Platelet Aggregation Is Inhibited by Nitrite Reduction to Nitric Oxide In Blood
Authors: Schechter Alan N. , Srihirun Sirada , Sriwantana Thanaporn , Unchern Supeenun , Noulsri Egarit , Pattanapanyasat Kovit , Fucharoen Suthat , Piknova Barbora , Sibmooh Nathawut 
Published: October 2019

Abstract
Abstract 3189
Nitrite is now recognized as a circulating and storage form of nitric oxide (NO), whose biological effect is largely mediated by its chemical reduction to NO via the nitrite reductase activity of heme-containing proteins and other enzymatic reactions. NO activates soluble guanylyl cyclase to produce cGMP, leading to vasodilation that facilitates tissue oxygen supply, but which is also known to strongly inhibit platelet aggregation. Although the vasorelaxation effect of nitrite has recently been clarified, the effect of nitrite, especially at physiologic levels, on platelet function has not been well studied. We hypothesized that platelet inhibition by nitrite could occur under the conditions known to promote its reduction to NO, such as the presence of deoxygenated erythrocytes. We found that nitrite up to 100 μM had no effect on platelet aggregation in plasma; however, in the presence of erythrocytes inhibition of platelet aggregation by nitrite was observed, which was dependent on hematocrit. At 20% hematocrit, nitrite at physiologic concentration (0.1 mM) significantly inhibited platelet aggregation induced by either ADP or collagen. In contrast, DEANONOate, an NO donor, at low concentrations inhibited platelet aggregation in plasma, but its effect was abolished by erythrocytes at even 1% hematocrit. C-PTIO, a membrane impermeable NO scavenger, completely inhibited the effect of nitrite plus erythrocytes on platelets, suggesting that nitrite was converted to NO in or on the surface of the erythrocytes and then entered the platelets. Furthermore, deoxygenation increased the inhibitory effect of nitrite plus erythrocytes on platelets as demonstrated by a decrease of P-selectin expression (a marker of platelet degranulation) and a corresponding increase in cGMP levels in the platelets. We also found negative correlations between platelet aggregation induced by ADP and nitrite concentrations in whole blood and erythrocyte suspensions from various individuals. These results suggest an essential role of endogenous nitrite as a source of NO, produced by erythrocytes and perhaps other cells in the vasculature, in modulating platelet activity in the circulation. Although nitrite alone at physiological concentrations does not have a direct inhibitory effect on platelets, nitrite plus erythrocytes, which simulate the normal circulating blood, inhibit platelet aggregation and the effect is promoted by deoxygenation. The net effect is modulated by the tendency of oxyhemoglobin in erythrocytes to destroy NO and will likely depend on relative concentrations and the kinetics of the competing reactions, including the mechanisms by which NO activity may leave the erythrocyte. We suggest that these reactions may contribute to differences in the mechanisms of hemostasis in arterial and venous blood and might be the basis of pharmacological approaches to preventing thrombosis.

Disclosures:
Schechter: National Institutes of Health: I am a co-inventor of a patent for the use of nitrites in the treatment of cardiovascular diseases., Patents & Royalties.