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

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.

Keywords: 3-isobutyl-1-methylxanthine; BP; IBMX; Inorganic nitrate; LTA; Nitrite; PBS; PPP; PRP; Platelet; Sper-NO; blood pressure; cGMP; light transmission aggregometry; phosphate-buffered saline; platelet-poor plasma; platelet-rich plasma; spermine-NONOate..

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. 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. 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. 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. 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. 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. 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. 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.