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.

Nitrate pharmacokinetics: Taking note of the difference

“…With a burgeoning awareness of the potential importance of NO 3 -in sport and exercise, an ever-increasing variety of NO 3 -fortified products are becoming commercialised, ranging from sport bars and lozenges to BR capsules. Importantly, NO 3 -salts (in solution or via capsule) are readily available for both oral and intravenous administration and ] ratio, as defined in Table 1) was relatively consistent (1.0-1.2) and is in keeping with the work of most using BR [11][12][13][14], but inconsistent with Webb et al hrs, and a plasma t½ of >6 hrs is found irrespective of KNO 3 dose (4 -24 mmol).…”

“…Of those utilising a similar BR source, in one or two repeated tests there is an apparent split between those exhibiting high relative yield [12][13][14] and those finding significantly lower yield [3,11]. In these examples, volume of NO 3 -administration does not appear to be the cause of the discrepancy, neither does this appear to be methodological (since all studies essentially use ozonebased chemiluminescence to measure plasma NO metabolites). ]…”

Globin X is a six-coordinate globin that reduces nitrite to nitric oxide in fish red blood cells

“…sGC activation catalyzes the formation of the second messenger cGMP and subsequent inhibition of intracellular calcium release, resulting in decreased platelet aggregation. Recent studies from multiple laboratories show that low concentrations of nitrite in the presence of RBCs inhibit human platelets activation in vitro and in vivo in mice and humans (10)(11)(12). It has been demonstrated that in low oxygen conditions, Hb functions as a nitrite reductase, generating NO from nitrite and thus producing an antiaggregation effect on platelets (13,14).…”

Pharmacology and therapeutic role of inorganic nitrite and nitrate in vasodilatation

“…Control subjects who consumed 140 ml nitrate-depleted beetroot juice had plasma nitrite levels of 271 nmol/l, whereas subjects consuming the same volume of beetroot juice (containing 8.7 mmol nitrate) showed a two-fold higher plasma nitrite level of 532 nmol/l (Cermak et al, 2012). It has also recently been demonstrated that nitrite/nitrate handling in humans shows sexual dimorphism: increases in plasma nitrate and nitrite levels as well as inhibition of platelet function after consumption of beetroot juice revealed a clear gender difference (Velmurugan et al, 2013). The consumption of dietary nitrate from beetroot juice in healthy volunteers has been shown by Wylie and colleagues to increase plasma nitrate and nitrite levels in a dose-dependent manner (Wylie et al, 2013).…”

“…It has been shown by a number of research groups that nitrite-derived bioactivity is cardioprotective by minimising cell death by apoptosis (Webb et al, 2004;Duranski et al, 2005;Dezfulian et al, 2007;Shiva et al, 2007aShiva et al, , 2007bTripatara et al, 2007), exerts anti-aggregatory effects (Srihirun et al, 2012;Corti et al, 2013;Park et al, 2013;Velmurugan et al, 2013), inhibits hypoxic and inflammatory pulmonary arterial hypertension (Baliga et al, 2012;Sparacino-Watkins et al, 2012;Bueno et al, 2013), and that it increases forearm blood flow (FBF) and decreases BP by acting as a vasodilator (Cosby et al, 2003;Dejam et al, 2007;Maher et al, 2008). In addition, nitrate has been shown to improve exercise performance by reducing the oxygen cost of exercise in skeletal muscle (Larsen et al, 2011).…”