Background: Oxidative stress is well-documented in people with hemolytic diseases and is reported in canine immune-mediated hemolytic anemia (IMHA). Oxidative stress induces erythrocyte membrane phosphatidylserine externalization in people, causing eryptosis and thrombus formation. This has not been evaluated in dogs. Acute thromboembolism is the leading cause of natural death in canine IMHA despite thromboprophylaxis, highlighting the need for novel therapeutic targets. Hypothesis/
Objectives: To determine if oxidative stress induces PS externalization in canine erythrocytes (objective 1), and if exposure to antioxidants prevents such changes (objective 2). Animals: Five healthy adult purpose-bred research Beagles.
Methods: In vitro, experimental study. Blood was collected from each dog and erythrocytes harvested. For objective 1, erythrocytes were exposed to pro-oxidant agents tert-butyl hydroperoxide (TBHP) at 2, 3, or 4 mM, or 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) at 30, 40, or 50 mM. For objective 2, erythrocytes were exposed to 3 mM TBHP and N-acetylcysteine-amide (NACA) at various concentrations (0, 1, or 3 mM). PS externalization was assessed using flow cytometry (Annexin V assay) with median fluorescence intensity (MFI) recorded.
Results: Tert-butyl hydroperoxide at 3 and 4 mM caused increased PS expression in erythrocytes (P < 0.05). AAPH at all concentrations caused increased PS expression (P < 0.01). NACA at all concentrations prevented significant PS expression from erythrocytes exposed to TBHP (P < 0.01). Conclusions and clinical importance: Oxidative stress causes PS externalization in canine erythrocytes and NACA ameliorates this effect. Future studies are needed to determine if this occurs in IMHA dogs and its role in promoting thromboembolism.
