Exposure of phosphatidylserine in the outer leaflet of human red blood cells. Relationship to cell density, cell age, and clearance by mononuclear cells

Abstract

Human red blood cells (RBC) were separated by density on self-forming Percoll gradients into five distinct populations. The transbilayer movement and equilibrium distribution of 1-oleoyl-2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4- yl)aminocaproyl)phosphatidylserine (NBD-PS) was slower in dense cells and equilibrated in the inner leaflet of these cells to a lesser degree than in light cells. Conversely, the outward movement of the lipid was slower in light cells. Assessment of endogenous PS in the cells' outer leaflet by the prothrombinase activity of externalized PS revealed an increase in its presence at the cell surface with increasing cell density. The presence of PS on the cell surface directly correlated with the propensity of the RBC to bound by autologous monocytes. To determine whether increased cell density is associated with increased cell age, the in vivo clearance of density-separated murine RBC was monitored in syngeneic mice. The T1/2 of circulation of light cells was about twice that of dense cells. The majority of the cleared cells localized in the spleen. Studies carried out in antibody-deficient SCID mice indicated that RBC were cleared via a mechanism that was independent of antibody. These data suggest that cell age is related to cell density and that cells of increasing age and density display higher amounts of PS in their outer leaflet.