PROJECT SUMMARY Recently, we have made the novel discovery of necroptosis in mature, anucleate erythrocytes (red blood cells, RBCs) in response to CD59-specific pore-forming toxins (PFTs) and complement. Even though eryptosis, a programmed cell death (PCD) exclusive to RBCs, occurs these cells have been thought to lack the machinery necessary for PCD that occur in nucleated cells. Our findings show that RBC necroptosis shares molecular features with nucleated cell necroptosis, challenging views on PCD and RBC biology. Phosphorylation of the kinases central to necroptosis in nucleated cells, RIP1 and RIP3, is driven by CD59 signaling in RBCs, leading to Syk kinase-dependent phosphorylation of integral membrane protein Band 3, vesiculation, and release of Fas ligand (FasL). RIP1/RIP3 phosphorylation depends on FasL in RBCs and necroptotic death requires membrane pore formation in addition to this. Death by RBC necroptosis is driven by glycolytic formation of advanced glycation end products (AGEs) and reactive oxygen species (ROS). RBC necroptosis is induced by complement and CD59 ligation suggesting relevance to complement-mediated transfusion reactions and clearance. Key events in RBC necroptosis include echinocyte formation (vesiculation) and in vivo clearance post-transfusion. We will analyze the mechanism of RBC necroptosis in vitro and in vivo specifically exploring the role of lipid rafts in the RBC necroptosis events of vesicle formation, RBC clearance, and cellular demise (Aim 1). In addition to exploring the mechanistic control of RBC necroptosis, we will address potential outcomes of this PCD (Aim 2). This will include determining the role of necroptotic vesicles in bystander cell death (Aim 2A) and bystander inflammatory responses in vitro and in vivo (Aim 2B). Defining the mechanisms and outcomes of RBC necroptosis may help explain components of complement interactions with RBCs and may reveal overlooked factors involved in vascular damage and inflammation associated with complement-mediated transfusion reactions.