CD47 on erythrocytes inhibits phagocytosis through interaction with the inhibitory immunoreceptor SIRP␣ expressed by macrophages. Thus, the CD47-SIRP␣ interaction constitutes a negative signal for erythrocyte phagocytosis. However, we report here that CD47 does not only function as a "do not eat me" signal for uptake but can also act as an "eat me" signal. In particular, a subset of old erythrocytes present in whole blood was shown to bind and to be phagocytosed via CD47-SIRP␣ interactions. Furthermore, we provide evidence that experimental aging of erythrocytes induces a conformational change in CD47 that switches the molecule from an inhibitory signal into an activating one. Preincubation of experimentally aged erythrocytes with human serum before the binding assay was required for this activation. We also demonstrate that aged erythrocytes have the capacity to bind the CD47-binding partner thrombospondin-1 (TSP-1) and that treatment of aged erythrocytes with a TSP-1-derived peptide enabled their phagocytosis by human red pulp macrophages. Finally, CD47 on erythrocytes that had been stored for prolonged time was shown to undergo a conformational change and bind TSP-1. These findings reveal a more complex role for CD47-SIRP␣ interactions in erythrocyte phagocytosis, with CD47 acting as a molecular switch for controlling erythrocyte phagocytosis. (Blood. 2012;119(23):5512-5521) IntroductionErythrocyte clearance has been studied for many years in the context of normal turnover, 1,2 enhanced clearance resulting from defects in erythrocyte metabolism or changes in membrane composition, [3][4][5] and in blood transfusion. 6 A large number of factors that may influence erythrocyte clearance have been proposed, ranging from autoantibody binding to band 3 after conformational changes resulting from aging (the "senescent cell antigen"), 7,8 to the expression of phosphatidylserine on the outer leaflet of the erythrocyte membrane. 9 In general, it is thought that erythrocytes are cleared after accumulating so-called "eat me" signals and are then phagocytosed by macrophages of the reticuloendothelial system, predominantly in the spleen and liver. 1,10,11 In contrast, one of the membrane proteins expressed by erythrocytes, CD47, has been shown to inhibit phagocytosis of erythrocytes by macrophages of the reticuloendothelial system. 12-16 CD47 exerts its inhibitory effect through binding to SIRP␣ on the macrophage, which induces inhibitory signaling by the immunoreceptor tyrosinebased inhibition motifs (ITIMs) residing in the cytoplasmic tail of SIRP␣. 17,18 On ligation of SIRP␣ by CD47, the SIRP␣ ITIMs recruit and activate tyrosine phosphatases SHP-1 and SHP-2, and this regulates, generally in a negative fashion, downstream signaling pathways and effector functions. The inhibitory effect of CD47 on erythrocyte clearance can be illustrated by transfusion of CD47-deficient erythrocytes into a wild-type recipient, which leads to rapid phagocytosis of the CD47-deficient erythrocytes by red pulp macrophages in the spleen. 12,19 ...

SummaryDuring storage, erythrocytes undergo changes that alter their clearance and function after transfusion and there is increasing evidence that these changes contribute to the complications observed in transfused patients. Stored erythrocytes were incubated overnight at 37°C to mimic the temperature after transfusion. After incubation, several markers for erythrocyte damage were analysed. After overnight incubation, stored erythrocytes showed increased potassium leakage, haemolysis, PS exposure and vesicle formation, and all these effects increased with increasing storage time. Furthermore, we demonstrated that long-term stored erythrocytes develop decreased flippase activity and increased scrambling activity after overnight incubation, leading to PS exposure and the release of vesicles. Reduced intracellular potassium was identified as the cause of the decreased flippase activity. Lastly, we provide evidence that erythrocytes can return to a PSnegative state by shedding parts of their membrane as PS-containing vesicles and that these vesicles can serve as a platform for the coagulation cascade. These findings reveal that potassium leakage, a well-known phenomenon of prolonged erythrocyte storage, primes erythrocytes for PS exposure. PS exposure will lead to vesicle formation and might have an important impact on the post-transfusion function and side effects of stored erythrocytes.

Storage of RBCs leads to an increased rate of hypoxia-induced nitrite reduction to NO and this is associated with increased methemoglobin formation. The increased methemoglobin formation and consequent decrease in oxygen delivery capacity might contribute to the storage-related impairment of aged RBCs to oxygenate the microcirculation.