Autophagic vesicles on mature human reticulocytes explain phosphatidylserine-positive red cells in sickle cell disease

Abstract

Key Points• Reticulocyte maturation involves the release of intact, inside-out autophagic vesicles with PS exposed on their surface.• Elevated levels of autophagic vesicles on circulating reticulocytes cause PS exposure in patients with SCD.During maturation to an erythrocyte, a reticulocyte must eliminate any residual organelles and reduce its surface area and volume. Here we show this involves a novel process whereby large, intact, inside-out phosphatidylserine (PS)-exposed autophagic vesicles are extruded. Cell surface PS is a well-characterized apoptotic signal initiating phagocytosis. In peripheral blood from patients after splenectomy or in patients with sickle cell disease (SCD), the number of circulating red cells exposing PS on their surface is elevated. We show that in these patients PS is present on the cell surface of red cells in large (∼1.4 mm) discrete areas corresponding to autophagic vesicles. The autophagic vesicles found on reticulocytes are identical to those observed on red cells from splenectomized individuals and patients with SCD. Our data suggest the increased thrombotic risk associated with splenectomy, and patients with hemoglobinopathies is a possible consequence of increased levels of circulating mature reticulocytes expressing inside-out PS-exposed autophagic vesicles because of asplenia. (Blood. 2015;126(15):1831-1834) IntroductionThe erythrocyte is one of the most abundant, accessible, and best characterized of human cells but until the recent development of in vitro erythroid culture systems 1,2 obtaining large numbers of its precursor cell, the human reticulocyte has been problematic. Reticulocytes are broadly grouped into R1, motile multilobular, and normally confined to bone marrow, and R2, which are nonmotile, much more mechanically stable, and released into peripheral circulation where they comprise ;2% of red blood cells.3,4 During maturation to an erythrocyte, the reticulocyte must lose ;20% of its surface area, reduce its volume, and degrade or eliminate residual cytosolic organelles. Current dogma considers that loss of plasma membrane is through the release of endocytosed plasma membrane as exosomes, whereas purging of cellular organelles is executed by autophagy. 5Surface phosphatidylserine (PS) exposure is a well characterized signal for initiating phagocytosis of unwanted cells or cellular material. 6PS is normally located on the intracellular surface of plasma membranes. Relocation to the extracellular surface may occur by activation of a scramblase 7 or a bidirectional trafficking process involving cytosolic vesicles.8 PS-exposed red cells are found in the peripheral blood of patients who have undergone splenectomy, or have sickle cell disease (SCD) or thalassemia. 9-13 Study designFor details of anonymized patient samples, antibodies used, and full methods, see supplemental Methods, available on the Blood Web site. Briefly, reticulocytes were derived from erythroid cultures and confocal microscopy was performed as described.2 PS was detected using Annexin V fluore...

Oxidative stress and phosphatidylserine exposure in red cells from patients with sickle cell anaemia

“…Thus, recently, the expulsion of membrane‐bound organelles by autophagy during maturation of erythrocyte precursors has been shown to result in punctuate externalised PS localised to discrete patches (Mankelow et al , 2015). Furthermore, PS may be expelled as microvesicles.…”

Comparison of the Proteome of Adult and Cord Erythroid Cells, and Changes in the Proteome Following Reticulocyte Maturation

“…Alterations in membrane proteins have been observed to occur during enucleation (10), and the role of autophagic vesicles in reticulocyte maturation has recently been described (1,11). However, a more comprehensive understanding of the final stages of reticulocyte maturation is now required in order to facilitate the study of inherited and acquired anemias exhibiting reticulocytosis (12) and aid in extending in vitro culture systems to create mature erythrocytes.…”

The heart in sickle cell disease, a model for heart failure with preserved ejection fraction

“…Rapid red cell turnover is partially buffered by increased erythropoiesis. Unfortunately, erythropoiesis is metabolically demanding, and increases procoagulant autophagic vesicles (4). In addition, developing erythroblasts produce a number of vasoactive signaling molecules, including placental growth factor, that are associated with airway hyperactivity, monocyte activation, and pulmonary hypertension (5).…”