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Cardiovascular Research 2007 75(4):793-802; doi:10.1016/j.cardiores.2007.05.021
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Copyright © 2007, European Society of Cardiology

Age decreases nitric oxide synthesis and responsiveness in human platelets and increases formation of monocyte–platelet aggregates

Iya Goubareva1, Eugenia Gkaliagkousi1, Ashish Shah1, Lindsay Queen, James Ritter and Albert Ferro*

Department of Clinical Pharmacology, Cardiovascular Division, School of Medicine, King's College London, London, UK

* Corresponding author. 3.07 Franklin–Wilkins Building, King's College London, 150 Stamford Street, London SE1 9NH, UK. Tel.: +44 20 7848 4283; fax: +44 20 7848 6220. albert.ferro{at}kcl.ac.uk

Received 18 December 2006; revised 20 March 2007; accepted 21 May 2007


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective Ageing is associated with an increase in atherothrombotic disease. Platelet-derived nitric oxide (NO) inhibits platelet activation, but the effect of age on platelet NO signaling is unknown. We investigated platelet NO biosynthesis and responsiveness in older (>45 years old) as compared with younger (<30 years old) healthy human subjects.

Methods Platelet NO synthase (NOS) activity was evaluated by l-[3H]-arginine to l-[3H]-citrulline conversion, and cGMP was determined by radioimmunoassay. Platelet expression of NOS3, phosphoserine-1177-NOS3 and soluble guanylyl cyclase (sGC) were quantified by Western blotting. Circulating monocyte–platelet aggregates (MPA) were measured by flow cytometry.

Results Basal NOS activity was similar in both groups. By contrast, whereas both albuterol and collagen stimulated platelet NOS in younger subjects, stimulation was absent in older subjects. Platelet NOS3 expression was similar in both age groups, but NOS3 serine-1177 phosphorylation was greater in younger subjects. Basal, albuterol- and collagen-stimulated cGMP, as well as sGC expression, were all greater in younger than older subjects, and within the younger group both cGMP (basal and stimulated) and sGC expression were greater in women than in men. Circulating MPA were greater in older subjects and, whilst NOS inhibition increased MPA further in both groups, it did so to a lesser extent in the older age bracket.

Conclusions These data suggest that platelet NO production and responsiveness decrease with age, and this is reflected in increased circulating MPA.

KEYWORDS Aging; Platelets; Monocyte–platelet aggregates; Nitric oxide

Abbreviations: NO, nitric oxide • NOS, nitric oxide synthase • sGC, soluble guanylyl cyclase • cGMP, cyclic guanosine-3',5'-monophosphate • cAMP, cyclic adenosine-3',5'-monophosphate • PKA, protein kinase A • PKC, protein kinase C • MPA, monocyte–platelet aggregates • AR, adrenoceptors • ER, estrogen receptors • GFP, gel-filtered platelets • PRP, platelet-rich plasma • L-NMMA, NG-monomethyl-l-arginine.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Nitric oxide (NO) is synthesized from l-arginine in a reaction catalyzed by NO synthase (NOS), with l-citrulline being the by-product [1]. NO activates soluble guanylyl cyclase (sGC), thereby increasing cyclic guanosine-3',5'-monophosphate (cGMP) in target cells. Endothelium-derived NO causes vasorelaxation, and also inhibits platelet adhesion and aggregation, thus maintaining blood fluidity and preventing thrombosis.

Platelets also produce NO [2], and express NOS2 and NOS3, NOS3 being the predominant isoform [3]. NOS3 activity is regulated by changes in intracellular calcium concentrations, which in turn affect the binding of calmodulin to NOS3. Activation of NOS3 can also be achieved by phosphorylation of Ser1177 by protein kinase Akt [4], or via phosphorylation of Ser633 or Ser1177 by protein kinase A (PKA) and/or cGMP-dependent protein kinase [5]. In contrast, phosphorylation of Thr495 of NOS3 by protein kinase C (PKC) decreases its activity [6]. Platelet-derived NO inhibits platelet aggregation [7], adhesion to vascular endothelium [8], recruitment to growing thrombi [9], and the formation of leukocyte–platelet aggregates [10]. Effects of NO on mononuclear (rather than polymorphonuclear) leukocytes have not been described; nor is it known whether platelet-derived NO modulates monocyte–platelet interactions. Monocyte–platelet aggregates (MPA) are of particular interest because they represent a better indicator of in vivo platelet activation than either circulating neutrophil–platelet aggregates or circulating P-selectin-positive non-aggregated platelets [11], and because they may themselves contribute to the initiation and progression of atherosclerosis [12,13].

Platelets express {alpha}2-and β2-adrenoceptors (AR), with {alpha}2-AR being pro-aggregatory and β2-AR anti-aggregatory [14–16]. β2-AR stimulation activates adenylyl cyclase, causing intracellular accumulation of cyclic adenosine-3',5'-monophosphate (cAMP) [14], thus inhibiting platelet aggregation [17]. Furthermore, stimulation of platelet β2-AR activates platelet NOS3 via a cAMP-dependent mechanism, with no detectable change in intracellular calcium, and this mediates the inhibitory effect of β2-AR on platelet adhesion [8].

Platelet activation contributes to the pathogenesis of atherosclerosis and thrombosis. Atherothrombotic disease becomes increasingly common with advancing age, and can occur in the absence of other known cardiovascular risk factors. The synthesis of NO by arterial endothelium decreases with age [18]. However, the effect of age on platelet NO biosynthesis has not been reported previously. We hypothesized that platelet NO biosynthesis and/or responsiveness decreases with age. The primary aim of the present study, therefore, was to investigate the effect of age on platelet NO signaling and associated functional responses (MPA formation). As a secondary aim, since the incidence of cardiovascular disease is lower in pre-menopausal women than in men of comparable age but increases sharply after the menopause [19], and given that platelets are known to express estrogen receptors (ER{alpha} and ERβ) [20], we also wished to determine whether sex differences exist in platelet NO signaling and MPA formation, especially in younger subjects, and whether any such differences may be abrogated following the menopause.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Subject recruitment
Subjects were healthy, asymptomatic, with no history of serious disease, with normal plasma biochemistry (electrolytes, fasting glucose, lipid, renal and liver profiles), and on no regular medication; in particular, they were not taking hormonal contraception, and had taken no aspirin or other anti-platelet medication for at least 2 weeks before study. The investigation conforms with the principles outlined in the Declaration of Helsinki (Cardiovascular Research 1997;35:2–4). All subjects gave informed consent. The study was approved by the St Thomas' Hospital Research Ethics Committee. In a preliminary study, 12 young women (age 20–29 years) were studied twice: once on day 1 and again on day 14 of the menstrual cycle. Subjects for the main study were recruited sequentially in response to advertisement and enrolled in one of two groups: younger (<30 years old; n=19) and older (>45 years old; n=20). Their characteristics are summarized in Table 1. Within each group, there was no significant difference in age, platelet count or biochemical characteristics between men and women. In the main study, young women were studied at any stage of the menstrual cycle, and all older women were post-menopausal.


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  		Table 1 Subject characteristics

 
2.2 Preparation of platelets
Subjects attended in the morning, having fasted overnight and refrained from smoking and caffeine since the previous evening. 70–80 mL venous blood was taken from a large antecubital vein using a 19G Butterfly® needle, collected into tri-sodium citrate (0.38% final concentration), and centrifuged (200 xg, 10 min, room temperature) to obtain platelet-rich plasma (PRP). Gel-filtered platelets (GFP) were obtained by eluting PRP through a Sepharose gel column as previously described [8,21]. Platelet count in the eluate was obtained using a Coulter counter.

2.3 Platelet NOS activity 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 [8,21]. NOS activity was taken as the difference in l-citrulline in the absence and presence of NG-monomethyl-l-arginine (L-NMMA). Similarly, the amount of cGMP produced in response to NO ("NO-attributable cGMP") was determined as the difference in measured cGMP in the absence and presence of L-NMMA.

2.4 Measurement of platelet superoxide anion production
Platelet-derived O2– was measured by pholasin-enhanced chemiluminescence, using a Plate Chameleon V microplate reader (Hidex, Turku, Finland). GFP (200 µL) was mixed with pholasin (0.5 µg/mL) and horseradish peroxidase (4 U/mL), in the presence of the O2– scavenger Tiron (1 mmol/L) or corresponding vehicle. Samples were allowed to dark adapt for 5 min prior to recording of luminescence.

2.5 Measurement of intraplatelet calcium
Platelets were loaded with fura 2-AM as previously described [22], then incubated with albuterol (1 µmol/L), collagen (8 µg/mL) or thrombin (0.01 U/mL, as a positive control). Changes in cytoplasmic Ca2+ were examined as a function of time, from the ratio of emission at 510 nm after excitation at 340 and 380 nm, in an LS50 luminescence spectrometer.

2.6 Western blotting
Expression of NOS3, phosphoserine-1177-NOS3, sGC and {alpha}-tubulin were determined in platelets, as follows. Aliquots of GFP (5 mL) were centrifuged (1400 xg, 20 min, 4 °C) to obtain platelet pellets, and lysed by the addition of 100 µL lysis buffer (composition in mmol/L: NaCl 150; Tris 25; NaF 50; Na orthovanadate 1.0; phenylmethylsulfonyl fluoride 1.0; aprotinin 1 µg/mL; leupeptin 10 µg/mL; pH 7.6). Lysates were subjected to SDS-PAGE on a 7.5% polyacrylamide gel, followed by Western blotting, enhanced chemiluminescence detection and densitometric quantitation of bands, as previously described [23]. The following primary antibodies (all affinity-purified) were used for detecting the proteins of interest: anti-NOS3 at 1:1000 dilution (rabbit polyclonal antibody, from Santa Cruz Biotechnology, CA, USA), anti-phosphoserine-1177-NOS3 at 1:500 dilution (rabbit polyclonal antibody, from Cell Signaling Technology, MA, USA), anti-sGC at 1:1000 dilution (rabbit polyclonal antibody to β1 subunit of sGC, from Chemicon, Hampshire, UK), and anti-{alpha}-tubulin at 1:1000 dilution (rat polyclonal antibody, from Chemicon, Hampshire, UK).

2.7 Platelet aggregometry
Aggregation responses of PRP were measured turbidimetrically using a Payton dual-channel 600B model aggregometer. 300 µL samples at 37 °C were stirred at 600 rpm with a magnetic stirrer. The aggregometer was calibrated using the light transmission of PRP and platelet-poor plasma to represent 0% and 100% aggregation respectively. Following the addition of Ca2+ (final concentration 1 mmol/L), in order to re-establish physiological ambient Ca2+ concentration, aggregation was stimulated with ADP (concentration range 0.01–30 µmol/L) and recorded for 2 min or until a plateau was reached.

2.8 Measurement of MPA
100 µL whole blood was incubated with either vehicle or L-NMMA (0.1 mmol/L) for 10 min, following which saturating concentrations of phycoerythrin-labeled anti-CD14 and fluorescein isothiocyanate-labelled anti-CD42b monoclonal antibodies (0.25 µg/10 µl for each), or isotype control antibody (0.5 µg/10 µl), were added (all from Becton Dickinson, UK) and incubation continued for 20 min at room temperature. Erythrocytes were lysed by adding 1 mL FACS lysing solution (BD Bioscience) for 10 min at room temperature, and samples washed twice with phosphate buffered saline/0.1% bovine serum albumin/0.01% azide. Samples were fixed with 1% paraformaldehyde and kept at 4°C until analyzed by flow cytometry (FACSscan, Becton Dickinson, Oxford, UK) using CellQuest software; all analyses were performed within 72 h of sample preparation. Gating was performed using forward and side light scatter characteristics to access the monocyte population. MPA were taken as the population positive for both CD14 and CD42b, and were quantified as a percentage of the total monocyte population.

2.9 Statistical analysis
All data were expressed as mean±standard error of the mean. Where data exhibited a Gaussian distribution, within-group and between-group changes were analyzed by ANOVA with or without repeated measures respectively; where data were non-Gaussian, the non-parametric Friedman and Kruskal–Wallis tests were used to compare within-and between-group changes respectively. In all cases, P<0.05 (two-tailed) was considered to be significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Platelet NOS activity
Since it was thought possible that hormonal influences might affect platelet NOS activity, we firstly measured platelet NOS activity, both at baseline and in response to β-AR stimulation (using isoproterenol 1 µmol/L), at days 1 and 14 of the menstrual cycle in young women aged 20–29 years (Fig. 1A). On each occasion, isoproterenol-stimulated platelet NOS activity. Basal and isoproterenol-stimulated NOS activity were each similar on the two occasions (95% CI for the difference –83.1 to +74.5 and –73.5 to +61.5 fmol l-citrulline/108 platelets respectively).


Figure 1
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  		Fig. 1 Platelet NO signaling as a function of age and sex. A, The effect of timing in the menstrual cycle on platelet NOS activity in young women (n=12). Platelets were isolated at days 1 and 14 of the menstrual cycle from young women, and NOS activity was determined from L-arginine to L-citrulline conversion, both under basal conditions (control) and in response to 1 µmol/L isoproterenol. B, NOS activation in response to albuterol and collagen in younger (n=19) and older (n=20) subjects. Platelets were incubated with albuterol 1 µmol/L, collagen 8 µg/mL or vehicle, and the resulting change in NOS activity (measured as L-arginine to L-citrulline conversion) was expressed as a percentage of basal activity (vehicle). ***P<0.001 as compared with basal. ##, ###P<0.01 and <0.001 respectively, as compared with younger subjects. C, NOS activation in response to albuterol and collagen in younger and older subjects, subdivided by gender. In the younger group, n=9 men and 10 women; in the older group, n=10 men and 10 women. D, Platelet NO-attributable cGMP levels at baseline and in response to albuterol and collagen in platelets from younger (n=19) and older (n=20) subjects. Platelets were incubated with albuterol 1 µmol/L, collagen 8 µg/mL or vehicle (control), and cGMP was then measured in platelet lysates by radioimmunoassay. ***P<0.001 as compared with basal. ###P<0.001 as compared with younger subjects. E, Platelet NO-attributable cGMP levels at baseline and in response to albuterol and collagen in platelets from younger and older subjects, subdivided by gender. In the younger group, n=9 men and 10 women; in the older group, n=10 men and 10 women. **, ***P<0.01 and <0.001 respectively as compared with men in same age bracket.

 
In the main study, basal NOS activity was similar in each group (younger subjects: 111.9±22.8, older subjects 105.9±15.2 fmol l-citrulline/108 platelets). In younger subjects, albuterol (1 µmol/L) stimulated platelet NOS activity, but such stimulation did not occur in platelets from older subjects (Fig. 1B). To determine whether these differences were specific to the β-AR pathway, or whether they reflected a more generalized defect in the ability of NOS to undergo stimulation in platelets from older subjects, we also measured NOS activation by collagen in these same subjects. Platelet NOS activity increased in platelets from younger subjects, but not in those from older subjects, in response to collagen (Fig. 1B).

Within each age bracket, basal NOS activity was not different between men and women (younger subjects: 105.5±28.4 vs 118.8±24.6 fmol l-citrulline/108 platelets; older subjects: 100.8±23.6 vs 107.3±27.2 fmol l-citrulline/108 platelets; P=NS for each). Additionally, we found no difference in NOS activation by either albuterol or collagen between men and women within each age group (Fig. 1C).

The age-related differences observed in NOS activation could not be explained by differences in total intraplatelet calcium, since no change in intraplatelet calcium could be detected following treatment with either albuterol or collagen (Fig. 2). Nevertheless, these experiments do not rule out the possibility of small localized changes in calcium in the vicinity of the NOS3 enzyme.


Figure 2
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  		Fig. 2 Effect of albuterol and collagen on intraplatelet calcium. A representative trace is shown here, for platelets from a younger man. Platelets were loaded with the calcium-sensitive fluorophore fura-2, and changes in intraplatelet calcium were detected as alterations in the ratio of emission at 510 nm following excitation at 340 nm and 380 nm. No change in calcium was detected in response to either albuterol 1 µmol/L or collagen 8 µg/mL, whereas thrombin 0.01 U/mL (used as a positive control) elicited a rise in calcium. For simplicity and clarity, a single trace is shown with sequential addition of albuterol, collagen and thrombin; in other experiments, when these drugs were used singly, similar results were obtained (no observable change with albuterol or collagen, increase in emission ratio with thrombin). Experimental results were the same when performed in younger men (n=3), younger women (n=3), older men (n=3) and older women (n=3).

 
3.2 Platelet cGMP production
NO-attributable cGMP was measured in platelets in the basal and stimulated states, as an index of bioactive NO, since this is determined not only by the rate of NO synthesis, but also by that of its breakdown, as well as by the responsiveness of platelets to NO. Platelets from younger subjects exhibited higher NO-attributable cGMP both basally and when stimulated with albuterol or collagen, as compared with older subjects (Fig. 1D). In subgroup analysis, we found no differences in basal, albuterol- or collagen-stimulated platelet NO-attributable cGMP between men and women in the older age bracket. By contrast, platelets from younger women exhibited higher NO-attributable cGMP levels, both basally and in response to albuterol and collagen, than did those from younger men (Fig. 1E).

3.3 Platelet superoxide anion generation
Since NO generated within platelets can be rapidly inactivated by O2–, producing peroxynitrite, O2– generation by platelets was measured in younger and older subjects by pholasin chemiluminescence. O2– production was greater in platelets from older as compared to younger subjects, and in both groups the chemiluminescent signal was almost entirely abolished by co-incubation with the O2– scavenger Tiron (1 mmol/l), confirming that the measured signal was indeed attributable to O2– (Fig. 3A). No difference was found in platelet O2– production between men and women in either age group.


Figure 3
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  		Fig. 3 Platelet superoxide anion production as a function of age, and its modulation of cGMP. A, Platelet O2– production in platelets from younger (n=6) and older (n=6) subjects, and the effect of co-incubation with Tiron 1 mmol/l, as measured by pholasin chemiluminescence. *P<0.05 as compared with younger subjects. B, Effect of Tiron 1 mmol/l on platelet NO-attributable cGMP levels, at baseline and in response to albuterol and collagen, in platelets from younger (n=6) and older (n=6) subjects. *, **P<0.05 and <0.01 respectively, as compared with basal. ###P<0.001 as compared with absence of Tiron.

 
To ascertain whether the observed decrease in intraplatelet NO-attributable cGMP in older subjects could be attributed to the increase in O2– generated by platelets from older subjects, we measured the effect of Tiron co-incubation on intraplatelet NO-attributable cGMP in younger and older subjects. In both age groups, Tiron substantially increased NO-attributable cGMP, both basal and that in response to albuterol or collagen (Fig. 3B). However, the degree of increase was not different between younger and older subjects (basal: 1070.3±157.2 vs. 1126.0±137.9 fmol cGMP/108 platelets; albuterol: 1086.7±147.6 vs. 1002.2±82.0 fmol cGMP/108 platelets; collagen: 1085.3±41.6 vs. 1056.8±118.9 fmol cGMP/108 platelets; P=NS for each). Within each age group, again the effect of Tiron on NO-attributable cGMP was not different between men and women.

3.4 Platelet NOS3 and phosphoserine-1177-NOS3 expression
In view of the observed difference in platelet NOS activation by albuterol and collagen according to age but not sex, with no apparent effect of either agonist on intraplatelet calcium in any group, we investigated the expression of NOS3 as well as its phosphorylation at Ser1177 in platelets from younger and older subjects, since phosphorylation of NOS3 at this residue by PKA and/or Akt is a well-described mechanism for sensitizing NOS3 to the activating effects of Ca2+. NOS3 expression did not differ between the two age brackets, when expressed as the densitometric ratio of NOS3 to {alpha}-tubulin (as a housekeeping protein control). By contrast, NOS3 in platelets from younger subjects underwent Ser1177 phosphorylation to a greater degree than did NOS3 in platelets from older subjects at baseline (Fig. 4). Ser1177 phosphorylation of NOS3 did not, however, increase from basal in response to either albuterol or to collagen, either in younger or older subjects (data not shown). Expression of {alpha}-tubulin was not different in platelets from the two age groups.


Figure 4
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  		Fig. 4 Expression of NOS3 and of phosphoserine-1177-NOS3 in platelets from younger and older subjects. A, Representative Western blot of NOS3 (arrowed) from younger (Y) and older (O) subjects. Underneath is shown the corresponding bands for {alpha}-tubulin. B, Accumulated results for NOS3 expression, shown as the densitometric ratio of NOS3 to {alpha}-tubulin bands, in younger (n=19) and older (n=20) subjects. C, Representative Western blot of phosphoserine-1177-NOS3 (arrowed) from younger (Y) and older (O) subjects. Underneath is shown the corresponding bands for {alpha}-tubulin. D, Accumulated results for phosphoserine-1177-NOS3, shown as the densitometric ratio of phosphoserine-1177-NOS3 to NOS3, in younger (n=19) and older (n=20) subjects. **P<0.01 as compared with younger subjects.

 
3.5 Platelet sGC expression
sGC is expressed as the {alpha}1β1 heterodimer (molecular mass 70 kDa and 86 kDa respectively) in human platelets. In view of the observed differences in cGMP, both basal and stimulated, between platelets from younger and older subjects, as well as the sex differences in cGMP within the younger group, we determined the expression of sGC in platelets by Western blotting for the β1 subunit. Data were corrected to {alpha}-tubulin content by expressing as the densitometric ratio of sGC to {alpha}-tubulin. Platelets from younger subjects exhibited higher sGC expression compared to older subjects; moreover, although no difference was seen in platelet sGC expression between men and women in the older group, we found a higher expression of sGC in platelets from younger women as compared with younger men (Fig. 5).


Figure 5
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  		Fig. 5 Expression of sGC in platelets from younger and older subjects. A, Representative Western blot of sGC (arrowed) from two younger (Y) and two older (O) subjects. Underneath is shown the corresponding bands for {alpha}-tubulin. B, Accumulated results for sGC expression, shown as the densitometric ratio of sGC to {alpha}-tubulin bands, in younger (n=19) versus older (n=20) subjects. ***P<0.001 as compared with younger subjects. C, Representative Western blot of sGC (arrowed) from two younger men (M) and two younger women (W). Underneath is shown the corresponding bands for {alpha}-tubulin. D, Accumulated results for sGC expression, shown as the densitometric ratio of sGC to {alpha}-tubulin bands, from younger and older subjects, subdivided by gender. In the younger group, n=9 men and 10 women; in the older group, n=10 men and 10 women. *P<0.05 as compared with men in same age bracket.

 
3.6 Platelet aggregometry
Aggregation responses of PRP from younger and older subjects to ADP are shown in Fig. 6. In both groups, a concentration-dependent aggregation response was observed to ADP, which was not affected by co-incubation with L-NMMA (0.1 mmol/L). No differences were seen in ADP aggregation responses between younger and older subjects.


Figure 6
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  		Fig. 6 Platelet aggregatory responses to ADP in younger and older subjects, and effects of NOS inhibition. Aggregation responses of PRP as a function of ADP concentration are shown for younger (A, n=15) and older (B, n=15) subjects, in the absence and presence of L-NMMA 0.1 mmol/L.

 
3.7 Monocyte–platelet aggregates
Percentages of MPA in blood from younger and older subjects are shown in Fig. 7. Older subjects exhibited a higher level of circulating MPA than did younger subjects (Fig. 7A and B). Incubation of blood with L-NMMA (0.1 mmol/L) increased MPA in both groups (Fig. 7B), whereas 1400 W, a selective NOS2 inhibitor, at concentrations up to 1 mmol/L, had no effect (data not shown). L-NMMA increased MPA in older subjects to a lesser degree than in younger subjects (Fig. 7B and C). No differences were seen in NO-attributable MPA between men and women within each age group (Fig. 7D).


Figure 7
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  		Fig. 7 MPA in younger and older subjects, and effects of NOS inhibition. A, Representative flow cytometric traces from one younger (left panel) and one older (right panel) man. MPA are represented by events in the top right hand quadrant. B, MPA levels in blood taken from younger (n=19) and older (n=20) subjects, incubated in vitro with either L-NMMA 0.1 mmol/L or corresponding vehicle. MPA were quantitated as a percentage of the total monocyte population. **, ***P<0.01 and <0.001 respectively as compared with vehicle. #P<0.05 as compared with younger subjects. C, NO-attributable MPA in younger (n=19) and older (n=20) subjects, expressed as the difference in measured MPA in the absence and presence of L-NMMA 0.1 mmol/L. ***P<0.001 as compared to younger subjects. D, NO-attributable MPA in younger and older subjects, again expressed as the difference in measured MPA in the absence and presence of L-NMMA 0.1 mmol/L, subdivided by gender. In the younger group, n=9 men and 10 women; in the older group, n=10 men and 10 women.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In the present study, we have investigated the effect of age on platelet-derived NO signaling including MPA formation. Upon activation, platelets express on their surface P-selectin which is constitutively stored in {alpha}-granules [24]; P-selectin mediates the adhesion of monocytes and neutrophils to platelets [25,26], via binding to the P-selectin glycoprotein ligand-1 (PSGL-1) which is expressed on leukocytes [27–29]. Increased MPA have been found in patients with both stable coronary disease [30] and acute coronary syndromes [31,32]; similar findings have been reported in patients with end-stage renal disease [33], type 1 diabetes mellitus [34], and in cigarette smokers [35]. Circulating MPA represent a sensitive marker for platelet activation [32], and have been demonstrated to be a better indicator of in vivo platelet activation than either circulating neutrophil–platelet aggregates or circulating P-selectin-positive non-aggregated platelets [11]. There is also evidence to suggest that MPA may play a pathogenetic role in the initiation and progression of atherosclerosis, since MPA have been demonstrated to promote formation of atherosclerotic lesions in apolipoprotein E-deficient mice [12], and support monocyte adhesion to endothelium with the formation of monocyte clusters at the vessel wall [13]. Therefore, as well as serving as a marker for increased platelet activation, an increase in circulating MPA may in itself play a causative role in atherosclerosis and its complications.

To investigate the effect of age on platelet NO signaling, we compared responses in younger (<30 years) and older (>45 years) subjects. Within each age group, as a secondary outcome, we wished to compare responses in men and women, since younger women are relatively protected from cardiovascular disease and events as compared with men of similar age, whereas following the menopause women rapidly lose this cardioprotection, so that their cardiovascular event rate is similar to that of men of similar age. All of the women in the older group were therefore chosen to be post-menopausal; and all those in the younger group were pre-menopausal, had no relevant gynecological history and were not taking hormonal contraception. Estrogen receptors have been identified on platelets [20], but it is unclear whether these influence platelet function. Since plasma estrogen concentrations reach their peak at the time of ovulation (usually at or around day 14 of the menstrual cycle), we measured platelet NOS activity on day 1 and day 14 of the menstrual cycle in a group of younger women. NOS activity (both basal and stimulated) was similar on day 1 and day 14, suggesting that changes in estrogen levels as occur during ovulation do not significantly affect platelet NO production. In subsequent experiments, blood samples for platelet isolation and testing were taken from younger women irrespective of time in their menstrual cycle.

In all our subjects, we excluded any clinically apparent atherosclerosis. We did not investigate in detail for the presence of subclinical atherosclerotic disease, and indeed it is likely that such subclinical disease was present in many of our subjects, especially the older ones. Nevertheless, our study represents a "real-life" cross-section of clinically healthy younger and older subjects.

Forearm vascular responsiveness to intra-arterial acetylcholine, an endothelium-dependent vasodilator, decreases with age [36]. The present study demonstrates for the first time that age influences NO biosynthesis and responsiveness in platelets. Basal NOS activity was unaffected by age and NOS3 expression was similar in younger and older subjects. However, activation of NOS in response to albuterol or to collagen was less marked in platelets from older as compared to younger subjects. Albuterol and collagen activate platelet NOS3 through quite different mechanisms, albuterol through β2-AR stimulation and consequent activation of adenylyl cyclase, cAMP and PKA [8], and collagen through a glycoprotein VI signaling cascade involving Src family kinases, phosphatidylinositol 3-kinase and PKC [37]. Thus, it appears that the age-related decrease in NOS3 activation is a general phenomenon, not specific to one pathway. We investigated albuterol and collagen because endogenous catecholamines (which are elevated during acute stress, including myocardial infarction) and collagen (exposed in the vessel wall, for example following plaque fissuring or rupture) are of pathophysiological importance in arterial thrombosis.

The age-related reduction of NOS3 activation by albuterol and collagen could not be explained by effects on cytosolic Ca2+, since we found that neither agonist caused a detectable change in intraplatelet Ca2+ levels (unlike thrombin, used as a positive control). On the other hand, it could be explained by our finding that, despite similar levels of NOS3 expression, basal Ser1177 phosphorylation of NOS3 was reduced in platelets from older as compared to younger subjects, even though NOS3 Ser1177 phosphorylation was not increased in either age group following treatment with albuterol or collagen. Ser1177 phosphorylation activates NOS3 by increasing its sensitivity to Ca2+ [38]; and although we found no change in intraplatelet Ca2+ in response to either agonist, both β2-adrenoceptor stimulation and collagen are described to increase Ca2+ entry at the platelet membrane [39,40]. Therefore it is likely that, despite no obvious increase in total intraplatelet Ca2+, the local concentration of Ca2+ in the vicinity of the membrane-bound NOS3 enzyme is increased by albuterol and collagen, and thus the age-related difference in basal Ser1177 phosphorylation of the enzyme would account for the difference in NOS3 activation by both agonists.

Since O2– rapidly combines with NO to produce ONOO–, thereby inactivating NO, we considered it possible that an increase in platelet O2– generation with age might explain the age-related suppression of bioactive NO. Our data confirm that, indeed, platelets from older subjects produce more O2– than do those from younger subjects (with no gender difference in either group). Moreover, we found that Tiron (an O2– scavenger) increases intraplatelet NO-attributable cGMP substantially in all groups studied, as would be expected; however, the degree of increase in NO-attributable cGMP did not differ between younger and older subjects, nor between men and women within each age group. This suggests that, whilst platelet O2– generation is indeed age-dependent, these age-related differences in O2– cannot explain the observed impairment of platelet NO signaling with age. On the other hand, we also found that sGC expression was reduced in platelets from older as compared to younger subjects; this can explain the suppression of basal and stimulated NO-attributable cGMP in platelets from older subjects. It remains unclear why sGC expression decreases with age. It may relate to a decrease in its synthesis in megakaryocytes, or an increase in its degradation in megakaryocytes and/or platelets, with age; indeed, given our finding that platelet O2– production increases with age, it is possible that sGC undergoes an increase in oxidation and subsequent degradation in platelets from older subjects.

Our data allow us to conclude that both NO biosynthesis and NO responsiveness decrease in platelets with age. In subgroup analysis, we found that both basal and stimulated platelet NOS activity were each similar in men and women within either age group. However, platelets from young women had higher levels both of basal and stimulated NO-attributable cGMP than did platelets from young men, consistent with the Western blotting data, which show that platelets from young women express higher levels of sGC than do those from young men. No such differences in cGMP or sGC were found between men and women within the older age group. Thus, despite producing similar amounts of NO, platelets from younger women may respond to NO by synthesizing more cGMP than platelets from younger men.

In the present study, we found that NOS inhibition in platelets using L-NMMA had no effect on platelet aggregation responses to ADP. Nor was platelet aggregation to ADP different in younger and older subjects. Platelet aggregometry may not reveal subtle differences in platelet function; by contrast, MPA are generally considered a sensitive and early marker of platelet activation, and in addition are believed to play an important part in the pathophysiology of atherosclerosis [12,13]. We found that L-NMMA has clear effects on MPA levels formed during incubation in vitro of whole blood from younger subjects, suggesting that platelet-derived NO may have important effects on platelet recruitment and adhesion, in agreement with previous reports [8,9]. In older subjects, L-NMMA had a significantly reduced effect on MPA, again consistent with reduced platelet NO bioactivity (secondary to both reduced NO synthesis and responsiveness) with age.

Leukocytes express only NOS2, whereas platelets express predominantly NOS3 but also a small amount of NOS2 [3]. Since L-NMMA increased MPA in all subject groups, whereas the selective NOS2 inhibitor 1400 W had no such effect, this suggests that NOS3 in blood is the predominant isoform regulating MPA formation, and by inference this is likely to be platelet NOS3. Our data suggest that platelet-derived NO has important effects on MPA formation, and that this effect decreases with age; this may contribute to the increase in circulating MPA observed with age, which may in turn have important pathophysiological consequences. Since circulating MPA were similar in blood from younger women and younger men, despite increased NO responsiveness of platelets from younger women, it is possible that, at the levels of NO synthesis and responsiveness seen in younger men, MPA inhibition by NO is at or near maximum.

In conclusion, age has an important influence on platelet NO signaling, which in turn may have important functional consequences, especially with regard to increased circulating MPA with age. This suggests an important mechanism by which ageing may contribute to atherosclerosis and thrombosis, independently of the presence of other traditional cardiovascular risk factors.

Time for primary review 33 days


   Acknowledgement
 
We thank Dr Valerie Corrigall (Academic Department of Rheumatology, King's College London) for her help with the flow cytometry assays.


   Notes
 
1 These authors contributed equally. Back

* Financial support: This study was supported by the Coronary Research Fund and the Greek Foundation for State Scholarships. Back


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

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E. Gkaliagkousi, V. Corrigall, S. Becker, P. de Winter, A. Shah, C. Zamboulis, J. Ritter, and A. Ferro
Decreased platelet nitric oxide contributes to increased circulating monocyte-platelet aggregates in hypertension
Eur. Heart J., August 17, 2009; (2009) ehp330v1.
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