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Sex-Dependent Effect of Platelet Nitric Oxide

Visual Abstract


SUMMARY
A nitrate-rich diet has many cardiovascular benefits, but the mechanism behind this is unclear. We hypothesized that the ingestion of nitrate augments nitrate to nitrite reduction, leading to nitric oxide (NO) production, which may suppress platelet reactivity. In a randomized, double-blinded, placebo-controlled study involving healthy individuals, ingestion of nitrate augmented saliva and plasma nitrite/nitrate concentration and enhanced platelet NO production disproportionately in women compared with men. The response of elevated platelet NO in men was increased platelet reactivity and the response of markedly elevated platelet NO in women slightly  (1,2). We previously demonstrated that the platelet phenotype in peripheral artery disease and myocardial infarction is dysregulated so that platelets behave in an unpredictable manner and may show resistance to antiplatelet medications (3)(4)(5). Considering sex as a biological variable was not a significant feature of early preclinical antiplatelet medication studies. Platelet reactivity in women may not be the same as men, yet this is not taken into account when prescribing antiplatelet medications (2,5,6).
A plant-based diet may be recommended for patients with established vascular disease (7). The benefits of increased nitrate ingestion from a plantbased diet may include augmented NO production.
NO was shown to inhibit platelet alpha granule and Weibel Palade body exocytosis in endothelial cells and in platelets through covalent modifications of Nethylmaleimide-sensitive factor (8,9). Platelets have a dual role of regulating hemostasis as well as thrombosis. Platelet activation, therefore, is tightly regulated through controlled release of plateletactivating molecules including adenosine diphosphate (ADP), and thromboxane A 2 , and platelet inhibitory molecules including prostaglandin E 2 , prostaglandin I 2 (PGI 2 ), and nitric oxide (NO) (10,11).
We hypothesized that healthy men and women would have diminished platelet reactivity following oral nitrate ingestion if platelet NO production increases (12). As proof of principle that augmenting platelet NO production has beneficial effects, we conducted an investigation in which healthy men and women were administrated an oral nitrate load or placebo on 1 of 2 visits. We then assessed the effect of oral nitrate on blood nitrate concentration, platelet NO production, and platelet reactivity.

METHODS
DEMOGRAPHICS. The randomized controlled trial was approved by the Institutional Review Board (IRB) at the University of Rochester. A separate protocol was approved by the IRB at the University of Rochester and also the IRB at the Cleveland clinic to study translational aspects of platelet biology in vitro.
We evaluated in a randomized, double-blinded, placebo-controlled manner whether capsules containing nitrate (2 Berkeley Life Professional tabs, each containing 490 mg potassium nitrate) alter platelet NO production and receptor agonist sensitivity. Given that age and obesity were previously reported to affect platelet activation (13,14) 22 healthy Caucasian individuals were recruited (12 men, 10 women) who were without medical conditions, of similar age and body mass index, who were nonsmokers, not taking medications, and not taking supplements ( Figure 1). Each subject presented twice; once for the administration of nitrate then placebo in a random order by an individual not involved in the study. One male subject had obesity class 1 and 1 female subject was postmenopausal. The washout period was a minimum of 2 weeks between treatments. This time period was chosen given that NO lasts for minutes in the body and this was a 1-time dose of nitrate. In      Figure 8B). A pictorial representation of the effect of platelet NO in men and women is shown in Figure 9.
The key mechanistic differences with respect to NO production in platelets are that women require enteric absorption and processing of nitrate to generate nitrite and liberate NO in platelets, whereas platelets from men harbor a mechanism whereby nitrate can be used as a substrate for NO production.
Conversely, NOS can readily use L-Arginine as a substrate to markedly augment NO production in platelets from healthy women but there is little effect in platelets from healthy men.

DISCUSSION
In this randomized and double-blinded investigation, we confirm that platelet reactivity is markedly greater in young healthy women compared with young healthy men and especially through platelet PAR1.
We now show that oral nitrate, a molecule found in many vegetables, increases platelet reactivity in men and attenuates platelet reactivity in women coincident with augmented platelet NO production.
A critical distinction in the data between men and women is that enteric processing and absorption of nitrate is required for nitrate to augment platelet NO production in women, which does not appear to be a We demonstrated 10-fold greater NO production in platelets from women compared with men after an oral nitrate load. Because plasma and saliva nitrate were similar in men and women, this also suggests the possibility of a nitrate shuttle that permits reduction to nitrite and NO release locally inside the platelet.
Facultative anaerobes in the gut are well-known to reduce nitrate to nitrite enzymatically (26). Another explanation for our data is that the composition of gut microbiota required for NO reduction from nitrate may differ in men and women, as suggested (27).
Our data confirm previous reports that NO has an inhibitory effect on platelet activation, although we which is a substrate for endothelial nitric oxide synthase (eNOS) known to be expressed in platelets. NO is released following conversion to L-Citrulline in the presence and absence of a competitive eNOS inhibitor L-NG-Nitro arginine methyl ester (L-NAME) (300 mM, 30 minutes).
Nitrate (NO 3 À ) capsules (1 mg/mL, 30 minutes) were also exposed to healthy platelets ex vivo and the effect on NO production in the platelet was assessed. In a study that did not reveal the sexual identity of experimental mice, manipulating the blood nitrite/nitrate balance through dietary restriction or antibiotic use alters platelet reactivity (30), invoking the gut microbiome as a potential explanation for sex-specific differences in platelet NO production.
Sex-dependent differences in platelet reactivity and responsiveness to antiplatelet medications were previously suggested (2,5,6,31). Because VASP phosphorylation is well-known to inhibit platelet function (23,32), and phosphorylation of VASP at serine 239 is reported as a mechanism of platelet inhibition through the NO-VASP-PKG module (33), we offer this pathway as a potential mechanism for the sex-dependent difference in platelet reactivity to NO observed.
STUDY LIMITATIONS. A limitation of our study lies in the inclusion of premenopausal women with blood draws throughout the menstrual cycle. One report suggests differences in plasma-soluble P-selectin and