Size Dependence of Ghosts from Stored Erythrocytes on Calcium and Adenosine‐Triphosphate

Heterogeneity within ghosts prepared from stored erythrocytes was studied by multichannel particle analysis of size distributions of reconstituted ghosts. On restoration of isotonicity of hemolysates from fresh erythrocytes, ghosts were smaller than the corres- ponding red cells. After subsequent incubation for 15 mm, they swelled to a final stable volume. The hemolysis of stored erythrocytes and restoration of isotonicity yielded ghosts similar to those from fresh cells, but, after sub- sequent incubation, a second popula- tion of small ghosts appeared. This population resulted from marked!y diminished ghost swelling, that is at- tributed to decreased sodium permea- bility and increased membrane rigidity, causing resistance to changes in volume. Erythrocyte ATP depletion during storage of ACD blood was as- sociated with an increase in the per- centage of small ghosts. Similar ghosts were produced from fresh cells when CaCI2 (1 X 10-6 M) was introduced into ghosts during hemolysis. The erythro- cyte calcium content increased during prolonged storage from 11.3 ± 6.3 izg atoms/liter of cells (stored for 1-2 wk) to 22.0 ± 7.0 g atoms/liter of cells, after 7-9 wk of storage. The introduc- tion of nucleotides (2 X 10 M) into ,ghosts during hemolysis of stored cells prevented the appearance of small ghosts. The inhibitory effect was greater than predicted from stability constants of calcium nucleotide cam- plexes, indicating the specific inter- action of nucleotides with the mem- brane. The percentage of small ghosts from red cells stored for various times agreed with the percentage of non- viab!e cells derived from the litera- ture. It is concluded that the decrease in ATP and the accumulation of cal- cium in stored erythrocytes induce conformational changes of membrane fibrous (contractile) proteins that re- sults in decreases in membrane elas- ticity and permeability, which is in turn reflected by formation of small ghosts. p ECENT STUDIES have suggested that the posttransfusion viability of red _I_ cells stored in ACD solution is determined by physical properties of erythrocyte membranes'.2 This has been indicated by correlations between red cell posttransfusion viability and filterability, deformability, osmotic fragil- ity, and shape of red cells and their ghosts.'4 Two significant observations emerged from these studies. (1) Shape abnormalities and bimodal derivative curves of osmotic fragility of stored red cells suggested that membrane