Effect of Blood Nitrite and Nitrate Levels on Murine Platelet Function

Ji Won Park,Alan N Schechter,Barbora Piknova,Constance T Noguchi,Paul L Huang,Andreas Zirlik

doi:10.1371/journal.pone.0055699

Abstract

Nitric oxide (NO) appears to play an important role in the regulation of thrombosis and hemostasis by inhibiting platelet function. The discovery of NO generation by reduction of nitrite (NO2 −) and nitrate (NO3 −) in mammals has led to increased attention to these anions with respect to potential beneficial effects in cardiovascular diseases. We have previously shown that nitrite anions at 0.1 µM inhibit aggregation and activation of human platelet preparations in vitro in the presence of red blood cells and this effect was enhanced by deoxygenation, an effect likely due to NO generation. In the present study, we hypothesized that nitrite and nitrate derived from the diet could also alter platelet function upon their conversion to NO in vivo. To manipulate the levels of nitrite and nitrate in mouse blood, we used antibiotics, NOS inhibitors, low nitrite/nitrate (NOx) diets, endothelial NOS knock-out mice and also supplementation with high levels of nitrite or nitrate in the drinking water. We found that all of these perturbations affected nitrite and nitrate levels but that the lowest whole blood values were obtained by dietary restriction. Platelet aggregation and ATP release were measured in whole blood and the results show an inverse correlation between nitrite/nitrate levels and platelet activity in aggregation and ATP release. Furthermore, we demonstrated that nitrite-supplemented group has a prolonged bleeding time compared with control or low NOx diet group. These results show that diet restriction contributes greatly to blood nitrite and nitrate levels and that platelet reactivity can be significantly affected by these manipulations. Our study suggests that endogenous levels of nitrite and nitrate may be used as a biomarker for predicting platelet function and that dietary manipulation may affect thrombotic processes.

Highlights

Platelets are discoid non-nucleated cells that circulate freely in the plasma and are highly reactive entities that play a critical role in hemostasis and thrombosis both as a surface for clotting of plasma proteins and as a component of the clot itself
Low NOx diet treatment for a week resulted in dramatic decreases in both nitrite (64%) and nitrate (72%) in WT mice and there was no clear difference between WT and endothelial NOS (eNOS) knock-out in nitrite and nitrate levels under low NOx diet
The endogenous NOS system partially contributes to the changes of nitrite and nitrate levels, a few animal studies [32,33,34], in which low NOx diet was used for dietary nitrite and nitrate depletion, show that blood and tissue nitrite and nitrate levels were significantly reduced under the low NOx diet suggesting the remarkable contribution of diet to steady-state levels of nitrite and nitrate

Summary

Introduction

Platelets are discoid non-nucleated cells that circulate freely in the plasma and are highly reactive entities that play a critical role in hemostasis and thrombosis both as a surface for clotting of plasma proteins and as a component of the clot itself. A number of stimuli are known to stimulate these platelet activities by modulating expression of surface glycoproteins, shape change, granule secretion, adhesion, and aggregation [1]. Collagen, one of the major components in the vessel wall, is exposed at the site of injury and initiates platelet adhesion by interacting with platelets through their glycoprotein and integrin receptors [2]. To maintain normal vascular flow and responses to injury, there must be a tight balance between proand anti-thrombotic signals within the circulation and platelet function is delicately regulated under physiological conditions. The ability of NO to regulate cGMP via activation of sGC and

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Results

Conclusion