CD47 mediates oxygen-responsive activation of RBC eNOS during circulatory transit

Abstract BACKGROUND: Red blood cells (RBCs) contain active type 3 endothelial nitric oxide synthase (eNOS); however, the precise regulation of RBC eNOS during circulation remains unclear. Our previous work demonstrated that RBC eNOS activity oscillates in synchrony with RBC traversal of oxygen (O2) gradients during circulatory transit. Upon RBC deoxygenation, eNOS associates with the membrane and is activated, with resultant S-nitrosylation of multiple key regulatory and structural proteins that optimize O2 delivery phenotype (RBC: Bohr effect, deformability, vasoregulation, energetics). eNOS translocation and activation is mechanistically linked to a signaling cascade involving: (1) docking of deoxyhemoglobin (deoxyHb) on the cytoplasmic domain of Band 3 (cdB3); (2) Piezo1-mediated calcium influx; (3) eNOS phosphorylation. We further identified a novel role for non-muscle myosin IIA (NMIIA) as a force-generating link between deoxyHb–cdB3 docking and activation of the mechanosensitive Piezo1 channel. Building on these findings, the present study investigates a potential novel role for CD47 as a bridge between Band 3–deoxyhemoglobin interaction and NMIIA activation via a kinase-dependent signaling pathway. Elucidating additional details of RBC eNOS regulation during circulation may reveal new mechanisms contributing to RBC-based O2delivery homeostasis. METHODS Heparinized red blood cells (RBCs) from human donors or murine models (eNOS(-/-), CD47(-/-), and WT (C57BL/6J)) were used as samples. RBCs were imaged using Stimulated Emission Depletion (STED) microscopy and fluorescence microscopy. Cells were oxygenated or deoxygenated, with or without pretreatment using the NMIIA inhibitor blebbistatin. RBCs were fixed (4% paraformaldehyde / 0.08% glutaraldehyde), permeabilized (0.1% Triton X-100), and incubated with primary antibodies targeting NMIIA, Band 3, Piezo1, spectrin, and actin, followed by incubation with secondary antibodies prior to visualization. Additional samples were treated with MLCK and SRC kinase inhibitors. Intracellular calcium levels in oxygenated and deoxygenated RBCs were assessed using the Ca+ and NO-sensitive probes Fluo-3 and DAF-FM, respectively, and quantified with an environmentally controlled fluorescent plate reader (Biotek Neo2, USA). Single-cell, ‘no-touch’ determination of O2responsive murine RBC (eNOS(-/-) or WT) deformability was determined by quantifying the Brillouin frequency shift. The Bohr effect in eNOS(-/-) and WT RBCs was evaluated by measuring O2 dissociation curves (ODCs). ODCs were determined at two fixed CO₂ levels using a thin-film rotating tonometer and a gas blender, with final measurements performed using an arterial blood gas analyzer (ABL90 Flex). Exercise capacity of Piezo 1 (-/-) mice was evaluated by treadmill running to exhaustion. RESULTS Upon RBC deoxygenation, colocalization between non-muscle myosin IIA (NMIIA) and Band 3, as well as between NMIIA and Piezo1, was significantly increased and blocked by NMIIA inhibition, which also prevented deoxygenation induced increase in intra-erythrocytic Ca and eNOS activation. Moreover, intracellular Ca+ levels (measured using Fluo-3) and NO levels (measured using DAF-FM) production, which increase upon deoxygenation in wild-type (WT) RBCs were markedly blunted in CD47(-/-) RBCs. Further, RBC deoxygenation-induced calcium influx and eNOS activation were attenuated by inhibition of MLCK and SRC kinases. RBCs from eNOS (-/-) mice exhibited altered O2 - responsive cytoskeletal organization with impaired deformability as well as a diminished Bohr effect. The Piezo 1 (-/-) mice had decreased exercise capacity. Summary These findings reveal a novel role for CD47 as a mechanistic bridge between Band 3–deoxyhemoglobin interaction and NMIIA activation through a SRC & MLC kinase-dependent signaling pathway. Additionally, we highlight the critical role of eNOS in maintaining the O2 responsive variation in RBC phenotype that is essential to O2 delivery homeostasis during physiologic stress.