RBC Hemolysis and Platelet Activation in Paroxysmal Nocturnal Hemoglobinuria Are Mediated by Oxidative Stress That Can Be Ameliorated by Antioxidants.

Paroxysmal Nocturnal Hemoglobinuria (PNH) is an acquired stem cell disorder characterized by the unique triad of intravascular hemolysis, bone marrow failure and thrombosis. Complement-mediated hemolysis in PNH is explained by a deficiency in glycosylphosphotidylinositol (GPI)-anchored proteins, CD55 and CD59 on the RBC surface.

Using flow cytometry for simultaneous measurement of oxidative-state markers in RBC and platelets, we studied blood samples obtained from normal donors and PNH patients. Reactive oxygen species (ROS), reduced glutathione (GSH), membrane lipid peroxidation and phosphatidylserine (PS) exposure were measured by staining with dichlorofuorescein, mercury orange, fluor-DHPE and Annexin V, respectively, followed by gating based on size and granularity. The results of blood samples obtained from normal donors (N=25) and PNH patients (N=11) indicated that the RBC from the patients had higher levels of ROS (1.4-fold) and lipid peroxides (3.5-fold) and lower GSH levels (two-fold) than those of normal RBC. Similar results were obtained with platelets from the same samples, indicating that both cell types are in a state of oxidative stress. Within each cell type, cells with the PNH immunophenotype (CD55⁻ CD59⁻) had higher ROS levels and PS exposure than cells with the normal (CD55⁺CD59⁺) phenotype. These results support the direct relationship between the PNH phenotype and intracellular oxidative stress. Oxidants, such as hydrogen peroxide, phenylhydrazine, hemin, ferric ammonium citrate, increased the oxidative status of the cells and caused concentration-dependent RBC lysis and platelet activation. Complement-containing plasma had a similar effect. The RBC lysis and platelet activation were preceded by a burst of ROS and were more prominent in cells with the PNH phenotype. Incubation of PNH-cells with antioxidants such as N-acetyl cysteine, vitamin C and tocotrinol, prevented the complement-mediated lysis.

We previously reported that cellular oxidative stress, prevalent in conditions such as thalassemia, may be associated with RBC hemolysis, platelet activation and a decreased PMN antibacterial potential. Oxidative stress in RBC, platelets and neutrophils in PNH could in part account for clinical manifestations such as hemolysis, a hypercoagulable state and recurrent bacterial infections. Our present results, indicating that incubation with antioxidants ameliorates such stress and its adverse consequences, suggest the possibility of using antioxidants for treatment of PNH and similar conditions.