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 degrees 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 (RBCPS), 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 RBCPS. Moreover, the active influx of Zn2+ into RBCs induced by their co-incubation with both ZnCl2 and A23187 did not increase the percent of RBCPS 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.