Storage of erythrocytes induces conformational changes in CD47 and TSP-1 binding. (A) CD47 on stored erythrocytes undergoes a conformational change, as detected by an increase in 2D3 binding, after overnight dilution in whole blood. (B) TSP-1 binding as detected by the A6.1 antibody on stored erythrocytes after overnight dilution in whole blood. (C) The 4N1K peptide binds to stored erythrocytes after overnight incubation in whole blood. The 4N1K peptide binding to aged erythocytes is shown for comparison. (D) Competition of CD47 antibody binding and 4N1K peptide binding was analyzed on stored erythrocytes after overnight. Stored erythrocytes were first incubated with either CD47 antibody or biotinylated TSP-1 peptides and subsequently incubated with biotinylated TSP-1 peptide and CD47 antibody, respectively. Subsequently, total TSP-1 peptide binding was determined by streptavidin binding (all experiments are n = 4). ns indicates not significant. *P < .05; ***P < .001.