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Transmembrane Mobility of Phospholipids in Sickle Erythrocytes:
Effect of Deoxygenation on Diffusion and Asymmetry
By Nadia Blumenfeld, Alain Zachowski, Frederic Galacteros, Yves Beuzard, and Philippe F. Devaux
We studied the effect of sickling on the transmembrane
reorientation and distribution of phospholipids in the red
blood cells of patients homozygous for sickle cell anemia
(SS). To this purpose, we followed the redistributionkinetics
of trace amounts of spin-labeled analogues of natural phospholipids first introduced in the membrane outer leaflet of
normal or sickle erythrocytes exposed to air or nitrogen.
Deoxygenation had no effect on the lipid redistribution
kinetics in normal (AA) cell membranes. At atmospheric PO,,
unfractionnated SS cells were not different from normal
cells. However, on deoxygenation inducing sickling, phosphatidylcholine passive diffusion was accelerated and the
rate of the adenosine triphosphate-dependenttransport of
aminophospholipids was reduced, especially for phosphatidylserine. The stationary distribution of the aminophospholipids between the two leaflets was slightly less asymmetric,
a phenomenon more pronounced with phosphatidylethanolamine. These changes were rapidly reversible on reoxygenation. When SS cells were separated by density, both dense
and light cells exhibited the propertiescited above. However,
dense cells exposed to air possesseda lower aminophospholipid transport rate. These data favor the relationship between aminophospholipid translocase activity and phospholipid transmembrane asymmetry. Sickle cell disease is the
first case of aminophospholipid translocase pathology.
o 1991 by The American Society of Hematology.
P
method shows that deoxygenation of SS cells is accompanied by a decrease in aminophospholipid translocase activity and asymmetry, as well as by an accelerated PC
diffusion. The major change in asymmetry is related to PE
distribution, which tends to be more symmetrical in sickled
erythrocytes. This change begins to reverse as soon as the
incubation conditions are modified.
HOSPHOLIPIDS ARE DISTRIBUTED over both
leaflets of the human erythrocyte membrane in an
asymmetric fashion.' Phosphatidylcholine (PC) and sphingomyelin are essentially located on the external monolayer,
while most of phosphatidylethanolamine (PE) and practically all of phosphatidylserine (PS) are found on the
cytoplasmic leaflet. In the late 1970s, it was proposed that
the asymmetry of PS is a consequence of interactions
between this lipid and the major cytoskeleton component,
spe~trin.~
Experimental
.~
support for this mechanism derived from studies on model systems in which spectrin
interacted with various phospholipids as monomolecular
films or liposome^.^ Since 1984, one has known that there
also exists an adenosine triphosphate (ATP)-dependent
translocation of PS and PE from the outer toward the inner
membrane leaflet.' This translocation is ensured by a
protein, the aminophospholipid translocase, which recognizes both PS and PE with a higher affinity for the former
Whether these two mechanisms act in concert with
each other or whether just one of them is implicated in
maintaining the asymmetry is still subject to deb at^.^.^
Reversible sickle red blood cells (RBCs) have a membrane structure that is modified on deoxygenation: in
sickled erythrocytes, the transbilayer diffusion of PC is
accelerated'0.'' and the aminophospholipids PS and PE are
also more exposed on the outer leaflet." This randomization was supposed to be the consequence of the formation
of spicules where the cytoskeleton is uncoupled from the
bilayer and where the accelerated diffusion takes place, and
thus was taken as an indication of the stabilizing role of
spectrin. In this study, we examined the transmembrane
reorientation at 37°C of spin-labeled phospholipid analogues after their incorporation in the outer layer. These
probes, in a normal RBC, behave as radioactive phospholipi d ~(ie,
' ~ there is no artifact because of the presence of the
nitroxide group) and adopt, with time, the distribution of
their endogenous counterparts. Previously, we performed
such a study at low temperature in which sickle cells
remained discocytic because of the absence of hemoglobin
polymerization, which indicated that phospholipid transport capacity of a sickle cell anemia (SS) cell is slightly
impaired.I4 Here, under incubation conditions in which
sickling occurs, the spin-labeled phospholipid analogue
Blood, Vol 77, No 4 (February 15). 1991: pp 849-854
MATERIALS AND METHODS
Preparation of erythrocytes. After obtaining informed consent,
15 to 20 mL of venous blood was collected in heparinized tubes
from normal (AA) controls and homozygous SS patients at
distance from crisis. The cells were pelleted by centrifugation (5
minutes, 1,500g) and the supernatant and buffy coat were removed
by aspiration. They were resuspended in a fourfold volume of a
buffer containing 20 mmol/L HEPES, 10 mmol/L HNa,PO,, 10
mmol/L glucose, 145 mmol/L NaCI, 5 mmol/L KCI, 1 mmol/L
EDTA, 10 mmol/L inosine, pH 7.4, 300 mOsm. The cells were
washed three times and resuspended in buffer at a 50% hematocrit.
In one set of experiments, dense cells (see below) were incubated
in a hypo-osmotic medium (200 mOsm) containing 95 mmol/L
NaCI. Conversely, light cells were then incubated in a hyperosmotic buffer (500 mOsm) containing 245 mmol/L NaCI.
Fractionation of erythrocytes. A Stractan gradient was prepared
according to the technique of Corash et ah'' A dense fraction and a
light fraction were obtained after centrifugation and washed three
times in buffer.
From the Laboratoire de Biochimie and INSERM U91, Hbpital
Henri Mondor, Crkteil; and the Institut de Biologie Physico-Chimique,
Paris, France.
Submitted January 2,1990; accepted October 11, 1990.
Supported by grants from the Institut National de la Santk et de la
Recherche Mkdicale (U 9I), the Centre National de la Recherche
Scientijique (UA 526 and UA 607), the Universitk Paris 7, and the
Fondation pour la Recherche Mkdicale.
Address reprint requests to N. Blumenfeld, MD, INSERM U152,
Pavillon Gustave Roussy, Hbpital Cochin, 27 rue du Faubourg
Saint-Jacques, 75014 Patis, France.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C.section I734 solely to
indicate this fact.
0 1991 by The American Sociey of Hematology.
0006-497119117704-0006$3.OO/O
849
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850
BLUMENFELD ET A1
Incubation conditions. When required, deoxygenation was obtained by a humid nitrogen flux. Oxygen partial pressure was in the
range of 20 to 30 mm Hg throughout the entire experiment (ie,
until the end of the kinetics assay) as verified by cooximetry.
Sickling of the SS cells was checked by observing the cells fixed in
2% formalin after a 30-minute deoxygenation of the sample.
Oxygenated or deoxygenated samples were incubated at 37°C for
30 minutes before spin labeling.
Spin labeling. The synthesis of the spin-labeled PC*, PE*, and
PS* has been described by Morrot et al.’ An aliquot of the desired
analogue (correspondingto 1%of the endogenousphospholipid in
the final incubation) from a chloroform solution was deposited in a
glass tube, dried under vacuum, and resuspended in buffer by
vortexing. Blood sample and lipid suspension were preheated at
37°C and the translocation assay initiated by mixing 1 vol of
phospholipid suspension to 2 vol of RBCs. Spin probes were
incorporated in the membrane bilayer within a minute as shown by
electron spin resonance (ESR) spectroscopy. Di-isopropylfluorophosphate, 5 mmolL, was added to all samples to minimize
the hydrolysis of spin-labeled phospholipids.
Kinetics assay. A kinetics assay was performed as described by
Morrot et al.’ Briefly, at various times, 80-pL aliquots were drawn
from the incubation, mixed with 80 pL of 2% fatty acid-free bovine
serum albumin (BSA) in buffer under the corresponding atmosphere, and incubated for 1minute on ice. After centrifugation(0.5
minutes, 10,OOOg in an Eppendorf tube), the supernatant was kept
and analyzed by ESR spectroscopy to determine the amount of
probe extracted by BSA. As only the probe exposed on the outer
layer was accessible to BSA, this measurement is a direct determination of the relocation of the phospholipid, originally on the outer
leaflet, on the cytoplasmiclayer. Before spectroscopy,each sample
was supplemented with 10 mmol/L (final concentration) of potassium hexaferricyanideto reoxidize all of the label.
100,
IIJ
0
1
I
B
v
m----
0
)
5 0
RESULTS
Transmembranereorientation in AA erythrocytes. Figure
1A displays the kinetics of relocation of PS*, PE*, and PC*
in the membrane of AA RBCs under oxygenated and
deoxygenated conditions. There is no significant difference
between these two states, and both the steady-state distribution of the analogues between the membrane leaflets and
the initial velocities of the inward motion (Table 1) are
identical to those previously reported.’
Transmembrane reorientation in SS erythrocytes. Under
oxygenated conditions, sickle cells behave as normal erythrocytes (Fig lB), with both the active transport of the
aminophospholipids and the passive diffusion of PC* being
unchanged (Table 1). However, important modifications
occur as soon as the SS erythrocytes are deoxygenated (Fig
IC). The asymmetry of PS* is slightly reduced and that of
PE* is more affected, whereas the transmembrane distribution of PC* is not changed (Table 1). The slowing down of
the aminophospholipid translocase activity is important for
PS* (a decrease of approximately 50%), while the initial
velocity of PE* remains unchanged. PC* diffusion is three
times faster after deoxygenation. Data presented in Table 1
are from a typical experiment. In fact, the values obtained
showed interpatient variability. For instance, the plateaus
obtained with PE* were in the range of 91.0% to 72.5%
under oxygenated conditions (initial velocity varying between 163.8% and 114.0% per hour), and in the range
65.7% to 57.1% under nitrogen atmosphere (initial velocity
0
Time
(h)
Fig 1. Kinetics of reorientation of spin-labeled phospholipid analogues (PS, circles; PE, diamonds; PC, triangles) in normal cells under
oxygenated (open symbols) or deoxygenated (closedsymbols) conditions (A) and in sickle cells under oxygenated (B) or deoxygenated (C)
conditions. Analogues were introduced as 1% of the endogenous
phospholipids in the outer layer of the cells and their equilibration
between the two layerswas assayed by back-exchangeon BSA. After
3 hoursof incubation (arrows in Band C), an aliquot of the oxygenated
sickle cells was transferred under nitrogen atmosphere and the
kinetics of PE analogue followed (closed diamonds in [e]).A parallel
experiment was performed with deoxygenated sickle cells transferred into a carbon monoxide atmosphere (open diamonds in [CI).
from 109.2% to 172.8% per hour). However, the same
pattern is found for each patient: deoxygenation lowered
the PE* equilibrium plateau by a mean factor of 0.74 (range
0.68 to 0.81) while the velocity varied by a factor of 1.02 f
0.15. We studied the reversibility between the two states for
PE* stationary distribution. When oxygenated cells are
transferred under a nitrogen atmosphere, there is an
immediate outward motion of PE*, and the asymmetry
characteristic of sickled erythrocytes is reached within 2
hours (arrow, Fig 1B), conversely, when deoxygenated and
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85 1
PHOSPHOLIPID MOBILITY IN SICKLE ERYTHROCYTES
Table 1. Stationary Distributionand Initial Velocity of Relocation of
PhospholipidAnalogues in AA or SS RBCs
Incubation
AA
PS oxy/deoxy
PE oxy/deoxy
PC oxyldeoxy
Plateau 1%)
Light Fraction
Initial Velocity (%/h)
1,466
126
5.4
94.0
80.5
33.1
1,322
700
137
129
6.7
18.8
88.6
77.3
84.4
61.2
32.8
32.6
The stationary distribution is expressed as the percentage of the
phospholipid analogue that is located on the cytoplasmic leaflet of the
membrane of each type of erythrocyte under air or nitrogen atmosphere. The initial velocity is deduced from curves as those displayed in
the figures. Data presented in this table are from a typical experiment,
and are representative of 15 independent couples of experiments.
Abbreviations: oxy, oxygenated; deoxy, deoxygenated.
sickled cells are exposed to carbon monoxide, sickling
disappears immediately and PE* asymmetry increases and
reaches the value obtained under oxygenated condition in
less than 1hour (arrow, Fig 1C).
Density-fractionated sickle cells. See Fig 2 and Table 2
for results. Here again, one can see interpatient variability;
however, a general trend exists that can be schematized as
follows: arithmetic mean ? range. Under air atmosphere,
light cells exhibit a normal steady-state asymmetry while the
membrane outer layer of the dense cells contains less PE*
(65.2% ? 10.2% compared with 82.3% ? 5.2%). The major difference between the two populations lies in the initial
rates of reorientation that are severely reduced in dense
1
h
E
S
W
CI
W
E:
Q
z
L
W
c
c
U
I=
0
4
a
Fig 2. Transmembrane reorientation of PS (circles), PE (diamonds), and PC (triangles) analogues in sickle cells separated on
Stractan gradient. Light cells under air (A) or nitrogen (B) atmosphere. Dense cells under air (C) or
nitrogen (D) atmosphere.
Dense Fraction
Plateau
Initial Velocity
Plateau
Initial Velocih/
(Oh)
(%/h)
(04
(%/h)
PS
OXY
deoxy
ss
PS oxy
deoxy
PE oxy
deoxy
PC oxy
deoxy
Table 2. Stationary Distributionand Initial Velocity of Relocation of
the PhospholipidAnalogues in Density-SeparatedSickle Cells
89.3
84.3
1,473
647
93.4
90.1
303
251
87.5
72.5
156
137
75.4
63.3
88
87
PE
OXY
deoxy
PC
OXY
deoxy
28.5
39.0
11.1
33.5
24.6
26.6
5.9
8.9
Values correspond to a density-separation of one SS blood sample
and are representative of three independent experiments.
Abbreviations: oxy, oxygenated; deoxy, deoxygenated.
cells when compared with their light counterparts (3.0- to
5.0-fold for PS*, 1.7- to 2.6-fold for PE*, and 1.4- to 2.0-fold
for PC*). When deoxygenated, the two fractions behave
differently. Exposure of light cells to nitrogen atmosphere
reduces the transport of PS by 50% to 60% and increases
the PC* diffusion rate three to six times. The plateau
reached by PS* is marginally affected by deoxygenation,
which lowers the plateau of PE* by 20% 2 4% and
increases the fraction of PC* present on the inner layer by
one third. Deoxygenated dense cells exhibit a smaller
decrease of PS* transport rate (approximately 20%) than
the light cells and a lesser increase of PC* diffusion velocity
(approximately 1.5 times). None of the transmembrane
distribution is affected with the exception of PE, which is
more symmetrically exposed.
Incubation of light cells under hypertonic conditions
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852
BLUMENFELD E T AL
rendered PC diffusion less sensitive to deoxygenation, as it
only doubled between nitrogen and air atmosphere. The
aminophospholipid transport was severely decreased (two
times) by the high ionic strength under oxygenated conditions and showed little further sensitivity to deoxygenation.
Incubation of dense cells under hypotonic conditions did
not further increase PC diffusion after deoxygenation as
compared with incubation under isotonic conditions.
DISCUSSION
In normal AA erythrocytes, neither the aminophospholipid translocase-catalyzed inward transport of PS* and
PE* nor the passive uptake of PC* is sensitive to the state of
oxygenation of the cell. Accordingly, the transmembrane
distribution of the phospholipids is unaffected, as previously described.'* Conversely, sickle cells are sensitive to
the incubation conditions. In oxygenated samples, both
translocation rate and stationary distribution of PS*, PE*,
and PC* are identical to those obtained with AA erythrocytes.12,16
When the cells are sickled, the salient feature is a
higher exposure of both aminophospholipids on the outer
membrane half, while the distribution of PC* is not
changed, in accordance with the results obtained with
endogenous PC.'"3'6Approximately 20% of PS* and 40% of
PE* are present on this monolayer. These values are
slightly higher than those reported by Lubin et a],'' but they
correspond to the degradation by phospholipase A, of
newly incorporated PS obtained by Middelkoop et a]."
These modifications cannot be accounted for by a decrease
in cell ATP during the incubation, as no significant differences were detected in ATP concentration over the incubation duration. Data presented in Table 1 show that the PS*
uptake by the aminophospholipid translocase is reduced
twofold in a sickled cell. The rate of movement in the
opposite direction (inner to outer layer) can be deduced
from the kinetic parameters.'8 For PS*, this inside-outside
rate increased by less than 50% with deoxygenation. Applying the same calculation to PE* indicates that the reduced
asymmetry depends on an increased (approximately two
times) outward relocation of the molecules together with an
unaffected initial velocity of inward motion depending on
aminophospholipid translocase activity. As for PC*, deoxygenation induced a threefold increase in passive diffusion
rate in both directions, as reported previously by Franck et
al." Middelkoop et all7 suggested that, in sickle cells, the
asymmetric distribution of PS is maintained by both the
translocase and the interaction with skeletal proteins; the
appearance of aminophospholipids on the outer layer
would be due to the disruption of the PS-cytoskeleton
interaction.'"." However, even when the cytoskeleton is
close to the lipid bilayer, it does not prevent PS from
diffusing to a measurable extent from the inner to the outer
membrane leaflet, showing that PS-cytoskeleton interaction
is weak.I8Furthermore, when the cytoskeleton is decoupled
from some areas of the membrane in deoxygenated sickled
cells, the outward diffusions of the three phospholipids
studied are similarly increased, indicating that there is no
head-group specificity in this phenomenon. The fact that
the asymmetric distribution of aminophospholipids is dominated by the action of the translocase was reported by
Calvez et a1,I9 who showed that, even in spectrin-poor
membranes, an almost normal lipid asymmetry can be
obtained.
The modification of the equilibrium distribution is totally
reversible, as changing the atmospheric composition (from
air to nitrogen or from nitrogen to carbon monoxide)
initiates transmembrane motion of PE*, leading to a
distribution equivalent to that detected when the final
conditions are prevailing from the beginning of the incubation. Note that the inward motion of PE* induced by
transferring deoxygenated cells into a carbon monoxide
atmosphere progressed at a rate compatible with the
uptake of PE* in oxygenated sickle cells. One could
hypothesize that the events occurring after deoxygenation
are the consequence of an elevated level of calcium in the
cytoplasm,'" an ion known to inhibit the aminophospholipid
translocase.6,z'However, cytoplasmic calcium depletion by
A 23187 ionophore and chelator is ineffective in modifying
the difference of kinetics between oxygenated and deoxygenated SS cells (data not shown). This is consistent with the
fact that, although total cell calcium increases in sickle cells,
the cytosolic, free calcium level remains normal.''
Fractionation of SS RBCs gives a light fraction mainly
populated by reticulocytes and discocytes (under normal
oxygen tension) that sickle into very spiculated cells, and a
dense fraction of mostly irreversibly sickled cells (ISC) that
exhibit minor morphologic changes on deoxygenation. At
normal PO,, the active uptake of PS and PE analogues is
slowed in dense cells and accompanied by a lower asymmetry of PE analogue, in accordance with the steady-state
distribution of the endogenous PE.'2.16This change cannot
be accounted for by the lower ATP content of dense cells
compared with other RBCs because ATP concentration in
free water is normal in the dense cells. This effect might be
explained by the membrane lipid and protein oxidation,
which is known to be abnormally elevated in dense
and affects the translocase activity?' In these dense cells
that show little further deformation on deoxygenation, no
drastic change appears in the absence of oxygen, other than
an increased exposure of PE* on the exofacial membrane
layer. In light cells, which under oxygen have phospholipid
diffusion characteristics similar to those of normal cells,
deoxygenation induces major modifications. The PS* translocation rate decreases by a factor of two and its transmembrane asymmetry seems slightly altered. PE* distribution
becomes less in favor of the cytoplasmic leaflet, mainly
because of an increased motion rate in the in-to-out
direction. PC* diffusion is stimulated threefold to sixfold by
the sickling process, and PC* appears to be more present
on the inner monolayer. Therefore, a correlation could
exist between the morphologic changes and the alteration
in phospholipid dynamics induced by deoxygenation, with a
site of enhanced passive diffusion of PC located in the
spicules formed on deoxygenation and especially in their
highly curved tip.".'* To test this hypothesis, we incubated
light cells under hypertonic conditions, in which hemoglo-
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853
PHOSPHOLIPID MOBILITY IN SICKLE ERYTHROCYTES
bin polymer formation still occurs after deoxygenation but
shape change is prevented. Deoxygenation induced a less
pronounced increase in PC* diffusion, comparable with the
one observed in dense cells. This would tend to support the
previously cited hypothesis. Incubation of dense cells under
hypotonic conditions did not modify the extent of PC*
diffusion change after deoxygenation; however, dense cells
deformed much less than light cells did in nitrogen atmosphere, arguing for the change in passive diffusion occurring solely in severely deformed cells.
The alteration in translocase activity expressed by light
cells according to the atmosphere composition might,
among other possibilities, also be related to the shape
change. However, no conclusion can be drawn from experiments performed with light cells under hypertonic conditions, as the high osmotic pressure by itself greatly reduced
the activity. Thus, the reasons for the change in translocase
activity under nitrogen atmosphere are not yet understood.
However, as the normal activity is recovered on reoxygenation, the alteration has to be temporary. If not related to
modifications of the physico-chemical properties of the
membrane on stretching, this alteration could arise from
changes occurring at the cytoplasmic leaflet, such as membrane-protein phosphorylation.26
We have previously studied phospholipid reorientation
in SS RBCs at low temperat~re.'~
A striking point, which we
have confirmed during this study using the BSA backexchange technique, is a lower PS* translocation rate in SS
cells at 4°C with respect to AA cells, while no difference
appears for PE*. The fact that, at high temperature, PS*
translocation seems to be identical in AA and SS cells might
be caused by the high motion rate, which precludes a
precise determination and only allows to reach default
values.
These results showing an altered distribution of PE and,
to a lesser extent, of PS in sickled cells may be used to
understand some aspects of the vaso-occlusive episodes
occurring in sickle cell anemia. Pathologic cells exhibit a
hypercoagulability27 related to the procoagulant activity
detected in vitro with sickled cells and ISCs,28 and tend to
adhere to endothelial cells of the vessels.29JoThese alterations are consistent with a reduced phospholipid asymmetry, as the appearance of internal lipids in the outer
membrane leaflet is accompanied by a procoagulant activity3' and a strong adherence to endothelial cells.32
In conclusion, sickle cell disease appears to be the first
described aminophospholipid translocase pathology.
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1991 77: 849-854
Transmembrane mobility of phospholipids in sickle erythrocytes:
effect of deoxygenation on diffusion and asymmetry
N Blumenfeld, A Zachowski, F Galacteros, Y Beuzard and PF Devaux
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