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Membrane Phospholipid Asymmetry in Human Thalassemia
By Frans A. Kuypers, Jie Yuan, Rachel A. Lewis, L. Michael Snyder, Charles R. Kiefer, Ahnond Bunyaratvej,
Suthat Fucharoen, Lisa Ma, Lori Styles, Kitty de Jong, and Stanley L. Schrier
Phospholipid asymmetry in the red blood cell (RBC) lipid
bilayer is well maintained during the life of the cell, with
phosphatidylserine (PS) virtually exclusively located in the
inner monolayer. Loss of phospholipid asymmetry, and consequently exposure of PS, is thought to play an important
role in red cell pathology. The anemia in the human thalassemias is caused by a combination of ineffective erythropoiesis
(intramedullary hemolysis) and a decreased survival of adult
RBCs in the peripheral blood. This premature destruction of
the thalassemic RBC could in part be due to a loss of
phospholipid asymmetry, because cells that expose PS are
recognized and removed by macrophages. In addition, PS
exposure can play a role in the hypercoagulable state reported to exist in severe b-thalassemia intermedia. We
describe PS exposure in RBCs of 56 comparably anemic
patients with different genetic backgrounds of the a- or
b-thalassemia phenotype. The use of fluorescently labeled
annexin V allowed us to determine loss of phospholipid
asymmetry in individual cells. Our data indicate that in a
number of thalassemic patients, subpopulations of red cells
circulate that expose PS on their outer surface. The number
of such cells can vary dramatically from patient to patient,
from as low as that found in normal controls (less than 0.2%)
up to 20%. Analysis by fluorescent microscopy of b-thalassemic RBCs indicates that PS on the outer leaflet is distributed either over the entire membrane or localized in areas
possibly related to regions rich in membrane-bound a-globin
chains. We hypothesize that these membrane sites in which
iron carrying globin chains accumulate and cause oxidative
damage, could be important in the loss of membrane lipid
organization. In conclusion, we report the presence of PSexposing subpopulations of thalassemic RBC that are most
likely physiologically important, because they could provide
a surface for enhancing hemostasis as recently reported, and
because such exposure may mediate the rapid removal of
these RBCs from the circulation, thereby contributing to the
anemia.
r 1998 by The American Society of Hematology.
T
b-thalassemic RBCs are phagocytosed by murine monocytes/
macrophages at a rate 20 times normal8 and by human
macrophages at a rate 2 to 3 times normal.9 Several mechanisms
have been implicated in the enhanced recognition and phagocytosis of thalassemic RBCs. One hypothesis is that the lower
sialic acid level of b-thalassemic RBCs leads to enhanced
recognition and phagocytosis.8 Another observation is that
RBCs from b-thalassemia intermedia patients show aggregated
clusters of hemichrome and band 3, presumably as a result of
oxidant injury. Immunoglobulin (perhaps autologous antibodies) and complement components localize at the membrane
exoface over these clusters.10 Macrophages recognize immunoglobulin as well as complement components and act to remove
these damaged cells.9 Loss of phospholipid (PL) asymmetry has
also been implicated in the recognition of RBCs. In particular,
phosphatidylserine (PS), which in normal cells is strictly
confined to the inner half of the membrane phospholipid bilayer,
is recognized as a first step before removal in vitro11 and in
vivo.12 It has been proposed that PS exposure might play a role
both in the removal of b-thalassemic RBCs and the hypercoagulable state thought to exist in severe b-thalassemia intermedia.13
b-thalassemic red cells induce increased thrombin formation in
vitro when incubated with purified prothrombinase factors,
indicating that a PS-containing surface is available.14 These
studies, however, do not resolve whether PS exposure correlates
with disease severity, or if it is similar in a- or b-thalassemic
patients with comparable anemia. Moreover, because PS exposure could lead to RBC removal, it is of importance to know if
loss of PL asymmetry occurs to some degree in all cells, or if
subpopulations of cells exist that expose PS while the rest of the
red cell population exhibits a normal phospholipid asymmetry.
We have recently developed a method to determine the
exposure of PS in individual red cells, using fluorescently
labeled annexin V (AV).15 This approach also allows the
selection of these cells from the population for further analysis.15 Identification and selection of RBCs using this method is
advantageous, because the PS-exposing cells are selected by
HE ANEMIA IN THE human thalassemias is caused by a
combination of ineffective erythropoiesis (intramedullary
hemolysis) and hemolysis of adult RBCs in the peripheral
blood. Although ineffective erythropoiesis might play a more
important role in b-thalassemic variants, destruction of RBCs in
the peripheral blood may be relatively more important in
comparably severe a-thalassemia.1,2 Several studies have proposed possible mechanisms that lead to destruction of RBCs in
the peripheral blood. Both a- and b-thalassemic RBCs have an
altered morphology and exhibit a decreased deformability as
measured by ektacytometry.3 This altered deformability is in
part due to rigidity of their membranes and the state of
hydration of these cells.3 Oxidative damage to the membrane
skeleton might in part be responsible for this membrane rigidity,
adversely affecting the deformability of the cell.4-6 The decreased deformability could impair passage of RBCs through
the sinusoidal walls of reticuloendothelial organs,7 and consequently trigger the removal of these cells from the circulation.
In addition to these well-described mechanical abnormalities,
From the Children’s Hospital Oakland Research Institute, Oakland,
CA; Stanford University, Stanford, CA; University of Massachusetts
Medical Center, Worcester, MA; Ramathibodi Hospital, Bangkok,
Thailand; and the Siriraj Hospital Bangkok, Thailand.
Submitted July 3, 1997; accepted November 26, 1997.
Supported by the National Institutes of Health Grants No. DK32094,
HL20985, HL55213, HL27059, and DK13682. Presented previously at
the 36th meeting of the American Society of Hematology (Blood
84:259a,1994 [abstr, suppl 1]).
Address correspondence to Frans A. Kuypers, PhD, Children’s
Hospital Oakland Research Institute, 747 52nd Street, Oakland, CA
94609.
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 1734 solely to indicate
this fact.
r 1998 by The American Society of Hematology.
0006-4971/98/9108-0044$3.00/0
3044
Blood, Vol 91, No 8 (April 15), 1998: pp 3044-3051
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PHOSPHOLIPID ASYMMETRY IN THALASSEMIA
binding to AV in the presence of physiologic concentrations of
calcium and can be isolated from the probe by simply lowering
the calcium concentration.
The purpose of this study was to determine if there are
differences in PS exposure on red cells in comparably anemic
patients with the different genetic background of the a- or
b-thalassemia phenotypes. Differences could suggest not only
the mechanism for an imbalanced hemostatic system, but also a
cause for early removal of these cells. Because individual cells
can be studied, we were able to identify colocalization in RBCs
of areas of PS exposure with areas of excess globin chain accumulation. Sites of globin chain accumulation carrying iron, heme, and
hemichromes could be involved in oxidant attack on the membrane
and thus result in membrane lipid organizational damage.
MATERIALS AND METHODS
Blood samples. Blood was collected from patients and controls in
Bangkok, Thailand, and in the San Francisco Bay Area at Children’s
Hospital, Oakland. Blood was withdrawn under protocols approved by
the Institutional Review Boards of the relevant institutions (Ramathibodi Hospital, Siriraj Hospital, both in Bangkok, Thailand, and
Children’s Hospital Oakland, Oakland, CA) into anticoagulant citrate
dextrose (ACD; Sigma, St Louis, MO) or citrate phosphate dextrose
(CPD; Sigma) and shipped from Thailand to Oakland or Stanford, CA
on ice along with control normal blood (shipment controls). Typically,
samples arrived within 48 hours and were immediately processed. The
RBC phenotypes studied included: Hemoglobin H (HbH), variants of
Hemoglobin Constant Spring (HbCS), and Hemoglobin E/b thalassemia (HbE/b-thal). The patients from Bangkok were ethnic Thai adults
between the ages of 20 and 40 and had not been transfused for at least 3
months before the samples were sent. The diagnoses were based on
globin gene analysis as in our prior publication.16 The severity of
anemia varied greatly in both the a- and b-thalassemics16 and some of
the most severely affected patients had been splenectomized. The
patients from Oakland were immigrants from Southeast Asia and China.
All data reported were from samples of different patients.
Erythrocytes. Erythrocyte suspensions were prepared from samples
as indicated above or from fresh human venous blood collected in ACD.
Erythrocytes were pelleted by centrifugation, washed twice with 0.9%
NaCl and once with incubation buffer, and finally diluted in incubation
buffer to the appropriate hematocrit. Either Hanks buffered salt solution,
pH 7.4 (HBSS, Sigma, St Louis, MO), or 10 mmol/L Tris/HCl buffered
saline, pH 7.4 (TBS), was used as buffer throughout the experiments;
similar results were found with both buffers. Additional ingredients, all
of reagent quality, such as CaCl2, N-ethyl maleimide (NEM, Sigma) and
Phenylhydrazine (Sigma) were added as indicated.
NEM and calcium and ionophore treatment of RBCs. To generate
RBCs that expose PS on their outer surface, which can serve as a
positive control, normal red cells were incubated with NEM and
calcium ionophore as described before.15 NEM inhibits the aminophospholipid translocase by reacting with a sulfhydryl group necessary for
its activity. RBCs at 30% hematocrit were incubated in buffer containing 10 mmol/L NEM (Sigma) for 30 minutes at room temperature and
subsequently washed in buffer without NEM. Calcium and ionophore
treatment will induce membrane lipid scrambling. RBCs at a 16%
hematocrit were equilibrated in incubation buffer with 1 mmol/L
calcium for 3 minutes at 37°C. Subsequently, calcium ionophore
A23187 was added to the RBC suspension to a final concentration of 4
µmol/L. The suspension was incubated for 1 hour at 37°C, washed with
5 mmol/L EDTA and buffer containing 1% bovine serum albumin
(BSA) to remove ionophore, and resuspended in buffer.
Phenylhydrazine treatment. Phenylhydrazine was used to generate
Heinz bodies as described before.5 RBCs at a 10% hematocrit were
3045
incubated in TBS with 0.2 mg/mL phenylhydrazine for 60 minutes at
37°C and subsequently washed five times in TBS. Microscopy showed
at least one Heinz body in each red cell.
AV purification and fluorescein-5-isothiocyanate (FITC) labeling.
Recombinant AV was purified from an Escherichia coli expression
system by phospholipid affinity chromatography. Purified AV was
incubated in 50 mmol/L Borate buffer, pH 9.0, at 4°C for 16 hours in the
dark at a final concentration of 1 mg AV/mL in the presence of 20 molar
equivalents of FITC (Molecular Probes, Eugene, OR). Subsequently,
unreacted FITC was removed by incubation with 1.0 mol/L Tris/HCl,
pH 8.0, and filtration on a PD-10 Sephadex G-25 column (Pharmacia,
Uppsala, Sweden). The heterogeneously labeled AV (AV-FITC) species
were separated by fast protein liquid chromatography (FPLC) and the
brightly labeled fractions were collected. The preparation used in our
studies had an average of 3 FITC molecules per protein molecule.
Alternatively, a commercially available AV-FITC was used (R&D
Systems). Virtually identical results were obtained with both probes.
AV labeling of RBC. RBCs were suspended in buffer to a final
concentration of 4 3 106 cells/mL. Four µL of a 500 µM FITC-labeled
AV solution was added to 0.5 mL of this suspension in the presence of 2
mmol/L Ca21. The samples were incubated for 30 minutes at room
temperature and subsequently resuspended to approximately 106 cells
per 250 µL in buffer with 2 mmol/L calcium for flow cytometric and
microscopic analysis as described before.15
Magnetic cell separation. Magnetic beads (average size 15 nm),
covered with an anti-FITC antibody, were supplied by Miltenyi Biotec
Inc (Auburn, CA). The stock solution of beads was fivefold diluted in
AV-labeling buffer containing 2 mmol/L calcium. Red cells labeled with
FITC-AV were washed and 6 3 107 cells were taken up in 80 µL of the
calcium containing labeling buffer. To the cell suspension, 20 µL of the
diluted beads were added. After a 10 minute incubation at room
temperature, the cells were separated in a magnetic separation setup
(Minimac, Miltenyi Biotec Inc, Auburn, CA), following the standard
protocol supplied by the manufacturer. The cells were eluted with HBSS
without calcium.
Flow cytometry. Samples were analyzed by flow cytometry as
described before.15 The instrument was calibrated according standard
protocols to achieve day-to-day reproducibility. The red cell population
was defined by size in forward and side scatter plots. Events that
correlated with intact RBC were analyzed for fluorescence intensity
using the same standard settings on a calibrated flow cytometer at each
measurement. Fluorescence intensities were expressed in logarithmic
mode. Each sample was incubated in the absence or presence of
FITC-AV. The control sample incubated without FITC-AV was used to
set the region for positive fluorescence such that the fraction of cells
with positive (auto-) fluorescence was lower than 0.2% of total. The
population of cells labeled with FITC-AV above background was
determined from the fraction of cells in this region in excess of that
obtained with the (unlabeled) control.
This approach was of particular importance because a number of
thalassemic RBCs exhibited an increased autofluorescence as compared
with normal control cells. An example is given in Fig 1. Normal control
cells show very low fluorescence in FACS analysis in the absence of an
added fluorophore (Fig 1A) and the marker M1 is set such that less than
0.2% of the cells are included in this region. Addition of FITC-AV
resulted in a shift of 0.26 6 0.2% (n 5 42) cells into this region,
indicating the presence of PS on the surface of these cells. Treatment of
control RBCs with NEM followed by incubation with calcium and
ionophore15 resulted in PS exposure, the binding of FITC-labeled AV to
the cells and a shift of the cells into the positive region M1 (Fig 1C). In
comparison with the control cells (Fig 1A), samples of thalassemic red
cells exhibited an increased background fluorescence in the absence of
FITC-AV (Fig 1B). Addition of FITC-AV led to a shift of a subpopulation of cells into the region marked by M1 (Fig 1D). This increase in the
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3046
KUYPERS ET AL
Fig 1. Typical flow cytometric analysis of (A) normal RBCs and (B) thalassemic RBCs (HbE/b-thal (splx), incubated without AV-FITC; (C) normal
RBCs incubated with 1 mmol/L calcium in the presence of 4 mmol/L ionophore A23187 labeled with AV-FITC, and (D) thal RBCs (HbE/b-thal (splx)
labeled with AV-FITC. RBCs were selected by their light scattering properties and the number of cells with fluorescence above background was
defined by gate M1.
number of cells was defined as cells that expose PS as indicated in the
result section.
Labeling of globin chains. A monoclonal antibody directed against
the unique 17-26 peptide sequence of human a-globin chains17 was
used to label a-globin chains bound to membranes. This antibody does
not label hemoglobin and has an apparent specificity for denatured
a-globin chains, such as those found in Heinz bodies.17,18 A secondary
Texas-Red–labeled goat antimurine immunoglobulin was used to show
the localization of this antibody under fluorescent microscopy as
described before.17,18
Fluorescence microscopy. Confocal scanning optical microscopy
(CSOM) was performed on AV-FITC–positive RBCs as described
before.17,18 When required, CSOM analyses provided optical sections at
0.5 µm cuts through the AV-FITC positive RBCs. Alcian Blue coated
glass was used to immobilize the cells during confocal scanning.
RBCs were incubated with AV-FITC and the AV-FITC positive cells
were purified with anti-FITC magnetic beads as described above. The
AV-FITC-magnetic bead complex was removed in buffer low in
calcium. The RBCs were subsequently incubated in labeling buffer, and
relabeled with AV-FITC. The population, enriched in AV-FITC labeled
cells was studied by fluorescent microscopy.
In experiments where we wished to simultaneously identify PS
exposure by AV-FITC and a-globin chains by a secondary Texas-Red–
labeled antibody, we lightly fixed and permeabilized RBCs.17 In
performing these experiments special care was taken that RBCs were
labeled with AV-FITC before this treatment. Hence, RBCs labeled with
AV-FITC (as above) were subsequently fixed, permeabilized, labeled
with a-globin antibody followed by the secondary Texas-Red–labeled
antibody, and analyzed by two-color immunofluorescence. Areas of
colocalization of red and green fluorescence appear yellow.
To rule out cross-reactivity of the Texas-Red–labeled goat antimurine
immunoglobulin with AV-FITC, we used normal red cells in which the
phospholipid organization was scrambled by treatment with NEM,
calcium, and ionophore. Although more than 95% of the cells were
fluorescent after AV-FITC labeling (Fig 1C), neither the scrambled cells
nor the AV-FITC–labeled cells showed any fluorescence in the TexasRed channel after incubation with Texas-Red–labeled goat antimurine
in the absence or presence of a-globin chain antibody (not shown).
These data indicate that neither a-globin chain antibody nor the
secondary Texas-Red–labeled goat antimurine immunoglobulin binds
to PS or AV-FITC. This apparent lack of cross-reactivity suggests that
the Texas-Red–labeled antibodies and the AV-FITC label recognize
distinct sites in or adjacent to the membrane, albeit sometimes in the
same region.
RESULTS
Flow cytometry. Figure 1 indicates typical flow cytometric
analyses of normal and thalassemic RBCs. In the absence of
AV-FITC, a number of thalassemic RBCs (Fig 1B) showed an
increased fluorescence as compared with normal control cells
(Fig 1A), as shown here for a HbE/b-thalassemic, splenectomized sample. This shift varied significantly between the
different samples. Although it was not observed in any of the
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PHOSPHOLIPID ASYMMETRY IN THALASSEMIA
3047
HbH RBCs, it was very pronounced in a number of the
b-thalassemic RBC samples. Hence, it was important to appropriately select a standard gate (M1) and correct for background
fluorescence in each individual sample to obtain the correct
fraction of AV-FITC–labeled cells as indicated in the Materials
and Methods section.
Figure 1C shows control cells treated with NEM, calcium,
and ionophore to scramble their phospholipid organization, and
virtually all RBCs (95% 6 5%) are found in gate M1 indicating
labeling with AV.
Although control cells show a very low increase in fluorescence when AV is added, the thalassemic RBCs shown (HbE/bthalassemia, splenectomized) exhibit an increase in fluorescence on addition of AV-FITC (Fig 1D). In this case, 10.1% of
cells increased their fluorescence into gate M1, indicating that
these cells label with AV-FITC and expose PS. The fluorescence
per thalassemic cell varies considerably, as indicated in Fig 1D.
Instead of a distinct peak, as observed after the scrambling with
calcium and ionophore (Fig 1C), merely a shift in the fluorescence of thalassemic cells is found. Although some cells exhibit
a significant fluorescence, others increase their fluorescence
only slightly above background. This wide range of fluorescence was observed in all thalassemic samples analyzed. We
determined the fraction of cells in the population that appear in
channel M1 and are positive for AV-FITC labeling as the result
of PS exposure. These data are given in Table 1. Fresh control
RBCs exhibit low numbers of cells in the population that label
with AV-FITC (0.26 6 0.2, n 5 42). The shipment controls
show no difference as compared with the fresh samples (range
0.23% to 0.42%). RBC membranes scrambled by treatment
with NEM, calcium, and ionophore showed high levels of
labeling (95% 6 5%). A good correlation was found between
the labeling with AV-FITC and stimulation of prothrombinase
activity as was reported before,15 confirming the exposure of PS
(not shown). Table 1 records results of 56 samples of a- and
b-thalassemic RBCs analyzed by flow cytometry. A wide range
in the number of cells in the population that exposed PS was
observed. The size of the subpopulation that could be labeled
with AV-FITC ranged from normal (0.2%) to significantly
increased above normal (up to 20%), indicating a wide variety
in the number of cells that expose PS. This wide range is further
emphasized by the graphic presentation of the data in Fig 2.
Table 1. PS Exposure in Normal and Thalassamic RBC
Mutation
AV Binding (%)
6SD
n
Normal control
HbH
HbH/CS
HbH/CS, splenectomized
HbCS/CS
HbE/b-thal
HbE/b-thal, splenectomized
b-thal intermedia
b-thal intermedia splenectomized
All thalassemic cells
0.26
0.53
1.00
1.11
0.85
0.45
9.5
0.07
3.4
1.42
0.20
0.60
1.66
42
17
16
1
5
10
4
1
2
56
0.61
0.37
7.5
3.7
1.01
AV-FITC labeling of normal controls (fresh and travel controls) and
patient samples. Indicated is the percentage of cells in the population
that were labeled with AV-FITC above background as indicated in
Fig 1.
Although many thalassemic samples were found in the normal
range, a number were significantly increased above normal.
These data suggest that certain thalassemic mutations are more
likely to result in PS exposure than others. More importantly,
samples from patients that had undergone splenectomy seemed
to show increased numbers of cells in the population that
exposed PS, in particular in the HbE/b-thalassemic variants.
These data indicate that in contrast with normal cells, thalassemic RBC might contain a substantial subpopulation of cells that
expose PS, in particular in severely affected patients.
Image analysis. When normal RBC phospholipid asymmetry is scrambled by treatment with NEM, calcium, and ionophore
they can be analyzed by fluorescent microscopy (Fig 3A, B).
Analysis by CSOM shows uniform labeling of the membrane
by AV-FITC (Fig 3C) of these spherocytic cells. Because a
simple wash in calcium-poor buffer removes all AV-FITC
labeling from the cell, the data indicate that the AV-FITC
complex is bound to PS on the outside of these cells, as was
reported before.15
Because the number of positive cells in the thalassemic RBC
samples was low, the population was enriched in AV-FITC–
positive cells by magnetic bead separation as described before.15 After separation, the cells were washed with calciumfree buffer, which removed all of the AV-FITC/magnetic bead
complex from the cells. These RBCs were reincubated in
AV-FITC in the presence of calcium to visualize the distribution
of PS on the membrane surface. These unfixed AV-FITC–
labeled RBCs were then studied by CSOM, and optical sections
taken at 0.5 µm cuts were made when indicated (Fig 4). When it
was necessary to simultaneously analyze the distribution of PS
on the red cell surface and a-globin deposits on the cytosolic
site of the membrane, AV-FITC–labeled cells were lightly fixed,
permeabilized, and incubated with the murine monoclonal
anti–a-globin antibody, followed by Texas-Red–labeled goat
antimurine immunoglobulin antibody.17,18
Figure 4 shows representative fields of RBCs from a splenectomized patient with HbE/b-thalassemia PS exposing RBCs,
enriched by magnetic bead separation, relabeled with AV-FITC,
and analyzed by CSOM, showed two patterns of fluorescence.
Approximately half of the AV-FITC–labeled thalassemic RBCs
show a smooth rim fluorescence over the entire membrane (Fig
4A), similar to normal cells with a scrambled membrane (Fig 3).
However, in other RBCs the AV-FITC membrane fluorescence
was more heterogeneously distributed. In addition to a rim
fluorescence, sites with increased fluorescence were observed,
indicating that PS was enriched in these areas (Fig 4B).
Interestingly, these AV-FITC–labeled sites at the surface of the
cell were localized to areas that seemed to bulge over an
inclusion body as shown in the equatorial cut of such labeled
cells shown in Fig 4B. Because this patient had severe
b-thalassemia intermedia, it was logical to suppose that the
inclusion was a membrane associated deposit of excess aglobin chains, which we tried to confirm using the monoclonal
antibody to denatured a-globin chains shown by Texas-Red–
labeled antimurine immunoglobulin.
The equatorial cut (midsection) of such labeled cells showed
the enrichment of Texas Red in distinct areas, indicating that
denatured a-globin chains were localized in domains adjacent
to the membrane, confirming results reported before.18 When
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3048
KUYPERS ET AL
Fig 2. Graphic presentation of
RBCs that expose PS as determined by AV-FITC labeling in normal controls and patient samples.
Indicated is the percentage of
cells in the population that were
labeled with AV-FITC above background (o) as well as the average
(x) and standard deviation.
such RBCs were analyzed for both AV-FITC (green) and
anti–a-globin/Texas-Red (red), fluorescence microscopy showed
a bright-yellow fluorescence in dual-color analysis, indicating
that both AV-FITC and the Texas-Red label were enriched in the
same membrane regions (Fig 4B and C). In contrast, the cells
that labeled homogeneously with AV-FITC did not label with
Texas Red. These data strongly suggest a colocalization of
denatured a-globin chains and PS in the same area.
To evaluate the effect of Heinz bodies on the (re)distribution
of PS, we treated normal cells with phenylhydrazine and
subsequently incubated them with AV-FITC. Although these
cells contained an abundance of Heinz bodies,5 they did not
expose their membrane PS as shown by the absence of
AV-FITC–labeled cells in flow cytometry. These findings and
the effect of other oxidants on the asymmetric distribution of
phospholipids in the normal red cell are published elsewhere.19
DISCUSSION
The increased autofluorescence observed in samples from
thalassemic patients suggests the presence of fluorescent products in the red cells, presumably as the result of increased levels
of oxidative damage.19 Our data indicates the presence of PS on
the outside of subpopulations of thalassemic RBCs. Rather than
a uniform loss of phospholipid asymmetry in all cells, distinct
subpopulations of RBCs that expose PS on their outer surface
were found in moderately severe thalassemics, particularly
Fig 3. Analysis of RBCs incubated with FITC-labeled AV after treatment with calcium in the presence of 4 mmol/L ionophore A23187 by
fluorescence microscopy. (A) A representative field of this population as observed in bright field and (B) in fluorescence; note the RBC ghost
indicated by arrow. (C) A calcium ionophore scrambled spherocytic red cell analyzed by serial optical sections in confocal fluorescent microscopy.
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PHOSPHOLIPID ASYMMETRY IN THALASSEMIA
3049
Fig 4. RBCs from a thalassemic patient (HbE/b-thalassemia, splenectomized) enriched from the population labeled with AV-FITC using
magnetic beads coated with an FITC antibody (see the Materials and Methods section). (A, B) Typical cells labeled with AV-FITC in the native
unfixed state and then analyzed by serial optical sections in confocal fluorescent microscopy. Panel A shows the equatorial section of a cell
homogeneously labeled with AV-FITC. Panel B shows three equatorial sections of a cell heterogeneously labeled with AV-FITC. (C,D) RBCs
initially labeled with AV-FITC in the unfixed state and then labeled with monoclonal anti–a-globin chain antibody, and secondary
Texas-Red–labeled goat antimurine antibody. This double-labeled cell was then analyzed by fluorescent microscopy. The areas where Texas Red
(red) and FITC (green) overlap are yellow.
splenectomized patients with HbE/b-thalassemia. In this study
we could not identify a direct correlation between the severity
of the anemia and the proportion of RBC-exposing PS. Similar
results were reported for sickle cells,15, 20 and as was observed in
sickle cell samples, the size of this subpopulation can vary
considerably. Although a number of thalassemic samples were
in the normal range, others exhibited a significant increase in
the population that exposes PS. The lack of appropriate
splenectomized normal controls and the relatively low numbers
of comparable splenectomized and nonsplenectomized patients
do not allow a conclusion on the statistical significance of
splenectomy on the presence of PS-exposing cells.
Importantly, there is a caveat that relates to the events that
produce the exposure of PS and the rapidity by which such
RBCs are possibly removed from the circulation. We noted that
splenectomized HbE/b-thal patients exhibited larger populations of AV-FITC–positive RBCs than nonsplenectomized HbE/
b-thal patients. This increased proportion of RBCs with an
altered phospholipid bilayer could reflect the increased severity
of the disease (anemia) that leads to the splenectomy. Alternatively, removal of the spleen, a major macrophagic organ,
would allow RBCs that expose PS to circulate longer whereas in
the presence of a functioning spleen they would have been
expeditiously removed. In other words, the AV-FITC–positive
cells observed in the peripheral blood of patients might very
well be a cohort of short-lived cells that appears as a subpopulation when the removal system is less than optimal.
The labeling with AV-FITC allows identification of individual cells that expose PS by fluorescent microscopy. AV-FITC
binds to the outer surface of cells that have lost their normal
phospholipid asymmetry as indicated by CSOM analysis. Those
cells that have lost their membrane integrity (ghosts) are
brightly labeled due to the fact that AV-FITC has access to the
PS in the inner monolayer. AV-FITC can be removed from intact
red cells by a simple wash in calcium-poor buffer, a further
indication that the FITC-labeled 38 kD protein has only access
to the outer surface of the cell. In order to increase the number
of AV-FITC–positive cells in the thalassemic red cell population
to make analysis by fluorescent microscopy more feasible, we
used a technique that selects these cells by magnetic bead
separation.15 Analysis after renewed labeling of unfixed RBC
with AV-FITC shows at least two types of PS exposure in
thalassemic red cells. On the one hand, a similar labeling is
found (Fig 4A) as with normal red cells scrambled with calcium
and ionophore as shown in Fig 3C. In these cells, uniform rim
fluorescence indicates a uniform distribution of PS on the outer
surface. Other cells showed enhanced domains of PS on the
surface of the thalassemic red cells (Fig 4B). The underlying
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3050
mechanism for these hot spots is not clear. These data suggest
that the normal maintained asymmetry of the PL bilayer can be
disrupted locally. However, based on the rapid diffusion rates of
phospholipids in the plane of the bilayer before AV-FITC labeling,
one would not expect these domains in the case of unrestricted
movement of PS on the surface of the cell. Hence, this would lead
to the conclusion that PS movement is restricted in the plane of
the bilayer of these cells confining PS to local areas that are
identified by AV-FITC labeling. Although the concept of lipid
domains as the result of restricted movement has been hypothesized, few reports have been able to indicate such regions.
Interestingly, in RBCs that had been labeled with AV-FITC in
the native unfixed state and then lightly fixed and permeabilized, antibodies against a-globin chains colocalize with AVFITC (Fig 4C and D), leading to similar pictures as found with
AV-FITC labeling of intact unfixed cells (Fig 4B). These data
suggest that in some cases PS is enriched on the outer surface in
areas of a-globin chain accumulation. It was recently reported
that endogenous red cell AV is found in regions where Heinz
bodies attach to the plasma membrane,21 thereby suggesting that
PS was enriched in these areas in the inner monolayer. Membrane skeletal proteins including spectrin22 and a band 4.1,23,24
have been shown to interact with PS. A change in the
distribution or lipid/protein interaction of these proteins could
be involved in the local accumulation of PS. However, the
underlying mechanism is not known at present.
One possibility for the localization of PS in these regions
could be found in the expected local oxidative damage of the
membrane induced by the accumulation of heme containing
a-globin chains.10 The local generation of oxygen radical
species could locally damage normal interactions in the membrane, as well as interactions of the bilayer with the cytoskeleton. Phenylhydrazine that oxidizes a-globin chains and produces a surrogate b-thalassemia lesion,5 did not reproduce the
PS exposing cells seen in b-thalassemic RBCs. Under the in
vitro conditions chosen, Heinz bodies were formed, and aglobin aggregates were formed adjacent to the membrane.
However, these conditions appeared not sufficient to reproduce
the events that occur in vivo. In addition, other means of
oxidative stress also failed to induce the exposure of PS as we
report elsewhere.19 These data suggest that oxidative damage
will not necessarily lead to the loss of phospholipid asymmetry.
It might very well be an important factor, but another at present
unknown factor also seems to play an important role in the
events that will lead to the exposure of PS, preferentially
localized to areas that seemed to bulge over an inclusion body.
In conclusion, we have shown that in thalassemic patients,
subpopulations of red cells circulate that expose PS on their
outer surface; there is not an overall loss of phospholipid
asymmetry in all cells. The number of such cells can vary
dramatically from patient to patient. PS is found to be either
distributed over the entire membrane or localized in areas
seemingly related to a-globin–rich regions. The presence of
these subpopulations of cells are physiologically important.
They could form an increased red cell–derived PS surface
responsible for the hypercoagulable state proposed to occur by
some investigators in severe b-thalassemia. Furthermore, it can
KUYPERS ET AL
be speculated that these cells will be rapidly removed from the
circulation, thereby contributing to the anemia.
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1998 91: 3044-3051
Membrane Phospholipid Asymmetry in Human Thalassemia
Frans A. Kuypers, Jie Yuan, Rachel A. Lewis, L. Michael Snyder, Charles R. Kiefer, Ahnond Bunyaratvej,
Suthat Fucharoen, Lisa Ma, Lori Styles, Kitty de Jong and Stanley L. Schrier
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