Platelet Nitric Oxide Production Research Articles

Dengue induces iNOS expression and nitric oxide synthesis in platelets through IL-1R.

Dengue is an arthropod-born disease caused by dengue virus (DENV), that may manifest as a mild illness or severe form, characterized by hemorrhagic fever and shock. Nitric oxide (NO) is a vasodilator signaling molecule and an inhibitor of platelet aggregation known to be increased in platelets from dengue patients. However, the mechanisms underlying NO synthesis by platelets during dengue are not yet elucidated. IL-1β is a pro-inflammatory cytokine able to induce iNOS expression in leukocytes and present in dengue patients at high levels. Nevertheless, the role of IL-1β in platelet activation, especially regarding iNOS expression, are not clear. We prospectively followed a cohort of 28 dengue-infected patients to study NO synthesis in platelets and its relationship with disease outcomes. We used in vitro infection and stimulation models to gain insights on the mechanisms. We confirmed that platelets from dengue patients express iNOS and produce higher levels of NO during the acute phase compared to healthy volunteers, returning to normal levels after recovery. Platelet NO production during acute dengue infection was associated with the presence of warning signs, hypoalbuminemia and hemorrhagic manifestations, suggesting a role in dengue pathophysiology. By investigating the mechanisms, we evidenced increased iNOS expression in platelets stimulated with dengue patients´ plasma, indicating induction by circulating inflammatory mediators. We then investigated possible factors able to induce platelet iNOS expression and observed higher levels of IL-1β in plasma from patients with dengue, which were correlated with NO production by platelets. Since platelets can synthesize and respond to IL-1β, we investigated whether IL-1β induces iNOS expression and NO synthesis in platelets. We observed that recombinant human IL-1β enhanced iNOS expression and dose-dependently increased NO synthesis by platelets. Finally, platelet infection with DENV in vitro induced iNOS expression and NO production, besides the secretion of both IL-1α and IL-1β. Importantly, treatment with IL-1 receptor antagonist or a combination of anti-IL-1α and anti-IL-1β antibodies prevented DENV-induced iNOS expression and NO synthesis. Our data show that DENV induces iNOS expression and NO production in platelets through mechanisms depending on IL-1 receptor signaling.

Inhibition of platelet aggregation by activation of platelet intermediate conductance Ca2+‐activated potassium channels

BackgroundWithin the vasculature platelets and endothelial cells play crucial roles in hemostasis and thrombosis. Platelets, like endothelial cells, possess intermediate conductance Ca2+‐activated K+ (IKCa) channels and generate nitric oxide (NO). Although NO limits platelet aggregation, the role of IKCa channels in platelet function and NO generation has not yet been explored. ObjectivesWe investigated whether IKCa channel activation inhibits platelet aggregation, and per endothelial cells, enhances platelet NO production. MethodsPlatelets were isolated from human volunteers. Aggregometry, confocal microscopy, and a novel flow chamber model, the Quartz Crystal Microbalance (QCM) were used to assess platelet function. Flow cytometry was used to measure platelet NO production, calcium signaling, membrane potential, integrin αIIb/β3 activation, granule release, and procoagulant platelet formation. ResultsPlatelet IKCa channel activation with SKA‐31 inhibited aggregation in a concentration‐dependent manner, an effect reversed by the selective IKCa channel blocker TRAM‐34. The QCM model along with confocal microscopy demonstrated that SKA‐31 inhibited platelet aggregation under flow conditions. Surprisingly, IKCa activation by SKA‐31 inhibited platelet NO generation, but this could be explained by a concomitant reduction in platelet calcium signaling. IKCa activation by SKA‐31 also inhibited dense and alpha‐granule secretion and integrin αIIb/β3 activation, but maintained platelet phosphatidylserine surface exposure as a measure of procoagulant response. ConclusionsPlatelet IKCa channel activation inhibits aggregation by reducing calcium‐signaling and granule secretion, but not by enhancing platelet NO generation. IKCa channels may be novel targets for the development of antiplatelet drugs that limit atherothrombosis, but not coagulation.

Sex-Dependent Effect of Platelet Nitric Oxide: Production and Platelet Reactivity in Healthy Individuals

A nitrate-rich diet has many cardiovascular benefits, but the mechanism behind this is unclear. We hypothesized that the ingestion of nitrate augments nitrate to nitrite reduction, leading to nitric oxide (NO) production, which may suppress platelet reactivity. In a randomized, double-blinded, placebo-controlled study involving healthy individuals, ingestion of nitrate augmented saliva and plasma nitrite/nitrate concentration and enhanced platelet NO production disproportionately in women compared with men. The response of elevated platelet NO in men was increased platelet reactivity and the response of markedly elevated platelet NO in women slightly inhibited platelet reactivity.

Role of the Platelets and Nitric Oxide Biotransformation in Ischemic Stroke: A Translative Review from Bench to Bedside.

Ischemic stroke remains the fifth cause of death, as reported worldwide annually. Endothelial dysfunction (ED) manifesting with lower nitric oxide (NO) bioavailability leads to increased vascular tone, inflammation, and platelet activation and remains among the major contributors to cardiovascular diseases (CVD). Moreover, temporal fluctuations in the NO bioavailability during ischemic stroke point to its key role in the cerebral blood flow (CBF) regulation, and some data suggest that they may be responsible for the maintenance of CBF within the ischemic penumbra in order to reduce infarct size. Several years ago, the inhibitory role of the platelet NO production on a thrombus formation has been discovered, which initiated the era of extensive studies on the platelet-derived nitric oxide (PDNO) as a platelet negative feedback regulator. Very recently, Radziwon-Balicka et al. discovered two subpopulations of human platelets, based on the expression of the endothelial nitric oxide synthase (eNOS-positive or eNOS-negative platelets, respectively). The e-NOS-negative ones fail to produce NO, which attenuates their cyclic guanosine monophosphate (cGMP) signaling pathway and—as result—promotes adhesion and aggregation while the e-NOS-positive ones limit thrombus formation. Asymmetric dimethylarginine (ADMA), a competitive NOS inhibitor, is an independent cardiovascular risk factor, and its expression alongside with the enzymes responsible for its synthesis and degradation was recently shown also in platelets. Overproduction of ADMA in this compartment may increase platelet activation and cause endothelial damage, additionally to that induced by its plasma pool. All the recent discoveries of diverse eNOS expression in platelets and its role in regulation of thrombus formation together with studies on the NOS inhibitors have opened a new chapter in translational medicine investigating the onset of acute cardiovascular events of ischemic origin. This translative review briefly summarizes the role of platelets and NO biotransformation in the pathogenesis and clinical course of ischemic stroke.

Deficiency of platelet adhesion molecule CD226 causes megakaryocyte development and platelet hyperactivity.

This study used constitutive CD226 gene knockout (KO) mice as a model to investigate the functions and mechanisms of CD226 in megakaryocyte (MK) maturation and platelet activation. Although CD226 deficiency did not cause MK polyploidization or platelet granule abnormalities, increased MK counts were detected in the femora bone marrow (BM) and spleen of CD226 KO mice. Particularly, CD226 KO mice have a more extensive membrane system in MKs and platelets than wild-type (WT) mice. We also demonstrated that CD226 KO mice displayed increased platelet counts, shortened bleeding time, and enhanced platelet aggregation. CD226 KO platelets had an increased mature platelet ratio compared to the control platelets. In addition, the observed reduction in bleeding time may be due to decreased nitric oxide (NO) production in the platelets. Platelet-specific CD226-deficient mice showed similar increased MK counts, shortened bleeding time, enhanced platelet aggregation, and decreased NO production in platelets. Furthermore, we performed middle cerebral artery occlusion-reperfusion surgery on WT and CD226 KO mice to explore the potential effect of CD226 on acute ischemia-reperfusion injury; the results revealed that CD226 deficiency led to significantly increased infarct area. Thus, CD226 is a promising candidate for the treatment of thrombotic disorders.

A Standardized Composition Comprised of Extracts from Rosmarinus officinalis, Annona squamosa and Zanthoxylum clava-herculis for Cellulite.

Background:Cellulite, characterized by changes in the skin morphology presented as dimpled or puckered skin appearance, is highly prevalent among postadolescent women. Cellulite management ranges from topical cream applications to invasive procedures. While some interventions showed improvements in physical appearances of affected areas, so far, none have reversed the condition to a full recovery. These unsuccessful measures signify the intricate nature of cellulite etiology highlighting its complexity leading to the possibility for a combination treatment approach to target multiple mechanisms.Materials and Methods:We screened our plant library for extracts that reduce cellular lipid accumulation, improve microcirculation, possess high total antioxidant capacity, significant anti-platelet aggregation, and anti-inflammatory activities using lipid accumulation assay in 3T3-L1 cells, Croton oil-induced hemorrhoid test in rats as a model for microcirculation, anti-platelet aggregation assay, nitric oxide (NO) inhibition assay, and 1,1-diphenyl-2-picrylhydrazyl assay.Results:Three known botanicals such as Rosemary officinalis, Annona squamosa and Zanthoxylum clava-herculis were identified as lead extracts in these tests. Treatment of 3T3 cell with A. squamosa at 1 μg/ml resulted in 68.8% reduction in lipid accumulation. In croton oil-induced hemorrhoid study, Z. clava-herculis reduced the recto-anus coefficient by 79.6% at 6 mg/kg indicating improvement in microcirculations. Similarly, R. officinalis caused inhibition of 82%, 71.8%, and 91.8% in platelet aggregation, NO production and free radical generation at 31.25 μg/ml, 6.2 μg/ml, and 40 μg/ml concentrations suggesting its anti-oxidant, and anti-inflammatory activities.Conclusions:Data depicted here suggest that formulation of these well-known botanicals at a specific ratio perhaps may yield a composition with a much wider spectrum of mechanisms of actions to impact the multiple pathways involved in cellulite onset, continuation, or exacerbations.SUMMARY Cellulite represents one of the main esthetic concerns of women with a likely cause of psychological insecurities. Its pathophysiology involves multiple pathways that include vascular, adipose tissues, inflammation, structural and physiological.Treatment strategies for cellulite comprises increasing microcirculation flow, reducing lipogenesis, promoting lipolysis, free radicals scavenging or formation reduction, anti-inflammation and other invasive procedures.We screened our plant library for extracts that reduces cellular lipid accumulation, improves microcirculation, possesses high total antioxidant capacity, inhibits platelet aggregation, and moderates inflammation.Botanical extracts from Rosmarinus officinalis, Annona squamosa and Zanthoxylum clava-herculis were identified as leads and formulated to yield a standardized composition designated as UP1307 and suggested its usage for cellulite. Abbreviations Used: GMP: Good Manufacturing Practice; CA: Carnosic acid; NF-kB: nuclear factor-kB; HPLC: high-performance liquid chromatography; EtOH: Ethanol; DMEM: Dulbecco's modified Eagle's medium; FBS: fetal bovine serum; SD: Sprague Dawley; RAC: recto-anus coefficient; LPS: Lipopolysaccharide; DPPH: 1,1-diphenyl-2-picryl-hydrazyl; TNF-α: tumor necrosis factor; NO: Nitric oxide

PHARMACOLOGICAL CHARACTERIZATION OF THE FUNCTIONAL ROLE OF CALCIUM-ACTIVATED POTASSIUM CHANNELS IN PLATELETS

In arteries, stimulation of endothelial cell small (SKCa) and intermediate (IKCa) conductance calcium-activated potassium channels provides a negative-feedback mechanism to limit agonist-induced vasoconstriction. Additionally, endothelial cell KCa channels in conjunction with nitric oxide (NO) mediate vasodilation in response to agonists and physical stimuli such as increases in blood flow. Platelets, like endothelial cells, possess KCa channels and have the capability to generate NO via endothelial nitric oxide synthase (eNOS). NO is known to limit platelet aggregation but the role of KCa channels in platelet function and NO-generation has not been explored. Our objective was to pharmacologically characterize SKCa and IKCa channel function in platelets, and to investigate their role in platelet NO production. Our hypothesis was that pharmacological activation of KCa channels would inhibit platelet aggregation and enhance platelet NO production.

Hyperaggregability and impaired nitric oxide production in platelets from postmenopausal women

ObjectiveCardiovascular mortality increases after menopause in women. Nitric oxide is essential for proper platelet function inhibiting its aggregation and maintaining vascular haemostasis. Here, we investigated whether platelet function and intraplatelet l-arginine–nitric oxide pathway are impaired in postmenopausal women. Study designCross-sectional. Main outcomes measuresBlood was collected from 16 premenopausal and 12 postmenopausal women without any additional risk factor for cardiovascular disease. Platelet reactivity was measured by light transmission aggregometry. l-Arginine–nitric oxide pathway was assessed measuring transmembrane l-[3H]-arginine transport, nitric oxide synthase activity by the citrulline assay, and arginase activity by the conversion of l-[14C]arginine to l-[14C]-urea. The activity of antioxidant enzymes was measured by spectrophotometric assays. Protein expression was determined by Western blotting. ResultsPlatelet aggregation was increased in postmenopausal compared to premenopausal women. Postmenopausal women demonstrated reduced plasma levels of l-arginine, a lower nitric oxide synthase activity, similar endothelial and inducible nitric oxide synthase expression, and a compensatory increase in l-arginine transmembrane transport. Arginase expression and activity did not differ between groups. In regard to oxidative stress, no differences between groups were observed NAPDH oxidase subunits expression and protein carbonylation. However, the activity of the antioxidant enzyme superoxide dismutase and catalase protein levels in platelets were higher in postmenopausal women. ConclusionPostmenopausal women present increased platelet reactivity, which may be due to a reduction in intraplatelet nitric oxide synthesis. Platelet hyperaggregability is known to be associated with arterial and venous thromboembolic event; therefore, it may contribute to the heightened risk of cardiovascular adverse events in this population.

Selective serotonin reuptake inhibitors and endothelial function in women.

Among women worldwide, major depression (MDD) and heart disease rank first and second, respectively, in burden of disease. Although selective serotonin reuptake inhibitors (SSRIs) are frequently prescribed, possible inhibition of nitric oxide (NO) function has caused concerns about their effects on protective vascular mechanisms. Our study aimed to determine the effect of SSRIs on flow-mediated vascular dilatation (FMD), platelet aggregation, and platelet NO production among women. Women (n=28) without known cardiovascular disease were recruited prior to undergoing SSRI treatment for MDD, postpartum depression (PPD), or premenstrual dysphoric disorder (PMDD). Symptoms were quantified using the Hamilton Depression/Anxiety and Beck Depression scales. FMD, platelet aggregation, and platelet NO production were measured before and after 1 month of SSRI (sertraline, fluoxetine, or paroxetine) therapy. Depression and anxiety symptoms decreased significantly with SSRI treatment (ps <0.01). FMD and platelet aggregation did not differ between pre- and posttreatment, although FMD rose to the normal range (≥ 8%) in two of three women with abnormal FMD prior to SSRI treatment. We observed a 21% decrease (p=0.024) in platelet NO production. SSRI treatment had little effect on FMD or platelet aggregation. The health impact of decreased NO production is unclear, particularly in this relatively young group of women without cardiovascular disease, but should be considered in future studies focusing on SSRI safety in patients with cardiovascular disease.

Stimulation of Platelet Nitric Oxide Production by Nebivolol Prevents Thrombosis

dl-Nebivolol, a selective β1-adrenergic receptor antagonist, besides its hypotensive activity exerts vasodilatory and platelet inhibitory effects in vitro by a mechanism involving nitric oxide (NO). Our aim was to evaluate whether nebivolol exerts in vivo antithrombotic effects, to unravel the mechanism of this action and to clarify the relative roles of its 2 enantiomers: d- and l-nebivolol. In wild-type mice, dl-nebivolol, l-nebivolol, and d-nebivolol, but not bisoprolol, reduced mortality consequent to platelet pulmonary thromboembolism induced by the intravenous injection of collagen plus epinephrine (-44%, -45%, -29%, respectively; P<0.05), whereas in eNOS(-/-) mice only dl-nebivolol and d-nebivolol were effective. dl-Nebivolol, l- and d-nebivolol reduced photochemical damage-induced femoral artery thrombosis in wild-type mice, whereas in eNOS(-/-) mice only dl-nebivolol and d-nebivolol were active. Moreover, dl-nebivolol and l-nebivolol increased plasma, urinary-, and platelet-derived nitrites and nitrates (NOx), NO degradation products, in wild-type but not in eNOS(-/-) mice. In vivo platelet activation, assessed by platelet P-selectin expression, was reduced by dl-nebivolol and l- and d-nebivolol in wild-type mice but only by dl-nebivolol and d-nebivolol in eNOS(-/-) mice. In bone marrow-transplanted, chimeric mice with only blood cells, and not the endothelium, producing NO dl-nebivolol and l-nebivolol maintained their antithrombotic activity, whereas they lose it in chimeras with only endothelium, and not blood cells, producing NO. In vitro, with isolated platelets, dl-nebivolol and l-nebivolol, but not d-nebivolol and bisoprolol, increased platelet cGMP and NOx formation. Treatment with dl-nebivolol and l-nebivolol increased phophorylated eNOS in platelets. Our data show that dl-nebivolol exerts an antithrombotic activity by stimulating the formation of NO by platelets, and that this effect is generated by its l-enantiomer, whereas the d-enantiomer exerts a weak antiplatelet effect because of β-adrenergic receptor-independent stimulation of adenyly cyclase. These results confirm that platelet-derived NO plays a role in thrombosis prevention and it may represent a target of pharmacological intervention.

In vitro effects of nitric oxide donors on apoptosis and oxidative/nitrative protein modifications in ADP-activated platelets

Nitric oxide (NO) is an important physiological signaling molecule. However, when produced in excessive amounts, NO can also have toxic effects. The aim of this study is to investigate the effects of exogenous- and endogenous-derived NO on oxidative modifications of proteins and apoptosis in activated platelets. Washed platelets were incubated with L-arginine or nitroso-glutathione (GSNO) in the presence of adenosine diphosphate (ADP). After incubation, caspase-3 activity, phosphatidylserine (PS) externalization and the potential of mitochondrial membrane as markers of apoptosis were measured. In addition, the alterations in protein carbonylation (PCO) and nitrotyrosine (NT) formation as markers of protein oxidation were examined. Platelet activation with ADP (20 µM) significantly increased PCO and NT levels and apoptotic events. After incubation with L-arginine, platelet NO production increased significantly. This L-arginine-induced increase caused decreases in formerly increased PCO and NT levels associated with ADP-induced platelet activation. Stimulation of NO production with L-arginine protected platelets from apoptosis. GSNO caused an increase in protein NT levels. Despite this change, GSNO was effective in inhibition of P-selectin expression, platelet aggregation, protein carbonylation and apoptosis. The results suggest that L-arginine and GSNO-mediated NO leads to the inhibition of key apoptotic processes including caspase-3 activation, PS exposure and low mitochondrial membrane potential in washed platelets. The inhibitory effect of platelet clearance of L-arginine and GSNO may be a novel useful therapeutic property in clinical application.

Dehydroepiandrosterone Prevents the Aggregation of Platelets Obtained from Postmenopausal Women with Type 2 Diabetes Mellitus through the Activation of the PKC/eNOS/NO Pathway

The steroid hormone dehydroepiandrosterone (DHEA), suggested to be a cardioprotector, prevents platelet aggregation in healthy humans. This hormone is reduced in postmenopausal women by 60% of its normal value. Platelets in patients with type 2 diabetes (T2D) are more sensitive to aggregation, which has been attributed to a reduced ability to produce nitric oxide (NO). In light of these precedents and considering that DHEA is able to increase the production of NO in cultured endothelial cells, we suggest that DHEA prevents the aggregation of platelet from postmenopausal women with T2D through the activation of PKC/eNOS/NO/cGMP pathway. To determine the effect of DHEA in platelet aggregation, platelet-rich plasma (PRP) obtained from postmenopausal women with T2D was preincubated with DHEA, and aggregation induced by ADP was determined in the presence or absence of L-NNA (LNG-nitroarginine), Rottlerin, NOS, or PKC delta inhibitors, respectively. Platelet NO production was measured with the fluorescent probe DAF2DA and eNOS activation was determined by Western blot, using an anti-p-eNOS (ser 1177) antibody. DHEA 1) prevented platelet aggregation by 40% compared to control, 2) increased NO production by 63%, 3) increased p-eNOS (phosphorylated endothelial nitric oxide synthase) levels, and 4) increased cGMP production. These effects were reduced in the presence of L-NNA or Rottlerin. DHEA prevents platelet aggregation induced by ADP. This effect is mediated by the activation of the PKCδ/eNOS/NO/cGMP pathway. Our results suggest that DHEA could be considered to be a potential therapeutic tool in the prevention of atherothrombotic processes in postmenopausal women with T2D.

Increased nitric oxide production in platelets from severe chronic renal failure patients

Nitric oxide (NO) production occurs through oxidation of the amino acid L-arginine by NO synthase (NOS). NO inhibits platelet activation by increasing the levels of cyclic guanosine monophosphate (cGMP), thus maintaining vascular homeostasis. Our group previously demonstrated (da Silva et al. 2005) an enhancement of the L-arginine-NO-cGMP pathway in platelets taken from chronic renal failure (CRF) patients on haemodialysis associated with reduced platelet aggregation. We investigate the platelet L-arginine-NO-cGMP pathway, platelet function, and inflammation from patients in CRF on conservative treatment. A total of 42 CRF patients and 42 controls (creatinine clearance = 27 ± 3 vs. 93 ± 1 mL per min per 1.73 m2, respectively) participated in this study. NOS activity and expression and cGMP concentration were measured in platelets. Platelet aggregation induced by collagen or ADP was evaluated and plasma levels of fibrinogen were determined by the Clauss method. A marked increase in basal NOS activity was seen in undialysed CRF patients compared with controls, accompanied by an elevation of fibrinogen plasma levels. There were no differences in expression of NOS and in cGMP levels. In this context, platelet aggregation was not affected. We provide the first evidence of increased intraplatelet NO biosynthesis in undialysed CRF patients, which can be an early marker of future haemostatic abnormalities during dialysis treatment.

MS358 ONE-MONTH CONSUMPTION OF AN OLIVE LEAF EXTRACT ENHANCES CARDIOVASCULAR STATUS IN HYPERCHOLESTEROLEMIC SUBJECTS

Aims: Assessment of the simultaneous effects of hypertension and hypercholesterolemia on platelet activation, oxidative stress, and nitric oxide (NO) production, and evaluation of the effect of irbesartan, an angiotensin II type 1 receptor antagonist employing an original hamster model. Material and Methods: The experiments were performed on platelets isolated from the blood of Golden Syrian hamsters divided in three experimental groups: controls, C, hypertensive-hypercholesterolemic, HH and hypertensivehypercholesterolemic treated with irbesartan, HHI (fed as HH group plus irbesartan, 4 months). Results: The new animal model, HH is characterized by: i. platelet activation as demonstrated by structural modifications, increased integrin 3 beta expression on plasma membrane and of proteins implicated in the activation and aggregation process (FAK, PI3K, Src); ii. a significantly higher endogenous generation of ROS in platelets, mostly produced by NADPH-oxidase; iii. enhanced expression of oxidase subunit gp91phox of NAD(P)H, 12lipoxygenase, cyclooxygenase-1, cPLA2, PKC protein expression, and iv. reduced platelet NO production (compared with C group). Compared with HH group, the treatment with irbesartan (HHI) significantly attenuates the level of all the molecules tested. Conclusions: Experimental hypertension associated with hypercholesterolemia induces major changes in blood platelets, which are significantly diminished by irbesartan administration. Moreover, this AT1-R antagonist reduces platelet ROS generation acting on the enzymatic sources involved in ROS production, suggesting an antioxidant effect of irbesartan.

Combination Antithrombotic Therapies

Atherothrombosis is the major pathophysiological process responsible for the occurrence of severe ischemic events in patients with cardiovascular diseases. In the United States, atherothrombosis strongly influenced mortality in 2004: One in 2.8 deaths was due to CVD, 1 in 5 deaths to coronary heart disease, and 1 in 17 deaths to stroke.1 Because cardiovascular disease is a progressive and systemic disease, long-term antithrombotic therapies that effectively target the entire arterial vasculature and modulate the key components responsible for thrombus generation are essential to improve patient outcomes. Because platelet activation is determined by multiple receptor-mediated signaling pathways, clinical studies have evaluated the efficacy of multidrug administration in the prevention of atherothrombotic complications.2,3 The major concern with these therapies is the critical balance between anti-ischemic effect and bleeding risk. This review summarizes our understanding of the role of combination antiplatelet therapies in the treatment and prevention of atherothrombosis. Platelet activation and aggregation play a pivotal role in the generation of occlusive thrombus at the site of coronary arterial plaque rupture. In addition, platelets influence various endothelial and inflammatory responses during the initiation and progression of atherosclerosis. Under normal conditions, anucleate circulating platelets are in a quiescent state. Healthy vascular endothelium prevents adhesion and activation of platelets by producing antithrombotic factors such as CD39 (ectoADPase), prostaglandin I2, nitric oxide, heparin, matrix metalloproteinase-9, protein S, and thrombomodulin.3,4 Endothelial activation and denudation and frank atherosclerotic plaque rupture expose the subendothelial matrix and release prothrombotic factors during acute coronary syndromes (ACS) and percutaneous interventions. These processes result in localized platelet adhesion and platelet activation. After adhesion to the exposed subendothelial matrix, platelets are activated by shear and the soluble agonists thromboxane A2 (TxA2), ADP, and thrombin. TxA2 is produced from arachidonic acid, which originates from membrane phospholipids and …

Apolipoprotein E genotype is related to nitric oxide production in platelets

The presence of the epsilon4 allele of apolipoprotein E (APOE) is considered a risk factor for sporadic Alzheimer's disease (AD). Our recent data demonstrated that the systemic modulation of oxidative stress in platelets and erythrocytes is disrupted in aging and AD. In this study, the relationship between APOE genotype and oxidative stress markers, both in AD patients and controls, was evaluated. The AD group showed an increase in the content of thiobarbituric acid-reactive substances (TBARS) and in the activities of nitric oxide synthase (NOS) and Na, K-ATPase, when compared to controls. Both groups had a similar cGMP content and superoxide dismutase activity. APOE epsilon4 allele carriers showed higher NOS activity than non-carriers. These results suggest a possible influence of APOE genotype on nitric oxide (NO) production that might enhance the effects of age-related specific factor(s) associated with neurodegenerative disorders.

Signaling-mediated Functional Activation of Inducible Nitric-oxide Synthase and Its Role in Stimulating Platelet Activation

Nitric oxide (NO) is a short lived secondary messenger, synthesized by nitric-oxide synthases (NOS). It is believed that the activity of inducible NOS (iNOS) is regulated primarily at the transcription level by inducing expression of iNOS mRNA and protein, which then continuously produces NO, until its degradation. Platelets do not have the nuclear transcriptional regulatory mechanisms of the iNOS gene and are believed to generate NO in response to agonist stimulation via endothelial NOS (eNOS). However, here we show that agonist-induced NO production is only partially eNOS-dependent and is also mediated by iNOS. Platelet agonist-induced NO production is significantly reduced in iNOS-knockout platelets. Platelet NO production occurs within seconds after agonist addition and is not accompanied by changes in iNOS protein levels, indicating a signaling-mediated functional activation mechanism of iNOS. Importantly, iNOS knock-out and iNOS inhibitors reduce agonist-induced platelet secretion and aggregation and cGMP levels, indicating that iNOS activation is important in stimulating platelets via the newly identified NO-cGMP-dependent platelet secretion pathway. Furthermore, iNOS knock-out mice have prolonged bleeding time, suggesting that this novel mode of regulation of iNOS activity plays a physiologically relevant role in hemostasis.

Resveratrol, at Concentrations Attainable with Moderate Wine Consumption, Stimulates Human Platelet Nitric Oxide Production 3

The mechanisms through which moderate wine consumption reduces ischemic cardiovascular events are not yet fully unraveled. Grape extracts or a mixture of the polyphenols contained in wine were previously shown to increase nitric oxide (NO); however, little information is available on the effect of resveratrol, one of the main polyphenols of wine, on platelet NO production. We assessed the effects of resveratrol, at the concentrations attainable after moderate wine intake, on platelet NO production and the mechanism of this activity. Twenty healthy volunteers were studied before and after 15 d of controlled white or red wine intake (300 mL/d). After wine intake, plasma resveratrol and the release of NO by stimulated platelets increased significantly. Resveratrol, at the concentrations detected in plasma after wine intake, was incubated in vitro with washed platelets and several variables related to NO production and to signal transduction were measured. Resveratrol in vitro enhanced significantly the production of NO by stimulated platelets, the activity of platelet NO synthase (NOS), phosphorylation of protein kinase B, an activator of the endothelial NOS (eNOS), and phosphorylation of vasodilator-activated protein (VASP), an expression of the biologic activity of NO in platelets. Simultaneously, we observed decreased phosphorylation of P38 mitogen-activated protein kinase (p38MAPK), a proinflammatory pathway in human platelets, a reduction of the activity of NADPH oxidase, a major source of reactive oxygen species (ROS) and of the generation of O2− radicals, as detected by cytochrome C reduction. In conclusion, resveratrol, at concentrations attainable after moderate wine intake, activates platelet eNOS and in this way blunts the proinflammatory pathway linked to p38MAPK, thus inhibiting ROS production and ultimately platelet function. This activity may contribute to the beneficial effects of moderate wine intake on ischemic cardiovascular disease.

Platelet nitric oxide production and IR: Relation with obesity and hypertriglyceridemia

Background and Aim Three NOS isoforms are responsible for nitric oxide production in various tissues. Endothelial constitutive NOS is expressed in vascular endothelium and in platelets, contributing to vascular tone regulation and platelet aggregation. The aim of the present work was to examine eNOS polymorphism, to find a correlation with platelet NO production and degree of insulin resistance (IR) in non-diabetic subjects and in patients affected by type 2 diabetes. Methods and Results Seventy-one non-diabetic subjects and 37 patients affected by Type 2 diabetes were recruited. The subjects were subdivided into 3 groups as cut-off for the definition of an insulin resistant state: IR non-diabetic subjects, insulin sensitive subjects, and insulin-resistant patients affected by Type 2 diabetes. Plasma glyco-metabolic parameters, platelet nitric oxide production, endothelial nitric oxide synthase (eNOS) gene polymorphism were measured in all subjects enrolled. Significant differences between groups were found in BMI, fasting glycaemia, fructosamine and HbA 1c, triglycerides and HDL cholesterol levels. Evaluating all the subjects, platelet NO production was significantly related with BMI, waist circumference, and triglycerides concentrations, thus suggesting an association between increased platelet NO production, obesity and hypertriglyceridemia, independent of the degree of insulin-resistance. Conclusion The modified platelet NO synthesis does not seem to be due to eNOS Glu298Asp polymorphism, while it can be hypothesized that it is caused by an iNOS induction, present in obesity, hypertriglyceridemia and in type 2 diabetes.

A study on the action of vitamin E supplementation on plasminogen activator inhibitor type 1 and platelet nitric oxide production in type 2 diabetic patients

Background and aim Type 2 diabetic (T2DM) patients show decreased fibrinolysis, mainly linked to high plasminogen activator inhibitor type 1 (PAI-1) production, together with a reduced bioavailability of nitric oxide and an impairment in Na +/K +-ATPase activity possibly involved in increased cardiovascular risk. Vitamin E is the major natural lipid-soluble antioxidant in human plasma. The present work was conducted in order to measure PAI-1, ICAM and VCAM-1 plasma levels, platelet nitric oxide production and membrane Na +/K +-ATPase activity in type 2 diabetic subjects treated with vitamin E (500 IU/day) for 10 weeks and then followed for other 20 weeks. Methods and results Thirty-seven T2DM patients (24 males and 13 females) were studied. None of them were affected by any other disease or diabetic complications. Significant differences were detected for PAI-1 antigen ( p < 0.001), PAI-1 activity ( p < 0.001), nitric oxide (NO) production ( p < 0.001), and Na +/K +-ATPase activity ( p < 0.001) among the 4 phases of the study. A significant decrease both in ICAM and VCAM-1 plasma levels was also found at the 10th week compared with baseline (respectively p < 0.001 and p < 0.05). Conclusion Our data suggest that vitamin E counteracts endothelial activation in T2DM patients possibly representing a new tool for endothelial protection.