title: 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.
authors.affiliation: Division of Allergy and Inflammation, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, United States of America.
authors.forename: Joseph
authors.initials: J
authors.lastname: Khoory
authors.affiliation: Division of Allergy and Inflammation, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, United States of America.
authors.forename: Jessica
authors.initials: J
authors.lastname: Estanislau
authors.affiliation: Department of Surgery, Brigham and Women's Hospital and Harvard Medical School, Boston, United States of America.
authors.forename: Abdallah
authors.initials: A
authors.lastname: Elkhal
authors.affiliation: Division of Allergy and Inflammation, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, United States of America.
authors.forename: Asmae
authors.initials: A
authors.lastname: Lazaar
authors.affiliation: Division of Allergy and Inflammation, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, United States of America.
authors.forename: Mark I
authors.initials: MI
authors.lastname: Melhorn
authors.affiliation: Division of Allergy and Inflammation, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, United States of America.
authors.forename: Abigail
authors.initials: A
authors.lastname: Brodsky
authors.affiliation: Department of Neurology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, United States of America.
authors.forename: Ben
authors.initials: B
authors.lastname: Illigens
authors.affiliation: Department of Synthetic Chemistry & Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Kyoto, Japan.
authors.forename: Itaru
authors.initials: I
authors.lastname: Hamachi
authors.affiliation: Department of Synthetic Chemistry & Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Kyoto, Japan.
authors.forename: Yasutaka
authors.initials: Y
authors.lastname: Kurishita
authors.affiliation: The Barnett Institute of Chemical and Biological Analysis, Northeastern University, Boston, United States of America.
authors.forename: Alexander R
authors.initials: AR
authors.lastname: Ivanov
authors.affiliation: Department of Biomedical Engineering, Cullen College of Engineering, University of Houston, Houston, United States of America.
authors.forename: Sergey
authors.initials: S
authors.lastname: Shevkoplyas
authors.affiliation: Department of Emergency Medicine and Center for Vascular Biology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, United States of America.
authors.forename: Nathan I
authors.initials: NI
authors.lastname: Shapiro
authors.affiliation: Division of Allergy and Inflammation, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, United States of America.
authors.forename: Ionita C
authors.initials: IC
authors.lastname: Ghiran
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000378:metabolism; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism*; D006021:Glycophorins / Q000378:metabolism*; D006801:Humans; D050356:Lipid Metabolism; D010084:Oxidation-Reduction; D010641:Phenotype; D010766:Phosphorylation; D017382:Reactive Oxygen Species / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000378:metabolism
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D006021:Glycophorins; D017382:Reactive Oxygen Species; D000255:Adenosine Triphosphate; D053148:Caspase 3; D020830:rac1 GTP-Binding Protein