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. 2014 Mar 18;9(3):e92435.
doi: 10.1371/journal.pone.0092435. eCollection 2014.

A flow cytometric analysis of the inhibition of platelet reactivity due to nitrite reduction by deoxygenated erythrocytes

Krittapoom Akrawinthawong  1 , Ji Won Park  2 , Barbora Piknova  2 , Nathawut Sibmooh  3 , Suthat Fucharoen  4 , Alan N Schechter  2
Affiliations

A flow cytometric analysis of the inhibition of platelet reactivity due to nitrite reduction by deoxygenated erythrocytes

Krittapoom Akrawinthawong et al. PLoS One. .

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.

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Conflict of interest statement

Competing Interests: The authors have the following interests: Dr. Alan Schechter is listed as a coinventor on several patents issued to the National Institutes of Health for the use of nitrite salts for the treatment of cardiovascular diseases. He receives royalties based on NIH licensing of these patents for clinical development but no other compensation. Gladwin, M.T., Cannon, III, R.O., and Schechter, A.N.: Use of nitrite salts for the treatment of cardiovascular conditions. U.S. Patent Appl. No. 10/563, 682 filed January 6, 2006, U.S. Patents 20,060,182,815; 20,070,154,569; 20,100,247,682. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Effect of DEANONOate on platelet activation in platelet-rich plasma + erythrocytes.
Platelet activation was induced by ADP (A, D, 20 μM), collagen (B, E, 20 μg/mL) and thrombin (C, F, 1 U/mL) in platelet-rich plasma + erythrocytes preincubated with DEANONOate at 37°C for 2 minutes. Platelet activation was analyzed by measuring the percentage of cells expressing P-selectin (A, B, C) and activated gpIIb/IIIa (D, E, F) on the membrane using specific antibodies (CD62P and PAC-1, respectively, mean Hct 40%, mean pO2 49 mmHg, n = 6).
Figure 2
Figure 2. Effect of nitrite on platelet activation in platelet-rich plasma + erythrocytes.
Platelet-rich plasma + erythrocytes were preincubated with nitrite (1 μM) at 37°C for 5 minutes and platelet activation was induced by ADP (A, D, 20 μM), collagen (B, D, 20 μg/mL) and thrombin (C, F, 1 U/mL). P-selectin (A, B, C) and activated gpIIb/IIIa expression (D, E, F) was monitored. (CD62P and PAC-1, respectively, mean Hct 40%, mean pO2 49 mmHg, n = 6).
Figure 3
Figure 3. Nitrite inhibits thrombin-induced platelet activation in the presence of deoxygenated erythrocytes.
Representative flow diagrams demonstrate the inhibitory effects of nitrite on the thrombin-induced activation of platelets. Platelet activation was analyzed by monitoring the percentage of cells expressing P-selectin (A, B, C) and activated gpIIb/IIIa (D, E, F) after thrombin (1 U/mL) stimulation in both control (blue) and nitrite (1 μM; orange) treated group in the presence of erythrocytes (Hct 42.6%) with different oxygen concentrations [pO2; 97.4±4.2 mmHg (A,D), 58.2±6.2 mmHg (B,E) and 26.7±2.8 mmHg (C,F), n = 6].
Figure 4
Figure 4. Effect of hematocrit changes on the thrombin-induced platelet activation in platelet-rich plasma + erythrocytes.
Nitrite (1 μM) was added to platelet-rich plasma + erythrocytes with various hematocrits (15.4±1.8, 23.0±2.3, 36.7±1.6, 40.2±1.2%) before thrombin (1 U/mL) stimulation and the percentage of cells expressing P-selectin and activated gpIIb/IIIa were measured (n = 6, * p<0.05).
Figure 5
Figure 5. Platelet inhibition by nitrite is mediated by its reduction to NO.
NO scavenger (CPTIO) or enzyme inhibitors (L-NAME for NOS and oxypurinol for XOR) were preincubated at 37°C for 5 minutes with platelet-rich plasma + erythrocytes before nitrite addition and then thrombin-induced P-selectin exposure and gpIIb/IIIa activation were analyzed (mean Hct 43.4%, pO2 55.4 mmHg, thrombin 1 U/mL, nitrite 1 μM, oxypurinol 100 μM, L-NAME 300 μM, CPTIO 200 μM, n = 6, * p<0.05).
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