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The Unusual Pathobiology of Hemoglobin Constant Spring Red Blood Cells
By S.L. Schrier, A. Bunyaratvej, A. Khuhapinant, S. Fucharoen, M. Aljurf, L.M. Snyder, C.R. Keifer, L. Ma,
and N. Mohandas
Hemoglobin Constant Spring (HbCS) is the most common
nondeletional a-thalassemic mutation and is an important
cause of HbH-like disease in Southeast Asia. HbCS variants
have an almost normal mean cell volume (MCV) and the
anemia is more severe when compared with other a-thalassemic variants. We explored the pathobiology of HbCS red
blood cells (RBCs) because the underlying cause(s) of this
MCV ‘‘normalizing’’ effect of HbCS and the more severe anemia are not fully explained. HbCS containing RBCs are distinctly overhydrated relative to deletional a-thalassemia
variants, and the derangement of volume regulation and cell
hydration occurs early in erythroid maturation and is fully
expressed at the reticulocyte stage. Furthermore, the membrane rigidity and membrane mechanical stability of HbCS
containing RBCs is increased when compared with HbH and
a-thalassemia-1 trait RBCs. In seeking the cause(s) underlying these cellular alterations we analyzed membranes from
HbCS and deletional a-thalassemic variants and found that
in addition to oxidized b-globin chains, oxidized acs-globin
chains are also associated with the membranes and their
skeletons in HbCS containing RBCs. We propose that the
membrane pathology of HbCS variants is caused by combination of the deleterious effects induced by membranebound oxidized acs- and b-globin chains. The membrane alterations induced by acs chains are more akin to those
induced by bA-globin chains than those induced by the aAglobin chains that accumulate in the b-thalassemias. Thus,
each globin chain, acs, aA, bA, appears to produce its own
form of membrane perturbation.
q 1997 by The American Society of Hematology.
T
specific oxidized globin chains on the K:Cl cotransporter
and possibly other transport pathways.11 Thus, our attention
was drawn to variants of Hb Constant Spring (CS), a nondeletional a-thalassemic mutation that is very common in
Southeast Asia.8-10,12-14 HbCS is caused by a mutation in the
stop codon of the a2 -globin gene that results in poor output
(1% of normal) of an a-globin which has 31 additional amino
acids,13,14 and when inherited along with a-thalassemia-1,
produces a variant of HbH disease which is generally characterized by a more severe anemia and by an almost normal
mean corpuscular volume (MCV) caused by increased cell
hydration.15,16 This paradoxical event is reinforced by the
finding that HbCS homozygosity, which should produce an
asymptomatic or very mild clinical picture like a-thalassemia trait, due to the persistence of two fully functional aglobin genes, instead produces a disease with hemolysis,
mild anemia, jaundice, and splenomegaly.15 These observations suggest that accumulation of acs could have deleterious
effects on RBCs leading to more severe hemolysis.
To understand the cellular events that may contribute to
these differences in HbCS disease severity and state of hydration, we performed a series of biophysical and biochemical analyses on erythrocytes obtained from patients with
severe HbH disease, HbH/CS, HbCS/CS, HbCS trait, and
a-thalassemia-1 trait. We discovered that the membrane pathology of HbCS variants, which includes increased membrane rigidity, increased membrane mechanical stability, and
altered cell hydration, is the result of perturbations induced
by association of oxidized acs with the membrane.
HE HUMAN thalassemias have proved to be very useful
models to study the different cellular and membrane
consequences of accumulation of excess unmatched a- or
b-globin chains. Highly specific effects of excess a- or bglobin chains on ineffective erythropoiesis, hemolysis,
targeted oxidant attack, state of cell hydration, membrane
mechanical stability, and deformability have been documented.1-3 Red blood cell (RBC) membranes in b-thalassemia intermedia are more mechanically unstable possibly
due to oxidation of protein 4.1, whereas in hemoglobin H
(HbH) disease the membranes are hyperstable and there is
no evidence of oxidation or dysfunction of protein 4.1.2-5
Ineffective erythropoiesis appears to be primarily responsible
for anemia in severe b-thalassemia intermedia, while peripheral RBC destruction appears to account for anemia in severe
a-thalassemia (HbH).6,7 Furthermore, the state of RBC dehydration is also different in a- and b-thalassemia. Accumulation of excess a-globin chains in b-thalassemia leads to
cellular dehydration whereas accumulation of b-globin
chains in severe a-thalassemia results in increased hydration
of RBCs.2,8-10 It is likely that these differential alterations in
cellular hydration are the result of the differing effect of
From the Division of Hematology, Stanford University, Stanford,
CA; Life Sciences Division, Lawrence Berkeley National Laboratory,
Berkeley, CA; University of Massachusetts Medical Center, Worcester; and Ramathibodi Hospital and Siriraj Hospital, Bangkok, Thailand.
Submitted April 22, 1996; accepted October 1, 1996.
Supported by grants from National Institutes of Health (DK
13682, DK 26263, and HL 31579); a grant from the European
Community, EEC TSS-CT92-0081 from NATSDA, Thailand; and a
grant from Prajadhipok Rambhai Barni Foundation.
Address reprint requests to S.L. Schrier, MD, Division of Hematology, Stanford University School of Medicine, Stanford, CA 943055112.
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.
q 1997 by The American Society of Hematology.
0006-4971/97/8905-0008$3.00/0
MATERIALS AND METHODS
Samples of venous blood were obtained from individuals with
HbH, HbH/CS, HbCS/CS, a-thalassemia-1 trait, and HbCS trait and
normal controls under protocols approved by the Thalassemia Center, Siriraj Hospital and the Department of Pathology, Ramathibodi
Hospital, Bangkok, Thailand. The diagnoses were based on globin
gene analysis.10 None of the patients had received a blood transfusion
for at least 3 months before blood collection. The subjects were
adults, ages 20 to 40 years, with equal numbers of each gender.
Very few of these patients had undergone splenectomy. The samples
were drawn into EDTA or citrate-phosphate-dextrose (CPD) and
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HEMOGLOBIN CONSTANT SPRING RED BLOOD CELLS
shipped on ice to California. Each shipment was accompanied by
blood drawn from at least one control subject in exactly the same
manner and under exactly the same conditions. Analysis of RBC
indices were done at the Ramathibodi Hospital, measurement of
membrane deformability and stability were performed at the Lawrence Berkeley National Laboratory, and all other analyses were
performed at Stanford University.
Determination of RBC volume, Hb content, and Hb concentration.
The Bayer H*3 automated hematology analyzer (Diagnostics, Tarrytown, NY) provides a cell-by-cell measurement of volume, Hb
concentration, and Hb content of individual RBCs and displays these
data in graphic form. Statistical analyses were performed using analysis of variance (ANOVA) and post-hoc analysis by Fisher’s PLSD.
From these histograms the proportion of RBCs outside specified
normal limits can also be determined.17 Furthermore, reticulocytes
are identified using an RNA-specific dye17,18 and their indices are
independently measured and displayed. By comparing histograms
of these parameters of RBCs and reticulocytes, one can determine
if reticulocytes freshly delivered from the bone marrow already show
abnormalities of volume, Hb concentration (cell hydration), or Hb
content or whether these changes occur relatively slowly during the
life span of RBCs in the peripheral circulation.
Membrane mechanical stability and deformability measurements.
Resealed membranes were prepared for mechanical stability and
deformability measurements as previously described.2 The erythrocytes were washed three times in 5 mmol/L Tris, 140 mmol/L NaCl
(pH 7.4), and then lysed in 40 vol of 7 mmol/L NaCl and 5 mmol/L
Tris (pH 7.4). The membranes were then pelleted by centrifugation,
resuspended in 10 vol of 5 mmol/L Tris and 140 mmol/L NaCl (pH
7.4), and incubated for 30 minutes at 377C for resealing.
For mechanical stability measurements, the resealed membranes
were pelleted by centrifugation and 100 mL of a 40% membrane
suspension was mixed with 3 mL dextran (40,000 molecular weight,
35 g/100 mL in 10 mmol/L phosphate buffer, pH 7.4, viscosity 95
cp) and subjected continuously to 750 dynes/cm2 in the ektacytometer. Under this stress, the membranes progressively fragment,
generating undeformable spheres. This process is detected as a timedependent decrease in the Deformability Index (DI). The time required for the DI to decrease to 60% of its maximum value is termed
T60 and is taken as a measure of membrane mechanical stability.2
For deformability measurements, resealed membranes, prepared
as described above, were suspended in 3 mL of Stractan (Arabinogalactan, St Regis Paper Co, Tacoma, WA) (290 mOsm, 22 cp, pH
7.4) and exposed to gradually increasing shear stress (0 to 125 dynes/
cm2) in the ektacytometer. For resealed membranes, the shear stress
required to obtain a defined value of DI is determined by the property
of membrane deformability without contributions from either internal viscosity or cell geometry. Analysis of the DI curve generated by
the ektacytometer provides a measure of membrane deformability.2
These studies were performed on 4 patients with a-thalassemia-1
trait, 3 with HbCS trait, 2 with homozygous Hb CS/CS, 4 with HbH,
and 3 with HbH/CS.
Analysis of ghosts and membrane skeletons. RBCs were washed
extensively and then pretreated with proteolysis inhibitors diisopropyl fluro-phosphate (DFP; Sigma, St Louis, MO) (2 mmol/L), pepstatin A (10 mg/mL), and leupeptin (10 mg/mL) for 30 minutes at
377C. Ghosts were then prepared by lysing 1 vol of packed erythrocytes with 40 vol of lysing buffer consisting of 5 mmol/L phosphate
buffer (pH 8.0) containing 0.5 mmol/L DFP. The membranes were
then pelleted by cetrifugation and washed three times with the lysing
buffer.2,3,19 Membrane skeletons were prepared from the washed
ghosts by Triton extraction exactly as previously described.2,3,19
Ghosts and skeletons were analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) using concave 6%
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to 18% PAGE gradients in the Laemmli separation system as previously described.2,3 Gels were stained with Coomassie Brilliant Blue
and then destained. Bands were identified and quantified, as required,
by densitometry using the LKB ultrascan XL Laser Densitometer.
(Pharmacia LKB Biotechnology AB, Bromma, Sweden). To confirm
the identity of the globin bands in the 15- to 20-kD region of the
gel as either a- or b-globin we performed Western blotting using
specific monoclonal antibodies (MoAbs).3 The MoAbs to human aand b-globin chain were developed by Dr L. Michael Snyder and
Dr C.R. Keifer of the University of Massachusetts. The reactions
were revealed by a peroxidase-conjugated rabbit anti-mouse Ig obtained from Dako Corp (Carpenteria, CA). The substrate for the
horseradish peroxidase was 4-chloro-1-naphthol (Sigma Chemical Co).
Thiol-disulfide exchange chromatography was performed to identify proteins that had undergone intramolecular sulfhydryl loss as
previously described.3 These measurements enabled us to identify
those specific proteins that had been oxidatively damaged.3 In performing these studies we analyzed 11 normal shipment controls, 3
a-thalassemia-1 trait, 5 HbCS trait, 9 homozygous HbCS/CS, 8 HbH,
and 10 HbH/CS.
RESULTS
RBC parameters. The Hb values, RBC indices, and proportions of hypochromic RBCs in the different variants of
a-thalassemia and HbCS are summarized in Fig 1. The degree of anemia as reflected by Hb levels ranged from being
very mild in individuals with HbCS trait to being quite severe
in individuals with HbH/CS. In general, the extent of decrease in Hb values was as follows: HbH/CS ú HbH ú
HbCS/CS ú a-thalassemia-1 trait ú HbCS trait. As expected, the mean cell Hb content of RBCs in these variants
was decreased. The smallest decrease in Hb content was
noted in HbCS trait while the largest decrease was found in
HbH and HbH/CS. RBCs in a-thalassemia-1 trait and Hb
CS/CS exhibited intermediate decrease in Hb content. The
largest decrease in cell volume was seen in a-thalassemia1 trait and in HbH and the extent of decrease in cell volume
was roughly proportional to the decrease in Hb content.
Thus, the mean cell Hb concentration of a-thalassemia-1
trait RBC was only slightly decreased compared with that
of normal RBCs. In contrast, RBCs in HbH and HbH/CS
had larger cell volumes in relation to their Hb content, resulting in marked reduction in the mean cell Hb concentration. RBCs in Hb CS/CS also showed considerable decrease
in mean cell Hb concentration. The discordance between cell
volume and cell Hb content in these variant RBCs is best
illustrated by quantitating the numbers of hypochromic
RBCs (Fig 1, bottom panel). Although less than 1% of normal RBCs are hypochromic (Hb concentration values õ28
g/dL), approximately 70% of RBCs in HbH/CS are hypochromic. The percentage of hypochromic RBCs proceeded
in the following order: HbH/CS ú HbH ú HbCS/CS ú athalassemia-1 trait ú HbCS trait. The difference between athalassemia-1 trait and HbCS trait was not significant. These
results imply that RBCs in these different variants are unable
to regulate their volume in accordance with their Hb content,
thereby leading to increased hydration. These results further
suggest that acs may have distinct effects on RBC volume
regulation.
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1764
SCHRIER ET AL
Fig 1. An analysis of patients with variant forms of a-thalassemia
from Bangkok, Thailand. The top panel shows the Hb concentration.
The subsequent four panels are parameters derived from the H*3 Bayer
Hematology Analyzer indicating the mean cell volume (MCV), the mean
cell Hb content (MCH), and the mean cell Hb concentration (MCHC),
while the bottom panel indicates the percent of cells that are hypochromic (percent of RBC where the MCHC is less than 28 g/dL). The error
bars indicate 1 standard deviation about the mean. Abbreviations: aT, a-thalassemia-1 trait; CS-T, heterozygous HbCS; CS/CS, homozygous CS; HbH, a-thalassemia-1 trait/a-thalassemia-2 trait; HbH/CS, a
thalassemia-1 trait/aCSa. The symbols indicate P values showing significant differences from the normal. The following symbols indicate
the respective P values: NS, not significant; #, Ú .01; L, Ú .0005; n, Ú
.0001. The number of analyses done for the top four panels are indicated within the bars in the top panel. The number of analyses done
to determine the extent of hypochromia (bottom panel) are indicated
within or on the bars in that panel.
Reticulocyte parameters. The volume, Hb concentration, and Hb content histograms of RBCs and reticulocytes
in representative normals, HbH, and HbH/CS blood samples
are shown in Fig 2 and values for all the variants studied
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are shown in Table 1. A distinguishing feature of these histograms is that reticulocytes in both HbH and HbH/CS exhibit
increased volume and decreased cell Hb concentration compared with their mature RBC counterparts, a feature that is
also characteristic of reticulocytes in normal blood. Thus,
the disproportionate increase in cell volume relative to its
Hb content is a feature that is expressed even at the reticulocyte stage in these variant RBCs. From these data we can
conclude that the deranged volume regulation of RBCs responsible for their increased hydration occurs early in their
evolution. The finding that these reticulocytes enter the circulation from the bone marrow already overhydrated and once
in circulation are not able to regulate their volume in relation
to their Hb content implies that the transport mechanisms
responsible for volume regulation of these RBCs has been
irreversibly damaged.
The Hb content histograms in HbH and HbH/CS also
exhibited an interesting feature. In normal blood, the Hb
content of reticulocytes and RBCs is very similar and the
distributions are symmetric.17 In contrast, while reticulocyte
Hb content histograms in both HbH and HbH/CS are symmetric, the RBC histograms are markedly asymmetric with
a characteristic tail extending to low values of Hb content
(Fig 2). This finding implies that during their circulatory life
span, but not during reticulocyte maturation, some RBCs
underwent fragmentation resulting in generation of mature
cells with decreased Hb content. This fragmentation may be
relatively greater in a-thalassemia-1 trait and in HbH because these variants showed the greatest reduction in MCV
from the reticulocyte stage to mature RBC (Table 1, MCV
ratios, respectively, of 1.35 P õ .05 and 1.41 P õ .0001
when compared with the normal).
Membrane material properties. Mechanical stability and
dynamic rigidity of resealed membranes prepared from these
variant a-thalassemic RBCs was measured by ektacytometry
and the data are shown in Fig 3. Membrane dynamic rigidity
was increased for the variant RBCs with membranes of
HbCS/CS RBCs exhibiting the highest increase in rigidity
(Fig 3A). CS trait RBCs, HbH, and HbH/CS RBCs exhibited
intermediate increases in membrane rigidity, whereas membranes of a-thalassemia-1 trait RBCs exhibited no increase
in rigidity (Fig 3). In addition to increased membrane rigidity, these variant RBCs also exhibited increased membrane
mechanical stability (Fig 3B). The greatest increase in mechanical stability was noted for membranes of HbCS/CS
RBCs, whereas membranes of HbH/CS and HbH RBCs exhibited intermediate increases in membrane mechanical stability. The membrane mechanical stability of CS trait RBCs
and a-thalassemia-1 trait RBCs was similar of that of normal
RBCs (data not shown). These findings suggest that acschain interaction with the membrane can induce changes in
material properties that mimic the membrane changes induced by excess b-globin chains interacting with the membrane in HbH RBCs.2
Analysis of membrane and skeletal protein components.
To determine the nature of the globin chains interacting
with membranes in these variant RBCs, we performed SDSPAGE analysis of ghosts from patients with HbH disease,
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HEMOGLOBIN CONSTANT SPRING RED BLOOD CELLS
1765
Fig 2. Parameters for the RBCs and reticulocytes from a normal patient, a patient with classical HbH, and a patient with classical HbH/CS.
The distribution curves for the entire RBC population are shown in gray, whereas the reticulocyte population is displayed in red.
Table 1. Comparison of Values in Reticulocytes and RBCs
RBCs (mean { SD)
Reticulocyte (mean { SD)
Ratio: Reticulocytes/RBCs (mean { SD)
MCV
Normal (10)
a-T (7)
CS-T (8)
CS/CS (6)
HbH (30)
HbH/CS (19)
89.27
75.49
85.38
82.05
74.79
85.74
{
{
{
{
{
{
3.89
4.53
4.12
3.72
7.81
5.65
113.98
102.00
106.38
98.68
104.89
107.48
{
{
{
{
{
{
4.97
4.96
4.81
5.45
8.00
5.81
1.28
1.35
1.25
1.20
1.41
1.26
{
{
{
{
{
{
0.04
0.08
0.03
0.06
0.10
0.06
MCHC
Normal
a-T
CS-T
CS/CS
HbH
HbH/CS
31.27
28.03
29.81
29.03
23.94
22.46
{
{
{
{
{
{
0.82
0.63
0.64
1.21
1.29
1.22
25.65
22.63
25.09
25.53
19.15
20.02
{
{
{
{
{
{
1.13
1.35
0.64
2.01
0.86
1.12
0.82
0.81
0.84
0.88
0.80
0.89
{
{
{
{
{
{
0.03
0.04
0.01
0.04
0.04
0.03
MCH
Normal
a-T
CS-T
CS/CS
HbH
HbH/CS
27.22
20.51
24.74
23.03
16.85
18.27
{
{
{
{
{
{
1.66
1.59
1.18
1.60
1.28
1.12
28.20
22.27
25.96
24.32
19.31
20.58
{
{
{
{
{
{
1.53
1.77
1.12
1.05
1.00
1.06
1.04
1.09
1.05
1.06
1.15
1.13
{
{
{
{
{
{
0.02
0.05
0.01
0.03
0.05
0.04
The number in parentheses in the top panel indicates the number of patient samples analyzed.
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1766
SCHRIER ET AL
Fig 3. Relative membrane rigidity and membrane mechanical stability of variant a-thalassemia RBCs. HbCS/CS RBCs exhibited the
greatest increase in membrane rigidity while HbH/CS, HbH, and CS
trait RBCs exhibited intermediate increases in membrane rigidity (A).
The a-thalassemia-1 trait RBC membranes exhibited normal membrane rigidity (A). Representative membrane mechanical stability
data for the variant RBCs are shown (B). The rate of decline of deformability index of membranes from HbCS/CS was the slowest, indicating that these membranes exhibit the greatest increase in membrane
mechanical stability. The rate of decline of deformability index of
membranes of HbH/CS and HbH RBCs was intermediate between
that of HbCS/CS and normal membranes, indicating somewhat less
of an increase in membrane mechanical stability. The abbreviations
are the same as those in Fig 1.
HbH/CS, and Hb CS/CS and the data are shown in Fig 4
(left panel). The HbCS trait was omitted from this analysis
because the band was too faint to be seen regularly. In all
of the ghost membranes from patients with HbCS, a polypeptide species migrating with a molecular weight of 19 kD
was detected. As acs with its additional 31 amino acids has
a molecular weight of approximately 19 kD, these data suggest that this polypeptide is likely to be membrane-associated
acs. To unequivocally document that this polypeptide is indeed acs, Western blotting of these proteins was performed
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using MoAbs to human a- and b-globin chains, and the data
are shown in Fig 4. The 19-kD polypeptide species reacted
with the a-globin MoAb but not with b-globin antibody,
confirming the identity of the membrane-associated polypeptide as acs. As we have previously shown, only b-globin
chains were found in association with HbH membranes (Fig
4). Interestingly, the amount of membrane-bound globin was
substantially increased in two splenectomized patients, one
with the HbH genotype and the other with HbH/CS genotype, compared to membranes from their nonsplenectomized
counterparts. Using laser densitometry we were able to measure the ratio of spectrin to acs in two patients with HbH/
CS and one patient with Hb CS/CS. Using relatively standard
assumptions regarding membrane protein content,20 we were
able to calculate that there were 0.16 and 0.25 mg of membrane associated acs per 1010 RBCs in HbH/CS and 0.4 mg
of acs per 1010 RBCs in the patient with HbCS/CS. Assuming
that the cytosolic HbCS content is about 2% in HbH/CS and
about 4% in Hb CS/CS,14,15 then about 7% of acs is membrane
bound in each case.
We had previously shown that much of the membranebound a-globin in severe b-thalassemia and b-globin in athalassemia is associated with membrane skeletons.3,19 To
determine if membrane-associated acs chains are also associated with membrane skeletons, Triton-extracted membranes
prepared from acs variant RBCs were analyzed by SDSPAGE. The 19-kD polypeptide corresponding to the molecular weight of acs was indeed found to be associated with
membrane skeletons in all HbCS variant RBCs (Fig 5) except HbCS-T, where the amounts were too small to be seen
regularly. We then determined by laser densitometry the
ratio of spectrin to acs in ghosts and skeletons in a patient
with Hb CS/CS and found that 55% of membrane-bound acs
remained with the skeleton fraction.
To determine if there had been oxidation of membraneassociated globin chains, we performed thiol-disulfide exchange chromatography, and the data are shown in Fig 6.
In this assay, the unbound fraction represents proteins devoid
of free thiols and, hence, oxidized proteins. The unbound
protein fraction of membranes prepared from HbCS/CS cells
contained acs-globin chains, indicating that the membraneassociated acs-globin chains were indeed oxidized. Membranes of HbH RBCs contained only oxidized b-globin
chains. In Fig 6, b-thalassemia intermedia membranes contained only oxidized a-globin chains. All HbCS-containing
RBCs had varying amounts of partially oxidized membraneassociated acs (not shown). Using laser densitometry we determined the ratio of acs to glycophorin A in two patients
with Hb CS/CS from which we calculated that 15% to 30%
of the membrane-associated acs had become oxidized. This
probably means that the single cysteine at position 10414 had
become oxidized.
DISCUSSION
The HbCS gene is very common in Southeast Asia and
its frequency approaches 5% to 8% in Thailand.8,9,21 The CS
mutation only affects the a2 gene, which accounts for about
2/3 of normal a-globin chain production.14 Furthermore, the
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HEMOGLOBIN CONSTANT SPRING RED BLOOD CELLS
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Fig 4. The left panel (Transfer
Gel) identifies the SDS-PAGE
analysis of solubilized membranes of normal controls,
patients with HbH, HbH/CS,
and patients with homozygous
HbCS/CS. Splenectomized patients are indicated by splx. The
globin band, running at approximately 15 kD, appears at the bottommost portion of the gel. The
arrow indicates bands seen only
in HbCS variants that run at a
position consistent with 19 kD
molecular weight. Western blotting of similar patients is shown
on the two righthand panels
with a monoclonal anti– a-globin in the center and anti– b-globin MoAb on the right. The anti–
b-globin antibody shows accumulations of b-globin in HbH
and HbH/CS, but the largest accumulations occurred in splenectomized patients with HbH
and HbH/CS. Of importance is
the fact that the anti– a-globin
antibody reacted specifically
with the 19-kD band.
mRNA of acs is very unstable compared with normal a
mRNA and as such accounts for less than 1% of protein
output of a normal a2 gene.14 Interestingly, in terms of pathophysiology, the synthesis of even small amounts of elongated
acs results in more severe anemia than is seen in analogous
deletional a-thalassemia-2.14,15 Therefore, it has been suggested that acs-chains may have deleterious effects on cellular and membrane properties of HbCS-containing RBCs and
these changes in turn could account for increased hemolysis.
To date the only well-documented pathobiologic feature of
some of the HbCS variant RBCs is their increased cell volume relative to their Hb content.15,16 The unique properties
of HbCS lead to studies indicating that the inclusion bodies
seen in HbH are distributed somewhat differently than the
inclusion bodies seen in HbH/CS.12 There is defective spectrin self association in HbH/CS but the same pattern is seen
in HbH.22 HbCS/CS skeletons dissociated more readily with
release of spectrin, actin, and band 4.1 than skeletons of
HbH/CS which in turn dissociated more readily than HbH.20
Other than these observations there is currently little information available on various membrane alterations of HbCScontaining RBCs and the mechanistic basis for these
changes.
The present study has enabled us to document a number
of cellular and membrane changes in HbCS variant RBCs
and also obtain some new insights into the mechanistic basis
for these changes. A distinguishing feature of all HbCS variant RBCs studied is their increased volume relative to their
Hb content (Fig 1), resulting in a large increase in the percentage of hypochromic (Hb concentration õ28 g/dL) RBCs
(Fig 1). Increased numbers of hypochromic RBCs were seen
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even in heterozygous HbCS RBCs, implying a role of acs in
cell volume regulation. The increased numbers of hypochromic RBCs in HbH/CS compared with HbH further validates
this thesis. Importantly, our finding that reticulocytes in both
HbH and HbH/CS are more hydrated than normal reticulocytes (Fig 2, Table 1) implies that hydration of these cells
occurs early in their development. We have previously documented increased hydration of HbH RBCs and proposed
that damage to membrane transport pathways induced by
membrane-associated oxidized b-chains may be responsible
for the deranged volume regulation of these RBCs.2 Our
present finding that increased hydration is also a feature of
reticulocytes in HbH allows us to consider that damage to the
K-Cl cotransport pathway might be the basis for increased
hydration of these RBCs without excluding other cellular
hydration systems. However, the K-Cl cotransport pathway
plays a major role in volume regulation of early erythroid
cells as well as reticulocytes; therefore, damage to this transport mechanism could prevent the volume loss that accompanies maturation of normal erythroblasts and reticulocytes to
RBCs.10,23,24 Furthermore, the finding that reticulocytes and
RBCs in HbH/CS are even more hydrated than comparable
HbH RBC suggests that acs interacting with the membrane
may accentuate the damage induced by b-globin chains to
the K-Cl cotransport, or other transport systems controlling
cellular hydration.23,24 These effects of acs on cell volume
regulation resemble those exerted by b-globin rather than
those induced by a-globin.2,11
Quantitation of membrane rigidity and mechanical stability showed that HbCS variant RBCs demonstrated considerable alterations in these membrane material properties. In-
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1768
SCHRIER ET AL
creased membrane rigidity and increased membrane
mechanical stability were features of all the HbCS variant
cells studied. These changes are identical in direction to
those induced by oxidized b-globin chains interacting with
the membrane and enhanced them.2 Thus, as with volume
regulation, the membrane alterations induced by acs were
akin to those induced by b-globin chains rather than those
induced by a-globin chains in severe b thalassemia.2
The exploration of the biochemical basis for these impressive cellular and membrane alterations in HbCS variant
RBCs showed that partially oxidized acs was associated with
the membranes of all HbCS variant RBCs (Figs 4 and 6).
Membrane skeletal preparations of HbCS variant RBC revealed that acs was associated with membrane skeletal structures (Fig 5). Thus, as in the case of HbH and b-thalassemia
intermedia where oxidized b-globin and oxidized a-globin,
respectively, are associated with the membrane and its skeletons,2,3 partially oxidized acs is associated with the skeletal
structure in all the HbCS variant RBCs. We do not know
why acs behaves in this manner. The 31 additional amino
Fig 6. Thiol-disulfide exchange chromatography was performed
on a normal control, a b-thalassemia intermedia patient who had
been splenectomized, an HbH patient who had been splenectomized,
and a patient with homozygous HbCS/CS. The unbound fraction was
analyzed by SDS-PAGE and the aCS was identified at a position consistent with a molecular weight of 19 kD. Note the presence of globin
bands at the bottom of the gel. These have previously been shown
to be aA in b-thalassemia intermedia and bA in HbH.2
Fig 5. Membrane skeletons, prepared as described, were subjected to SDS-PAGE analysis. Characteristic of membrane skeletons
is the prominence of a- and b-spectrin, band 4.1, and actin. Relatively
faint bands identified as aCS, because of their position at 19 kD, are
seen only in the two patients with HbH/CS and the patient with
HbCS/CS.
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acids do not contain an extra cysteine which could be oxidized. But this stretch of amino acids contains 14 relatively
hydrophobic amino acids and thus might lead to insertion
of acs into membrane transport sites. By comparing the cellular and membrane alterations documented in HbH, HbH/CS,
HbCS/CS, and b-thalassemia intermedia, we come to the
conclusion that oxidized acs induced changes that mimic
those induced by oxidized b-globin.
The mechanism(s) by which these highly specific globinchain–induced abnormalities are produced remains incompletely understood. Each of these globin chains contains
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HEMOGLOBIN CONSTANT SPRING RED BLOOD CELLS
heme and its associated iron. The role of this membraneassociated iron in producing localized oxidant damage in
sickle disease and in thalassemia has been extensively explored by Shalev et al.25 A likely hypothesis is that each of
these partially oxidized globin chains (aA, acs, and bA) produce highly specific sorts of oxidant damage relating to their
underlying physical chemistry and special sites of localization. We believe that we have begun to unravel the unique
pathophysiology of the HbCS variants that accounts for the
increased severity of the anemia and the related alterations
in cellular hydration and in membrane mechanical properties. In these HbCS variants of a-thalassemia, in addition to
the membrane accumulation of the excess b-globin chains
characteristic of all a-thalassemias, there is membrane-associated acs as well. Surprisingly, this additional impact produced by acs leads to membrane damage more akin to that
produced by bA than the aA accumulation seen in b-thalassemia. The mechanistic basis for the effects of oxidized acs
on cell volume regulation and membrane material properties
awaits further detailed studies of the specific protein targets
perhaps undergoing oxidant damage, including the K-Cl
transporter, spectrin, and band 3.
NOTE ADDED IN PROOF
While the manuscript was being prepared Prof Prapon
Wilairat informed us that he had also noted the presence of
aCS in the membrane of RBC containing HbCS.26
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1997 89: 1762-1769
The Unusual Pathobiology of Hemoglobin Constant Spring Red Blood Cells
S.L. Schrier, A. Bunyaratvej, A. Khuhapinant, S. Fucharoen, M. Aljurf, L.M. Snyder, C.R. Keifer, L. Ma
and N. Mohandas
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