title: Active caspases-8 and -3 in circulating human erythrocytes purified on immobilized annexin-V: a cytometric demonstration.
abstract: Human red blood cells (RBCs) have a normal life span of 120 days in vivo and might be primed in vitro to die in response to apoptotic stimuli through a caspase-independent pathway. It is well known that, in vivo, aging RBCs externalize phosphatidylserine residues but is unknown whether these cells express active caspases at this stage. We isolated RBCs expressing phosphatidylserine on their surface from human blood by applying an original method of affinity chromatography using annexin-V fixed on gelatin or on magnetic beads. The isolated RBCs were then analyzed by flow cytometry for morphological changes (dot-plot forward scatter versus side scatter), phosphatidylserine externalization (annexin-V test), cell viability (calcein-AM test), and caspase activities using fluorescent substrates specific for caspases-3 and -8. In addition, cells were systematically visualized using phase contrast, fluorescence, and confocal microscopy. We found that the population of RBCs fixed on annexin-V is a mixture of discocytes and shrunken cells. This annexin-V-positive population showed a dramatic loss of viability based on esterase activity determination (calcein-AM test). Moreover, we demonstrated that circulating RBCs express both active caspases-8 and -3 in half of the annexin-V-positive cells. All of these results were confirmed by phase contrast, fluorescence, and confocal microscopy. Our results demonstrate active caspases in RBC isolated from blood suggesting that caspases may participate in the regulation of in vivo RBC half-life. This finding open the door to fruitful investigations in the field of RBC pathology.
authors.affiliation: National Institute for Biological Science Research and Development, Bucharest, Romania. bratosind@yahoo.com
authors.forename: Daniela
authors.initials: D
authors.lastname: Bratosin
authors.forename: Luminita
authors.initials: L
authors.lastname: Tcacenco
authors.forename: Manuela
authors.initials: M
authors.lastname: Sidoroff
authors.forename: Coralia
authors.initials: C
authors.lastname: Cotoraci
authors.forename: Christian
authors.initials: C
authors.lastname: Slomianny
authors.forename: Jérôme
authors.initials: J
authors.lastname: Estaquier
authors.forename: Jean
authors.initials: J
authors.lastname: Montreuil
mesh_terms: D017304:Annexin A5 / Q000737:chemistry / Q000378:metabolism*; D053148:Caspase 3 / Q000097:blood*; D053181:Caspase 8 / Q000097:blood*; D002469:Cell Separation / Q000379:methods*; D002846:Chromatography, Affinity; D004912:Erythrocytes / Q000201:enzymology*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D008856:Microscopy, Fluorescence
chemical_list: D017304:Annexin A5; D053148:Caspase 3; D053181:Caspase 8