Effect of high potassium diet on endothelial function.

BACKGROUND AND AIMS: Increased potassium intake is related to reduced blood pressure (BP) and reduced stroke rate. The effect of increased dietary potassium on endothelial function remains unknown. The aim was to determine the effect of increased dietary potassium from fruit and vegetables on endothelial function. METHODS AND RESULTS: Thirty five healthy men and women (age 32 ± 12 y) successfully completed a randomised cross-over study of 2 × 6 day diets either high or low in potassium. Flow mediated dilatation (FMD), BP, pulse wave velocity (PWV), augmentation index (AI) and a fasting blood sample for analysis of Intercellular Adhesion Molecule-1 (ICAM-1), E-selectin, asymmetric dimethylarginine (ADMA) and endothelin-1 were taken on completion of each intervention. Dietary change was achieved by including bananas and potatoes in the high potassium and apples and rice/pasta in the low potassium diet. Dietary adherence was assessed using 6 day weighed food diaries and a 24 h urine sample. The difference in potassium excretion between the two diets was 48 ± 32 mmol/d (P = 0.000). Fasting FMD was significantly improved by 0.6% ± 1.5% following the high compared to the low potassium diet (P = 0.03). There were no significant differences in BP, PWV, AI, ICAM-1, ADMA or endothelin-1 between the interventions. There was a significant reduction in E-selectin following the high (Median = 5.96 ng/ml) vs the low potassium diet (Median = 6.24 ng/ml), z = -2.49, P = 0.013. CONCLUSION: Increased dietary potassium from fruit and vegetables improves FMD within 1 week in healthy men and women but the mechanisms for this effect remain unclear. CLINICAL TRIAL REGISTRY: ACTRN12612000822886.

Beneficial clinical effects of perhexiline in patients with stable angina pectoris and acute coronary syndromes are associated with potentiation of platelet responsiveness to nitric oxide.

AIMS: To examine whether the prophylactic antianginal agent perhexiline potentiates platelet responsiveness to nitric oxide (NO) in patients with stable angina pectoris (SAP) and acute coronary syndromes (ACS: unstable angina pectoris or non-Q-wave myocardial infarction). METHODS AND RESULTS: Blood samples were obtained from patients before and after initiation of treatment with perhexiline. ADP-induced platelet aggregation and its inhibition by the NO donor sodium nitroprusside (SNP) were determined via impedance aggregometry in whole blood (WB) and platelet-rich plasma (PRP). Intraplatelet cGMP content was assayed by RIA, and superoxide (O(2)(-)) level by lucigenin-derived chemiluminescence. In patients with ACS not receiving perhexiline (n=12), platelet responsiveness to SNP did not vary significantly over the first 3 days post admission to hospital. Therapy with perhexiline for 3 days was associated with increases in SNP-induced inhibition of aggregation from 29+/-2% to 43+/-4% (n=50,P <0.001) in WB and from 20+/-5% to 42+/-7% (n=12, P<0.01) in PRP. Resolution of symptomatic ischaemia (n=39) was associated with significantly greater (P<0.01) increases than non-resolution (n=11). Similar increases in SNP responsiveness (P<0.001) occurred following institution of perhexiline therapy in patients with SAP (n=30), associated with a 85% decrease in anginal frequency. Treatment with perhexiline potentiated the cGMP-elevating effects of SNP in platelets (n=9,P =0.03). Although perhexiline did not alter whole blood O(2)(-) concentration ex vivo, it inhibited (P<0.01) O(2)(-) release from neutrophils in vitro. CONCLUSION: Perhexiline potentiates platelet responsiveness to NO both in SAP and ACS patients; in the latter group this improvement was predictive of resolution of ischaemic symptoms. The predominant mechanism of perhexiline effect is an increase in platelet cGMP responsiveness. Perhexiline also may reduce the potential for NO clearance by neutrophil-derived O(2)(-).

Induction of platelet formation from megakaryocytoid cells by nitric oxide.

Although the growth factors that regulate megakaryocytopoiesis are well known, the molecular determinants of platelet formation from mature megakaryocytes remain poorly understood. Morphological changes in megakaryocytes associated with platelet formation and removal of senescent megakaryocytes are suggestive of an apoptotic process. Previously, we have established that nitric oxide (NO) can induce apoptosis in megakaryocytoid cell lines. To determine whether there is an association between NO-induced apoptosis and platelet production, we exposed Meg-01 cells to S-nitrosoglutathione (GSNO) with or without thrombopoeitin (TPO) pretreatment and used flow cytometry and electron microscopy to assess platelet-sized particle formation. Meg-01 cells treated with TPO alone produced few platelet-sized particles (<3% of total counts), whereas treatment with GSNO alone produced a significant percentage of platelet-sized particles (22 +/- 4% of total counts); when combined with TPO pretreatment, however, GSNO led to a marked increase in platelet-sized particle production (48 +/- 3% of total counts). Electron microscopy confirmed that Meg-01 cells treated with TPO and GSNO yielded platelet-sized particles with morphological features specific for platelet forms. The platelet-sized particle population appears to be functional, because addition of calcium, fibrinogen, and thrombin receptor-activating peptide led to aggregation. These results demonstrate that NO facilitates platelet production, thereby establishing the essential role of NO in megakaryocyte development and thrombopoiesis.

Heparin reacts with and inactivates nitric oxide.

Although heparin is a well-known anticoagulant, in some cases it promotes a prothrombotic state and does so through both antibody-dependent and antibody-independent platelet activation. In this study, heparin was found to reverse the antiplatelet effect of an NO donor. S-nitroso-glutathione (SNO-Glu), with an EC(50) of 1.8 U/mL. Ultraviolet/visible spectral analysis and the Griess assay showed that increasing heparin concentrations on a dose-dependent basis eliminated acidified NO(x) species. Since heparin is a heterogeneous mixture of glycosaminoglycans, the effects of six different heparin disaccharides were compared with various substitutions on the hexose rings to determine which functional group(s) of the polysaccharide interact with acidified NO(x). Among the six disaccharides tested, only types I-S and II-S had the effect, suggesting that the sulfamino-group at the C2 position of the glucosamine moiety was critical for the elimination of acidified NO(x) species. Mass spectrometry experiments gave results consistent with these observations, indicating that only the I-S and II-S heparin disaccharides were modified upon treatment with NaNO(2)/HCl. Negative-ion electro-spray ionization MS and tandem MS analyses of the native compounds and their deuterium-labeled analogs confirmed that the reaction products from nitrosation of these N-sulfated disaccharides had eliminated the C2-sulfamino-moiety and replaced it with methoxide derived from the solvent. Participation of the 6-sulfato-substituent appears to facilitate the elimination reaction. These data show that heparin can impair the antiplatelet properties of nitric oxide by interacting with the nitrosating species, and suggest that heparin-like glycosamino-glycans may interact with endothelium-derived nitric oxide in vivo to regulate the bioactivity of this important antiplatelet and vasorelaxant substance.

Stable angina and acute coronary syndromes are associated with nitric oxide resistance in platelets.

OBJECTIVES: The study examined possible clinical determinants of platelet resistance to nitric oxide (NO) donors in patients with stable angina pectoris (SAP) and acute coronary syndromes (ACS), relative to nonischemic patients and normal subjects. BACKGROUND: We have shown previously that platelets from patients with SAP are resistant to the antiaggregating effects of nitroglycerin (NTG) and sodium nitroprusside (SNP). METHODS: Extent of adenosine diphosphate (1 micromol/liter)-induced platelet aggregation (impedance aggregometry in whole blood) and inhibition of aggregation by NTG (100 micromol/liter) and SNP (10 micromol/liter) were compared in normal subjects (n = 43), nonischemic patients (those with chest pain but no fixed coronary disease, (n = 35) and patients with SAP (n = 82) or ACS (n = 153). Association of NO resistance with coronary risk factors, coronary artery disease (CAD), intensity of angina and current medication was examined by univariate and multivariate analyses. RESULTS: In patients with SAP and ACS as distinct from nonischemic patients and normal subjects, platelet aggregability was increased (both p < 0.01), and inhibition of aggregation by NTG and SNP was decreased (both p < 0.01). Multivariate analysis revealed that NO resistance occurred significantly more frequently with ACS than with SAP (odds ratio [OR] 2.3:1), and was less common among patients treated with perhexiline (OR 0.3:1) or statins (OR 0.45:1). Therapy with other antianginal drugs, extent of CAD, intensity of angina and coronary risk factors were not associated with variability in platelet responsiveness to NO donor. CONCLUSIONS: Patients with symptomatic ischemic heart disease, especially ACS, exhibit increased platelet aggregability and decreased platelet responsiveness to the antiaggregatory effects of NO donors. The extent of NO resistance in platelets is not correlated with coronary risk factors. Pharmacotherapy with perhexiline and/or statins may improve platelet responsiveness to NO.

Inhibition of long-chain fatty acid metabolism does not affect platelet aggregation responses.

A number of anti-anginal agents (perhexiline, amiodarone, trimetazidine) have been shown to inhibit myocardial carnitine palmitoyltransferase-1, which controls access of long-chain fatty acids to mitochondrial sites of beta-oxidation. In view of clinical data suggesting that perhexiline improves symptomatic status in unstable angina pectoris, and the known role of mitochondrial beta-oxidation in platelet metabolism, we compared the platelet carnitine palmitoyltransferase-1 inhibitory and putative anti-aggregatory effects of perhexiline, amiodarone and trimetazidine with those of specific carnitine palmitoyltransferase-1 inhibitors: etomoxir and hydroxyphenylglyoxylate in both normal subjects and patients with stable angina. All of the compounds examined inhibited platelet carnitine palmitoyltransferase-1 activity; rank order of potency etomoxir > malonyl-CoA > hydroxyphenylglyoxylate > amiodarone > or = perhexiline > trimetazidine. However, only perhexiline, amiodarone and trimetazidine inhibited platelet aggregation. We conclude that (a) the carnitine palmitoyltransferase-1 inhibitors perhexiline, amiodarone and trimetazidine exert significant anti-aggregatory effects which may be therapeutically relevant and, (b) these effects are independent of carnitine palmitoyltransferase-1 inhibition.

Multiple agonist induction of aggregation: an approach to examine anti-aggregating effects in vitro.

We tested the hypothesis that utilisation of multiple agonists in physiological concentrations is more appropriate for platelet aggregation in vitro than a single agonist in high concentration. Utilising impedance aggregometry in whole human blood (normal subjects and patients with coronary artery disease) we observed potentiation of pro-aggregatory effects of ADP by the combination of adrenaline, serotonin and thrombin in subthreshold concentrations (multiple agonist approach). In blood samples from the patients, verapamil (Ver, an L-type Ca(2+) channel blocker), nitroglycerine (NTG, a stimulator of cGMP formation) and prostaglandin E(1) (PGE(1) a stimulator of CAMP formation) inhibited platelet aggregation in vitro. With NTG and PGE(1) there was increased sensitivity to multiple agonists in comparison with ADP alone. For example, inhibition of aggregation with 10(-4)M NTG increased from 37 ± 5% with ADP alone to 86 ± 13% (P < 0.01) with multiple agonists. Threshold effects of NTG were seen at 10(-6) M with ADP alone and 10(-7) M with multiple agonists; whilst threshold for PGE(1) was reduced from 10(-10) to 10(-11) M. However, responses to Ver were unchanged by multiple agonists, demonstrating that the potentiation of anti-aggregating effects utilising the multiple agonist approach is not a non-specific phenomenon. The ability of multiple agonists to enhance the anti-aggregating effects of NTG and PGE(1) provides an in vitro experimental method mimicking the in vivo situation.