD62+ platelets: 7.3 +/- 1.2 versus 3.7 +/- 1; means +/- SEM), whereas the expression of GPIIb/IIIa did not differ in the two groups (percentage of CD41a+ platelets: 72.5 +/- 4.5 versus 70.4 +/- 3.9). Moreover, flow cytometry showed an increased size of platelets in hypertensive patients compared with that in control subjects (forwards scattering: 46.5 +/- 1.5 versus 38.9 +/- 1.1; means +/- SEM). Flow-cytometric evaluation of washed platelet suspensions showed no statistically significant differences between the expression of GMP-140 and GPIIb/IIIa in the two groups. beta-Thrombo-globulin plasma levels were higher in hypertensive patients than they were in normal subjects (36.3 +/- 2.0 versus 28.2 +/- 1.3 ng/ml; means +/- SEM). Von Willebrand factor plasma levels were not significantly different in the two groups (101.2 +/- 10.3 versus 86.3 +/- 5.6 U/dl). CONCLUSIONS: These findings provide further evidence that there is a significant, albeit weak, platelet activation in hypertensive patients compared with normal subjects.

Adult↗

PubMed1996

The antiplatelet effects of a new nitroderivative of acetylsalicylic acid--an in vitro study of inhibition on the early phase of platelet activation and on TXA2 production.

We studied in vitro the antiplatelet activity of a new nitroderivative chemically related to acetylsalicylic acid: 2 acetoxybenzoate 2-[1-nitroxy-methyl]-phenyl ester (NCX 4016), in order to identify any effects due to the release of nitric oxide and the blockade of cyclo-oxygenase. The effects of scalar doses of NCX 4016 on the early phase of platelet activation, platelet aggregation and thromboxane A2 production were investigated. We observed inhibitory effects of NCX 4016 on platelet adhesion (IC50 = 7.3 x 10(-5) M), platelet cytosolic calcium concentration, assayed by fluorescent probe Fura 2, and the expression of glycoprotein IIb/IIIa (CD41/alpha IIb beta 3) (IC50 = 3.4 x 10(-5) M) and P-selectin (CD62/GMP-140) (IC50 = 4.9 x 10(-5) M) measured by flow cytometry. NCX 4016 also prevented thrombin-induced platelet aggregation (IC50 = 3.9 x 10(-5) M). None of these parameters were affected by acetylsalicylic acid. These inhibitory activities of NCX 4016 were abolished by oxyhaemoglobin and methylene blue. Intracellular cyclic GMP observed during thrombin-induced aggregation was increased by incubation with NCX 4016. These results appear to be attributable to the release of nitric oxide, which activates soluble platelet guanylylcyclase and promotes intracellular cyclic GMP increase. NCX 4016 almost completely inhibited platelet thromboxane A2 production and arachidonic acid-induced platelet aggregation. This also occurred in the presence of oxyhaemoglobin and methylene blue, indicating that its antiplatelet activity can be attributed not only to nitric oxide release but also to cyclo-oxygenase inhibition.

Adult↗