Effects of cell-free hemoglobin and NO exposure on platelet activation. (A) The in vitro exposure of purified human cell-free hemoglobin to platelets from healthy donors increased the percentage of activated platelets as indicated by cell-surface expression of activated GPIIb/IIIa (Plt + Hb; ■). The NO donor MAHMA-NONOate (50 μM) inhibited baseline activation, but this inhibitory effect was overcome by the NO-scavenging activity of cell-free hemoglobin in a dose-dependent manner (Plt + Hb + NO; ▲). (B) Platelet activation was induced significantly by cell-free hemoglobin. Activation was inhibited by the NO donor, but this inhibition was eliminated by the addition of 50 μM cell-free hemoglobin. The markers and bars represent the mean percentages (± SEM) of activated platelets after 3 experiments of healthy donor samples (*P < .05 compared with baseline samples; †P < .05 compared with NO alone; paired t test). (C) The induction of platelet activation by ADP was nearly abolished by the NO donor, but this action was again inhibited by cell-free hemoglobin (50 μM).