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Cited by 114Nitric Oxide and Endothelin EffectsPlatelet Disorders and TreatmentsAntiplatelet Therapy and Cardiovascular Diseases

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Antiplatelet effects of dietary nitrate in healthy volunteers: Involvement of cGMP and influence of sex

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Velmurugan, Shanti, Kapil, Vikas, Ghosh, Suborno M., Davies, Sheridan, McKnight, Andrew, Aboud, Zainab, Khambata, Rayomand S., Webb, Andrew J., Poole, Alastair, Ahluwalia, Amrita·2013·Free Radical Biology and Medicine
DOI 10.1016/j.freeradbiomed.2013.06.031PMC PMC3878381
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Abstract

Ingestion of vegetables rich in inorganic nitrate has emerged as an effective method, via the formation of a nitrite intermediate, for acutely elevating vascular NO levels. As such a number of beneficial effects of dietary nitrate ingestion have been demonstrated including the suggestion that platelet reactivity is reduced. In this study we investigated whether inorganic nitrate supplementation might also reduce platelet reactivity in healthy volunteers and have determined the mechanisms involved in the effects seen. We conducted two randomised crossover studies each in 24 (12 of each sex) healthy subjects assessing the acute effects of dietary nitrate (250 ml beetroot juice) or potassium nitrate capsules (KNO3, 8 mmol) vs placebo control on platelet reactivity. Inorganic nitrate ingested either from a dietary source or via supplementation raised circulating nitrate and nitrite levels in both sexes and attenuated ex vivo platelet aggregation responses to ADP and, albeit to a lesser extent, collagen but not epinephrine in male but not female volunteers. These inhibitory effects were associated with a reduced platelet P-selectin expression and elevated platelet cGMP levels. In addition, we show that nitrite reduction to NO occurs at the level of the erythrocyte and not the platelet. In summary, our results demonstrate that inorganic nitrate ingestion, whether via the diet or through supplementation, causes a modest decrease in platelet reactivity in healthy males but not females. Our studies provide strong support for further clinical trials investigating the potential of dietary nitrate as an adjunct to current antiplatelet therapies to prevent atherothrombotic complications. Moreover, our observations highlight a previously unknown sexual dimorphism in platelet reactivity to NO and intimate a greater dependence of males on the NO-soluble guanylate cyclase pathway in limiting thrombotic potential.

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Fig. 1 Dietary nitrate supplementation elevates plasma nitrite levels and attenuates platelet reactivity in healthy male volunteers. Ex vivo platelet (PRP) aggregation assessed by light transmission aggregometry in response to ADP (0.1–0.3 µmol/L), collagen (0.1–30 µg/ml), and epinephrine (0.001–100 µmol/L), before and 3 h post placebo (A–C) or dietary nitrate (beetroot juice, ∼3.1 mmol nitrate, D–F) (n=12). Effect of dietary nitrate on plasma nitrate (G) and nitrite (H) concentrations. Data are expressed as mean±SEM. Significance shown as ###P<0.001 for two-way repeated-measures ANOVA, and ⁎P<0.05,⁎⁎P<0.01, and ⁎⁎⁎P<0.001 for Bonferroni post hoc tests following one-way or two-way repeated-measures ANOVA as appropriate. (ADP, adenosine diphosphate; PRP, platelet-rich plasma).
Fig. 1 Dietary nitrate supplementation elevates plasma nitrite levels and attenuates platelet reactivity in healthy male volunteers. Ex vivo platelet (PRP) aggregation assessed by light transmission aggregometry in response to ADP (0.1–0.3 µmol/L), collagen (0.1–30 µg/ml), and epinephrine (0.001–100 µmol/L), before and 3 h post placebo (A–C) or dietary nitrate (beetroot juice, ∼3.1 mmol nitrate, D–F) (n=12). Effect of dietary nitrate on plasma nitrate (G) and nitrite (H) concentrations. Data are expressed as mean±SEM. Significance shown as ###P<0.001 for two-way repeated-measures ANOVA, and ⁎P<0.05,⁎⁎P<0.01, and ⁎⁎⁎P<0.001 for Bonferroni post hoc tests following one-way or two-way repeated-measures ANOVA as appropriate. (ADP, adenosine diphosphate; PRP, platelet-rich plasma).CC BY-NC-ND · PubMed Central
Fig. 2 Dietary nitrate supplementation elevates plasma nitrite levels but does affect platelet reactivity in healthy female volunteers. Ex vivo platelet (PRP) aggregation assessed by light transmission aggregometry in response to (A, B) ADP (0.1–0.3 µmol/L), (C, D) collagen (0.1–30 µg/ml), and (E, F) epinephrine (0.001–100 µmol/L), and (G) plasma nitrate and (H) nitrite levels before and 3 h post placebo or dietary nitrate (beetroot juice, ∼3.1 mmol nitrate) consumption in females (n=12). Data are expressed as mean±SEM. Significance shown as ⁎⁎⁎P<0.001 for Bonferroni post hoc tests following one-way ANOVA. (ADP, adenosine diphosphate; PRP, platelet-rich plasma).
Fig. 2 Dietary nitrate supplementation elevates plasma nitrite levels but does affect platelet reactivity in healthy female volunteers. Ex vivo platelet (PRP) aggregation assessed by light transmission aggregometry in response to (A, B) ADP (0.1–0.3 µmol/L), (C, D) collagen (0.1–30 µg/ml), and (E, F) epinephrine (0.001–100 µmol/L), and (G) plasma nitrate and (H) nitrite levels before and 3 h post placebo or dietary nitrate (beetroot juice, ∼3.1 mmol nitrate) consumption in females (n=12). Data are expressed as mean±SEM. Significance shown as ⁎⁎⁎P<0.001 for Bonferroni post hoc tests following one-way ANOVA. (ADP, adenosine diphosphate; PRP, platelet-rich plasma).CC BY-NC-ND · PubMed Central
Fig. 3 Spermine-NONOate but not KNO2 inhibits aggregation of platelet-rich plasma (PRP). Platelet aggregation induced by ADP (0.1–0.3 µmol/L) or collagen (0.1–30 µg/ml) assessed by light transmission aggregometry in PRP from healthy untreated male volunteers incubated ex vivo for 30 min with either KNO2 (A, B) or spermine-NONOate (C, D) or for 10 min with KNO2 (1 µmol/L, E, F) or spermine-NONOate (10 µmol/L G, H). Data are expressed as mean±SEM of n=5–13. Significance shown as #P<0.05, ##P<0.01, and ###P<0.001 for two-way repeated-measures ANOVA followed by Bonferroni post tests shown as ⁎⁎P<0.01 and ⁎⁎⁎ for P<0.001. (ADP, adenosine diphosphate; PRP, platelet-rich plasma; Sper-NO, Spermine-NONOate).
Fig. 3 Spermine-NONOate but not KNO2 inhibits aggregation of platelet-rich plasma (PRP). Platelet aggregation induced by ADP (0.1–0.3 µmol/L) or collagen (0.1–30 µg/ml) assessed by light transmission aggregometry in PRP from healthy untreated male volunteers incubated ex vivo for 30 min with either KNO2 (A, B) or spermine-NONOate (C, D) or for 10 min with KNO2 (1 µmol/L, E, F) or spermine-NONOate (10 µmol/L G, H). Data are expressed as mean±SEM of n=5–13. Significance shown as #P<0.05, ##P<0.01, and ###P<0.001 for two-way repeated-measures ANOVA followed by Bonferroni post tests shown as ⁎⁎P<0.01 and ⁎⁎⁎ for P<0.001. (ADP, adenosine diphosphate; PRP, platelet-rich plasma; Sper-NO, Spermine-NONOate).CC BY-NC-ND · PubMed Central
Fig. 4 KNO2 reduces platelet aggregation in whole blood with an associated rise in platelet cGMP in males. Platelet aggregation assessed by impedance aggregometry of whole blood of healthy male volunteers incubated ex vivo with KNO2 (0.3—3 µmol/L) in response to (A) ADP (10 µmol/L, n=13), (B) collagen (3 µg/ml, n=11), and (C) epinephrine (10 µmol/L, n=11). Platelet cGMP levels in whole blood of males incubated with KNO2 (D, 1 µmol/L, n=12). Effect of ex vivo incubation of whole blood with KNO2 (0.3–3 µmol/L) on (E) ADP-induced (10 µmol/L, n=7) aggregation and (F) platelet cGMP levels (n=6) collected from untreated healthy female volunteers. Data are expressed as mean±SEM. Significance shown as ⁎P<0.05, ⁎⁎P<0.01 for Dunnett's post hoc test compared to control (PBS) following one-way ANOVA. For cGMP data, significance shown as ⁎P<0.05 following paired t test. White bars represent PBS and black bars nitrite (ADP, adenosine diphosphate).
Fig. 4 KNO2 reduces platelet aggregation in whole blood with an associated rise in platelet cGMP in males. Platelet aggregation assessed by impedance aggregometry of whole blood of healthy male volunteers incubated ex vivo with KNO2 (0.3—3 µmol/L) in response to (A) ADP (10 µmol/L, n=13), (B) collagen (3 µg/ml, n=11), and (C) epinephrine (10 µmol/L, n=11). Platelet cGMP levels in whole blood of males incubated with KNO2 (D, 1 µmol/L, n=12). Effect of ex vivo incubation of whole blood with KNO2 (0.3–3 µmol/L) on (E) ADP-induced (10 µmol/L, n=7) aggregation and (F) platelet cGMP levels (n=6) collected from untreated healthy female volunteers. Data are expressed as mean±SEM. Significance shown as ⁎P<0.05, ⁎⁎P<0.01 for Dunnett's post hoc test compared to control (PBS) following one-way ANOVA. For cGMP data, significance shown as ⁎P<0.05 following paired t test. White bars represent PBS and black bars nitrite (ADP, adenosine diphosphate).CC BY-NC-ND · PubMed Central
Fig. 5 KNO3 but not KCl (8 mmol) supplementation elevates systemic nitrate and nitrite levels in healthy male volunteers. The effects of KNO3 or KCl ingestion 3 h prior on circulating plasma (A,B), salivary (C,D), and urinary (E,F) nitrate and nitrite levels. Data are expressed as mean±SEM of n=12. Significance shown for Bonferroni post hoc tests between groups as ⁎⁎P<0.01 and ⁎⁎⁎P<0.001 following one-way ANOVA.
Fig. 5 KNO3 but not KCl (8 mmol) supplementation elevates systemic nitrate and nitrite levels in healthy male volunteers. The effects of KNO3 or KCl ingestion 3 h prior on circulating plasma (A,B), salivary (C,D), and urinary (E,F) nitrate and nitrite levels. Data are expressed as mean±SEM of n=12. Significance shown for Bonferroni post hoc tests between groups as ⁎⁎P<0.01 and ⁎⁎⁎P<0.001 following one-way ANOVA.CC BY-NC-ND · PubMed Central
Fig. 6 KNO3 but not KCl (8 mmol) supplementation elevates systemic nitrate and nitrite levels in healthy female volunteers. The effects of KNO3 or KCl ingestion 3 h prior on circulating plasma (A, B), salivary (C, D), and urinary (E, F) nitrate and nitrite levels. Data are expressed as mean ±SEM of n=12. Significance shown for Bonferroni post hoc tests between groups as ⁎⁎P<0.01 and ⁎⁎⁎P<0.001 following one-way ANOVA.
Fig. 6 KNO3 but not KCl (8 mmol) supplementation elevates systemic nitrate and nitrite levels in healthy female volunteers. The effects of KNO3 or KCl ingestion 3 h prior on circulating plasma (A, B), salivary (C, D), and urinary (E, F) nitrate and nitrite levels. Data are expressed as mean ±SEM of n=12. Significance shown for Bonferroni post hoc tests between groups as ⁎⁎P<0.01 and ⁎⁎⁎P<0.001 following one-way ANOVA.CC BY-NC-ND · PubMed Central
Fig. 7 KNO3 (8 mmol) supplementation attenuates platelet aggregation in healthy male but not female volunteers. Platelet aggregation assessed by impedance aggregometry of whole blood collected from healthy male volunteers in response to ADP (10 µmol/L) and epinephrine (10 µmol/L) before and 3h post KNO3 (A, C) and KCl (B, D) supplementation and female volunteers (E, G and F, H respectively). Data are expressed as mean±SEM of n=12. Significance shown for Bonferroni post hoc tests between groups as ⁎P<0.05 following one-way ANOVA (ADP, adenosine diphosphate).
Fig. 7 KNO3 (8 mmol) supplementation attenuates platelet aggregation in healthy male but not female volunteers. Platelet aggregation assessed by impedance aggregometry of whole blood collected from healthy male volunteers in response to ADP (10 µmol/L) and epinephrine (10 µmol/L) before and 3h post KNO3 (A, C) and KCl (B, D) supplementation and female volunteers (E, G and F, H respectively). Data are expressed as mean±SEM of n=12. Significance shown for Bonferroni post hoc tests between groups as ⁎P<0.05 following one-way ANOVA (ADP, adenosine diphosphate).CC BY-NC-ND · PubMed Central
Fig. 8 KNO3 supplementation elevates platelet cGMP levels and simultaneously suppresses unstimulated platelet P-selectin expression in healthy male but not female volunteers. Platelet count (A, n=12), platelet cGMP levels (B, n=9) and platelet P-selectin expression (C and D, n=11–12) in healthy males following KNO3 or KCl (8 mmol) supplementation and in healthy females (E–H, respectively). Erythrocytic nitrite reductase activity at pH 7.4 (I) and pH 6.8 (J), in males (n=7) and females (n=5). All data are expressed as mean±SEM. Significance shown as ⁎P<0.05 and ⁎⁎P<0.01 following paired t test for two groups. No significant differences in nitrite reductase activity assessed using two-way repeated-measures ANOVA.
Fig. 8 KNO3 supplementation elevates platelet cGMP levels and simultaneously suppresses unstimulated platelet P-selectin expression in healthy male but not female volunteers. Platelet count (A, n=12), platelet cGMP levels (B, n=9) and platelet P-selectin expression (C and D, n=11–12) in healthy males following KNO3 or KCl (8 mmol) supplementation and in healthy females (E–H, respectively). Erythrocytic nitrite reductase activity at pH 7.4 (I) and pH 6.8 (J), in males (n=7) and females (n=5). All data are expressed as mean±SEM. Significance shown as ⁎P<0.05 and ⁎⁎P<0.01 following paired t test for two groups. No significant differences in nitrite reductase activity assessed using two-way repeated-measures ANOVA.CC BY-NC-ND · PubMed Central

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Authors
Velmurugan, S., Kapil, V., Ghosh, S. M., Davies, S., McKnight, A., Aboud, Z., Khambata, R. S., Webb, A. J., Poole, A., & Ahluwalia, A.
Affiliations
  • Queen Mary University of London (GB)
  • University of Bristol (GB)
Corresponding author
Ahluwalia, Amrita
ORCID
Venue
Free Radical Biology and Medicine
Publisher
Elsevier BV
Published
2013-12
Volume
65
Pages
1521-1532
ISSN
0891-5849 (ISSN-L)
DOI
10.1016/j.freeradbiomed.2013.06.031
PMC
PMC3878381
OpenAlex
W2028916136
Cited by
114
References
60
Citations by year
2026: 3 · 2025: 4 · 2024: 4 · 2023: 6 · 2022: 7 · 2021: 7 · 2020: 5 · 2019: 8 · 2018: 15 · 2017: 9 · 2016: 18 · 2015: 10 · 2014: 15 · 2013: 2
FWCI
10.42
Type
journal article
Language
en
Update notices
erratum (2015-07-01)
Open access
Licence
Locations
  • Free Radical Biology and Medicine (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • Research Portal (King's College London) (submittedVersion, other-oa)
Funders
  • British Heart Foundation (FS/11/41/28749)
  • Medical Research Council
Topics
  • Health Sciences › Medicine › Physiology (medical) › Nitric Oxide and Endothelin Effects
  • Health Sciences › Medicine › Hematology › Platelet Disorders and Treatments
  • Health Sciences › Medicine › Cardiology and Cardiovascular Medicine › Antiplatelet Therapy and Cardiovascular Diseases
Keywords
dietary nitrate, inorganic nitrate supplementation, platelet reactivity, platelet aggregation, nitric oxide, cyclic GMP, soluble guanylate cyclase, nitrite reduction, erythrocytes, P-selectin, sex differences, beetroot juice, potassium nitrate, atherothrombosis, antiplatelet therapy
MeSH terms
Adolescent, Adult, Blood Platelets, Cardiovascular Diseases, Collagen, Diet, Epinephrine, Erythrocytes, Female, Cyclic GMP, Guanylate Cyclase, Humans, Male, Middle Aged, Nitrates, Nitric Oxide, Platelet Aggregation, Platelet Aggregation Inhibitors, Sex Factors, Vegetables, Potassium Compounds, Cross-Over Studies, P-Selectin, Dietary Supplements, Beta vulgaris, Young Adult

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