Aging-associated changes in CD47 arrangement and interaction with thrombospondin-1 on red blood cells visualized by super-resolution imaging

CD47 serves as a ligand for signaling regulatory protein alpha (SIRP alpha) and as a receptor for thrombospondin-1 (TSP-1). Although CD47, TSP-1, and SIRP alpha are thought to be involved in the clearance of aged red blood cells (RBCs), aging-associated changes in the expression and interaction of these molecules on RBCs have been elusive. Using direct stochastic optical reconstruction microscopy (dSTORM)-based imaging and quantitative analysis, we can report that CD47 molecules on young RBCs reside as nanoclusters with little binding to TSP-1, suggesting a minimal role for TSP-1/CD47 signaling in normal RBCs. On aged RBCs, CD47 molecules decreased in number but formed bigger and denser clusters, with increased ability to bind TSP-1. Exposure of aged RBCs to TSP-1 resulted in a further increase in the size of CD47 clusters via a lipid raft-dependent mechanism. Furthermore, CD47 cluster formation was dramatically inhibited onthbs1(-/-)mouse RBCs and associated with a significantly prolonged RBC lifespan. These results indicate that the strength of CD47 binding to its ligand TSP-1 is predominantly determined by the distribution pattern and not the amount of CD47 molecules on RBCs, and offer direct evidence for the role of TSP-1 in phagocytosis of aged RBCs. This study provides clear nanoscale pictures of aging-associated changes in CD47 distribution and TSP-1/CD47 interaction on the cell surface, and insights into the molecular basis for how these molecules coordinate to remove aged RBCs.