Storage Primes Erythrocytes for Necroptosis and Clearance

BACKGROUND/AIMS: Like nucleated cells, erythrocytes (red blood cells, RBCs) are capable of executing programmed cell death pathways. RBCs undergo necroptosis in response to CD59-specific pore-forming toxins (PFTs). The relationship between blood bank storage and RBC necroptosis was explored in this study. METHODS: Human RBCs were stored in standard blood bank additive solutions (AS-1, AS-3, or AS-5) for 1 week and hemolysis was evaluated in the context of necroptosis inhibitors and reactive oxygen species (ROS) scavengers. Activation of key factors including RIP1, RIP3, and MLKL was determined using immunoprecipitations and western blot. RBC vesiculation and formation of echinocytes was determined using phase-contrast microscopy. The effect of necroptosis and storage on RBC clearance was determined using a murine transfusion model. RESULTS: Necroptosis is associated with increased RBC clearance post-transfusion. Moreover, storage in AS-1, AS-3, or AS-5 sensitizes RBCs for necroptosis. Importantly, storage-sensitized RBCs undergo necroptosis in response to multiple PFTs, regardless of specificity for CD59. Storage-sensitized RBCs undergo necroptosis via NADPH oxidase-generated ROS. RBC storage led to RIP1 phosphorylation and necrosome formation in an NADPH oxidase-dependent manner suggesting the basis for this sensitization. In addition, storage led to increased RBC clearance post-transfusion. Clearance of these RBCs was due to Syk-dependent echinocyte formation. CONCLUSION: Storage-induced sensitization to RBC necroptosis and clearance is important as it may be relevant to hemolytic transfusion reactions.