Terminal density reversal and the role of Ca2+ in red blood cells clearance of healthy individuals

Density reversal of senescent red blood cells (RBCs) has been known for more than ten years, yet the identity of the candidate protein(s) is still elusive. While performing Percoll density gradient separation of RBCs from healthy individuals and their subsequent characterization, we identified a fraction of cells in the low-density fraction (~0.025% compared to total RBCs population) which shows reversal in their densities along with the characteristics of cellular senescence such as loss of membrane Band 3 protein and the phosphatidylserine exposure to the outer membrane leaflet. Our subsequent analysis showed that these cells are overloaded with Ca²⁺. We further measured intracellular [Na⁺] in individual RBCs by flow cytometry utilizing the dye CoroNa Green-AM.  Our findings showed that the cells with senescent characteristics lost their transmembrane Na⁺ gradient despite maintaining the membrane integrity. Consequently, these findings lead us to designate these cells as “senescent-like” cells. Our data further demonstrated altered activities of nonselective cation channels and pumps in these cells. In addition to a facilitated Na+ extrusion by Na⁺, K+-ATPase, our findings indicated altered ion transport via Piezo1 in these cells. Pharmacological modulation of Piezo1 with Yoda1/GsMTx4 showed that Piezo1 and, possibly, other nonselective cation channels by promiscuously transporting Na⁺ and Ca²⁺ play an important role in producing these low density “senescent like” cells.