In vitro stored red blood cells (RBC) and RBC artificially induced to vesiculate by incubation with dilauroylphosphatidyl-choline were monitored for age- and vesiculation-dependent changes in cell density, membrane lipid asymmetry, and their ability to be recognized and cleared by reticuloendothelial cells. RBC demonstrated a progressive increase in density on self-forming Percoll gradients upon vesiculation and in vitro “aging.” Uptake of vesiculated RBC by in vitro cultivated macrophages was increased threefold over non-vesiculated control RBC. The clearance rate of dense vesiculated RBC was biphasic and contained a rapid component and a slower second component consistent with the clearance rates of normal control populations. Determination of phosphatidylserine (PS) in the outer leaflet of RBC by the PS-dependent prothrombinase assay revealed that PS redistributed to the cell's outer leaflet upon in vitro storage and vesiculation. Inhibition of PS movement by oxidation of membrane sulfhydryls with pyridyldithioethylamine resulted in higher prothrombinase levels and enhanced clearance of vesiculated RBC. These experiments suggest that vesiculation contributes to alterations in membrane lipid asymmetry and cell density characteristic of the aged RBC phenotype.

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