The use of phospholipase C to detect structural changes in the membranes of human erythrocytes aged by storage

Human blood was stored under blood transfusion conditions for up to 10 weeks. At various times samples were removed, erythrocytes isolated and the susceptibility of the erythrocyte membrane lipids to non-lytic concentrations of phospholipase C from either Bacillus cereus or Clostridium perfringens tested. The morphology of the cells at various times and the release of microvesicles from the erythrocytes were also assessed. Initially the cells were attacked very little by the phospholipases at the concentrations chosen, but their susceptibility increased markedly after about 2 weeks, stabilised until 5 weeks, and then increased again to approach a nearly stable value after 8–10 weeks. The first rise accompanied the conversion of most of the cells to crenated and echinocytic configurations and was reversed if cells were incubated in a ‘rejuvenating’ medium designed to restore their energy supplies. The second rise occurred during the period when the cells underwent extensive microvesiculation and eventually became spherocytes: this phase involved, in particular, an increase in availability of phosphatidylethanolamine for hydrolysis by phospholipase C and was not reversed by attempts at ‘rejuvenation’. When microvesicles released from the cells were harvested and their phospholipase susceptibility compared with that of the residual cells it was found that the microvesicles were the more susceptible. These changes in phospholipase susceptibility presumably reflect subtle changes in membrane organization that occur during storage and vesiculation of erythrocytes; the possible nature of such changes is discussed.