Clara Lechi

The F2-Isoprostane 8-epiprostaglandin F2α 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.

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/llla (CD41/α2β3) and GMP- 140 (CD62/P-selectin/PADGEM). Other markers of platelet (β-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/llla 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/llla 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/llla in the two groups. β-Thromboglobulin 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.

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.

Altered excretion of prostaglandin and thromboxane metabolites in pregnancy-induced hypertension.

The renal and systemic metabolites (the latter as 2,3-dinor derivatives) of prostacyclin and thromboxane A2 were measured, along with renal prostaglandin E2 and kallikrein, in the urine of 15 patients with pregnancy-induced hypertension, 15 normotensive pregnant women matched for both age and gestational age, and 15 normotensive nonpregnant control women. Urinary excretion of all prostaglandin and thromboxane metabolites studied proved significantly higher in normotensive pregnant women than in controls. Prostaglandin E2, 6-keto-prostaglandin F1 alpha, and 2,3-dinor-6-keto-prostaglandin F1 alpha were significantly lower in pregnancy-induced hypertensive women than in normotensive pregnant women, whereas thromboxane B2 and 2,3-dinor-thromboxane B2 showed no significant differences in the two groups. A significant negative correlation (r = -0.636, p less than 0.01) was found between urinary 2,3-dinor-6-keto-prostaglandin F1 alpha and mean blood pressure in the two groups of pregnant women taken as a whole. These data indicate that, in pregnancy-induced hypertension, there is an imbalance between vasodilator and vasoconstrictor factors, not only in the kidneys, but also at the systemic vascular level. This imbalance, which may in itself produce vasoconstriction, may also potentiate the hypertensive effect of catecholamines and angiotensin II.

Antiaggregating and vasodilatory effects of a new nitroderivative of acetylsalicylic acid

We studied in vitro the effects on platelet aggregation and vascular tone of a new nitrocompound (nitroxy-butyl-acetylsalicylate: NO-ASA). In order to elucidate any possible activity due to the release of nitric oxide or the inhibition of platelet cyclo-oxygenase we compared NO-ASA to acetylsalicylic acid. NO-ASA 1 mM inhibited arachidonic acid-induced platelet aggregation (basal 75.4 +/- 2.35%; NO-ASA 22 +/- 3.46%; M +/- SEM; P < 0.001; n = 6), but proved less active than acetylsalicylic acid (complete inhibition at 2 x 10(-5) M). NO-ASA also significantly reduced thrombin-induced (0.04-0.08 U/ml) platelet aggregation in acetylsalicylic acid-treated platelets (basal 70.5 +/- 1.7%; NO-ASA 35.4 +/- 2.2%; P < 0.001; n = 10; IC50 7 x 10(-5) M). Methylene blue reduced the effects of NO-ASA on thrombin-induced (NO-ASA 46.7 +/- 5.25%; NO-ASA+MB 59.1 +/- 4.3%; P < 0.01; n = 8), but not arachidonic acid-induced platelet aggregation. The inhibitory effects of NO-ASA on platelet aggregation were partially removed by oxyhaemoglobin. Platelet thromboxane A2 production (TXB2 concentration in the supernatant of the aggregate 35.38 +/- 7.81 ng/ml; n = 8), was totally abolished by acetylsalicylic acid (0.17 +/- 0.04 ng/ml; P < 0.001; n = 8) and reduced by NO-ASA (8.3 +/- 4.05 ng/ml; P < 0.01; n = 8). In vitro studies on isolated rat aortic rings showed NO-ASA 10(-3) M, but not ASA up to 10(-3) M, induce a dose dependent vasorelaxation (100% of epinephrine-induced contraction) both in intact and endothelium denuded arteries (IC50 5 x 10(-5) M). Addition of methylene blue reversed this relaxation. In conclusion these data demonstrate that NO-ASA acts through a double mechanism: a) by inhibiting cyclo-oxygenase and b) by releasing NO active on guanylyn cyclase both in platelets and in vascular smooth muscle cells.

Erythrocyte Na+-H+ exchanger kinetics and Na+-Li+ countertransport activity in essential hypertensive patients

The authors measured Na+–H+ exchanger kinetics together with Na+–Li+ countertransport Vmax in the erythrocytes of 21 subjects with essential hypertension and 16 normotensive control subjects. Na+–H+ exchanger Vmax appeared to be increased in patients with essential hypertension, while the Na+–H+ exchanger affinity for intracellular proton sites (K50%) proved to be unchanged and the index of cooperativity among intracellular proton binding sites as measured by Hills coefficient (Hills n) decreased as compared with normotensive control subjects. Na+–Li+ countertransport Vmax appeared to be higher in patients with essential hypertension than in control subjects. The authors were unable to find any correlations between Na+–H+ exchanger kinetic parameters and metabolic variables such as parameters of insulin resistance and plasma lipids. On the basis of the data obtained, erythrocyte Na+–H+ exchanger activity was found to be abnormal in two kinetic variables in essential hypertensive patients and showed no simple linear correlations with the main variables of glucose metabolism, plasma lipids, renin or aldosterone.

Increased platelet aggregation and intracellular calcium in hypertensive patients: effects of cyclo-oxygenase blockade.

Increased platelet aggregation induced by ADP and arachidonic acid was observed in 12 patients with essential hypertension compared with 12 control subjects, but not after pretreatment with acetylsalicylic acid. The increase in intracellular calcium induced by ADP was also greater in the hypertensive patients, and again this difference disappeared after cyclo-oxygenase blockade. Urinary excretion of 2,3-dinor-thromboxane B2, the main metabolite of platelet thromboxane A2, was slightly, but not significantly increased in the hypertensive patients. These data suggest that thromboxane system activity is increased in the platelets of hypertensive patients.

Increased sodium-lithium countertransport in red cells of patients with Bartter’s syndrome

Erythrocyte sodium-lithium countertransport was evaluated in three adult patients with Bartter’s syndrome, diagnosed on clinical and histopathological basis and on laboratory tests. As compared with age-matched control subjects, lithium effluxes to both sodium medium and to sodum-free medium were high in two patients and Na+-Li+ countertransport was significantly greater in all three patients. The relationship between increased Na+-Li+ countertransport and the proposed primary disorder of Bartter’s syndrome (i.e., a primary tubular defect of chloride reabsorption) is not apparent. However, this finding favours the hypothesis of a generalized cellular membrane dysfunction of ion transport in Bartter’s syndrome.

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.

Measurement by bioluminescence technique of erythrocyte membrane Na+,K+-ATPase activity in hypertensive patients

Erythrocyte membrane Na+,K+-ATPase activity was measured using a bioluminescence technique in 28 hypertensive patients (24 with essential hypertension, 2 with renovascular hypertension and 2 with hypertension secondary to primary hyperaldosteronism) and in 28 normotensive control subjects matched for age and sex. Erythrocyte Na+,K+-ATPase activity was significantly reduced in the patients with essential hypertension (130.9 +/- 11.4 vs. 186.6 +/- 19.5 nmol ATP/mg prot per h; mean values +/- SEM; p less than 0.05) and in the patients with secondary hypertension. A significant negative correlation was found between erythrocyte Na+,K+-ATPase and systolic blood pressure (r = -0.603; p less than 0.01), but not between Na+,K+-ATPase and plasma renin activity or plasma aldosterone levels. These data confirm the findings of a number of previous studies reporting reduced activity of erythrocyte Na+,K+-ATPase possibly related to the presence of a circulatory inhibitor of sodium pump. The method, based on ATP assay by bioluminescence, presents a high degree of specificity as well as simple, rapid execution.