Non-oxidative band-3 clustering agents cause the externalization of phosphatidylserine on erythrocyte surfaces by a calcium-independent mechanism

Aging of red blood cells (RBCs) is associated with alteration in a wide range of RBC features, occurring each on its own timescale. A number of these changes are interrelated and initiate a cascade of biochemical and structural transformations, including band-3 clustering and phosphatidylserine (PS) externalization. Using specific band-3 clustering agents (acridine orange (AO) and ZnCl₂), we examined whether treatment of RBCs with these agents may affects PS externalization and whether this process is Ca²⁺-dependent. RBCs were isolated from the blood of eight healthy donors upon obtaining their informed consent. The suspension was supplemented with increasing concentrations of AO or ZnCl₂ (from 0.5 to 2.0 mM) and incubated at 25 °C for 60 min. To detect PS at the RBC surface, we used allophycocyanin-conjugated recombinant human Annexin V. We demonstrated, that treatment of RBCs with both clustering agents caused an elevation in the percent of cells positively labeled by Annexin-V (RBC^PS), and that this value was not dependent on the presence of calcium in the buffer: RBCs treated with AO in the presence of either EDTA, EGTA or calcium exhibited similar percentage of RBC^PS. Moreover, the active influx of Zn²⁺ into RBCs induced by their co-incubation with both ZnCl₂ and A23187 did not increase the percent of RBC^PS as compared to RBCs incubated with ZnCl₂ alone. Taken together, these results demonstrate that the band-3 clustering agents (AO or ZnCl₂) induce PS externalization in a Ca²⁺ independent manner, and we hereby suggest a possible scenario for this phenomenon.