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.