In this issue of Blood, Mankelow et al link phosphatidylserine (PS) exposure in sickle erythrocytes to a physiological event in reticulocyte maturation.1  This discovery has implications for efforts to prevent thrombosis in sickle cell disease (SCD).

Possible pathways of PS exposure in red blood cells (RBCs). PS is represented by blue circles. Starting from the upper left, in normal and sickle reticulocytes, plasma membrane internalization creates inside-out endocytic vesicles. These can merge with autophagic vacuoles in 2 ways: (1) their membranes can fuse to create a hybrid vacuole. Egress of this structure intact from the cell would require an autophagy-like event (as depicted) or a novel process. In the former case, breakdown of the limiting membrane (dashed line) would expose PS; and (2) alternatively, the plasma membrane-derived vesicle could provide a source of isolation membrane and merge (but not fuse) with the autophagic vacuole. In this case, loss of membrane asymmetry would be required for PS exposure. As applies to older cells, and shown at the top, sickling-dependent processes lead to changes in aminophospholipid translocase and phospholipid scramblase activities, and PS exposure diffusely in the plasma membrane.

Possible pathways of PS exposure in red blood cells (RBCs). PS is represented by blue circles. Starting from the upper left, in normal and sickle reticulocytes, plasma membrane internalization creates inside-out endocytic vesicles. These can merge with autophagic vacuoles in 2 ways: (1) their membranes can fuse to create a hybrid vacuole. Egress of this structure intact from the cell would require an autophagy-like event (as depicted) or a novel process. In the former case, breakdown of the limiting membrane (dashed line) would expose PS; and (2) alternatively, the plasma membrane-derived vesicle could provide a source of isolation membrane and merge (but not fuse) with the autophagic vacuole. In this case, loss of membrane asymmetry would be required for PS exposure. As applies to older cells, and shown at the top, sickling-dependent processes lead to changes in aminophospholipid translocase and phospholipid scramblase activities, and PS exposure diffusely in the plasma membrane.

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Reticulocyte maturation from enucleation to the disappearance of reticulin is a time of active cellular remodeling. During a brief developmental window, divided into R1 (first) and R2 (second) stages, reticulocytes downregulate a subset of plasma membrane proteins, shed plasma membrane, reduce their volume, and eliminate their mitochondria, ribosomes, and internal membranes.2  The end result is an erythrocyte: a cell that is flexible but durable, devoid of internal structure, and optimized for months of gas transport within the circulation.

In general, little is known about mechanisms of cellular remodeling in differentiation; in this regard, reticulocytes are a useful physiological model. For example, reticulocytes have been used to show that mitochondria are eliminated through an autophagy-related process.3  Employing an in vitro model of human reticulocyte development, Mankelow et al in the Anstee laboratory have provided insight into the mechanism of plasma membrane reduction. Earlier studies of Griffiths et al showed that glycophorin A (GPA)-containing vesicles are internalized at the R2 stage of development and merge with autophagic vacuoles (see figure)4 ; plasma membrane loss coincides with exocytosis of these hybrid vacuoles. Erythrocytes of hypercholesterolemic mice contain large autophagic inclusions, which also supports this model.5  Based on these studies, it is probable that hybrid endocytic-autophagic vacuoles are formed and eliminated during normal reticulocyte maturation, and that elimination of these structures plays a role in plasma membrane reduction.

In the earlier study by Griffiths et al, it was suggested that fusion of a vacuole with the plasma membrane leads to exocytosis of the vacuole contents, budding of the plasma membrane, and membrane loss.4  This model predicts retention of the normal membrane orientation and deposition of the vacuolar contents in the extracellular space. However, elegant immunofluorescence experiments by Mankelow et al show that intracellular epitopes of the anion exchanger 1 and GPA are expressed on the external face of the vesicle, together with the inner leaflet lipid PS, and the vacuolar contents are inside. Loss of membrane asymmetry in the vesicle prior to or during fusion with the plasma membrane or vacuole could partially explain these findings. A similar mechanism has been proposed for PS exposure in apoptotic cells.6  Another possibility is the involvement of an autophagy-like step leading to topological inversion upon fusion with the plasma membrane. Other, more esoteric, explanations are possible, and the precise mechanism of vesicle elimination from reticulocytes remains to be resolved.

Regardless of the mode of elimination, the study by Mankelow et al raises questions about the fate of the inside-out vesicles. Elimination of the vesicles occurs at the R2 stage; in vivo this would be predicted to occur in the circulation, possibly in the spleen due to macrophages with PS receptors. It has long been known that sickle erythrocytes externalize PS,7,8  and there is evidence that PS exposure plays a role in thrombosis via enhanced interactions with receptors on other circulating cells. Notably, in the current study, PS-positive cells sorted from the circulation display PS-positive surface foci, rather than a homogeneous surface of PS that has been shown previously with drug-treated RBCs.8  The authors attribute the presence of these cells in the blood of SCD patients to hyposplenism, which could reflect a desensitization of macrophages. Consistent with this interpretation, splenectomies of patients with immune thrombocytopenia leads to an increased number of circulating cells with associated PS-positive vesicles.

PS exposure in sickle red cells correlates with age and dehydration, and inversely correlates with fetal hemoglobin content; thus, sickling is implicated in the process.9  However, PS exposure is increased in both the low-density fractions and the high-density fractions, with the former enriched in reticulocytes.9,10  The current study should direct some attention to the role of reticulocytes in thrombosis, while also raising basic questions such as why the PS foci do not re-enter the plasma membrane and become more homogeneous.

A link between PS exposure and reticulocyte maturation has important implications. First, if most vesicles are released cell autonomously, as the authors’ in vitro studies suggest, and the spleen participates in the clearance of PS-positive vesicles after their release, then the prothrombotic effects of extruded vesicles may be greater than predicted based on the percentage of PS-positive erythrocytes alone. Further evidence and characterization of such free vesicles in vitro and in vivo is now crucial. Second, if it is determined that PS-positive vesicles, in isolation or tethered to erythrocytes, contribute to thrombosis in SCD, then strategies aimed at removing these vesicles or cells from the circulation could be therapeutically useful. Some of the uniquely exposed inside-out epitopes might, for example, provide a means to overcome the desensitization of hyposplenism; the “don't eat me” signal conferred by CD47 should be inside-out and make targeted clearance effective for nano-vesicles.11  The study of developmental processes can thus improve our understanding of disease pathogenesis and also suggest novel therapeutic strategies.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

1
Mankelow
 
TJ
Griffiths
 
RE
Trompeter
 
S
, et al. 
Autophagic vesicles on mature human reticulocytes explain phosphatidylserine-positive red cells in sickle cell disease.
Blood
2015
 
126(15):1831-1834
2
Ney
 
PA
Normal and disordered reticulocyte maturation.
Curr Opin Hematol
2011
, vol. 
18
 
3
(pg. 
152
-
157
)
3
Schweers
 
RL
Zhang
 
J
Randall
 
MS
, et al. 
NIX is required for programmed mitochondrial clearance during reticulocyte maturation.
Proc Natl Acad Sci USA
2007
, vol. 
104
 
49
(pg. 
19500
-
19505
)
4
Griffiths
 
RE
Kupzig
 
S
Cogan
 
N
, et al. 
Maturing reticulocytes internalize plasma membrane in glycophorin A-containing vesicles that fuse with autophagosomes before exocytosis.
Blood
2012
, vol. 
119
 
26
(pg. 
6296
-
6306
)
5
Holm
 
TM
Braun
 
A
Trigatti
 
BL
, et al. 
Failure of red blood cell maturation in mice with defects in the high-density lipoprotein receptor SR-BI.
Blood
2002
, vol. 
99
 
5
(pg. 
1817
-
1824
)
6
Lee
 
SH
Meng
 
XW
Flatten
 
KS
Loegering
 
DA
Kaufmann
 
SH
Phosphatidylserine exposure during apoptosis reflects bidirectional trafficking between plasma membrane and cytoplasm.
Cell Death Differ
2013
, vol. 
20
 
1
(pg. 
64
-
76
)
7
Wood
 
BL
Gibson
 
DF
Tait
 
JF
Increased erythrocyte phosphatidylserine exposure in sickle cell disease: flow-cytometric measurement and clinical associations.
Blood
1996
, vol. 
88
 
5
(pg. 
1873
-
1880
)
8
Kuypers
 
FA
Lewis
 
RA
Hua
 
M
, et al. 
Detection of altered membrane phospholipid asymmetry in subpopulations of human red blood cells using fluorescently labeled annexin V.
Blood
1996
, vol. 
87
 
3
(pg. 
1179
-
1187
)
9
Yasin
 
Z
Witting
 
S
Palascak
 
MB
Joiner
 
CH
Rucknagel
 
DL
Franco
 
RS
Phosphatidylserine externalization in sickle red blood cells: associations with cell age, density, and hemoglobin F.
Blood
2003
, vol. 
102
 
1
(pg. 
365
-
370
)
10
de Jong
 
K
Larkin
 
SK
Styles
 
LA
Bookchin
 
RM
Kuypers
 
FA
Characterization of the phosphatidylserine-exposing subpopulation of sickle cells.
Blood
2001
, vol. 
98
 
3
(pg. 
860
-
867
)
11
Rodriguez
 
PL
Harada
 
T
Christian
 
DA
Pantano
 
DA
Tsai
 
RK
Discher
 
DE
Minimal “self” peptides that inhibit phagocytic clearance and enhance delivery of nanoparticles.
Science
2013
, vol. 
339
 
6122
(pg. 
971
-
975
)
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