Red blood cell ATP export and transfusion in sepsis - ABSTRACT O2 uptake in the lung and O2 delivery peripherally depend on the efficient matching of blood flow to regional O2 availability in the lung and O2 consumption in tissues. To achieve this matching, red blood cell (RBC) hemoglobin allostery regulates not only trans-erythrocytic O2 flux but also RBC export of vasoactive mediators that respond to O2 demand. RBCs export vasoregulatory ATP basally and in response to O2 deficit. This RBC-based vascular control of the uptake and delivery of O2 may be disrupted by endogenous (e.g., in sepsis) and exogenous (via storage for transfusion) RBC injuries. Septic persons with moderate anemia are frequently transfused, but infrequently benefit. Yet conversely any anemia is a negative risk factor. Lung morbidity is frequent after RBC transfusion, possibly due to impaired RBC ability to export ATP, disrupting O2 uptake via pulmonary RBC- endothelial adhesion. We show in septic mice (cecal ligation/puncture, CLP) that the RBC’s ability to produce and export ATP is impaired. We will test the hypothesis that the O2-transport dysfunction in sepsis is mediated in part by impaired export by RBCs of vasoregulatory ATP, a dysfunction compounded by transfused RBCs, exacerbating acute lung injury (ALI) and hypoxemia, by achieving these Aims: Aim 1. Determine the role of RBC ATP export in mortality and ALI in a mouse model of sepsis and transfusion. Exchange transfusion of CLP RBCs into healthy mice exposed to hypoxia drives mortality. RBC ATP export takes place via pannexin 1 (Px1). We will determine the role and mechanism of depressed RBC ATP export via Px1 in the mortality, ALI, and O2 transport responses to sepsis (CLP or severe influenza) and transfusion in mice using genetic and pharmacological approaches. Aim 2. Determine the influence of augmenting transfusate RBC ATP content and/or export on organ function and O2 transport in septic mice. RBC ATP export can be augmented via clinically available approaches: hypoxic RBC storage; transfusate incubation with PIPA (phosphate, inosine, pyruvate, adenine) solution that preserves stored-RBC ATP and DPG; or using an activator of RBC pyruvate kinase (PKR), which augments RBC ATP with little effect on DPG or P50. We will test these approaches to augment or preserve RBC ATP content on ATP export in mice transfused during sepsis. Aim 3. Determine the influence of human sepsis on RBC vasoregulatory function ex vivo, and the functional influence of candidate modulators of ATP content and export in septic RBCs. We validated a novel RBC cryopreservation scheme with superior phenotype fidelity. We built a unique biobank of RBC specimens from septic children and adults. We prospectively sampled over 150 patients with severe sepsis; most subjects have ALI. We will determine the influence of translation-ready lead candidates identified in Aim 2 to augment RBC ATP export on key RBC respiratory functions: vasoactivity, anti-adhesivity, and O2 transport. We will model the effects of transfusate intervention in admixed septic and stored RBCs. Our novel focus on disrupted signaling by RBCs will produce novel, practical, and relevant insight into respiratory dysfunction in sepsis and transfusion.
