title: A flow cytometric analysis of the inhibition of platelet reactivity due to nitrite reduction by deoxygenated erythrocytes.
abstract: Nitric oxide (NO), a small gas molecule, has long been known to be a potent inhibitor of platelet function but the physiological and pathological implications of platelet inhibition by NO have not been well clarified. We recently showed that the addition of nitrite to platelet-rich plasma in the presence of erythrocytes could inhibit platelet aggregation and this inhibitory effect of nitrite + erythrocytes was enhanced by deoxygenation of erythrocytes as measured by P-selectin expression and cGMP production. In order to study the nitrite effect on platelets at different oxygen levels, we used the flow cytometric assays to detect platelet membrane surface markers upon activation. The P-selectin and activated gpIIb/IIIa expression on platelet membranes in response to ADP, collagen and thrombin stimulation was measured at various hematocrit and oxygen levels. Nitrite (0.1 to 1.0 μM) significantly decreased the percentage of these surface markers on the platelet membrane at the hematocrit values above 23% and oxygen levels lower than 49 mmHg. The inhibitory effect of nitrite was augmented by increasing hematocrit values and decreasing oxygen saturation. C-PTIO (an NO scavenger) prevented the platelet inhibition by nitrite + erythrocytes whereas the inhibitors of NO synthase and xanthine oxidoreductase had no effect. These results support the proposal that circulating nitrite decreases platelet reactivity in the presence of partially deoxygenated erythrocytes through its reduction to NO, which may also explain certain differences between arterial and venous thrombosis and support directly the role of deoxyhemoglobin in this process. We believe that our flow cytometric assays offer a possibility to identify the individual molecular process involved in these effects.
authors.affiliation: Molecular Medicine Branch, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States of America; Department of Biochemistry, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand.
authors.forename: Krittapoom
authors.initials: K
authors.lastname: Akrawinthawong
authors.affiliation: Molecular Medicine Branch, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Ji Won
authors.initials: JW
authors.lastname: Park
authors.affiliation: Molecular Medicine Branch, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Barbora
authors.initials: B
authors.lastname: Piknova
authors.affiliation: Department of Pharmacology, Faculty of Science, Mahidol University, Bangkok, Thailand.
authors.forename: Nathawut
authors.initials: N
authors.lastname: Sibmooh
authors.affiliation: Thalassemia Research Center, Institute of Science and Technology for Research and Development, Mahidol University, Nakhonpathom, Thailand.
authors.forename: Suthat
authors.initials: S
authors.lastname: Fucharoen
authors.affiliation: Molecular Medicine Branch, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Alan N
authors.initials: AN
authors.lastname: Schechter
mesh_terms: D000328:Adult; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology*; D015687:Cell Hypoxia; D004912:Erythrocytes / Q000378:metabolism*; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D006834:Hydrazines / Q000494:pharmacology; D008297:Male; D009569:Nitric Oxide / Q000502:physiology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D010084:Oxidation-Reduction; D015539:Platelet Activation; D053657:Platelet-Rich Plasma / Q000502:physiology; D013917:Thrombin / Q000502:physiology
chemical_list: D006834:Hydrazines; D020030:Nitric Oxide Donors; D009569:Nitric Oxide; C084012:1,1-diethyl-2-hydroxy-2-nitrosohydrazine; D013917:Thrombin