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European Journal of Heart Failure 2004 6(1):47-54; doi:10.1016/S1388-9842(03)00038-2
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© 2004 European Society of Cardiology

Determinants of platelet responsiveness to nitric oxide in patients with chronic heart failure

R.A. Andersona,*, G.R. Ellisb, Y.Y. Chirkovc, A.S. Holmesc, N. Paynea, D.J. Blackmana, S.K. Jacksona, M.J. Lewisa, J.D. Horowitzc and M.P. Frenneauxa

a Wales Heart Research Institute University of Wales College of Medicine, Cardiff, Wales, UK
b Department of Cardiology Royal Glamorgan Hospital, Llantrisant, Rhondda Cynon Taf, Wales, UK
c Department of Cardiology Queen Elizabeth Hospital, University of Adelaide, Adelaide, Australia

* Corresponding author. Wales Heart Research Institute, Health Park, Cardiff, Wales, UK CF14 4XN. Tel.: +44-02920-618625; fax.: +44-02920-743739. E-mail address: raanderson{at}ukgateway.net


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Congestive heart failure (CHF) is associated with oxidative stress. Platelet responsiveness to nitric oxide (NO) donors, are impaired in patients with angina pectoris, possibly by increasing oxidative stress. We investigated the occurrence of platelet resistance to NO in patients, with ischaemic or non-ischaemic cardiomyopathy compared with normal subjects. Anti-aggregatory effects of sodium nitroprusside (SNP), oxidative stress and whole blood superoxide anion content were determined, with correlates of responsiveness to SNP. Inhibition of platelet aggregation by SNP was 65.4±3.55% in controls and 59.3±4.1% in CHF (P=ns) despite increased oxidative stress and post-aggregation O2– in CHF patients. However, subsets of CHF patients have NO-resistant platelets: this is associated with increasing age and/or increased oxidative stress (both p<0.05).

Key Words: Heart failure • Platelets • Free radicals

Received June 19, 2002; Revised November 11, 2002; Accepted January 23, 2003


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Chronic congestive heart failure (CHF), whether ischaemic or non-ischaemic in origin, is frequently associated with disturbances of vasomotor homeostasis including the phenomenon of ‘impaired endothelial function’ [1,2]. While, the occurrence of endothelial dysfunction in the presence of a variety of coronary risk factors [3,4], and that of symptomatic or asymptomatic coronary atheromatous disease [5] has been documented extensively, pathogenetic mechanisms underlying the conditions are incompletely understood. It has been proposed widely that increased oxidative stress may play a role, perhaps via accelerated clearance of endothelial-derived nitric oxide (NO) by superoxide (O2–) anion, and/or a switch from NO towards O2– generation by nitric oxide synthase [6]. The consequences of endothelial dysfunction at a vascular level include increased vasomotor tone and ‘paradoxical’ vasoconstrictor responses to acetylcholine, bradykinin, thrombin and catecholamines.

A number of recent investigations have demonstrated that factors associated with endothelial dysfunction also may induce diminution of tissue responsiveness to NO, both at vascular [7–9] and platelet levels [10,11]. This has led to the developing concept that, endothelial dysfunction represents one component of a multi-tissue disorder of vascular homeostasis, in which smooth muscle and platelet dysfunction are equally important as abnormal endothelial physiology [7,8,11]. Furthermore, there are increasing evidences of correlation between the extent of endothelial and smooth muscle dysfunctions in both coronary [9] and peripheral [7] vascular beds, raising the possibility of common pathogenetic mechanisms. Furthermore, resistance to anti-aggregatory effects of NO donors in platelets appears to be engendered by reversible inactivation of soluble guanylate cyclase together with ‘scavenging’ of NO by O2– [11], mechanisms potentially applicable to smooth muscle dysfunction.

To date, endothelial dysfunction in patients with CHF has been examined primarily as an isolated phenomenon, linked to oxidative stress [12]. There are some evidences for the occurrence of de novo resistance to hemodynamic effects of NO donors in a minority of patients with CHF [13]. On the other hand, platelet responsiveness to NO has not previously been examined in such patients. Therefore, the objectives of the current study was: (1) to compare responses to the NO donor sodium nitroprusside (SNP) in platelets from subjects with stable CHF with those from normal subjects, and (2) to identify correlates of platelet hypo-responsiveness to NO. Factors to be examined as possible modulators of platelet responsiveness included age, severity of CHF, treatment modalities and indices of oxidative stress.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Subjects
Studies were performed in the following groups:

  1. Normal subjects (n=43, 22 men and 21 women, aged: 23–76, mean: 40.8±14.2 years), who were not taking medications known to affect platelet aggregation or antioxidants.
  2. Patients with stable symptomatic CHF (n=56), due, either to dilated cardiomyopathy (DCM), (n=28) or to ischaemic cardiomyopathy (ICM), (n=28).

Subjects with CHF (NYHA Class II–IV) had impaired systolic left ventricular function (LVEF<40%) confirmed by echocardiography or radionuclide ventriculography techniques. The patients were maintained on stable medical therapy during the study period (see Table 1). Therapies included diuretics and ACE inhibitors in most cases, digoxin in 32% and only 13% of patients were receiving β-adrenoceptor antagonists. Patients taking antioxidant preparations and/or those with a concurrent medical illness were excluded.


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  		Table 1 Functional severity and therapy of CHF subjects

 
Exclusion of ischaemic heart disease as the substrate for CHF in patients with DCM was based on coronary angiographic findings; all patients with ICM had documented myocardial infarction and/or angiographic evidence of significant coronary disease. The local research ethics committee's approved the study, and each subject provided written informed consent.

2.1.1. Blood sampling and preparation of platelets
All measurements of oxidative stress and platelet reactivity were assessed in the fasting state. Precautions were taken to minimise the potential for activation of the clotting system or platelets during blood sampling. Venous blood was collected from the antecubital fossa in plastic tubes containing 1:10 volume of acid citrate anticoagulant; acidified citrate was utilised in order to minimise deterioration of platelet function during experiments [14].

2.1.2. Platelet aggregation studies
Aggregation in whole blood was examined utilising a whole blood impedance aggregometer (Model 560, ChronoLog Corporation), as previously described [11]. Tests were performed at 37 °C and stirring speed of 900 rpm. Samples of blood were diluted two-fold with normal saline (final volume 1 ml) and pre-warmed for 5 min at 37 °C. Aggregation was induced with adenosine 5'-diphosphate (ADP) (1 µmol/l) and responses monitored continually for 7 min, responses were recorded as electrical impedance in Ohms. Inhibition of aggregation was assessed with the exogenous NO-donor, SNP (final concentration 10 µmol/l), added to samples 1 min before ADP. The duration of incubations were estimated as those optimal in preliminary experiments. In control tests, physiological saline was added in appropriate volumes. Inhibition of aggregation was evaluated as a percentage comparing the extent of maximal aggregation in the presence and absence of SNP.

2.2. Measurements of oxidative stress
2.2.1. Venous lipid-derived free radicals
We used the technique of ex vivo spin trapping to investigate the formation of secondary free radicals in venous blood [15–18] ex vivo [19], as previously described. Briefly lipid derived free radicals were measured ex vivo in venous blood, in the fasting state. Blood was taken directly into a vacuum-sealed foil-covered glass tube (to prevent photolytic degradation of the spin trap), containing 1 ml of the spin trap, {alpha}-phenyl N-tert-butyl nitrone (PBN) (0.125 mol/l). Following centrifugation at 2000 rpm for 5 min the PBN adduct was extracted twice from the plasma supernatant with equal volumes of toluene, dried under nitrogen gas and reconstituted in 100 ml of degassed chloroform. Electron paramagnetic resonance (EPR) spectra were recorded on a Varian E104 spectrometer operating at 9.1 GHz at 10 mW powers, 1 Gauss modulation, 0.25 s time constant and 100 G scan range. EPR spectral parameters were obtained from data acquisition and processing using in-house EPR computational software. EPR spectral peak heights were taken as a good correlation of spin-adduct concentration after confirmation of peak-to-peak line width conformity and double integration on selected samples. The separation between sets of peaks in the EPR spectrum (coupling constant) was used to identify the lipid radical species trapped. These were identified as carbonyl (L–) and alkoxyl (LO–) free radicals.

2.2.2. Thiobarbituric acid-reactive substances (TBARS)
The plasma level of TBARS, a second indicator of lipid peroxides in plasma, was also determined using a spectrophotometric assay (Oxis) [20]. These results were expressed as µmol/l and gives composite values for malonaldehyde (MDA) and 4-hydroxyalkenals (4-HNE) combined. The coefficient of variability for replicate estimates of TBARS was 7%, (n=20).

2.2.3. Whole blood superoxide (O2–) assay
In a subset of patients (n=19) and normals (n=7), the detection of O2– in whole blood was performed using a lucigenin-enhanced chemiluminescence technique, as previously described [11,22]. Blood samples were diluted two-fold with normal saline and pre-warmed for 5 min at 37 °C before the addition of lucigenin (12.5 mol/l). Chemiluminescence was measured using a photoluminometer component of a dual-channel lumi-aggregometer (Model 560, Chrono-log) equipped with a computer interface system. Chemiluminescence intensity was expressed in millivolts. O2– detection specificity was checked using superoxide dismutase (SOD), addition of which instantly cancelled the lucigenin signal. Chemiluminescence was estimated both prior to induction of aggregation and at peak aggregation.

2.3. Effects of treatment with ACE inhibitors
After analysis of results from the main patient cohort, further experiments were performed in order to detect an interaction between therapy with ACE inhibitors and platelet function; virtually all of the main cohort of patients were receiving ACE inhibitors or angiotensin receptor antagonists.

In 10 consecutive patients with stable CHF (NYHA Class II–IV), not previously treated either with ACE inhibitors or angiotensin receptor antagonists, the effects of therapy with perindopril (2–4 mg/day for 3.3±2.7 days) were examined. Platelet aggregation studies were performed with simultaneous determination of whole blood superoxide content.

Furthermore, in the view of previous data linking platelet resistance to NO with impaired function of platelet soluble guanylate cyclase [11], in four patients we examined platelet responsiveness to the cGMP-elevating effects of SNP. Intraplatelet cGMP content was assayed after incubation of platelet-rich plasma at 37 °C with SNP (10 µmol/l) for 1 min as previously described; these studies were repeated after initiation of therapy with perindopril.

2.4. Data analysis
The principal hypothesis was tested via comparison of SNP responses for normal subjects and those with CHF. Differences between normal subjects and patients with CHF, where data that was normally distributed, were examined via non-paired t-test; otherwise, comparison utilised a non-paired Wilcoxon test. Similar methodology was utilised to compare patients with ICM and DCM. Comparison of categorical variables utilised Chi-square test with Yates continuity correction or Fisher's exact test.

Evaluation of possible correlates of platelet responsiveness to SNP in patients with CHF were performed via stepwise multiple regression, utilizing the following parameters as possible correlates: age, gender, aetiology of CHF, NYHA functional class, diabetes mellitus, oxidative stress as measured via TBARS and EPR spectroscopy, long-acting nitrate therapy and β-adrenoceptor antagonist therapy. In the view of possible interdependence of TBARS and EPR spectroscopy data, the model was also evaluated with one of these parameters excluded in turn. Results were expressed as mean±SEM unless otherwise stated.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Patients/normal subjects’ characteristics
Comparisons between patients with CHF and normal subjects (Table 1) indicated that, there was no significant difference in gender distribution (P=0.14), but that patients were older than normals (P<0.001). Patients with ICM and DCM were similar with regard to gender distribution, but ICM patients were older (P<0.01). NYHA functional class and proportion of diabetic patients did not differ; however, as regards therapy, aspirin and perhexilene use were greater in patientswith ICM.

3.2. Platelet reactivity
Platelet reactivity to 1 µM, ADP did not vary significantly between CHF and normal subjects (Table 2). There was no significant inter-gender difference in ADP responses, and while ADP responses tended to be greater in ICM than DCM, this difference was not statistically significant.


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  		Table 2 Platelet aggregation (in Ohms) in response to 1 µmol/l ADP in whole blood

 
Mean inhibition of platelet aggregation by SNP was 65.4±3.5% in normal controls and 59.3±4.1% in CHF (P=ns). There was no significant difference between SNP responses in ICM (55.9±5.7%) and DCM (62.6±5.9%) (Table 3). Individual responses to SNP were normally distributed in normal subjects, but in patients with DCM, distribution was skewed (Fig. 1) with data suggesting a subgroup of CHF patients with reduced SNP responses.


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  		Table 3 Inhibition of platelet aggregation by 10 µmol/l SNP (% of control)

 


Figure 1
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  		Fig. 1 Relationships of SNP responses and markers of oxidative stress in each Groups: (a) DCM, (b) ICM, (c) Controls.

 
3.3. Measures of oxidative stress
Two indices of plasma oxidative stress were used in this study, they were: lipid derived free radicals directly sampled from venous blood, which gives a snapshot of oxidative stress, and longer lived markers of lipid peroxidation, (TBARS) as composites of background oxidative stress. Analysis of the EPR spectra from spin-trapped radicals derived from venous blood samples, suggested that the radicals trapped were alkoxyl radicals (coupling constants aN=13.9 Gauss, aβH=2.2 Gauss) and carbonyl radicals (aN=14.1 Gauss; aβH=4.0 Gauss). These assignments, which agree with our previous studies [15,17,19], suggest that these radicals were derived from decomposition of lipid hydroperoxides in the extracellular compartment. Detection of secondarily formed lipid free radicals strongly supports the presence of continuing peroxidative damage in vivo. Lipid derived free radicals and markers of lipid peroxidation were elevated in both CHF groups compared to healthy controls (Table 4), but did not differ between ICM and DCM aetiology.


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  		Table 4 Markers of oxidative stress

 
3.3.1. EPR spectroscopy and lipid peroxidation products
Results are summarised in Table 4. Concentrations of secondary free radicals were significantly elevated (P<0.001) in CHF vs. normals. There was no significant difference between ICM and DCM patients.

Concentrations of TBARS were also elevated in CHF patients (P<0.001) without significant difference between ICM and DCM. On univariate analysis, concentrations of TBARS (r=–0.53, P<0.01) but not of secondary free radicals (r=–0.34, P=0.11) were significant inverse correlates of SNP response (Fig. 2).


Figure 2
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  		Fig. 2 Relationships of SNP responses and markers of oxidative stress in CHF vs. TBARS: {square} – dilated cardiomyopathy, bullet – ischaemic cardiomyopathy.

 
3.3.2. Lucigenin-enhanced chemiluminescence
Mean basal and post-aggregation chemiluminescence intensity was 8.5±1.0 and 40.8±11.2 mV, respectively, in normal subjects and 11.1±6.3 (P=0.17 vs. normals) and 74±42 (P=0.04 vs. normals) in CHF patients. There was no significant difference in either basal or post-aggregation chemiluminescence between patients with ICM and DCM.

3.4. Relationship between SNP response and markers of oxidative stress
On univariate analysis, there tended to be an inverse relationship between SNP response in the CHF patients and both secondary free radicals (r=–0.340, P=0.105) and TBARS (Fig. 2; r=–0.531, P<0.01) concentrations. This was not the case in controls.

3.5. Determinants of SNP response: multiple regression
Multivariate analysis revealed that the only significant correlates of SNP response were increasing age (Fig. 3; t=–2.138; P=0.05) and TBARS concentration (t=–2.382; P=0.028), only in the CHF group. If TBARS were excluded from the regression model, secondary free radicals did not become a significant correlate irrespective of initial inclusion/exclusion. Secondary free radical concentration, and those of TBARS were correlated with each other (r=0.459; P=0.028).


Figure 3
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  		Fig. 3 Relationships of SNP responses and markers of oxidative stress in CHF vs. Age: {square} – dilated cardiomyopathy, bullet – ischaemic cardiomyopathy.

 
3.6. Effects of treatment with ACE inhibitors
Prior to initiation of therapy with perindopril, platelet aggregation in response to ADP was 10.6±5.9 Ohms (p=ns vs. entire CHF group). However, inhibition of aggregation by 10 µm/l was 36±7% (P<0.01 vs. pre-perindopril; Fig. 4). Initiation of perindopril therapy was associated with a decrease in whole blood O2– content from 20±4 to 16±2 mV (P=ns). Furthermore, cGMP formation in platelets on incubation with SNP increased from 122±12 to 170±15% (P=0.08).


Figure 4
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  		Fig. 4 Effects of treatment with ACE inhibitors on SNP responses. § – P<0.05 compared to baseline.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Finding that platelet responsiveness to NO donors was relatively normal in the majority of patients with CHF, even if the CHF were of ischaemic origin, was unexpected. We therefore, considered the possibility that therapy with ACE inhibitors or angiotensin antagonists, might have contributed to this finding. Hence, an additional cohort of patients was studied, before and after initiation of treatment with perindopril. These additional experiments demonstrated that platelets from CHF patients, not treated with ACE inhibitors, are resistant to the anti-aggregatory effects of NO donors. Thus, CHF can be classified, together with acute and chronic ischaemia [9,21], as a disorder associated with platelet resistances to NO. Furthermore, therapy with perindopril induced marked potentiation of platelet responses, to the extent that these were within the ‘normal’ range. Thus, ACE inhibitor therapy represents the major factor responsible for limitation of platelet NO resistance in CHF patients.

The additional experiments provided some information about the mechanism(s) of interaction between ACE inhibitors and platelet responsiveness to NO. As ACE inhibitor therapy may reduce activity of the p22 phox component of NADPH oxidase [23], it was possible that this might result in the reduction, in whole blood O2– content. However, this change did not reach statistical significance. Platelet NO resistance has also been associated with inactivation of soluble guanylate cyclase [9]: hence, experiments exploring cGMP generation were performed. These suggested that restoration of soluble guanylate cyclase activity might be the major mechanism of the beneficial effect of ACE inhibitor therapy.

Numerous previous studies have established correlations between the occurrence of oxidative stress and the presence of endothelial dysfunction, without demonstrating conclusively that this was a cause and effect relationship [22]. The current investigation extends previous observations in patients with chronic CHF, confirming the presence of oxidative stress on the basis of elevated plasma concentrations of secondary free radicals and of TBARS relative to those in normal subjects. Similarly, platelet concentrations of O2–, as measured by lucigenin-enhanced chemiluminescence, were elevated post aggregation in CHF patients relative to normal subjects.

However, the study demonstrated that, as a group, platelets from treated patients with CHF are not hyporesponsive to NO. This applied irrespective of ischaemic vs. non-ischaemic aetiology of CHF. Nevertheless, there was considerable variability within the CHF group, and multivariate analysis identified advanced age and elevated plasma concentrations of TBARS as significant correlates of resistance to NO in individual patients.

Our observations in this group of patients with stable CHF are consistent with the hypothesis, that impaired platelet responsiveness to NO, which may be induced by oxidative stress. The implication however, is of heterogeneity of oxidative stress, with mean extent of O2– generation less marked than that associated with stable angina pectoris [11], irrespective of whether CHF was of ischaemic origin or not. It is possible that, treatment of CHF may have limited oxidative stress in this group of patients, especially in view of the known effects of ACE inhibitors on NAD(P)H oxidase [23,24]. Since, virtually the entire patients group were being treated with ACE inhibitors, such an effect could not be explored in the current study.

The identification of increasing age as a correlate of resistance to anti-aggregatory effects of NO on multivariate analysis is consistent with these hypothesis that factors linked with oxidative stress may predispose towards NO resistance; it remains possible that the association of age with NO resistance is paraphenomenological. Nevertheless, no other coronary risk factors were correlated with impaired platelet responsiveness to NO.

The major limitation of the current study is that platelet functions was not compared with reactivity of vascular smooth muscle in response to endothelium-dependent or endothelium-independent vasodilator agents. It therefore, remains to be established whether CHF is associated with vascular NO resistance or its analogue of ‘nitrate resistance’ as suggested by the original observations of Armstrong et al. [13], or whether endothelial dysfunction is predictive of platelet dysfunction in such patients.

The finding in the current study that some patients with stable, treated CHF exhibit hyporesponsiveness to NO at the level of platelet aggregation is potentially relevant to clinical outcomes in such patients. The extent of impairment of platelet production of NO has been shown to predict the development of acute coronary syndromes [24], while in patients with symptomatic myocardial ischaemia, the extent of impairment of platelet responsiveness to NO was independently associated with the presence of acute coronary syndromes [25].

Furthermore, impairment of coronary vascular responsiveness to nitroglycerin in patients with stable angina pectoris was independently predictive of increased risk of thrombotic events during follow-up [26]. As acute myocardial infarction represents, a very common means of demise in patients with CHF treated with ACE inhibitors [27], identification of individuals at particular risk may prove useful.

Finally, the current data may provide some indication of possible means of optimising platelet responsiveness to NO (and to NO donors) in patients with CHF. The results raise the possibility that amelioration of oxidative stress may prove beneficial. Future prospective studies evaluating the therapeutic effects of agents exerting antioxidant effects should address possible correlations between normalisation of platelet function and outcomes.


   Acknowledgements
 
This work was supported by the British Heart Foundation (BHF). RAA and GRE are BHF Junior Research Fellows, MPF holds the BHF Sir Thomas Lewis Chair of Cardiology. The authors wish to thank Mr. Peter Gapper and Catherine Mumford for their technical support.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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