Summary Mature, circulating erythrocytes undergo senescence, which limits their life span to approximately 120 d. Upon injury, erythrocytes may undergo suicidal erythrocyte death or eryptosis, which may accelerate senescence and shorten their survival. Eryptosis is defined as cell shrinkage and exposure of phosphatidylserine at the cell surface. Triggers of eryptosis include oxidative stress. The present study addresses the impact of erythrocyte age on the relative susceptibility to eryptosis. Erythrocytes were separated into five fractions, based on age‐associated differences in density and volume. Cell membrane scrambling was estimated from binding of annexin V to phosphatidylserine at the erythrocyte surface, the cell volume from forward scatter, and the Ca2+ level from Fluo‐3‐dependent fluorescence. In addition, glutathione (GSH) concentrations were measured by an enzymatic/colourimetric method. After 48 h incubation in Ringer solution, Annexin V binding increased significantly with erythrocyte age. The differences were not accompanied by altered GSH concentrations, but were reversed by addition of the antioxidant N‐acetyl‐l‐cysteine in vitro. Also, N‐acetyl‐l‐cysteine significantly prolonged the half‐life of circulating mouse erythrocytes in vivo. Thus, the susceptibility to eryptosis increases with the age of the erythrocytes, and this effect is at least partially due to enhanced sensitivity to oxidative stress.

Inflammation enhances the secretion of sphingomyelinases (SMases). SMases catalyze the hydrolysis of sphingomyelin into phosphocholine and ceramide. In erythrocytes, ceramide formation leads to exposure of the removal signal phosphatidylserine (PS), creating a potential link between SMase activity and anemia of inflammation. Therefore, we studied the effects of SMase on various pathophysiologically relevant parameters of erythrocyte homeostasis. Time-lapse confocal microscopy revealed a SMase-induced transition from the discoid to a spherical shape, followed by PS exposure, and finally loss of cytoplasmic content. Also, SMase treatment resulted in ceramide-associated alterations in membrane–cytoskeleton interactions and membrane organization, including microdomain formation. Furthermore, we observed increases in membrane fragility, vesiculation and invagination, and large protein clusters. These changes were associated with enhanced erythrocyte retention in a spleen-mimicking model. Erythrocyte storage under blood bank conditions and during physiological aging increased the sensitivity to SMase. A low SMase activity already induced morphological and structural changes, demonstrating the potential of SMase to disturb erythrocyte homeostasis. Our analyses provide a comprehensive picture in which ceramide-induced changes in membrane microdomain organization disrupt the membrane–cytoskeleton interaction and membrane integrity, leading to vesiculation, reduced deformability, and finally loss of erythrocyte content. Understanding these processes is highly relevant for understanding anemia during chronic inflammation, especially in critically ill patients receiving blood transfusions.

During storage, RBCs develop an increased susceptibility to stress-induced loss of phospholipid asymmetry that is especially associated with an aging phenotype. This increased susceptibility may be responsible for the rapid disappearance of a considerable fraction of the RBCs during the first 24 hours after transfusion.

Red blood cells (RBCs) undergo extensive deformation when travelling through the microcapillaries. Deformability, the combined result of properties of the membrane-cytoskeleton complex, the surface area-to-volume ratio, and the hemoglobin content, is a critical determinant of capillary blood flow. During blood bank storage and in many pathophysiological conditions, RBC morphology changes, which has been suggested to be associated with decreased deformability and removal of RBC. While various techniques provide information on the rheological properties of stored RBCs, their clinical significance is controversial. We developed a microfluidic approach for evaluating RBC deformability in a physiologically meaningful and clinically significant manner. Unlike other techniques, our method enables a high-throughput determination of changes in deformation capacity to provide statistically significant data, while providing morphological information at the single-cell level. Our data show that, under conditions that closely mimic capillary dimensions and flow, the capacity to deform and the capacity to relax are not affected during storage in the blood bank. Our data also show that altered cell morphology by itself does not necessarily affect deformability.