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

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...

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Bibliographic Details
Published in:	PloS one Vol. 11; no. 1; p. e0141206
Main Authors:	Khoory, Joseph, Estanislau, Jessica, Elkhal, Abdallah, Lazaar, Asmae, Melhorn, Mark I, Brodsky, Abigail, Illigens, Ben, Hamachi, Itaru, Kurishita, Yasutaka, Ivanov, Alexander R, Shevkoplyas, Sergey, Shapiro, Nathan I, Ghiran, Ionita C
Format:	Journal Article
Language:	English
Published:	United States Public Library of Science 01-01-2016 Public Library of Science (PLoS)
Subjects:	Activation Adenosine Adenosine Triphosphate - metabolism Anion Exchange Protein 1, Erythrocyte - metabolism Antibodies ATP Binding sites Blood Blood cells Blood levels Capillary flow Cascades Caspase Caspase 3 - metabolism Caspase-3 Cell activation Chemistry Cloning Complement activation Deformability Deformation mechanisms Endothelial cells Engineering schools Erythrocyte Membrane - metabolism Erythrocytes Erythrocytes - drug effects Erythrocytes - metabolism Fluorescence Fluorescence recovery after photobleaching Formability Glutathione Glycophorin - metabolism Humans Immune response Immunoglobulins Inflammation Innate immunity Lipid Metabolism Medical schools Microchannels NAD(P)H oxidase Oxidation-Reduction Oxidative stress Oxygen Phenotype Phosphorylation Photobleaching rac1 GTP-Binding Protein - metabolism Reactive oxygen species Reactive Oxygen Species - metabolism Red blood cells Sepsis United States > US Japan
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Summary:	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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Conceived and designed the experiments: JK AE MIM NS ICG. Performed the experiments: JK JE AL MIM AB NS. Analyzed the data: JK JE AL MIM BI AI NS ICG. Contributed reagents/materials/analysis tools: IH YK SS. Wrote the paper: JK ICG. Competing Interests: The authors have declared that no competing interests exist.
ISSN:	1932-6203 1932-6203
DOI:	10.1371/journal.pone.0141206