Abstract

Aims

Heart failure is associated with deficient endothelial nitric oxide (NO) production as well as increased oxidative stress and accelerated NO degradation. The aim of this study was to evaluate platelet NO biosynthesis and superoxide anion (O2) production in patients with heart failure.

Methods and results

In platelets from patients with heart failure due to idiopathic dilated cardiomyopathy (n= 16) and healthy control subjects (n= 23), NO synthase (NOS) activity was evaluated by L-[3H]-arginine to l-[3H]-citrulline conversion, cGMP was determined by radioimmunoassay, vasodilator-stimulated phosphoprotein (VASP: total and serine-239-phosphorylated) was assessed by western blotting, and O2 production and O2 scavenging capacity were measured by pholasin-enhanced chemiluminescence. In platelets from patients with heart failure, basal NOS activity was higher than in those from controls; furthermore, whereas platelet NOS activity increased as expected in response to albuterol or collagen in controls, no increase occurred in platelets from heart failure subjects. Despite this, basal intraplatelet NO-attributable cGMP was lower in heart failure than in control subjects, as was serine-239 phosphorylation of VASP, suggesting a decrease in bioactive NO. Platelets from heart failure subjects exhibited higher basal and collagen-stimulated O2 production and impaired O2 scavenging capacity, resulting in higher oxidative stress, consistent with the observed decrease in bioactive NO.

Conclusion

In heart failure, despite activation of NOS, platelets produce less bioactive NO, probably as a result of NO scavenging due to increased O2 production. This functional defect in the platelet l-arginine/NO/guanylyl cyclase pathway could contribute to the platelet activation observed in heart failure.

Introduction

Despite the considerable recent advances made in the treatment of heart failure, its prevalence is increasing, the quality of life of patients remains impaired and its mortality rate remains high.1 Much attention has focused on the role of endothelium-derived nitric oxide (NO) in this condition, since it plays an important role in regulation of vascular tone,2,3 modulation of inflammation,4 inhibition of vascular remodeling,5,6 suppression of platelet adhesion to endothelium,7 aggregation, and thrombus formation as well as leucocyte adhesion to vessel wall.8 Traditional cardiovascular risk factors such as hypercholesterolemia, smoking, diabetes, and obesity are associated with impaired bioactive NO, an important characteristic of endothelial dysfunction, and independently of these risk factors, heart failure in itself may cause endothelial dysfunction.9–11

Despite reduced bioactive NO, conditions such as diabetes and hypertension have been reported to be associated with increased endothelial NO synthase type 3 (NOS3) activity and/or expression in the vasculature, but also with increased vascular superoxide anion (O2) production.12–14 O2 can react with NO very rapidly to form peroxynitrite (ONOO), a potent oxidant and nitrating agent, thereby both reducing NO availability and increasing oxidative and nitrative stress.

Although vascular NOS3 is mainly localized to the endothelium, platelets have also been reported to possess a functional l-arginine/NO pathway and to express NOS3.15–19 In the context of heart failure, platelets may play a pivotal role in much of the morbidity and mortality associated with the condition. Patients with heart failure are at increased risk of stroke and sudden cardiac death. These thrombosis-related complications have been attributed to a prothrombotic state in heart failure, the exact cause of which is unclear.20

The pathophysiological role of NO in heart failure remains the subject of much controversy. There are contradictory reports in the literature concerning NO production in patients with heart failure, some suggesting increased production,21–23 others no change,24 and yet others reduced production.25 Very little is currently known about platelet-derived NO and O2 in patients with heart failure.

The overall aim of the present study was to investigate platelet NO signalling in patients with heart failure. This was achieved by determining platelet NOS activity (to assess rate of NO biosynthesis), intraplatelet cyclic guanosine-3′,5′-monophosphate (cGMP, an index of bioactive NO), and platelet O2 production and O2 scavenging capacity.

Methods

Subjects

The study conforms with the Declaration of Helsinki. The King's College Hospital Research Ethics Committee granted approval for the study and all subjects gave written informed consent. Sixteen patients with heart failure were recruited from the heart failure clinics at St. Thomas’ Hospital and King's College Hospital, London, UK. All of these had heart failure diagnosed on clinical and echocardiographic criteria, with clinical features of heart failure coupled with a measured ejection fraction <40% on echocardiography, and were free of significant coronary atherosclerosis as determined by coronary angiography. Based on exclusion of other known causes of heart failure, the diagnosis in all cases was of idiopathic dilated cardiomyopathy. Other exclusion criteria were a history of hypertension, current blood pressure >140 mm Hg systolic and/or >90 mm Hg diastolic, evidence of other cardiovascular disease, use of recreational or other non-prescribed drugs, and the presence of any significant co-morbidities. Healthy control subjects of similar age and sex were recruited from the database of healthy volunteers held by the Department of Clinical Pharmacology, King's College London, London, UK. They were free of any cardiovascular risk factors, were not on any regular medications, and in particular had taken no medication including anti-platelet drugs in the 7 days prior to the study. Subject characteristics are shown in Table 1. Patients with heart failure were on a variety of medications, which are shown in Table 2.

Table 1

Subject characteristics

 Heart failure subjects (n = 16) Controls (n = 23) 
Age (years) 51.3 ± 4.1 50.9 ± 3.9 
Males/females 12/4 17/6 
NYHAI/II/III/IV 4/6/4/2 – 
Caucasian/African/Asian 12/3/1 13/4/6 
Pulse (beats/min) 70 ± 3 68 ± 5 
Systolic blood pressure (mmHg) 127 ± 3 130 ± 4 
Diastolic blood pressure (mmHg) 74 ± 2 77 ± 2 
BMI (kg/m230.5 ± 3.0* 26.4 ± 4.1 
Haemoglobin (g/dL) 13.8 ± 0.5 14 ± 0.3 
WCC (×109/L) 6.7 ± 0.7 6.1 ± 0.3 
Platelets (×109/L) 240.3 ± 20.8 260.1 ± 9.8 
MPV (fl) 7.3 ± 0.2* 6.8 ± 0.1 
Sodium (mmol/L) 139.9 ± 1.2 139.1 ± 0.5 
Potassium (mmol/L) 4.3 ± 0.1 4.1 ± 0.1 
Urea (mmol/L) 7.8 ± 1.1* 4.2 ± 0.3 
Creatinine (µmol/L) 97.4 ± 7.0* 79.8 ± 3.2 
eGFR (mL/min/1.73 m272.0 ± 5.0* 90.0 ± 5.0 
Total cholesterol (mmol/L) 4.1 ± 0.2* 5.5 ± 0.2 
LDL-cholesterol (mmol/L) 2.4 ± 0.2* 3.2 ± 0.2 
HDL-cholesterol (mmol/L) 1.1 ± 0.1 1.3 ± 0.1 
Triglycerides (mmol/L) 1.0 ± 0.1* 1.7 ± 0.2 
Glucose (mmol/L) 5.2 ± 0.2 5.4 ± 0.3 
HbA1C (%) 6.1 ± 0.3* 5.7 ± 0.2 
Homocysteine (µmol/L) 21.7 ± 3.3 18.9 ± 1.6 
hs-CRP (mg/L) 3.5 ± 1.0 3.1 ± 1.0 
vWF:Ag (IU/dL) 214.8 ± 14.6* 165.0 ± 26.0 
Beta-thromboglobulin (ng/mL) 32.1 ± 7.6* 18.9 ± 6.5 
NT-proBNP (ng/L) 3275.0 ± 1621.1* 58.3 ± 9.4 
Echocardiographic parameters   
 LVEF (%) 31.2 ± 2.6 – 
 LVESV (mL) 128.2 ± 14.3 – 
 LVEDV (mL) 169.2 ± 15.0 – 
 LVIDs (cm) 4.9 ± 0.2 – 
 LVIDd (cm) 5.7 ± 0.2 – 
 Heart failure subjects (n = 16) Controls (n = 23) 
Age (years) 51.3 ± 4.1 50.9 ± 3.9 
Males/females 12/4 17/6 
NYHAI/II/III/IV 4/6/4/2 – 
Caucasian/African/Asian 12/3/1 13/4/6 
Pulse (beats/min) 70 ± 3 68 ± 5 
Systolic blood pressure (mmHg) 127 ± 3 130 ± 4 
Diastolic blood pressure (mmHg) 74 ± 2 77 ± 2 
BMI (kg/m230.5 ± 3.0* 26.4 ± 4.1 
Haemoglobin (g/dL) 13.8 ± 0.5 14 ± 0.3 
WCC (×109/L) 6.7 ± 0.7 6.1 ± 0.3 
Platelets (×109/L) 240.3 ± 20.8 260.1 ± 9.8 
MPV (fl) 7.3 ± 0.2* 6.8 ± 0.1 
Sodium (mmol/L) 139.9 ± 1.2 139.1 ± 0.5 
Potassium (mmol/L) 4.3 ± 0.1 4.1 ± 0.1 
Urea (mmol/L) 7.8 ± 1.1* 4.2 ± 0.3 
Creatinine (µmol/L) 97.4 ± 7.0* 79.8 ± 3.2 
eGFR (mL/min/1.73 m272.0 ± 5.0* 90.0 ± 5.0 
Total cholesterol (mmol/L) 4.1 ± 0.2* 5.5 ± 0.2 
LDL-cholesterol (mmol/L) 2.4 ± 0.2* 3.2 ± 0.2 
HDL-cholesterol (mmol/L) 1.1 ± 0.1 1.3 ± 0.1 
Triglycerides (mmol/L) 1.0 ± 0.1* 1.7 ± 0.2 
Glucose (mmol/L) 5.2 ± 0.2 5.4 ± 0.3 
HbA1C (%) 6.1 ± 0.3* 5.7 ± 0.2 
Homocysteine (µmol/L) 21.7 ± 3.3 18.9 ± 1.6 
hs-CRP (mg/L) 3.5 ± 1.0 3.1 ± 1.0 
vWF:Ag (IU/dL) 214.8 ± 14.6* 165.0 ± 26.0 
Beta-thromboglobulin (ng/mL) 32.1 ± 7.6* 18.9 ± 6.5 
NT-proBNP (ng/L) 3275.0 ± 1621.1* 58.3 ± 9.4 
Echocardiographic parameters   
 LVEF (%) 31.2 ± 2.6 – 
 LVESV (mL) 128.2 ± 14.3 – 
 LVEDV (mL) 169.2 ± 15.0 – 
 LVIDs (cm) 4.9 ± 0.2 – 
 LVIDd (cm) 5.7 ± 0.2 – 

BMI, body mass index; WCC, white cell count; MPV, mean platelet volume; eGFR, estimated glomerular filtration rate; vWF: Ag, von Willebrand Factor antigen; LDL, low-density lipoprotein; HDL, high-density lipoprotein; HbA1c, glycated haemoglobin; hs-CRP, high-sensitivity C-reactive protein; NYHA, New York Heart Association class; NT-proBNP, N-terminal pro-B-type natriuretic peptide; LVEF, left ventricular ejection fraction; LVESV, left ventricular end-systolic volume; LVEDV, left ventricular end-diastolic volume; LVIDs, left ventricular internal dimension in systole; LVIDd, left ventricular internal dimension in diastole.

*P < 0.05 vs. controls.

Table 2

Medications taken by heart failure subjects (n= 16)

Medication
 
β-Blocker 15 
Calcium channel blocker 
Angiotensin II receptor blocker 
Angiotensin-converting enzyme inhibitor 14 
Statin 
Aspirin 
Clopidogrel 
Warfarin 10 
Furosemide 13 
Spironolactone 
Digoxin 
Medication
 
β-Blocker 15 
Calcium channel blocker 
Angiotensin II receptor blocker 
Angiotensin-converting enzyme inhibitor 14 
Statin 
Aspirin 
Clopidogrel 
Warfarin 10 
Furosemide 13 
Spironolactone 
Digoxin 

Blood was drawn in the fasting state for determination of full blood count, biochemical profile, lipid profile, glucose, glycated haemoglobin (HbA1C), high-sensitivity C-reactive protein (CRP), and homocysteine (Table 1). These measurements were carried out by the Departments of Hematology and Clinical Chemistry, St. Thomas’ Hospital. Additionally, von Willebrand Factor antigen (vWF:Ag) was measured by the Department of Hemostasis and Thrombosis, St Thomas’ Hospital.

Preparation of platelets

Subjects attended the Department of Clinical Pharmacology in the morning, having fasted overnight, and refrained from alcohol and caffeine since the previous evening. One hundred millilitres of blood was drawn from a large antecubital vein using a 19G butterfly® needle, collected into tri-sodium citrate (0.38% final concentration), and centrifuged (200 g, 10 min, room temperature) to obtain platelet-rich plasma (PRP). Gel-filtered platelets were obtained by eluting PRP through a Sepharose gel column as previously described.26

Platelet NOS and cGMP measurement

NOS activity was measured from the rate of conversion of l-[3H]-arginine to l-[3H]-citrulline, and cGMP was determined by radioimmunoassay, as previously described.26 cGMP assays were all done in the presence of 3-isobutyl-1-methylxanthine 500 µmol/L as described.26 NOS activity was measured as the difference in values in the absence and presence of the NOS inhibitor NG-monomethyl-l-arginine (l-NMMA, 100 µmol/L). Similarly, the amount of cGMP produced in response to NO (‘NO-attributable cGMP’) was determined as the difference in measured intraplatelet cGMP in the absence and presence of l-NMMA (100 µmol/L).

Measurement of platelet O2 production and antioxidant capacity

Platelet-derived O2 was measured by pholasin-enhanced chemiluminescence, as described in detail in the Supplementary material online.

Measurement of serum arginine, symmetric and asymmetric dimethylarginine levels, and arginase activity

Serum levels of arginine, symmetric dimethylarginine (SDMA) and asymmetric dimethylarginine (ADMA) were measured by high-performance liquid chromatography following extraction using a solid-phase cation exchange column, as previously described.27

Serum arginase activity was determined as described by Sopi et al.28 Briefly, 50 µl serum was pre-activated by addition of 10 mmol/L MnCl2 followed by heating at 55°C in a total volume of 100 µl. An equal volume of 250 mmol/L l-arginine was added to samples (yielding a final concentration of 125 mmol/L l-arginine), and incubated for 30 min at 55°C. The arginase reaction was stopped by addition of 1 ml diacetyl monoxime/acid solution prepared as follows: 1 ml diacetyl monoxime reagent (3% in absolute ethanol) was added to 29 ml of an acid mixture containing H2SO4, H3PO4, and H2O (1:3:7 v/v). Samples were heated at 100°C for 30 min and maintained in the dark for 10 min, before the absorbance of the urea (formed from arginine by the action of arginase) was measured spectrophotometrically at 490 nm and the concentration determined from a standard urea concentration calibration curve. One unit (U) of enzymatic activity is defined as the amount of enzyme that catalyzes the formation of 1 µmol urea/min. Data were expressed in U/L serum.

Determination of serine-239 phosphorylation of vasodilator-stimulated phosphoprotein

The degree of phosphorylation of vasodilator-stimulated phosphoprotein (VASP) at serine-239 was measured in platelet lysates by western blotting, as previously described.29

Statistical analysis

All data were expressed as mean ± SEM. Since data demonstrated a non-Gaussian distribution, within-group and between-group changes were analyzed by the non-parametric Friedman and Kruskal–Wallis tests, respectively, with Dunn's post-hoc testing where significance was found. In all cases, P< 0.05 (two-tailed) was considered to be significant. Data analysis was performed using GraphPad Prism version 5.

Results

Subject characteristics

Patients and healthy controls were well matched in terms of age, sex, and race distribution. Heart failure subjects had higher body mass index in comparison to healthy controls. Mean platelet volume (MPV) was higher in heart failure subjects in comparison to healthy controls, as were vWF:Ag, β-thromboglobulin and HbA1c (known diabetes was one of the exclusion criteria for the study). Urea and creatinine were higher, and estimated glomerular filtration rate (calculated using the abbreviated Modification of Diet in Renal Disease Study equation) was lower, in heart failure subjects, suggesting that they had impaired renal function as compared with healthy controls, as might be expected. Total and low-density lipoprotein cholesterol, as well as triglycerides, were lower in heart failure subjects in comparison to healthy controls, and this is most likely attributable to the high usage of statin therapy in these patients. As expected, N-terminal pro-B-type natriuretic peptide levels were higher in heart failure subjects than controls.

Platelet NOS activity and bioactive NO

Basal platelet NOS activity was considerably higher in heart failure subjects in comparison to healthy controls (Figure 1A). Despite this, we found intraplatelet NO-attributable cGMP to be markedly less in the former group compared with the latter: 0.7 ± 0.6 vs. 121.4 ± 35.4 fmol cGMP/108 platelets, respectively, (P < 0.05). In healthy subjects, platelet NOS activity significantly increased from baseline, upon stimulation with albuterol (10−5 mol/L) or collagen (0.8 µg/ml); although these agents are known to stimulate platelet NOS,18,26,30 in the present study the levels of platelet NOS activity reached after stimulation with either agent did not reach that found basally in subjects with heart failure (Figure 1A). In platelets from subjects with heart failure, no further increase was elicited in NOS activity in response to either albuterol or collagen. Platelet NOS activity, both basal and agonist-stimulated, and NO-attributable cGMP were not different between heart failure patients receiving or not receiving anti-platelet medications, warfarin, digoxin or statins. Platelet expression of NOS3 and of soluble guanylyl cyclase were not different between platelets from heart failure and control subjects, whilst neither NOS1 nor NOS2 expression were detectable, by western blotting (data not shown).

Figure 1

Basal and stimulated NOS activity, and serine-239 phosphorylation of VASP, in platelets from heart failure and healthy control subjects. (A) NOS activity at baseline and after stimulation with albuterol (10−5 mol/L) and collagen (0.8 µg/mL), in platelets from heart failure subjects (n= 16; open bars) and healthy controls (n= 23; solid bars). ***P < 0.001 vs. healthy controls. ##P < 0.01 vs. basal. (B) Densitometric ratio of serine-239-phospho-VASP to total VASP, in platelets from these same subjects. **P < 0.01 vs. healthy controls.

Figure 1

Basal and stimulated NOS activity, and serine-239 phosphorylation of VASP, in platelets from heart failure and healthy control subjects. (A) NOS activity at baseline and after stimulation with albuterol (10−5 mol/L) and collagen (0.8 µg/mL), in platelets from heart failure subjects (n= 16; open bars) and healthy controls (n= 23; solid bars). ***P < 0.001 vs. healthy controls. ##P < 0.01 vs. basal. (B) Densitometric ratio of serine-239-phospho-VASP to total VASP, in platelets from these same subjects. **P < 0.01 vs. healthy controls.

To confirm that the decrease in bioactive NO, as assessed by cGMP levels, in platelets from heart failure subjects gives rise to a functional consequence, we measured serine-239 phosphorylation of VASP in platelet lysates by western blotting, since phosphorylation of this residue is highly dependent on the activity of cGMP-dependent protein kinase. We found that, in platelets from heart failure subjects, serine-239 phospho-VASP was markedly reduced as compared with platelets from control subjects (Figure 1B).

Arginine levels were lower, and ADMA levels higher, in serum from heart failure as compared with control subjects (Table 3), with no difference seen in serum SDMA. Serum arginase activity was not different between the two groups (Table 3).

Table 3

Serum arginine, SDMA and ADMA levels and arginase activity

 Heart failure subjects (n= 16) Controls (n= 23) 
Arginine (µmol/L) 109 ± 7*** 139 ± 6 
SDMA (nmol/L) 590 ± 74 571 ± 25 
ADMA (nmol/l) 556 ± 17* 514 ± 9 
Arginase activity (U/L serum) 2.2 ± 0.5 2.7 ± 0.4 
 Heart failure subjects (n= 16) Controls (n= 23) 
Arginine (µmol/L) 109 ± 7*** 139 ± 6 
SDMA (nmol/L) 590 ± 74 571 ± 25 
ADMA (nmol/l) 556 ± 17* 514 ± 9 
Arginase activity (U/L serum) 2.2 ± 0.5 2.7 ± 0.4 

*,***P < 0.05 and <0.001, respectively vs. controls.

Platelet O2 production and scavenging capacity

Baseline platelet O2 production was increased in subjects with heart failure as compared with healthy controls (Figure 2). In both groups, incubation with collagen increased platelet O2 production and collagen-induced O2 production was greater in heart failure than in control subjects, when measured after 5 min of collagen co-incubation (Figure 2). This was reflected also by total platelet O2 production over 30 min after addition of collagen, as assessed by the area under the curve of luminescence vs. time over this period (97 620 ± 48 900 vs. 1 306 000 ± 674 000 (luminescence units/108 platelets) × min in healthy vs. heart failure subjects, respectively, P< 0.0001).

Figure 2

Basal and stimulated platelet O2 production in heart failure and healthy control subjects. Pholasin-enhanced chemiluminescence was measured in platelets, both at baseline and after stimulation with collagen (0.8 µg/mL); the difference in light signal in the absence and presence of Tiron was taken as an index of O2 production. Data are shown for platelets from heart failure subjects (n= 16; open bars) and healthy controls (n= 19; solid bars). *P < 0.05, **P < 0.01.

Figure 2

Basal and stimulated platelet O2 production in heart failure and healthy control subjects. Pholasin-enhanced chemiluminescence was measured in platelets, both at baseline and after stimulation with collagen (0.8 µg/mL); the difference in light signal in the absence and presence of Tiron was taken as an index of O2 production. Data are shown for platelets from heart failure subjects (n= 16; open bars) and healthy controls (n= 19; solid bars). *P < 0.05, **P < 0.01.

Addition of platelets to Na-tyrode containing HRP and pholasin resulted in a decrease in light signal. This might be expected to occur simply due to the physical presence of platelets, but additionally as a result of O2 scavenging by both enzymatic and non-enzymatic antioxidant systems present in platelets. Although a decrease in signal was found following addition of platelets from either group, the decrease in signal was attenuated with platelets from heart failure subjects as compared with those from healthy subjects (Figure 3). Since the data were normalized for number of platelets, this difference suggests a true difference in O2 scavenging capacity.

Figure 3

O2 scavenging capacity of platelets from heart failure and healthy control subjects. Pholasin-enhanced chemiluminescence was measured and the percent decrease in light signal following addition of platelets (normalized for platelet count) was taken as an index of platelet O2 scavenging capacity. Data are shown for platelets from heart failure subjects (n= 16; open bar) and healthy controls (n= 19; solid bar). ***P < 0.0005 vs. controls.

Figure 3

O2 scavenging capacity of platelets from heart failure and healthy control subjects. Pholasin-enhanced chemiluminescence was measured and the percent decrease in light signal following addition of platelets (normalized for platelet count) was taken as an index of platelet O2 scavenging capacity. Data are shown for platelets from heart failure subjects (n= 16; open bar) and healthy controls (n= 19; solid bar). ***P < 0.0005 vs. controls.

Discussion

In the present study, we examined l-arginine/NO signalling in platelets from subjects with heart failure. Platelet-derived NO inhibits several aspects of platelet function, including their aggregation31 and recruitment following aggregation.32 In a mouse model deficient in platelet-derived NO, bleeding time is reduced, underlining its importance in modulation of platelet function and thrombus formation.33 Therefore, deficient platelet NO production in heart failure may contribute to the increased thrombotic tendency seen in this condition.

Elevated MPV has previously been found to be a marker of platelet activation.34 We found that MPV was higher in heart failure subjects, consistent with platelet activation in these subjects. This is supported by the concomitant elevation of vWF:Ag and more specifically of β-thromboglobulin in this group. We also found that HbA1c was higher in subjects with heart failure, which may indicate underlying impaired glucose tolerance or insulin resistance (even though diabetic subjects were specifically excluded from this study). β-blocker therapy is now routine in the modern management of patients with heart failure, but their long-term use is associated with increased incidence of diabetes.35

We found that platelet NOS activity was higher in heart failure subjects in comparison to healthy controls, suggesting that platelet NO biosynthesis is increased. Most studies that have assessed NO production in heart failure have either measured total body NO production (not specific to the site of NO production) or assessed different vascular beds, in both human and animal models. We have studied NO production only from platelets and the literature to date has very few studies of platelet NO production in heart failure. Similar to the findings of Drexler et al. and Habib et al.,22,23 we found that, despite markedly increased platelet NO production in heart failure subjects at baseline, there was no further rise in response to stimulation by standard NOS agonists. Our findings suggest that in heart failure subjects, platelet NOS activity is already at maximum, with no further increase possible. The overall picture, therefore, is of an increase in NO biosynthesis in platelets from heart failure subjects, despite a decrease in plasma concentration of the principal substrate for NOS3 (l-arginine) and an increase in the plasma concentration of the principal endogenous inhibitor of NOS3 (ADMA), which is likely due to an increase in NOS3 activity despite unchanged levels.

On the other hand, despite elevated NOS activity, basal intraplatelet NO-attributable cGMP was reduced in subjects with heart failure, suggesting that, despite augmented NO biosynthesis, there is accelerated NO clearance in these subjects. That this decrease in bioactive NO translates to a decreased functional response was confirmed by the observation that phosphorylation of VASP on serine-239, which is highly dependent on cGMP-dependent protein kinase, and which regulates filamentous actin formation with resultant effects on cell adhesion and motility, was markedly reduced in the platelets of patients with heart failure.

One of the most important means by which NO is degraded is by reaction with O2. We therefore postulated that the apparent increase in NO clearance (and hence decrease in bioactive NO, as manifested by NO-attributable cGMP) in platelets might be explained by increased platelet O2 generation, in heart failure. We measured platelet O2 generation by pholasin-enhanced chemiluminescence in the absence and presence of Tiron. Tiron is a superoxide dismutase-mimetic and a cell membrane-permeable-specific scavenger of O236; therefore, the difference in luminescence in the absence and presence of Tiron is a specific index of O2 generation. We found that, indeed, platelets from heart failure subjects exhibit increased basal O2 production in comparison to those from healthy subjects. Upon incubation with collagen, O2 production increased in both heart failure and healthy subjects, but to a considerably greater extent in the former. Collagen was chosen as the stimulus, since platelet exposure to collagen is a major stimulus for platelet activation and thrombosis locally at sites of vascular damage.

Increasing evidence suggests that generation of reactive oxygen species (ROS), including O2, is increased in heart failure.37 ROS, and in particular O2, can directly inactivate NO and cGMP,9 the second messenger of NO. O2 reacts rapidly with NO to form peroxynitrite, which can uncouple NOS3,9 thereby resulting in a further decrease in NO production and a further increase in O2 production, setting up a vicious cycle of decreased NO and increased O2. Although the observed increase in oxidative stress is likely to explain the apparent reduction in bioactive NO in platelets from heart failure subjects in this study, we cannot exclude the possibility that the observed decrease in NO-attributable cGMP is in part caused by deficient production of cGMP (due to a reduction in activity of soluble guanylyl cyclase) and/or increased degradation of cGMP (due to increased phosphodiesterase activity). The latter possibility is unlikely, since in all our experiments cGMP measurements were performed in platelets pre-incubated with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. However, the possibility remains that soluble guanylyl cyclase activity is reduced in platelets from patients with heart failure; this in turn could conceivably arise as a result of oxidative damage to soluble guanylyl cyclase, although existing evidence suggests that soluble guanylyl cyclase oxidation, if anything, causes its activation rather than inhibition.38 Nevertheless, this possibility merits further study.

Various enzymatic and non-enzymatic sources of platelet ROS production have been characterized. It has been shown that collagen-induced platelet aggregation is associated with a burst of H2O2,39 a reactive oxygen species, which produces OH through a Fenton-type reaction.40 H2O2 is also known to activate arachidonic acid metabolism,41,42 through phospholipase A2 stimulation.40 De et al.43 have suggested that increased platelet O2 in patients with heart failure is mediated by tumour necrosis factor-α, via activation of arachidonic acid metabolism and the NADPH-oxidase pathway. Platelets exposed to anoxia-reoxygenation generate O2 and OH, which in turn activate arachidonic acid metabolism via phospholipases A2 and C, resulting in production of further O2.44 Yamagishi et al.45 have shown that hyperglycaemia potentiates collagen-induced platelet activation through mitochondrial O2 production. Our finding of increased HbA1c in heart failure subjects may therefore potentially create an environment for platelets to generate more O2 upon stimulation with collagen.

An excess of O2 may result from either increased O2 production or impairment in O2 scavenging capacity, or both, resulting in increased oxidative stress. Platelets are known to have reducing and antioxidant capacity,46,47 and to scavenge O2 when co-incubated with stimulated polymorphonuclear cells generating an oxidative burst.48 We found that platelets from heart failure subjects have impaired O2 scavenging capacity in comparison to those from healthy subjects. Impairment of antioxidant enzyme systems has been demonstrated in platelets from patients with coronary artery disease,49,50 as well as in myocardial cells in animal models of heart failure.51 The major antioxidant enzymes present in platelets are superoxide dismutase, catalase, and glutathione peroxidase.46,49,50,52 Whether the observed reduction in platelet O2 scavenging capacity results from a deficiency of one or more of these enzyme systems, or in other non-enzymatic antioxidant systems, remains to be determined.

An important limitation of this study is that all subjects with heart failure, unlike the controls, were on medications (often multiple). Indeed, 15 of 16 heart failure subjects were on β-blocker therapy, and all were either receiving an angiotensin-converting enzyme inhibitor or an angiotensin receptor blocker (and, in the case of one subject, both). Additionally, all heart failure subjects received one or more diuretics. Therefore, we cannot exclude the possibility that our results were influenced by use of these medications. It is possible, for example, that bradykinin release in response to angiotensin-converting enzyme inhibition may cause stimulation of NOS activity in platelets, and may even be a source of increased reactive oxygen species. However, for ethical reasons, it was not possible to study patients after a period of stopping these drugs. As for the other medications, we found that platelet NOS activity, both basal and agonist-stimulated, and NO-attributable cGMP were not different between heart failure patients receiving or not receiving anti-platelet medications, warfarin, digoxin, or statins. Although it remains possible that medications may have influenced our results, the existing literature suggests that most heart failure medications have little if any effect on platelet function (reviewed by Malinin et al.53).

Additionally, we did not study any possible differences in platelet uptake of l-arginine in the present work. Platelet l-arginine uptake occurs purely through system y+L and not system y+. To date, no literature exists on platelet l-arginine transport in heart failure in humans, although very recently Matsuura et al.54 have reported no change in platelet l-arginine transport in a rat model of doxorubicin-induced heart failure (a widely used model of human dilated cardiomyopathy). Moreover, in erythrocytes from humans with heart failure, it has been reported that, whilst overall arginine uptake is increased, this is due to an increase in transport via system y+ with no change in transport via system y+L.55 We therefore consider it unlikely that platelet l-arginine transport is altered in heart failure, although we did not specifically examine this in the present work since this was not the primary focus of the study.

Another limitation is that the present experiments do not allow us to distinguish between an increase in platelet O2 generation, a decrease in platelet antioxidant capacity, or both. To do this will require further experiments to measure individual antioxidant systems, both enzymatic and non-enzymatic, as well as to determine chemiluminescence signals in the presence of specific inhibitors of O2-generating pathways: mitochondrial electron transport, NADPH oxidase, and indeed NOS3 itself. Further work is also required to confirm that the increase in platelet generation of NO in parallel with the increase in its inactivation by O2 translates to an increase in nitrosative stress, as manifested by an increase in nitrotyrosine, as well as to explore alternative mechanisms influencing local activity of ROS.

In conclusion, we have demonstrated that, in platelets from subjects with heart failure, there is impaired bioactive NO despite increased NOS activity, which can be explained by an increase in platelet oxidative stress and resultant increased O2 generation.

Further studies are needed to evaluate the source of increased platelet O2 production, as well as to examine the precise nature of the defect in platelet O2 scavenging capacity, in heart failure.

Supplementary material

Supplementary material is available at Cardiovascular Research online.

Funding

This work was supported by a Clinical Research Fellowship to Ashish Shah by Pfizer plc.

Acknowledgement

We are grateful to Professor Neil Dalton (Professor of Paediatric Biochemistry, King's College London) for performing the ADMA, SDMA, and arginine assays reported here.

Conflict of interest: none declared.

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Author notes

These authors contributed equally to this work.