Recovery of membrane micro-vesicles from human erythrocytes stored for transfusion: a mechanism for the erythrocyte discocyte-to-spherocyte shape transformation.

It has been shown for fresh human erythrocytes that when intracellular concentrations of calcium are elevated a form of echinocytosis is induced (Allan & Michell, 1975; White, 1974). The resulting echinocytes then lose cytosol-filled vesicles to the medium (Allan et al., 1976). It therefore seemed possible that the more slowly developing echinocytosis which occurs in human erythrocytes during storage for transfusion at 4°C (Rumsby er al., 1974) might also be followed by the loss of membrane microvesicles into the surrounding plasma and that this process might provide the mechanism by which lipid is lost from aging erythrocytes undergoing the disc-to-sphere shape change, in which some 20% or more of the erythrocyte total phospholipid is lost (Fig. 1). This would also explain the irreversible nature of the disc-to-sphere transformation shown by aged cells. Therefore a search was made for membrane micro-vesicles in human blood aged in vitro in citric acid/sodium citrate/dextrose at 4°C. Donations of blood (Group 0 and A, Rhesus positive) in acid/citrate/dextrose were stored under Blood Transfusion Service conditions at 4°C for up to 8 weeks. Periodically, samples of blood were taken and tested for bacterial contamination (none found throughout the study) before the erythrocytes and leucocytes were sedimented by centrifugation at l000g for l0min. The plasma was withdrawn and centrifuged at 7000g for 15min to ensure the removal of leucocyte fragments and platelets. Subsequently the 7000g supernatant was centrifuged at 20000g for 30min and the supernatant from this step was then centrifuged at l00000g for 30min. Membrane microvesicles would be expected to sediment under these conditions (Allan et al., 1976). The pellets obtained by this procedure were washed by resuspension in 0 . 1 5 ~ NaCl at 4°C and recovered by re-centrifugation. With this procedure no pellets were obtained with either freshly drawn blood or with blood stored for 1 week. However, from week 2 to week 8 increasing amounts of a red pellet were obtained at 20000g and at 100000g, with about twice as much material in the lower-speed pellet. Electron microscopy of both pellets showed that they consisted of membrane-bounded micro-vesicles. These were approx. 100 nm in diameter, had electrondense interiors and appeared essentially identical with the cytosol-aled membrane micro-vesicles which are released from echinocytes induced by high intracellular calcium concentrations (Allan ef al., 1976). During the aging of human erythrocytes in vitro the molar cholesterol/phospholipid ratio of the membrane decreases slightly from the normal value of about 0.8 to around 0.65 (M. G. Rumsby, unpublished work). Analysis of the lOOOOOg micro-vesicles from blood aged in uitro showed that these had a molar cholesterol/phospholipid ratio similar to that of the parent erythrocytes. The phospholipid composition of the microvesicles was similar to that of the erythrocytes, except that they were slightly enriched in sphingomyelin. The micro-vesicles were enriched in 1 ,2-diacylglycerol, a lipid which also accumulates to a marked extent in the microvesicles released by erythrocytes having an elevated intracellular calcium content (Allan et al., 1976). Diacylglycerol concentrations were about four times as high in the micro-vesicles as in theerythrocytes from which they were derived. Analyses of erythrocyte ‘ghosts’ and of the micro-vesicle fractions from whole blood by gel electrophoresis show (Fig. 2) that the polypeptides of the spectrin complex (bands 1 and 2) are almost absent from the micro-vesicle membranes, as they were from micro-vesicles formed from erythrocytes with high internal calcium concentrations.