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Blood, Vol. 91 No. 8 (April 15), 1998:
pp. 3044-3051
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
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.
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ABSTRACT |
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  -thalassemia intermedia. We describe PS exposure in RBCs of 56 comparably anemic patients with different genetic backgrounds of the  - or
-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 -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 -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
PS-exposing 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.
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INTRODUCTION |
THE 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
-thalassemic variants, destruction of RBCs in the peripheral blood
may be relatively more important in comparably severe
-thalassemia.1,2 Several studies have proposed possible
mechanisms that lead to destruction of RBCs in the peripheral blood.
Both - and -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,
-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
-thalassemic RBCs leads to enhanced recognition and
phagocytosis.8 Another observation is that RBCs from
-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 -thalassemic RBCs and the
hypercoagulable state thought to exist in severe -thalassemia
intermedia.13 -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
- or -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 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 - or -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.
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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/ thalassemia (HbE/-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 - and -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 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 × 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 Ca2+. 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 × 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 ± 0.2% (n = 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 number of cells was defined as cells
that expose PS as indicated in the result section.
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| Fig 1.
Typical flow cytometric analysis of (A) normal RBCs and
(B) thalassemic RBCs (HbE/-thal (splx), incubated without AV-FITC; (C) normal RBCs incubated with 1 mmol/L calcium in the presence of 4 µmol/L ionophore A23187 labeled with AV-FITC, and (D) thal RBCs
(HbE/-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.
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Labeling of globin chains.
A monoclonal antibody directed against the unique 17-26 peptide
sequence of human -globin chains17 was used to label
-globin chains bound to membranes. This antibody does not label
hemoglobin and has an apparent specificity for denatured -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 -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 -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 Texas-Red channel
after incubation with Texas-Red-labeled goat antimurine in the absence
or presence of -globin chain antibody (not shown). These data
indicate that neither -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.
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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/-thalassemic,
splenectomized sample. This shift varied significantly between the
different samples. Although it was not observed in any of the HbH RBCs,
it was very pronounced in a number of the -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% ± 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/-thalassemia, 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 ± 0.2, n = 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% ± 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 - and -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. 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/-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.
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| 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.
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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
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| Fig 3.
Analysis of RBCs incubated with FITC-labeled AV after
treatment with calcium in the presence of 4 µmol/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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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 calcium-free 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 -globin deposits on the cytosolic site of the membrane,
AV-FITC-labeled cells were lightly fixed, permeabilized, and
incubated with the murine monoclonal anti--globin antibody,
followed by Texas-Red-labeled goat antimurine immunoglobulin antibody.17,18
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| Fig 4.
RBCs from a thalassemic patient (HbE/-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--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.
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Figure 4 shows representative fields of RBCs from a splenectomized
patient with HbE/-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 -thalassemia intermedia, it was logical to
suppose that the inclusion was a membrane associated deposit of excess
-globin chains, which we tried to confirm using the monoclonal
antibody to denatured -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
-globin chains were localized in domains adjacent to the membrane,
confirming results reported before.18 When such RBCs were
analyzed for both AV-FITC (green) and anti--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 -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
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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 splenectomized patients with
HbE/-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/-thal
patients exhibited larger populations of AV-FITC-positive RBCs than
nonsplenectomized HbE/-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 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 -globin chains colocalize with AV-FITC (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 -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 -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 -globin
chains and produces a surrogate -thalassemia lesion,5 did not reproduce the PS exposing cells seen in -thalassemic RBCs.
Under the in vitro conditions chosen, Heinz bodies were formed, and
-globin 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 -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 -thalassemia. Furthermore, it can be
speculated that these cells will be rapidly removed from the
circulation, thereby contributing to the anemia.
| |
FOOTNOTES |
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.
| |
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Abstract
Introduction
Abstract