Phosphatidylserine exposure and red cell viability in red cell aging and in hemolytic anemia

Abstract

Phosphatidylserine (PS) normally localizes to the inner leaflet of cell membranes but becomes exposed in abnormal or apoptotic cells, signaling macrophages to ingest them. Along similar lines, it seemed possible that the removal of red cells from circulation because of normal aging or in hemolytic anemias might be triggered by PS exposure. To investigate the role of PS exposure in normal red cell aging, we used N-hydroxysuccinimide-biotin to tag rabbit red cells in vivo, then used phycoerythrin-streptavidin to label the biotinylated cells, and annexin V-fluorescein isothiocyanate (FITC) to detect the exposed PS. Flow cytometric analysis of these cells drawn at 10-day intervals up to 70 days after biotinylation indicated that older, biotinylated cells expose more PS. Furthermore, our data match a simple model of red cell senescence that assumes both an age-dependent destruction of senescent red cells preceded by several hours of PS exposure and a random destruction of red cells without PS exposure. By using this model, we demonstrated that the exposure of PS parallels the rate at which biotinylated red cells are removed from circulation. On the other hand, using an annexin V-FITC label and flow cytometry demonstrates that exposed PS does not cause the reduced red cell life span of patients with hemolytic anemia, with the possible exception of those with unstable hemoglobins or sickle cell anemia. Thus, in some cases PS exposure on the cell surface may signal the removal of red cells from circulation, but in other cases some other signal must trigger the sequestration of cells.

Figure 1

Density plots of exposed PS (measured by FITC fluorescence) vs. biotin (measured by PE fluorescence) in red cells before and after irreversible in vivo biotinylation (rabbit 2). The quadrants indicate cutoffs for determining biotinylation and exposed PS. Notice that biotinylated cells steadily decrease in number after biotinylation, and a greater proportion of them have exposed PS than do nonbiotinylated cells. These experiments were done in duplicate assays (only one shown) for both rabbit 1 and rabbit 2.

Figure 2

Graphs on the left show that older, biotinylated cells expose more PS in both rabbit 1 (gray points and curves) and rabbit 2 (black points and curves). The parameters for our model of red cell senescence were chosen to provide a least-squares fit to these data on exposed PS in biotinylated cells (r² > 0.98). Graphs on the right show that the model correctly predicts the rate at which biotinylated cells are removed from circulation (r² > 0.97). This supports the model, which assumes that the amount of exposed PS is proportional to the rate of senescent death.

Figure 3

Red cell aging in rabbit 1 (gray points and curves) and rabbit 2 (black points and curves). The parameters shown in Table 1 characterizing these curves were fitted to the experimental data [i(x) and j(x) above] and agree with results from Brown and Eadie (17). The best fit curves for i(x) and j(x) match the experimental data closely (r² > 0.98).