Ligation of Glycophorin A Generates Reactive Oxygen Species Leading to Decreased Red Blood Cell Function

Abstract

Acute, inflammatory conditions associated with dysregulated complement activation are characterized by significant increases in blood concentration of reactive oxygen species (ROS) and ATP. The mechanisms by which these molecules arise are not fully understood. In this study, using luminometric- and fluorescence-based methods, we show that ligation of glycophorin A (GPA) on human red blood cells (RBCs) results in a 2.1-fold, NADPH-oxidase-dependent increase in intracellular ROS that, in turn, trigger multiple downstream cascades leading to caspase-3 activation, ATP release, and increased band 3 phosphorylation. Functionally, using 2D microchannels to assess membrane deformability, GPS-ligated RBCs travel 33% slower than control RBCs, and lipid mobility was hindered by 10% using fluorescence recovery after photobleaching (FRAP). These outcomes were preventable by pretreating RBCs with cell-permeable ROS scavenger glutathione monoethyl ester (GSH-ME). Our results obtained in vitro using anti-GPA antibodies were validated using complement-altered RBCs isolated from control and septic patients. Our results suggest that during inflammatory conditions, circulating RBCs significantly contribute to capillary flow dysfunctions, and constitute an important but overlooked source of intravascular ROS and ATP, both critical mediators responsible for endothelial cell activation, microcirculation impairment, platelet activation, as well as long-term dysregulated adaptive and innate immune responses.

Biocompatible coupling of therapeutic fusion proteins to human erythrocytes

“…30 In targeting RBCs for delivery of therapeutics, the present findings suggest that dose-and target-dependent changes in membrane physiology, and ultimately, circulatory behavior should be carefully considered. [24][25][26][27][28][29]64 As increases in RBC rigidity can result in an override of the CD47/SIRPA interaction, 65 these factors may also play a role in RBC interactions with host defenses and immune response.…”

“…30 Although antibodies to GPA and band 3 generally reduce membrane deformability, in early studies of the membrane deformability of antibody-coated RBCs, others observed that for some antigens, nonspherocytic antibody-sensitized RBCs could maintain normal deformability. 31 Membrane effects vary even among epitopes within the same target protein.…”

Global transformation of erythrocyte properties via engagement of an SH2-like sequence in band 3

“…Whereas the function of this regulatory pathway in healthy cells remains uncertain, it should be noted that dramatically more tyrosine phosphorylation of band 3 is seen in pathologies such as sickle cell disease and β-thalassemia (32), G6PD deficiency (33), sepsis (34), and malaria parasitemia (35). Not surprisingly, most of these diseases are also characterized by an abnormal erythrocyte membrane stability/morphology, an altered rate of erythrocyte glucose metabolism, and aberrant ion transport properties (36)(37)(38)(39)(40), raising the question of whether tyrosine phosphorylation of band 3 might also contribute to the development of these diseases.…”

Plasmodium falciparum erythrocyte-binding antigen 175 triggers a biophysical change in the red blood cell that facilitates invasion

“…These factors include energy gains from tight adhesion to the host cell (via receptor-ligand interactions) or manipulation of the host cell cytoskeletal or membrane properties, each of which could facilitate wrapping of the merozoite. Beyond malaria invasion biology, binding of two critical red cell receptors used by the merozoite during invasion (CR1 and GPA) have been shown to transmit biophysical changes to the RBC when bound by certain ligands (16,17). To date, it has not been assessed whether parasite ligands during invasion transmit similar changes, potentially influencing invasion efficiency.…”