Inhibition of platelet aggregation by nitric oxide donors improves with rest

It has been suggested that the platelets of patients with acute coronary syndromes (ACS) exist in a more activated state than those of patients with stable coronary heart disease (CHD) or healthy individuals. “Platelet nitrate responsiveness” (PNR) has been suggested as a measure of platelet activation, and has been shown to be reduced in both ACS and stable CHD. We examined the effect of a short period of undisturbed supine rest, an intervention aimed at reducing levels of “stress”, on PNR. In 8 healthy subjects we found that 45?minutes of rest led to a highly significant reduction in platelet aggregation and increase in PNR. Our finding supports the hypothesis that stress contributes to platelet activation as reflected in reduced PNR, which may contribute to the link between acute stress and cardiovascular events. It also emphasises that standardisation of sampling conditions in vitally important in studies utilizing PNR.     

Inhibition of platelet aggregation by nitric oxide donors improves with rest

It has been suggested that the platelets of patients with acute coronary syndromes (ACS) exist in a more activated state than those of patients with stable coronary heart disease (CHD) or healthy individuals. "Platelet nitrate responsiveness" (PNR) has been suggested as a measure of platelet activation, and has been shown to be reduced in both ACS and stable CHD. We examined the effect of a short period of undisturbed supine rest, an intervention aimed at reducing levels of "stress", on PNR. In 8 healthy subjects we found that 45 minutes of rest led to a highly significant reduction in platelet aggregation and increase in PNR. Our finding supports the hypothesis that stress contributes to platelet activation as reflected in reduced PNR, which may contribute to the link between acute stress and cardiovascular events. It also emphasises that standardisation of sampling conditions in vitally important in studies utilizing PNR.     

Inhibition of platelet aggregation by aspirin progressively decreases in long-term treated patients

OBJECTIVES: We sought to investigate, during a two-year follow-up period, the effects of aspirin on platelet aggregation. BACKGROUND: The platelets of patients given aspirin may be less sensitive to antiplatelet treatment, although the extent of such phenomenon over long-term follow-up is unclear. METHODS: Adenosine diphosphate (ADP) and collagen-induced platelet aggregation was periodically monitored before and after 2, 6, 12, and 24 months of treatment with aspirin (n = 150) or ticlopidine (n = 80) in patients matched for gender, age, and risk factors for atherothrombosis. RESULTS: Compared with baseline values, two months of aspirin treatment significantly inhibited platelet aggregation; thereafter, this inhibitory effect progressively decreased. At 24-month follow-up, collagen-induced platelet aggregation was significantly higher than that observed at two months (p < 0.05); a more pronounced difference was observed when collagen-induced lag phase was considered (p < 0.01). Restoration of platelet aggregation was less evident when ADP was used as an agonist. Conversely, the inhibition induced by ticlopidine was constant throughout follow-up with both agonists. CONCLUSIONS: The study demonstrates that a long-term treatment with aspirin is associated with a progressive reduction in platelet sensitivity to this drug.     

The role of nitric oxide in aspirin induced thrombolysis in vitro and the purification of aspirin activated nitric oxide synthase from human blood platelets

Aspirin, a well-known inhibitor of platelet aggregation, is extensively used for the prevention/treatment of coronary artery disease. The beneficial and antithrombotic effects of the compound are related to the inhibition of platelet cyclooxygenase. It is currently believed that aspirin has no effect on the formed thrombus, which results in coronary artery disease. It was found that the exposure of platelets to 4.0 microM aspirin either in vitro or in vivo resulted in fibrinolysis of the formed "clot" produced by the recalcification of platelet-rich plasma due to the production of NO in these cells by the compound. The lysis of clot in the presence of aspirin was found to be related to the fibrinolysis with simultaneous appearance of fibrin degradation products due to the generation of serine proteinase activity by NO in the assay mixture. The aspirin activated nitric oxide synthase that catalyzed the synthesis of NO in platelets was solubilized by Triton X-100 treatment and purified to homogeneity by chromatography on DEAE cellulose and Sephadex G-50 columns. The enzyme was found to be a single chain polypeptide with M.W. 19 kDa. The treatment of human plasminogen with NO was found to directly activate the zymogen to plasmin with the production of preactivation peptide in the absence of cofactors, or cells without the formation of cyclic GMP in the assay mixture.     

The effect of triflusal on human platelet aggregation and secretion: the role of nitric oxide

INTRODUCTION AND AIMS: The thrombotic process is a multicellular phenomenon in which not only platelets are involved but also neutrophils are involved. Recent in vitro studies performed in our laboratory have demonstrated that triflusal reduced platelet aggregation by stimulating nitric oxide (NO) production by neutrophils. The aim of the present study was to evaluate whether the in vivo treatment with triflusal could also modify the ability of neutrophils to produce NO. Furthermore, the role of NO released by neutrophils on platelet aggregation and secretion was also tested. METHODS: The study was performed in 12 healthy volunteers of 32 +/- 6 years of age. The volunteers were treated with triflusal (600 mg/day) for 5 days and platelets and neutrophils were isolated before and after treatment. The ability of neutrophils to produce NO and the capacity of inhibiting platelet aggregation and secretion of transforming growth factor-beta (TGF-beta) were assessed. RESULTS: After the treatment with triflusal we obtained the following results: a) an increase in NO production by neutrophils; b) potentiation of the inhibition of platelet aggregation by neutrophils, an effect that was reverted by incubating neutrophils with an L-arginine antagonist, L-NAME, and c) the presence of neutrophils reduced the release of TGF-beta by platelets measured as index of platelet secretion by a NO-independent mechanism. CONCLUSIONS: Triflusal (600 mg/day/5 days) stimulated NO production by neutrophils. After the treatment with triflusal, neutrophils inhibited both platelet aggregation and secretion. The antiaggregating effect of neutrophils was an NO-dependent mechanism while the inhibition of platelet secretion mediated by neutrophils after the treatment with triflusal was an NO-independent mechanism.     

Inhalation of nitric oxide inhibits ADP-induced platelet aggregation and alpha-granule release

To gather further information about the effects on blood platelet activation of in vivo exposure to nitric oxide (NO), platelet reactivity was studied in blood from healthy, non-smoking male volunteers before and after 30 min inhalation of 40ppm NO. Whole blood was stimulated in vitro with adenosine diphosphate or thrombin receptor activation peptide (TRAP-6). In an ex vivo perfusion model, non-anticoagulated blood was exposed to immobilised collagen at arterial blood flow conditions (2600 s -1 ). Blood samples from both the in vitro and ex vivo experiments were stained with fluorochrome-labelled Annexin-V and antibodies against CD42a, CD45, CD49b, CD61, CD62P and fibrinogen, and analysed with a three-colour flow cytometry technique. NO inhalation reduced the platelet activation response to adenosine diphosphate (ADP) stimulation by decreasing platelet-platelet aggregation, alpha-granule release and platelet-leukocyte conjugate formation. TRAP-stimulated platelet activation, collagen-induced platelet activation and thrombus growth was unaffected by NO inhalation. We therefore suggest an ADP receptor inhibitor mode of action of inhaled NO, selective on the newly suggested G protein- and phospholipase C-coupled P2Y1 receptor. Our results demonstrate that blood platelet activation in healthy subjects is modulated by inhalation of NO in therapeutically relevant doses, although the clinical impact of our findings remains unclear.     

Inhalation of nitric oxide inhibits ADP-induced platelet aggregation and alpha-granule release

To gather further information about the effects on blood platelet activation of in vivo exposure to nitric oxide (NO), platelet reactivity was studied in blood from healthy, non-smoking male volunteers before and after 30 min inhalation of 40 ppm NO. Whole blood was stimulated in vitro with adenosine diphosphate or thrombin receptor activation peptide (TRAP-6). In an ex vivo perfusion model, non-anticoagulated blood was exposed to immobilised collagen at arterial blood flow conditions (2600 s(-1)). Blood samples from both the in vitro and ex vivo experiments were stained with fluorochrome-labelled Annexin-V and antibodies against CD42a, CD45, CD49b, CD61, CD62P and fibrinogen, and analysed with a three-colour flow cytometry technique. NO inhalation reduced the platelet activation response to adenosine diphosphate (ADP) stimulation by decreasing platelet-platelet aggregation, alpha-granule release and platelet-leukocyte conjugate formation. TRAP-stimulated platelet activation, collagen-induced platelet activation and thrombus growth was unaffected by NO inhalation. We therefore suggest an ADP receptor inhibitor mode of action of inhaled NO, selective on the newly suggested G protein- and phospholipase C-coupled P2Y1 receptor. Our results demonstrate that blood platelet activation in healthy subjects is modulated by inhalation of NO in therapeutically relevant doses, although the clinical impact of our findings remains unclear.     

Inhibition of nitric oxide synthesis accentuates blood pressure elevation in hyperinsulinemic rats.

OBJECTIVES: To examine the role of endogenous nitric oxide (NO) in the pathogenesis of hypertension and insulin resistance in chronic hyperinsulinemic rats. METHODS: Sustained hyperinsulinemia was achieved by insulin infusion (21.5 pmol/kg per min) via subcutaneous osmotic minipump for 6 weeks. NO synthase inhibitor, Nomega-nitro-L-arginine methyl ester (L-NAME, 5 mg/kg per day) was given orally after 4 weeks of vehicle or insulin infusion. The systolic blood pressure (SBP) was measured under conscious state by an electrosphygmomanometer before and after drug treatments. RESULTS: Insulin infusion alone significantly increased SBP from 134 +/- 3 to 156 +/- 2 mmHg by week 4 and further to 158 +/- 3 mmHg by week 6 of insulin infusion. The insulin-infused rats had markedly decreased insulin sensitivity, as reflected by an elevated steady-state plasma glucose level estimated by the insulin suppression test. There were no significant differences in plasma glucose and triglyceride levels between rats with and without insulin infusion. When hypertension had been established in rats receiving insulin infusion for 4 weeks, superimposed L-NAME on insulin infusion for additional 2 weeks further increased SBP by 18 +/- 2 mmHg (from 157 +/- 2 to 175 +/- 2 mmHg). Plasma levels of NO metabolites (NOx) significantly decreased from 13.7 +/- 1.1 micromol/l during the control period to 6.1 +/- 0.6 micromol/l after 4 weeks of insulin infusion and further reduced to 4.1 +/- 0.5 micromol/l after combined infusion of L-NAME and insulin. L-NAME treatment alone for 2 weeks in control rats significantly increased SBP by 33 +/- 2 mmHg (from 133 +/- 2 to 166 +/- 2 mmHg) and plasma insulin levels, as a consequence of insulin resistance. Despite marked increases in blood pressure due to infusion of insulin alone or in combination with L-NAME, the sodium balance, urinary sodium and water excretions, water intake and body weight gain of insulin/L-NAME-treated rats were not significantly different from rats without insulin infusion. CONCLUSIONS: Sustained hyperinsulinemia causes partial impairment of NO production that may contribute to the development of insulin resistance and hypertension. Additional inhibition of NO synthesis by L-NAME accentuates the blood pressure elevation and insulin resistance in hyperinsulinemic rats. Furthermore, a rightward shift of the renal arterial pressure-natriuretic function relationship occurred in this hypertensive model.     

In vivo aspirin supplementation inhibits nitric oxide consumption by human platelets

Antiplatelet therapies improve endothelial function in atherosclerosis, suggesting that platelets regulate vascular nitric oxide (NO) bioactivity in vivo. Herein, washed platelets consumed NO on activation in an aspirin-sensitive manner, and aspirin enhanced platelet NO responses in vitro. To examine whether in vivo aspirin can inhibit platelet NO consumption, a double-blind placebo-controlled study was conducted. After a 2-week nonsteroidal anti-inflammatory drug (NSAID)-free period, healthy men were randomly assigned and administered aspirin (75 mg/d orally) or identical placebo for 14 days, then crossed over to the opposite arm. Following in vivo aspirin, NO consumption by platelets was inhibited 91%. Rate of onset and recovery following aspirin withdrawal was consistent with cyclooxygenase 1 (COX-1) inhibition. In a small substudy, NO consumption by platelets from postmenopausal women was faster in hypercholesterolemics and less sensitive to aspirin (ie, 39% versus 76% inhibition for hypercholesterolemics or normocholesterolemics, respectively). However, 150 mg aspirin/day increased inhibition of NO consumption by platelets of hypercholesterolemics to 80%. Comparisons of platelet COX-1 or -2 expression and urinary 11-dehydro-thromboxane B2 excretion suggested that aspirin was less able to block platelet activation in vivo in hypercholesterolemia. In conclusion, aspirin inhibits NO consumption by platelets from healthy subjects, but its beneficial effects on NO bioactivity may be compromised in some hypercholesterolemic patients.     

Testosterone modulates platelet aggregation and endothelial cell growth through nitric oxide pathway

The aim of the present study was to investigate the effect of testosterone on the modulation of cellular events associated with vascular homeostasis. In rat aortic strips, 5-20 min treatment with physiological concentrations of testosterone significantly increased nitric oxide (NO) production. The rapid action of the steroid was suppressed by the presence of an androgen receptor antagonist (flutamide). We obtained evidence that the enhancement in NO synthesis was dependent on the influx of calcium from extracellular medium, because in the presence of a calcium channel blocker (verapamil) the effect of testosterone was reduced. Using endothelial cell (EC) cultures, we demonstrated that androgen directly acts at the endothelial level. Chelerythrine or PD98059 compound completely suppressed the increase in NO production, suggesting that the mechanism of action of the steroid involves protein kinase C and mitogen-activated protein kinase pathways. It is known that endothelial NO released into the vascular lumen serves as an inhibitor of platelet activation and aggregation. We showed that testosterone inhibited platelet aggregation and this effect was dependent on endothelial NO synthesis. Indeed, the enhancement of NO production elicited by androgen was associated with EC growth. The steroid significantly increased DNA synthesis after 24 h of treatment, and this mitogenic action was blunted in the presence of NO synthase inhibitor N-nitro-l-arginine methyl ester. In summary, testosterone modulates vascular EC growth and platelet aggregation through its direct action on endothelial NO production.     

Angiotensin II AT1 receptor antagonists inhibit platelet adhesion and aggregation by nitric oxide release

This study investigated the process of nitric oxide (NO) release from platelets after stimulation with different angiotensin II type 1 (AT1)-receptor antagonists and its effect on platelet adhesion and aggregation. Angiotensin II AT1-receptor antagonist-stimulated NO release in platelets was compared with that in human umbilical vein endothelial cells by using a highly sensitive porphyrinic microsensor. In vitro and ex vivo effects of angiotensin II AT1-receptor antagonists on platelet adhesion to collagen and thromboxane A2 analog U46619-induced aggregation were evaluated. Losartan, EXP3174, and valsartan alone caused NO release from platelets and endothelial cells in a dose-dependent manner in the range of 0.01 to 100 micro mol/L, which was attenuated by NO synthase inhibitor N(G)-nitro-L-arginine methyl ester. The angiotensin II AT1-receptor antagonists had more than 70% greater potency in NO release in platelets than in endothelial cells. The degree of inhibition of platelet adhesion (collagen-stimulated) and aggregation (U46619-stimulated) elicited by losartan, EXP3174, and valsartan, either in vitro or ex vivo, closely correlated with the NO levels produced by each of these drugs alone. The inhibiting effects of angiotensin II AT1-receptor antagonists on collagen-stimulated adhesion and U46619-stimulated aggregation of platelets were significantly reduced by pretreatment with N(G)-nitro-L-arginine methyl ester. Neither the AT2 receptor antagonist PD123319, the cyclooxygenase synthase inhibitor indomethacin, nor the selective thromboxane A2/prostaglandin H2 receptor antagonist SQ29,548 had any effect on angiotensin II AT1-receptor antagonist-stimulated NO release in platelets and endothelial cells. The presented studies clearly indicate a crucial role of NO in the arterial antithrombotic effects of angiotensin II AT1-receptor antagonists.     

Inhibition of platelet aggregation by a monoclonal antibody against human fibronectin.

A monoclonal antibody (A3.3) has been generated against human platelet fibronectin (FN). A3.3 reacts with human plasma FN but with no other plasma proteins. A3.3 was found to inhibit thrombin- or ionophore A23187-stimulated aggregation of gel-filtered platelets in a concentration-dependent manner in both an aggregometer assay and a sensitive well plate aggregation assay. The antibody does not block secretion of serotonin. Four other anti-FN monoclonal antibodies that recognize different epitopes on FN than A3.3 does have no effect on platelet aggregation. A3.3 does not block the adhesion of CHO cells to FN-coated surfaces, indicating that it does not bind to the identified cell-binding domain of FN. A3.3 reacts with a 160/140-kDa doublet, known to contain the cell-binding domain, that is produced by digestion of FN with elastase or thermolysin. However, the antibody does not react with lower molecular weight species that also contain the cell-binding domain or with any of the other identified domains of FN. The A3.3 epitope is extremely protease sensitive and the smallest fragment found in any digest that retains reactivity with A3.3 is a 70-kDa peptide produced in low yield by mild thermolytic cleavage of FN. These data suggest that A3.3 defines a functional site present on both the platelet and plasma FN molecule that has a direct role in platelet aggregation.     

Thrombospondin-1 stimulates platelet aggregation by blocking the antithrombotic activity of nitric oxide/cGMP signaling

Platelet alpha-granules constitute the major rapidly releasable reservoir of thrombospondin-1 in higher animals. Although some fragments and peptides derived from thrombospondin-1 stimulate or inhibit platelet aggregation, its physiologic function in platelets has remained elusive. We now show that endogenous thrombospondin-1 is necessary for platelet aggregation in vitro in the presence of physiologic levels of nitric oxide (NO). Exogenous NO or elevation of cGMP delays thrombin-induced platelet aggregation under high shear and static conditions, and exogenous thrombospondin-1 reverses this delay. Thrombospondin-1-null murine platelets fail to aggregate in response to thrombin in the presence of exogenous NO or 8Br-cGMP. At physiologic concentrations of the NO synthase substrate arginine, thrombospondin-1-null platelets have elevated basal cGMP. Ligation of CD36 or CD47 is sufficient to block NO-induced cGMP accumulation and mimic the effect of thrombospondin-1 on aggregation. Exogenous thrombospondin-1 also reverses the suppression by NO of alphaIIb/beta3 integrin-mediated platelet adhesion on immobilized fibrinogen, mediated in part by increased GTP loading of Rap1. Thrombospondin-1 also inhibits cGMP-mediated activation of cGMP-dependent protein kinase and thereby prevents phosphorylation of VASP. Thus, release of thrombospondin-1 from alpha-granules during activation provides positive feedback to promote efficient platelet aggregation and adhesion by overcoming the antithrombotic activity of physiologic NO.     

The influence of organic peroxides on platelet aggregation and sensitivity to nitric oxide

The effects of oxidative stress, induced by water-soluble and lipid peroxides, on platelet reactivity and platelet sensitivity to nitric oxide were investigated. Hydrogen peroxide and cumene hydroperoxide potentiated thrombin-induced platelet aggregation. In contrast, 15(S)-hydroperoxyeicosatetraenoic acid had no such effect, while 12(S)-hydroperoxyeicosatetraenoic acid inhibited platelet reactivity. All of the peroxides tested were found to decrease platelet sensitivity to nitric oxide, although the mechanisms by which the various peroxides altered platelet sensitivity to nitric oxide were different. The water-soluble peroxides opposed the actions of nitric oxide without affecting cyclic GMP levels, while 15(S)-hydroperoxyeicosatetraenoic acid caused a significant reduction in the concentration of cyclic GMP formed in response to NO. The data from this study demonstrate that water-soluble and lipid peroxides both affect platelet reactivity and regulation, but by different mechanisms. Thus, caution should be exercised when selecting peroxides to be used as models of oxidative stress.