P40: Platelet activation in the presence of nitrite, nitric oxide and heme

Abstract

Background NO stimulates intercellular cGMP production leading to a reduction in platelet activation. Nitrite is reduced to nitric oxide (NO) by deoxygenated hemoglobin and therefore acts as a potential source of biological NO. Nitrite in the presence of red blood cells (RBCs) has been shown to reduce platelet aggregation and this reduction in aggregation is enhanced when RBCs are deoxygenated. In addition nitrite in the presences of deoxygenated RBCs has been shown to reduce platelet expression of p-selectin when platelets are activated by ADP. Methods Platelet rich plasma was separated from whole blood by centrifugation at 120 g for 10 min. Plasma was diluted 10-fold into phosphate buffered saline and exposed to nitrite, NO, and/or RBC, as indicated. Platelets were then activated by ADP After 10 min of incubation with ADP, platelets were labeled with fluorescent antibodies for platelet recognition (CD41 or CD61) and activation (Pac-1 or CD62P) and measured using flow cytometry. Results Here we confirm NO reduces platelet activation. However, in the presence of oxyhemoglobin (Hb) NO no longer reduces platelet activation. MetHb, which does not rapidly scavenge NO does not affect platelet activation reduction by NO. In our research we see similar trends in decreased platelet activation with increasing concentrations of nitrite in the presences on RBCs. However our results are not as sensitive to nitrite concentrations as previously published data. In addition we see trends of decreasing platelet activation with increasing nitrite concentration in the presence of Hb. Conclusions Our results verify the effect of NO on platelet activation. They also show a trend which agrees with previously published data showing a decrease in platelet activation in the presence of nitrite and red blood cells. Additionally we see the same trend in the presence of nitrite and cell free Hb. Disclosure Supported by NIH Grants HL058091 and HL098032.