Stefania Gaino

Increased Oxidative Stress and Platelet Activation in Patients With Hypertension and Renovascular Disease

Background—Hypertensive patients with renovascular disease (RVD) may be exposed to increased oxidative stress, possibly related to activation of the renin-angiotensin system. Methods and Results—We measured the urinary excretion of 8-iso-prostaglandin (PG) F2&agr; and 11-dehydro-thromboxane (TX) B2 as indexes of in vivo lipid peroxidation and platelet activation, respectively, in 25 patients with RVD, 25 patients with essential hypertension, and 25 healthy subjects. Plasma renin activity in peripheral and renal veins, angiotensin II in renal veins, cholesterol, glucose, triglycerides, homocysteine, and antioxidant vitamins A, C, and E were also determined. Patients were also studied 6 months after a technically successful angioplasty of the stenotic renal arteries. Urinary 8-iso-PGF2&agr; was significantly higher in patients with RVD (median, 305 pg/mg creatinine; range, 124 to 1224 pg/mg creatinine) than in patients with essential hypertension (median, 176 pg/mg creatinine; range, 48 to 384 pg/mg creatinine) or in healthy subjects (median, 123 pg/mg creatinine; range, 58 to 385 pg/mg creatinine). Urinary 11-dehydro-TXB2 was also significantly higher in RVD patients compared with healthy subjects. In RVD patients , urinary 8-iso-PGF2&agr; correlated with 11-dehydro-TXB2 (rs=0.48;P <0.05) and renal vein renin (rs=0.67;P <0.005) and angiotensin II (rs=0.65;P =0.005) ratios. A reduction in 8-iso-PGF2&agr; after angioplasty was observed in RVD patients with high baseline levels of lipid peroxidation. Changes in 8-iso-PGF2&agr; were related to baseline lipid peroxidation (rs=−0.73;P <0.001), renal vein angiotensin II (rs=−0.70;P <0.01) and renin (rs=−0.63;P <0.05) ratios. Conclusions—Lipid peroxidation is markedly enhanced in hypertensive patients with RVD and is related to activation of the renin-angiotensin system. Moreover, persistent platelet activation triggered or amplified by bioactive isoprostanes may contribute to the progression of cardiovascular and renal damage in this setting.

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.

Determinants of Platelet Activation in Human Essential Hypertension

Abstract—Experimental data suggest that oxidative stress might be enhanced in hypertension and contribute to platelet activation. We hypothesized that both oxidative stress and platelet activation could be related to the clinical characteristics of hypertensive patients. The urinary excretion of 11-dehydrothromboxane (TX) B2, reflecting in vivo platelet activation, was measured in 75 patients with mild to severe essential hypertension and 75 pair-matched, healthy controls. The urinary excretion of 8-iso-prostaglandin (PG) F2&agr; was determined as an index of in vivo lipid peroxidation. Urinary 11-dehydro-TXB2 was significantly higher in essential hypertensives compared with controls. Although no statistically significant difference in urinary 8-iso-PGF2&agr; was observed between patients and controls, plasma vitamin C was lower and plasma homocysteine higher in hypertensive patients than in controls. Both urinary 11-dehydro-TXB2 and 8-iso-PGF2&agr; were higher in patients with advanced hypertensive retinopathy compared with patients without retinopathy. Multivariate linear regression analysis identified urinary 8-iso-PGF2&agr;, plasma fibrinogen, homocysteine, and vitamin E as the only variables independently correlated with urinary 11-dehydro-TXB2. Logistic regression analysis showed that high urinary 8-iso-PGF2&agr;, plasma fibrinogen, and homocysteine, as well as low plasma vitamin E, advanced retinopathy, elevated diastolic blood pressure, and the absence of antihypertensive treatment, were predictors of high urinary 11-dehydro-TXB2. We demonstrated increased oxidative stress and persistent platelet activation in essential hypertensives with advanced vascular lesions. These findings might help identify hypertensive patients who are at increased risk of cardiovascular events and who might benefit from long-term antiplatelet therapy.

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.

Rapid stimulation of tyrosine phosphorylation signals downstream of G-protein-coupled receptors for thromboxane A2 in human platelets

Signals ensuing from trimeric G-protein-coupled receptors synergize to induce platelet activation. At low doses, the thromboxane A2 analogue U46619 does not activate integrin alphaIIbbeta3 or trigger platelet aggregation, but it induces shape changes. In the present study, we addressed whether low doses of U46619 trigger tyrosine phosphorylation independently of integrin alphaIIbbeta3 activation and ADP secretion, and synergize with adrenaline (epinephrine) to induce aggregation in acetylsalicylic acid (aspirin)-treated platelets. Low doses of U46619 triggered tyrosine phosphorylation of different proteins, including FAK (focal adhesion kinase), Src and Syk, independently of signals ensuing from integrin alphaIIbbeta3 or ADP receptors engaged by secreted ADP. The G(12/13)-mediated Rho/Rho-kinase pathway was also increased by low doses of U46619; however, this pathway was not upstream of tyrosine phosphorylation, because this occurred in the presence of the Rho-kinase inhibitor Y-27632. Although low doses of U46619 or adrenaline alone were unable to trigger platelet aggregation and integrin alphaIIbbeta3 activation, the combination of the two stimuli effectively induced these responses. PP2, a tyrosine kinase inhibitor, and Y-27632 inhibited platelet activation induced by low doses of U46619 plus adrenaline and, when used in combination, totally suppressed this platelet response. In addition, the two inhibitors selectively blocked tyrosine kinases and the Rho/Rho-kinase pathway respectively. These findings suggest that both tyrosine phosphorylation and the Rho/Rho-kinase pathway are required to activate platelet aggregation via G(12/13) plus G(z) signalling.

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) A2 metabolite TXB2, tumour necrosis factor‐α (TNF‐α), interleukin‐6 (IL‐6), expression and activity of tissue factor (TF) in stimulated, adherent human monocytes. Both ASA and NCX4016 1 – 1000 μmol l−1 dose‐dependently reduced TXB2 concentration, measured by RIA in the supernatant of 10 μg ml−1 LPS‐stimulated cells. NCX4016 activity was comparable to that of equimolar ASA when incubation lasted 6 h (NCX4016 30 μmol l−1: −86.0±10.1%, NCX4016 300 μmol l−1: −92.2±9.0%, ASA 30 μmol l−1: −92.3±7.5%, ASA 300 μmol l−1: −97.3±1.0%, n=6, M±s.d.). Most of the activity of NCX4016 up to 100 μmol l−1 was prevented by 10 μmol l−1 ODQ, inhibitor of cyclic GMP. NCX4016 100 – 300 μmol l−1 reduced TNF‐α (NCX4016 300 μmol l−1=−77.2±19.9%, n=6) and IL‐6 (NCX4016 300 μmol l−1: −61.9±15.2%, n=6) in LPS stimulated monocytes while ASA had no significant effects. TF activity (NCX4016 300 μmol l−1: 53.7±39.9%, n=4) and immunoreactive TF (NCX4016 300 μmol 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 TXA2 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.

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.

Study on Paradoxical Effects of NSAIDs on Platelet Activation

We recently described a stimulatory effect of high doses (>100 μmol/L) diclofenac on platelet adhesion. In this study we extend our research to the possible biochemical mechanisms of the observed effects, to other non steroidal anti-inflammatory drugs (NSAIDs) (flurbiprofen, indomethacin, acetylsalicylic acid, ibuprofen, nitrofenac and nitroflurbiprofen) and to the effect of high doses diclofenac and flurbiprofen on platelet aggreation. We observed that high doses of diclofenac and of flurbiprofen, but not of the other tested NSAIDs, increased platelet adhesion at doses ranging from 100 to 500 μmol/L, an effect completely removed by the 12-lipoxygenase-inhibitor nordihydroguaiaretic acid. Moreover, they had no pro-aggregating effect, inhibiting platelet aggregation induced by 10 μmol/L arachidonic acid and dose-dependently increasing the [Ca2+]i. Finally, whereas no basal nitric oxide release by washed platelets was detected, when platelets were incubated by 500 μmol/L diclofenac or flurbiprofen, the production of nitric oxide, as measured by amounts of nitrite released, was 4.4 ± 0.5 and 3.8 ± 0.4 pmol/5 × 108 platelets/min, respectively. Our data indicate that high doses diclofenac and flurbiprofen are promoters of the early phases of platelet activation, probably through the 12-lipoxygenase pathway.

EFFECTS OF A NEW CLASS OF NO-RELEASING NSAIDs ON PLATELETS AND ISOLATED ARTERIES

A new class of nitroderivatives of non-steroidal anti-inflammatory drugs has recently been synthesized (Nicox Ltd., London, UK). In order to improve gastric tolerance of the parent compound, a side-chain, able to release nitric oxide, has been added to the core structure of the molecule. We studied in vitro the effects of nitrofenac and two NO-aspirins (NCX 4215 and NCX 4016) on platelets and isolated arteries to identify any possible effect due to the release of nitric oxide or to the inhibition of cyclo-oxygenase activity. Nitrofenac induced a dose-dependent relaxation both with intact (46% with 1 × 10-3 mol/L) and endothelium-denuded (75% with 1 × 10-3 mol/L) rings of rat aorta precontracted with epinephrine, while diclofenac did not affect this contraction (0% relaxation in intact and 22% in rubbed arteries). Pretreatment with diclofenac 1 × 10-3 mol/L significantly increased the vasorelaxant effects of nitrofenac at each drug concentration, both in intact (86% with 1 × 10-3 mol/L) and rubbed preparations (89%). NO-aspirins, unlike acetylsalicylic acid, were able to relax both intact and endothelium-denuded rings of rat aorta (100% relaxation). Methylene blue and oxyhaemoglobin completely reversed the relaxation induced by nitrofenac and NO-aspirins, both in rubbed and intact aortic rings.