Phosphatidylserine Exposure in Human Red Blood Cells Depending on Cell Age

Abstract

Background/Aims: The exposure of phosphatidylserine (PS) on the outer membrane leaflet of red blood cells (RBCs) serves as a signal for suicidal erythrocyte death or eryptosis, which may be of importance for cell clearance from blood circulation. PS externalisation is realised by the scramblase activated by an increase of intracellular Ca²⁺ content. It has been described in literature that RBCs show an increased intracellular Ca²⁺ content as well as PS exposure when becoming aged up to 120 days (which is their life span). However, these investigations were carried out after incubation of the RBCs for 48 h. The aim of this study was to investigate this effect after short-time incubation using a variety of stimulating substances for Ca²⁺ uptake and PS exposure. Methods: We separated RBCs by age in five different fractions by centrifugation using Percoll density gradient. The intracellular Ca²⁺ content and the PS exposure of RBCs with different age has been investigated after treatment with lysophosphatidic acid (LPA) as well as after activation of protein kinase C (PKC) using phorbol-12 myristate-13 acetate (PMA). For positive control RBCs were treated with 4-bromo-A23187. Measurement techniques included flow cytometry and live cell imaging (fluorescence microscopy). Results: The percentage of RBCs showing increased Ca²⁺ content as well as the PS exposure did not change significantly in dependence on cell age after short-time incubation in control experiments (without stimulating substances) or using LPA or PMA. However, we confirm findings reported that Ca²⁺ content and the PS exposure of RBCs increased after 48 h incubation. Conclusion: No significant differences of intracellular Ca²⁺ content and PS exposure can be seen for RBCs of different age in resting state or after stimulation of Ca²⁺ uptake at short-time incubation.