Platelet inhibition by nitrite is dependent on erythrocytes and deoxygenation

Abstract

Background: Nitrite is a nitric oxide (NO) metabolite in tissues and blood, which can be converted to NO under hypoxia to facilitate tissue perfusion. Although nitrite is known to cause vasodilation following its reduction to NO, the effect of nitrite on platelet activity remains unclear. In this study, the effect of nitrite and nitrite+erythrocytes, with and without deoxygenation, on platelet activity was investigated.

Methodology/finding: Platelet aggregation was studied in platelet-rich plasma (PRP) and PRP+erythrocytes by turbidimetric and impedance aggregometry, respectively. In PRP, DEANONOate inhibited platelet aggregation induced by ADP while nitrite had no effect on platelets. In PRP+erythrocytes, the inhibitory effect of DEANONOate on platelets decreased whereas nitrite at physiologic concentration (0.1 µM) inhibited platelet aggregation and ATP release. The effect of nitrite+erythrocytes on platelets was abrogated by C-PTIO (a membrane-impermeable NO scavenger), suggesting an NO-mediated action. Furthermore, deoxygenation enhanced the effect of nitrite as observed from a decrease of P-selectin expression and increase of the cGMP levels in platelets. The ADP-induced platelet aggregation in whole blood showed inverse correlations with the nitrite levels in whole blood and erythrocytes.

Conclusion: Nitrite alone at physiological levels has no effect on platelets in plasma. Nitrite in the presence of erythrocytes inhibits platelets through its reduction to NO, which is promoted by deoxygenation. Nitrite may have role in modulating platelet activity in the circulation, especially during hypoxia.

Figure 1. Nitrite had no effect on platelet aggregation in plasma.

(A) Nitrite had no effect on ADP and collagen-induced platelet aggregation. Nitrite was pre-incubated in PRP for 5 min. Platelet aggregation was induced by 8 µM ADP or 2.5 µg/mL collagen. (B) DEANONOate inhibited ADP-induced platelet aggregation. DEANONOate was pre-incubated in PRP for 5 min. Platelet aggregation was induced by 8 µM ADP. (C) C-PTIO inhibited the anti-platelet activity of DEANONOate. PRP was incubated with 1 µM DEANONOate and 200 µM C-PTIO for 5 min before the induction of aggregation by 8 µM ADP. * P<0.05 compared with the others (ANOVA). All experiments were performed at 37°C. Data are means ± SEM (n≥3).

Figure 2. Nitrite+erythrocytes inhibited platelet aggregation.

(A) ADP induced platelet aggregation in PRP+erythrocytes (20% hematocrit) in the concentration dependent manner. (B) Erythrocytes at 0, 1, 10 and 20% hematocrit did not have effect on ADP-induced platelet aggregation in the absence of nitrite. (C) Nitrite+erythrocytes (20% hematocrit) inhibited ADP-induced platelet aggregation. Nitrite was incubated in PRP or PRP+erythrocytes in the presence or absence of 200 µM C-PTIO for 5 min before induction of aggregation by ADP. ^* P<0.05 compared with PRP and PRP+erythrocytes+C-PTIO (ANOVA). (D) The inhibitory effect of 0.1 µM nitrite was dependent on hematocrits. PRP and PRP+erythrocytes (1, 10, and 20% hematocrit) were incubated with 0.1 µM nitrite for 5 min before induction of platelet aggregation by 20 µM ADP. ADP-induced platelet aggregation in the absence of nitrite was shown in the control group. ^* P<0.05 compared with PRP+nitrite at 0% hematocrit (ANOVA). (E) Nitrite+erythrocytes inhibited collagen-induced platelet aggregation. PRP or PRP+erythrocytes samples (20% hematocrit) were incubated with nitrite in the presence and absence of 200 µM C-PTIO for 5 min and then the aggregation was induced by 2.5 µg/mL collagen. ^* P<0.05 compared with PRP and PRP+erythrocytes+C-PTIO (ANOVA). (F) Nitrite+erythrocytes inhibited U46619-induced platelet aggregation. 0.1 µM nitrite was incubated in PRP or PRP+erythrocytes (20% hematocrit) in the presence or absence of 200 µM C-PTIO for 5 min. Then, the aggregation was induced by 1 µM U46619. ^* P<0.05 compared with PRP+erythrocytes and PRP+erythrocytes+C-PTIO (ANOVA). All experiments were performed at 37°C. Data are means ± SEM (n≥3).

Figure 3. Nitrite+erythrocytes inhibited ATP release from platelets induced by ADP (A), collagen (B), and U46619 (C).

PRP+erythrocytes (20% hematocrit) were incubated with 0.1 µM nitrite in the presence and absence of 200 µM C-PTIO for 5 min. The ATP release was induced by 20 µM ADP, 2.5 µg/mL collagen or 1 µM U46619. ^* P<0.05 compared with PRP+erythrocytes and PRP+erythrocytes+nitrite+C-PTIO (ANOVA). All experiments were performed at 37°C. Data are means ± SEM (n = 5).

Figure 4. Erythrocytes abolished the effect of DEANONOate on platelet aggregation.

(A) Inhibition of platelet aggregation by DEANONOate decreased in the presence of erythrocytes. PRP and PRP+erythrocytes (20% hematocrit) were incubated with DEANONOate for 5 min. (B) Dependence of platelet aggregation in the presence of 0.1 µM DEANONOate on hematocrit. PRP and PRP+erythrocytes (1, 10, and 20% hematocrit) were incubated with 0.1 µM DEANONOate for 5 min. Platelet aggregation was induced by 20 µM ADP. ADP-induced platelet aggregation in the absence of nitrite was shown in the control group ^* P<0.05 compared with 0% hematocrit (ANOVA). All experiments were performed at 37°C. Data are means ± SEM (n≥3).

Figure 5. Deoxygenation enhanced platelet inhibition by nitrite+erythrocytes.

PRP was pre-incubated with nitrite (0.1 or 1 µM) or nitrite+C-PTIO (200 µM) in the presence of oxygenated or deoxygenated erythrocytes (20% hematocrit) for 5 min. Then, 20 µM ADP was added to the cell suspension and incubated for 10 min. Platelet activation was determined by the flow cytometry as an expression of P-selectin. ^* P<0.05 compared with the values of oxygenated erythrocytes. ^# P<0.05 compared with deoxygenated erythrocytes+C-PTIO (ANOVA). (B) Nitrite+erythrocytes increase the cGMP levels in platelets. Washed platelets+erythrocyte samples (20% hematocrit) were incubated with 0.1 µM nitrite for 5 min. ^* P<0.05 compared with platelets. ^# P<0.05 compared with platelets+nitrite+erythrocytes (ANOVA). All experiments were performed at 37°C. Data are means ± SEM (n≥4).

Figure 6. Platelet aggregation in whole blood showed inverse correlation with the nitrite levels in whole blood and erythrocytes.

Platelet aggregation in whole blood (20% hematocrit) was induced by 10 µM ADP and monitored for 5 min using the impedance aggregometry. The degree of platelet aggregation was correlated with the nitrite levels in whole blood (A) and erythrocytes (B). Each dot represents mean of duplicate measurement (n = 15).