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ASH 50th anniversary review
Red cell membrane: past, present, and future
Narla Mohandas1 and Patrick G. Gallagher2
1Red
Cell Physiology Laboratory, New York Blood Center, New York, NY; and 2Department of Pediatrics, Yale University School of Medicine, New Haven, CT
As a result of natural selection driven by
severe forms of malaria, 1 in 6 humans in
the world, more than 1 billion people, are
affected by red cell abnormalities, making
them the most common of the inherited
disorders. The non-nucleated red cell is
unique among human cell type in that the
plasma membrane, its only structural
component, accounts for all of its diverse
antigenic, transport, and mechanical characteristics. Our current concept of the red
cell membrane envisions it as a composite structure in which a membrane envelope composed of cholesterol and phospholipids is secured to an elastic network
of skeletal proteins via transmembrane
proteins. Structural and functional characterization of the many constituents of the
red cell membrane, in conjunction with
biophysical and physiologic studies, has
led to detailed description of the way in
which the remarkable mechanical proper-
ties and other important characteristics
of the red cells arise, and of the manner in
which they fail in disease states. Current
studies in this very active and exciting
field are continuing to produce new and
unexpected revelations on the function of
the red cell membrane and thus of the cell
in health and disease, and shed new light
on membrane function in other diverse
cell types. (Blood. 2008;112:3939-3948)
History
Introduction
Jan Swammerdam, a Dutch biologist and microscopist, first observed
and described red cells in 1668 but it was some years before his
observations, recorded in his notebooks, were publicly disseminated.
Antonie van Leeuwenhoek, another brilliant Dutch microscopist, was
the first to publish, in Philosophical Transactions of the Royal Society in
1675, a remarkable description of the unique features of human red
blood cells.1 He stated, “when he was greatly disordered, the globules of
his blood appeared hard and rigid, but grew softer and more pliable as
his health returned: whence he infers that in a healthy body they should be
soft and flexible, that they may be capable of passing through the capillary
veins and arteries, by easily changing their round figures into ovals, and
also reassuming their former roundness when they come into vessels
where they find larger room.” This striking observation made more than
300 years ago has proven both prescient and accurate.An excellent review
by Bessis and Delpech provides a comprehensive description of priorities
and credits for the discovery and description of red cells.2
George Gulliver, following the work of William Hewson,
published the primary features of red cell membranes in Blood of
Vertebrata in 1862, “Not withstanding the current observations that
the red corpuscle is absolutely homogeneous, it is really composed
of 2 very different parts. One of these is membranous, colourless
and insoluble in water; the other is semifluid or viscid, containing
the color, and very soluble in water.”3 Gorter and Grendel in 1925
provided the first insights into the structure of the membrane, and
indeed biologic membranes generally, by the brilliant deduction
that there are “bimolecular layers of lipids on the chromocytes of
blood.”4 This model has continually evolved over the past 80 years,
thanks to a succession of seminal contributions that included
outlining of the fluid mosaic model of the structure of cell
membranes by Singer and Nicolson,5 isolation of spectrin by
Marchesi and Steers,6 and the definition of the topology of red cell
membrane proteins by Steck and colleagues.7 This progress has
benefited greatly from key discoveries that included methods for
Submitted July 30, 2008; accepted August 15, 2008. DOI 10.1182/blood-200807-161166.
BLOOD, 15 NOVEMBER 2008 䡠 VOLUME 112, NUMBER 10
isolation of membranes (ghosts); protein component analysis
through the development of gel electrophoresis and mass spectrometry; advances in imaging technologies; biochemical, structural,
and functional characterization of the various protein components
of the membrane; defining of asymmetric distribution of phospholipids in the membrane; and delineating the nature of interactions
among various membrane proteins and between proteins and lipids.
While a very large number of investigators contributed to the many
exciting advances made in our understanding of the structural
organization of the red cell membrane during these intervening
years, a few deserve special mention including Peter Agre, Jane
Barker, Daniel Branton, Vann Bennett, Jean Delaunay, Bernard
Forget, Walter Gratzer, Joseph Hoffman, Philip Low, Samuel Lux,
Vincent Marchesi, Jon Morrow, Jiri Palek, Eric Ponder, and
Theodore Steck.
The non-nucleated erythrocyte is unique among human cells in that
the plasma membrane, its only structural component, accounts for all of
its diverse antigenic, transport, and mechanical characteristics. The
discoid shape of the red cell evolves from the multilobulated reticulocyte
during 48 hours of maturation first in the bone marrow and then in the
circulation (Figure 1). An important and distinguishing feature of the
discoid human red cell is its ability to undergo large passive deformations during repeated passage through the narrow capillaries of the
microvasculature, with cross-sections one-third its own diameter, throughout its 120-day life span. The most dramatic manifestations of this
property are seen during red cells’ transit in vivo from the splenic cords to
the splenic sinus and during flow-induced deformation in vitro (Figure 1).
Several elegant studies from the 1940s through the 1960s by, among
others, John Dacie, Lawrence E. Young, Thomas Hale Ham, James H.
Jandl, and William B. Castle, documented that splenic sequestration of
abnormal red cells with reduced deformability accounts for the decreased
life span and resulting hemolytic anemia in several red cell disorders.
Although a key role for cellular deformability in regulating red cell
function and survival has long been recognized, quantitative characterization of deformability began in 1964 with the publication of the
© 2008 by The American Society of Hematology
3939
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BLOOD, 15 NOVEMBER 2008 䡠 VOLUME 112, NUMBER 10
Figure 1. Multilobular reticulocyte (top left panel), the precursor of the mature discoid red cell (top right panel). A red cell traversing from the splenic cord to splenic
sinus (bottom left panel). Note the marked deformation the cell undergoes during its passage through the narrow endothelial slit separating the cord from the sinus. Ellipsoidal
cells generated in vitro by flow-induced deformation in vitro of discoid cells (bottom right panel).
seminal study by Rand and Burton, based on the micropipette aspiration
technique to measure “stiffness” of the membrane.8 Subsequently, a
variety of experimental strategies including rheoscopy, flow channels,
ektacytometry, and optical trapping have been used to generate quantitative descriptions of the material characteristics that regulate the ability of
red cells to undergo deformation, and of the role of various membrane
components in this process. Paul LaCelle, Evan Evans, Robert Hochmuth, Dennis Discher, and several other investigators developed both
quantitative measures of deformability and the essential theoretical
underpinnings.
Studies during the past 3 decades on red cells from healthy
people and from patients with various inherited red cell disorders
have illuminated the molecular processes underlying normal and
aberrant red cell membrane function.9-13 We will survey the current
state of understanding of the structural organization of the normal
red cell membrane and describe how anomalous structural organization accounts for altered features of membrane and cell in the
known red cell membrane disorders.
Present view of structural organization of normal red cell
membrane
The structural organization of the human red cell membrane
enables it to undergo large reversible deformations while maintaining its structural integrity during its 4-month sojourn in the
circulation. The red cell membrane exhibits unique material
behavior. It is highly elastic (100-fold softer than a latex membrane
of comparable thickness), rapidly responds to applied fluid stresses
(time constants in the range of 100 milliseconds), and is stronger
than steel in terms of structural resistance. While a normal red cell
can deform with linear extensions of up to approximately 250%, a
3% to 4% increase in surface area results in cell lysis. Hence an
important feature of induced red cell deformations, both in vitro
and in vivo, is that they involve no significant change in membrane
surface area. These unusual membrane material properties are the
result of an evolution-driven “engineering” process resulting in a
composite structure in which a plasma membrane envelope composed of cholesterol and phospholipids is anchored to a 2dimensional elastic network of skeletal proteins through tethering
sites on cytoplasmic domains of transmembrane proteins embedded in the lipid bilayer (Figure 2).11 Direct interaction of several
skeletal proteins with the anionic phospholipids affords additional
attachments of the skeletal network to the lipid bilayer.14,15
Membrane lipids. The lipid bilayer is composed of equal
proportions by weight of cholesterol and phospholipids.16 While
cholesterol is thought to be distributed equally between the
2 leaflets, the 4 major phospholipids are asymmetrically disposed.
Phosphatidylcholine and sphingomyelin are predominantly located
in the outer monolayer, while most phosphatidylethanolamine and
all phosphatidylserine (PS), together with the minor phosphoinositide constituents, are confined to the inner monolayer.17,18 Several
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BLOOD, 15 NOVEMBER 2008 䡠 VOLUME 112, NUMBER 10
Ankyrin complex
Rh
Band 3
GPA
LW
RhAG
CD47
Band 3
GPA
Protein 4.2
α-spectrin
4.1R complex
Duffy
Rh Kell
Band 3
XK
Glut1
GPC
p55
Dematin
Adducin
Ankyrin
β-spectrin
4.1R
Tropomyosin
Actin protofilament
Tropomodulin
Figure 2. A schematic representation of red cell membrane. The membrane is a
composite structure in which a plasma membrane envelope composed of amphiphilic
lipid molecules is anchored to a 2-dimensional elastic network of skeletal proteins
through tethering sites (transmembrane proteins) embedded in the lipid bilayer.
Illustration by Paulette Dennis.
different types of energy-dependent and energy-independent phospholipid transport proteins have been implicated in generating and
maintaining phospholipid asymmetry.19,20 “Flippases” move phospholipids from the outer to the inner monolayer while “floppases”
do the opposite against a concentration gradient in an energydependent manner. In contrast, “scramblases” move phospholipids
bi-directionally down their concentration gradients in an energyindependent manner. Although several different membrane proteins have been said to exert these different lipid transport activities
in human red cells, there is still considerable debate about their
identity. Recent studies have described “lipid rafts” enriched in
cholesterol and sphingolipids in association with specific membrane proteins that include flotillins, stomatin, G-proteins, and
␤-adrenegic receptors in the red cell membrane.21,22
The maintenance of asymmetric distribution of phospholipids,
in particular exclusive localization of PS and phosphoinositides to
the inner monolayer, has several functional implications. Because
macrophages recognize and phagocytose red cells that expose PS at
their outer surface, the confinement of this lipid in the inner
monolayer is essential if the cell is to survive its frequent encounter
with macrophages of the reticuloendothelial system, especially the
spleen. Loss of lipid asymmetry leading to exposure of PS on the
outer monolayer has been suggested to play a role in premature
destruction of thalassemic and sickle red cells.23-25 Furthermore,
the restriction of PS to the inner monolayer also inhibits the
adhesion of normal red cells to vascular endothelial cells, thereby
ensuring unimpeded transit through the microvasculature.26 By
reason of their interactions with skeletal proteins, spectrin, and
protein 4.1R, both PS and phosphatidylinositol-4,5-bisphosphate
(PIP2) can regulate membrane mechanical function.27,28 Recent
studies have established that binding of spectrin to PS enhances
membrane mechanical stability.27 PIP2 enhances the binding of
4.1R to glycophorin C but decreases its interaction with band 3,
and thereby may modulate the linkage of the bilayer to the
membrane skeleton.29 Lipid rafts that have been implicated in cell
signaling events in nonerythroid cells have been shown in erythroid
cells to mediate ␤2-adregenic receptor signaling and increase
cAMP levels, and thus regulating entry of malarial parasites into
normal red cells.30
RED CELL MEMBRANE
3941
Membrane proteins. More than 50 transmembrane proteins of
various abundance ranging from a few hundred to a million copies
per red cell have been well characterized. A large fraction—some
25—of transmembrane proteins define the various blood group
antigens. The membrane proteins exhibit diverse functional heterogeneity, serving as transport proteins, as adhesion proteins involved
in interactions of red cells with other blood cells and endothelial
cells, as signaling receptors, and other still undefined activities.
Membrane proteins with transport function include band 3
(anion transporter), aquaporin 1 (water transporter), Glut1 (glucose
and L-dehydroascorbic acid transporter), Kidd antigen protein
(urea transporter), RhAG (gas transporter, probably of carbon
dioxide), Na⫹-K⫹-ATPase, Ca⫹⫹ ATPase, Na⫹-K⫹-2Cl⫺ cotransporter, Na⫹-Cl⫺ cotransporter, Na⫹-K⫹ cotransporter, K⫹-Cl⫺
cotransporter, and Gardos Channel. Membrane proteins with
adhesive function include ICAM-4, which interacts with integrins
and Lu, the laminin-binding protein. Detailed discussions of the
defined as well as presumptive functions of various membrane
proteins can be found in several excellent recent reviews.31-34 Of
direct relevance to structural integrity of the membrane are
2 macromolecular complexes of membrane proteins, one ankyrinbased, and the other protein 4.1R-based. Band 3 and RhAG link the
bilayer to the membrane skeleton through the interaction of their
cytoplasmic domains with ankyrin, and glycophorin C, XK, Rh,
and Duffy through their interaction with protein 4.1R.35-39 Recent
studies have indicated that 2 other members of the spectrin-actinprotein 4.1R junctional complex, adducin and dematin, can also
serve as linking proteins by interacting with band 3 and Glut1,
respectively.39,40 These membrane protein linkages with skeletal
proteins may play a role in regulating cohesion between lipid
bilayer and membrane skeleton and thus enable the red cell to
maintain its favorable membrane surface area by preventing
membrane vesiculation. In addition to its linking function, band 3
also assembles various glycolytic enzymes, the presumptive CO2
transporter, and carbonic anhydrase into a macromolecular complex termed a “metabolon,” which may play a key role in regulating
red cell metabolism and ion and gas transport function.41
Skeletal proteins. The principal protein constituents of the
2-dimensional spectrin-based membrane skeletal network are ␣and ␤-spectrin, actin, protein 4.1R, adducin, dematin, tropomyosin,
and tropomodulin.16,42-44 A unique structural feature of the long
filamentous spectrin is its large number of triple-helical repeats of
106 amino acids, 20 in ␣-spectrin and 16 in ␤-spectrin. First
discovered in erythrocytes,45 these triple-helical bundles define a
spectrin super family of proteins that includes dystrophin, actinin,
and utrophin.46 ␣- and ␤-spectrin form an antiparallel heterodimer
through strong lateral interaction between repeats 19 and 20 near
the C-terminus of ␣-spectrin with repeats 1 and 2 near the Nterminus of ␤-spectrin. The 36 triple-helical repeats of spectrin are
structurally heterogeneous in terms of their thermal stability.47
Spectrin tetramer, the major structural component of the 2-dimensional skeletal network, is formed by the lateral interaction of a
solitary helix at the N-terminus of the ␣-chain from 1 dimer with
2 helices at the C-terminus of the ␤-chain from the other to create a
stable triple-helical repeat (Figure 3).48-50 While the spectrin dimerdimer interaction was long thought to be static, recent studies have
revealed that dissociation of spectrin tetramers can be induced by
membrane deformation.51 The other end of the 100 nm-long
spectrin dimer forms a junctional complex with F-actin and protein
4.1R.28,52,53 While actin interacts weakly with N-terminus of
␤-spectrin; the interaction is greatly enhanced by protein 4.1R.54
The length of the actin filaments in the red cell membrane appears
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3942
MOHANDAS and GALLAGHER
BLOOD, 15 NOVEMBER 2008 䡠 VOLUME 112, NUMBER 10
Figure 3. The horizontal linkages between spectrinspectrin dimers and between spectrin, actin, and
protein 4.1R in the junctional complex in the spectrinbased membrane skeleton. The repeats of ␣-spectrin
are colored gray while those of ␤-spectrin are colored
light green. The single helical repeat at N-terminus of
␣-spectrin of 1 dimer interacts with the 2 helical repeat at
C-terminus of ␤-spectrin of the second dimer to constitute
spectrin dimer-dimer interaction indicated in pink. The PS
binding spectrin repeats are colored in dark blue while
repeats with low thermal stability (Tm ⬍ 37°C) are shown
in red. The various components of the junctional complex
at the end of spectrin dimer are also shown. EF hands at
C-terminus of ␣-spectrin and the actin-binding domain
(ABD) at N-terminus of ␤-spectrin are also indicated.
Illustration by Paulette Dennis.
to be tightly regulated, probably by tropomyosin, and is made up of
14 to 16 actin monomers.55 Adducin and tropomodulin cap actin
filaments at opposite ends, while the function of the actin bundling
protein dematin in the junctional complex has yet to be fully
defined.56-58 The spectrin dimer-dimer interaction and the spectrinactin-protein 4.1R junctional complex are key regulators of membrane mechanical stability and play a critical role in preventing
deformation-induced membrane fragmentation as the cell encounters high fluid shear stresses in circulation.
While much progress has indeed been made in our understanding of the structural organization of the various lipid and protein
components of the normal red cell membrane, the current models
are far from comprehensive and continue to evolve. A recent study
using state-of-the-art proteomic approaches has generated a comprehensive catalog of red cell proteins and has identified more than
300 proteins including 105 integral membrane proteins.59 The
current membrane models account for fewer than 15% of these
molecules!
Implications of the structural organization of various
membrane components for normal red cell function
In performing its primary function of oxygen delivery to the
tissues, the red cell must absorb continuous mechanical punishment throughout its lifetime without structural deterioration. Extensive biophysical studies have identified 3 constitutive features as
the primary regulators of the ability of the cell’s capacity to
undergo the necessary deformations. These are: (1) the geometry of
the cell, particularly cell surface area to volume ratio; (2) the
cytoplasmic viscosity determined by intracellular hemoglobin
concentration; and (3) membrane deformability.9-11
Cell geometry. The normal biconcave human red cell with a
volume of 90 fL and surface area of 140 ␮m2 possesses an excess
surface area of 40% compared with a sphere of the same volume.
Without excess surface area to volume ratio the cell cannot deform,
for any deviation from the spherical state at constant volume
implies an increase in surface area, which is forbidden by the lipid
bilayer properties. Maintenance of membrane surface area is
mediated by strong cohesion between the bilayer and the membrane skeleton that prevents membrane vesiculation, and by a
mechanically stable spectrin-based membrane skeleton that prevents membrane breakup. Maintenance of cell volume is mediated
by various membrane-associated ion transporters. Surface area loss
as a result of membrane vesiculation due to decreased membrane
cohesion, or cell fragmentation as a consequence of reduced
membrane mechanical stability, as well as increase in cell volume
due to defective ion transporters, will all compromise the ability of
the cell to deform and lead to its premature removal from
circulation.
The determinant of normal membrane cohesion is the system of
“vertical” linkages between bilayer and membrane skeleton, formed
by the interactions of the cytoplasmic domains of various membrane proteins with the spectrin-based skeletal network (Figure 2).
Band 3 and RhAG provide such links by interacting with ankyrin,
which in turn binds to ␤-spectrin. Protein 4.2 binds to both band 3
and ankyrin and can regulate the avidity of the interaction between
band 3 and ankyrin. Glycophorin C, band 3, XK, Rh, and Duffy all
bind to protein 4.1R, the third member of the ternary junctional
complex with ␤-spectrin and actin.35-39 Recent studies reveal that
band 3 and Glut1 can also link the bilayer to the skeleton through
their interactions with the skeletal proteins, adducin and dematin,
respectively.39,40 Of the various linkages documented, those mediated by band 3 appear to be the dominant determinant of membrane
cohesion followed by the linkage mediated by RhAG.
A dominant regulator of membrane mechanical stability that
enables the red cell to maintain its structural integrity through the
vicissitudes of the circulation is the stability of the network.
Network stability is critically dependent on both the avidity of
interaction between spectrin dimers60,61 and the interactions that
define the junctional complex at the distal ends of spectrin
tetramers.62,63 While the spectrin tetramer is formed through the
association of the solitary helix at the N-terminus of the ␣-chain
from one dimer with 2 helices at the C-terminus of the ␤-chain
from the other to create a stable triple helical repeat, the adjacent
triple helical repeats are needed for effective interaction between
the dimers.50 The dimer-dimer interaction is not static, but dynamic
in the sense that it opens reversibly under tensile forces imposed by
deformation, and indeed flickers continuously between the open
and closed states, as reflected by the relatively low association
constant.51 This association constant is further strengthened by the
binding of ankyrin to the ␤-chain.64 The principal constituents of
the junctional complex at the end of spectrin tetramers are spectrin,
F-actin, and protein 4.1R and avidity of this complex is another
major determinant of membrane stability.16,63 At least 4 other
proteins, tropomyosin, adducin, tropomodulin, and dematin, are
present at lower copy numbers of 1 to 2 per junction.65 These
proteins appear to stabilize the short actin filaments in the complex, but
their influence on membrane stability appears modest compared with
the primary constituents, spectrin, actin and protein 4.1R.
While a major role for protein-protein interactions in regulating
mechanical stability has been well documented, the contribution of
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BLOOD, 15 NOVEMBER 2008 䡠 VOLUME 112, NUMBER 10
protein-lipid interactions has received much less attention. Recent
studies have determined that certain of the triple-helical repeats of
both ␣- and ␤-spectrin bind PS (Figure 3) and this binding in situ
increases membrane mechanical stability.15,27 In contrast, PIP2
binds to N-terminus of ␤-spectrin and decreases the propensity
spectrin to form a ternary complex with actin and protein 4.1R and
may thereby decrease membrane mechanical stability.28
Cytoplasmic viscosity and cell volume regulation. The ability
of normal red cells to rapidly change their shape in response to fluid
shear stresses is governed by cytoplasmic viscosity, which is determined
by intracellular hemoglobin concentration. Non-nucleated mammalian
red cells regulate their mean cell hemoglobin concentration within a
very narrow range (30-35 g/dL) while their mean cell volumes range
can vary widely (20-200 fL). While the mean cell hemoglobin concentration of normal human red cells is 33 g/dL, the distribution of
hemoglobin concentrations in individual red cells in whole blood ranges
from 27 to 37 g/dL. The viscosity of hemoglobin solution increases
steeply starting at 37 g/dL. While hemoglobin viscosity is only 5 centipoise (cp) [5 times greater than water] at 27 g/dL increasing to 15 cp at
37 g/dL, it rises abruptly to 45 cp at 40 g/dL, up to 170 cp at 45 g/dL and
650 cp at 50 g/dL.10,66 By tightly regulating hemoglobin concentration
within a narrow range, red cells minimize the cytoplasmic viscous
dissipation during cell deformation. Increases in hemoglobin concentrations above 37 g/dL markedly decrease the rate at which the cell
recovers its initial shape after both extensional and bending deformations.67 Thus the ability of the cell to accommodate rapidly to a narrow
capillary in the microcirculation will be compromised by increased
cytoplasmic viscosity, and with it, its efficacy in tissue oxygen delivery.
Of note, however, cell dehydration and the resultant increase in
cytoplasmic viscosity only minimally affect red cell survival.
The ability of the red cell to regulate its hemoglobin concentration within narrow limits is critically dependent on its ability to
control its volume. As red cell volume is primarily determined by
total cation content, a host of transport proteins, which transport
sodium and potassium across the membrane play a role in
regulating cytoplasmic viscosity.68-70
Membrane deformability. A unique feature of the normal red
cell membrane is its high elasticity, which enables the cell to
rapidly respond to applied fluid stresses in the circulation. While
both theoretical and experimental evidence implicates a critical
role for the spectrin-based skeletal network in general, and spectrin
in particular, in determining membrane elasticity, the precise
structural basis of the effect remains uncertain. An important
structural feature of the long filamentous spectrin dimer is the
succession of 36 spectrin repeats, 20 in ␣-spectrin and 16 in
␤-spectrin, which behave in part as independently folding units. A
recent study revealed that the thermal stabilities of the 36 individual repeats, expressed in terms of the mid-point unfolding
transition, vary widely, ranging from 21 to 72°C.47 It was inferred
that unfolding of the least stable spectrin repeats might affect
membrane elasticity. A recent elegant study, based on labeling by
fluorophores of sterically shielded cysteines of spectrin in intact
membranes in conjunction with quantitative mass spectrometry,
demonstrated that such cysteines indeed became available to the
reagent when the cell was mechanically stressed, and that these
groups were located in repeats of low stability.71 These findings
support the concept that the unfolding and refolding of distinct
spectrin repeats make a major contribution to the elasticity of the
normal red cell membrane.
During senescence, normal red cells lose surface area and
volume with little loss of hemoglobin and as a consequence cell
density progressively increases during the red cell’s 120-day life
RED CELL MEMBRANE
3943
span. Recent studies have documented that after extensive dehydration, normal red cells as well as sickle red cells, lose their ability to
maintain their cation homeostasis and cell volume increases.72 It
has been suggested that this cell population may represent the
“end-stage” normal red cells destined to be eliminated from cell
circulation. Alternate postulated mechanisms for removal of senescent normal red cells include phagocytosis of senescent cells by
macrophages either through recognition of clustered band 3 or PS
exposure on the outer monolayer of this cell population.73,74 While
it is difficult to assign the relative contribution of each of the
various documented cellular changes to removal of senescent red
cells from circulation, it is likely that all of them play some role in
the process.
Thus, our understanding of the mechanistic basis for normal red
cell deformability appears reasonably well developed and comprehensive. We may anticipate that ongoing structural work aimed at
determining the structures of major red cell membrane proteins at
atomic resolution will provide more refined insights into normal
red cell membrane structure and function.
Mechanistic basis for altered membrane and cell function in
inherited red cell disorders
Inherited red cell disorders with altered membrane and cell
function can be broadly divided into 2 classes. (1) Altered function
due to mutations in various membrane or skeletal proteins. Such
conditions include hereditary spherocytosis (HS), hereditary elliptocytosis (HE), hereditary ovalocytosis, and hereditary stomatocytosis. (2) Altered function due to secondary effects on the membrane resulting from mutations in globin genes; these conditions
include sickle cell disease, Hb SC disease, Hb CC disease, unstable
hemoglobins and thalassemias. A consistent feature of red cells in
all of these disorders is decreased cell deformability. We will
discuss briefly these various inherited disorders based on the
dominant cellular features responsible for impaired cell deformability, and on our current understanding of the molecular and
mechanistic basis for the documented cellular alterations.
Altered cell geometry. Decreased cell surface area to volume
ratio and consequent increased cell sphericity is a distinguishing
feature of red cells in HS, HE, and overhydrated hereditary
stomatocytosis (OHS). In the case of HS and HE, increased
sphericity is the result of loss of cell surface area while in the case
of OHS it results from increased cell volume.
HS is a common inherited hemolytic anemia affecting all ethnic
groups, but is particularly common in people of northern European
ancestry (1 in 3000).75,76 HS is usually associated with dominant
inheritance (75%), although nondominant inheritance (25%) is not
uncommon. “Typical” HS is characterized by evidence of hemolysis with anemia, jaundice, splenomegaly, reticulocytosis, gallstones, and the presence of spherocytes on peripheral blood smears.
However, the clinical manifestations of HS are highly variable
ranging from mild to very severe anemia. A common feature of all
forms of HS is loss of membrane surface area and resultant change
in cell shape from discocytes to stomatocytes to spherocytes
(Figure 4). As red cells with decreased membrane surface area are
unable to effectively traverse the spleen, they are sequestered and
removed from circulation by the spleen. Importantly, the severity of
anemia is related to extent of decrease in membrane surface area.
Splenectomy reduces the severity of anemia by increasing the
survival of spherocytic red cells. The mechanistic basis for
membrane loss in HS is decreased membrane cohesion due to a
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BLOOD, 15 NOVEMBER 2008 䡠 VOLUME 112, NUMBER 10
RhAG
Rh
Band 3
Band 3
Protein 4.2
Ankyrin
β-spectrin
α-spectrin
Figure 5. Membrane defects in HS affect the “vertical” interactions anchoring
the membrane skeleton to the lipid bilayer. Deficiency in any one of the protein
components (band 3, RhAG, ankyrin, protein 4.2, or spectrin) involved in the
anchoring process leads to HS. Illustration by Paulette Dennis.
Figure 4. Red cell morphology. Hereditary spherocytosis (HS; top panel); nonhemolytic hereditary elliptocytosis (HE; middle panel); elliptocytes, poikilocytes, and
fragmented red cells in hemolytic HE (bottom panel).
reduced number of “vertical” linkages between bilayer and membrane skeleton.77 Reduced anchoring results from deficiencies of
transmembrane proteins that link the bilayer to the membrane
skeleton (band 3 or RhAG), or of anchoring proteins (ankyrin or
protein 4.2) or of spectrin due to too little membrane skeleton
available for linkage (Figure 5). Thus, HS is the result of defects in
genes encoding any of the protein components involved in vertical
linkages between skeletal network and the membrane.75,78-83
HE is a relatively common, clinically and genetically heterogeneous disorder, characterized by presence of elliptically shaped red
cells on peripheral blood smear.84 HE has a worldwide distribution,
but is more common in malaria endemic regions with prevalence
approaching 2% in West Africa.85 Inheritance of HE is autosomal
dominant. The overwhelming majority of HE is asymptomatic but
approximately 10% of patients have moderate to severe anemia
including a few reported cases of hydrops fetalis. Typically, people
heterozygous for an elliptocytic variant are asymptomatic while
people with homozygosity or compound heterozygosity for HE
variants experience mild to severe anemia, including the severe
variant hereditary pyropoikilocytosis. A common feature of all
forms of HE is a mechanically unstable membrane that results in
progressive transformation of cell shape from discocyte to elliptocyte with time in the circulation (Figure 4), and in severe cases,
membrane fragmentation and cells with reduced membrane surface
area (Figure 4). Importantly, the severity of the disease is related to
extent of decrease in membrane mechanical stability and resultant
loss of membrane surface area.86 While very few patients with HE
need splenectomy, as with HS, splenectomy reduces the severity of
anemia in patients with severe anemia by increasing the circulatory
life span of fragmented red cells. The mechanistic basis for
decreased membrane mechanical stability in HE is weakened
“horizontal” linkages in membrane skeleton due either to defective
spectrin dimer-dimer interaction or a defective spectrin-actinprotein 4.1R junctional complex. Thus, HE is the result of defects
in genes encoding for ␣-spectrin, ␤-spectrin or protein 4.1R, all of
which are involved in “horizontal” linkages in the skeletal network
(Figure 3).62,63,79,84,87-90
OHS is a rare disorder characterized by presence of large
numbers of stomatocytes on blood smears in association with
moderately severe to severe anemia.91 Inheritance pattern of OHS
is autosomal dominant. The distinctive feature of red cells is their
increased sphericity due to increased cell volume without concomitant increase in membrane surface area. Stomatocytes with increased sphericity are sequestered by the spleen. However, while
splenectomy is highly beneficial in the management of HS and HE
patients, it is contraindicated in OHS, because it leads to increased
risk of venous thromboembolic complications. The mechanistic
basis for increased volume is the cell’s inability to regulate its
cation homeostasis. Stomatocytes exhibit a marked increase in total
cation content due to elevation of intracellular sodium, resulting in
increased cell volume. The molecular basis for OHS has not yet
been defined.92,93
Increased cytoplasmic viscosity. Red cell dehydration and
hence increased cytoplasmic viscosity is a feature of the inherited
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BLOOD, 15 NOVEMBER 2008 䡠 VOLUME 112, NUMBER 10
red cell membrane disorder, dehydrated hereditary stomatocytosis
(xerocytosis; DHS).91 Inheritance of DHS is autosomal dominant.
The distinctive feature of DHS is the increased MCHC of red cells
as a consequence of decreased total cation content and loss of cell
water. However, in contrast to significantly compromised survival
of hydrated cells of OHS, cell dehydration has only a marginal
effect on survival of DHS red cells. DHS is therefore associated
with well-compensated anemia with only a mild to moderately
enlarged spleen. The mechanistic basis for decreased volume is the
red cell’s inability to regulate cation homeostasis resulting in decreased
total cation content due to decreased intracellular potassium. The
molecular basis for DHS is, like OHS, still unknown.
Failure to regulate cell volume with ensuing cell dehydration
has long been recognized as a feature of red cells in sickle cell
disease, Hb SC disease and Hb CC disease.94,95 However, in
contrast to DHS, in which cell dehydration is the only dominant
feature, red cells in hemoglobinopathies are not only dehydrated
but also exhibit significant membrane alterations, including increased membrane rigidity.67 The membrane disturbances are the
result of oxidation-induced structural reorganization. While the
Gardos channel and K-Cl cotransporter have been implicated in an
altered state of hydration of red cells in hemoglobinopathies, the
mechanistic understanding of disordered volume regulation in
these disorders is far from complete.
Altered membrane deformability. Decreased membrane deformability is a distinguishing feature of red cells in hereditary ovalocytosis.96
Ovalocytosis is very common in SoutheastAsia where in malaria endemic
areas its prevalence ranges from 5% to 25%.97 The condition is characterized by the presence of oval-shaped red cells with 1 or 2 transverse ridges
or a longitudinal slit on blood smears. Inheritance of ovalocytosis is
autosomal dominant and to date only heterozygotes have been identified
in regions of high incidence implying that homozygosity is embryonic
lethal.98 Membranes of ovalocytes are 4 to 8 times less elastic than normal
membranes as assessed by ektacytometry and by micropipette aspiration.96,99 In terms of clinical manifestations, it is important to note that
despite a marked increase in membrane rigidity most affected people
experience minimal hemolysis. In all cases of ovalocytosis studied to date,
only 1 mutation has been identified: a genomic deletion of 27 bp encoding
amino acids 400 to 408 of band 3.100-102 Thus hereditary ovalocytosis is
unique among red cell membrane disorders in that the same mutation in a
single gene is responsible for the morphologic phenotype. Although
several hypotheses have been proposed regarding how a mutation in
band 3 could lead to a marked increase in membrane rigidity, the
mechanism of the effect has yet to be established.
Our current understanding of molecular basis for inherited red
cell membrane disorders, hereditary spherocytosis, hereditary
elliptocytosis and hereditary ovalocytosis is wide-ranging, yet
there are still cases where the molecular and genetic pathobiology
are unknown. In contrast, the molecular and genetic basis for red
cell disorders due to membrane transport defects such as the
dehydrated and overhydrated hereditary stomatocytosis syndromes
are largely unknown, remaining a continuing challenge.
Future
Studies on the red cell membrane have shed much new, often
unexpected light on structure and function of plasma membranes
RED CELL MEMBRANE
3945
generally, and have, we would argue justified the claims, so often
made, of its broad paradigmatic value. There can, at the same time,
be no doubt as to the advances in hematology that have flowed from
this work, for now we have a far-reaching understanding of the
causes of membrane defects in genetic diseases. As result of these
many advances, it might be felt that we have a complete understanding of all aspects of red cell membrane structure and function and
very few new and novel insights will be forthcoming from future
endeavors. This narrow point of view ignores the great continued
potential of red cell research as revealed by reports published in
2008 that outlined unexpected new and novel insights into red cell
membrane structure and function. Furthermore, just a few examples of the great unexhausted potential of red cell research
concern: nature and function of macromolecular complexes in the
membrane; dynamic regulation of skeletal protein and membrane
protein complexes by phosphorylation, and other posttranslational
modifications, and by phosphoinositides; dynamics of assembly
and function of the membrane microdomains, for which there is
now strong evidence; molecular basis for cell volume regulation;
detailed clarification of the sequence of events in regulated
assembly of plasma membrane, and its associated protein skeleton;
regulation of membrane properties by biodynamic ligands, including adrenergic agents, nucleotides and hormones; the nature of
membrane perturbations in diabetes and many other diseases; the
problems presented by membrane damage in sickle cell and related
diseases; interaction of red cells with endothelial surfaces in health
and disease; and of course the many problems, basic and practical,
presented by malaria. While it is clear these or related topics are
currently under intensive investigation in many other cell types,
because of its simplicity and elegance, it is very likely that the red cell
will continue to provide deep insights into these complex issues.
Acknowledgments
We very much regret that many important papers from several very
talented people who made significant contributions to red cell
research could not be cited or discussed due to strict space
limitations. We sincerely apologize for this omission. We gratefully
acknowledge the camaraderie and friendship of a very large
number of investigators associated with red cell research who over
the years have enriched our careers in red cell research. This work
is dedicated to the memory of Sally Marchesi, a dear friend and a
wonderful colleague.
This work was supported by the National Institutes of Health
(DK26263, DK32094, and HL31579 to N.M.; DK62039 and
HL65488 to P.G.G.).
Authorship
Contribution: N.M. and P.G.G. cowrote the paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Narla Mohandas, New York Blood Center,
310 East 67th Street, New York, NY 10065; e-mail: MNarla@
NYBloodcenter.org.
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From www.bloodjournal.org by guest on September 30, 2016. For personal use only.
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BLOOD, 15 NOVEMBER 2008 䡠 VOLUME 112, NUMBER 10
I arrived in the United States in 1968 with a degree in chemical engineering to begin graduate studies at the School of Engineering at Washington University, St Louis. It was there it all started—and
not the way I had envisioned. My thesis supervisor, Robert Hochmuth, introduced me to the field
of red cell research. It became my first professional love and I have stayed faithful to it ever since.
After completing my DSc, I spent 3 formative years in Paris working with Marcel Bessis. Using the
ektacytometer that we developed together, we found interesting differences in the deformability of
red cells in a variety of disorders. Realizing that my engineering background had not equipped me
for a broad understanding of the pathophysiologic implications of the phenomenon I was studying, in 1976 I began to look for collaborators with expertise in hematology, biochemistry, biophysics, cell biology, molecular biology, and genetics. This journey, spanning the past 3 decades, has
been extraordinarily rewarding both professionally and personally. During these years I have had
the privilege of working closely with an array of wonderful colleagues who have become close
friends. I would like especially to acknowledge my collaborators of many years, Xiuli An, Joel Ann
Chasis, Margaret Clark, John Conboy, Ross Coppel, Dennis Discher, Evan Evans, Gil Tchernia,
and Yuichi Takakuwa, and my many good friends, always generous with their support and help,
including Peter Agre, David Anstee, Anthony Baines, Jane Barker, Vann Bennett, David Bodine,
Pat Gallagher, Walter Gratzer, Kasturi Haldar, Bob Hebbel, Joe Hoffman, Phil Low, Sam Lux, Jon
Morrow, Luanne Peters, and Stan Schrier. A happy feature of the red cell field is its collegiality, and
it has been a particular pleasure to associate with so many excellent scientists, whose friendship
and support has meant so much to me. My fascination with red cells is still unabated after devoting the past 4 decades of my professional life to it.
Narla Mohandas
The little boy with sickle cell disease in Room 316A was hospitalized with the diagnosis of “impending crisis.” “What did his peripheral smear show?” Dr Beatrice Lampkin asked me on my
fourth day of internship at Cincinnati Children’s Hospital. “Let’s go look.” Listening to her describe the morphology and its significance and discuss the clinical manifestations and pathobiology of his disease and what it meant to this boy on a daily basis was a life lesson in hematology.
People in the field of hematology care, they are thoughtful, they want to understand how things
work, and they want to improve people’s lives. I also learned another lesson of hematology: you
can learn a lot more sitting around a microscope with a group of hematologists than just what’s on
the peripheral smear. After residency, I moved to Yale University for fellowship training, where I
began training in the laboratory of Dr Bernard Forget. The research training I received from Bernie
was priceless, going from experimental conception, design, execution, and interpretation to presentation. Group meetings in the Hematology Division at Yale at that time were exciting, with presentations on hemoglobin switching, gene expression, receptor signaling, gene structure and
function, membrane biology, and inherited and acquired human disease, which instilled an interest in a broad range of topics that continues today. I also greatly benefitted from William Tse’s
laboratory tutelage, for which I am most grateful. I think of my experiences in hematology as
vignettes. Bernie Forget coaching the art of peer review: “First, always say something nice.”
Watching the influence of Edward Benz’s smile and encouraging words on the people around him.
Sitting in an airport listening to Peter Agre comparing academics to drug addiction, explaining
that no matter the ups and downs, we just love academics and can’t stop. Introducing myself to
Samuel Lux when he was visiting New Haven; he replied, “I know who you are. Are you putting in a
K?” Meeting my collaborator and friend David Bodine at a Gordon Conference reception to discuss erythroid-specific promoters, and the Red Sox. David Nathan rescuing me from a tough
questioner at a Children’s Health Research Center meeting. Reading the name badge of the kind
Patrick G. Gallagher
man who came over to talk to me about my poster at ASH while I was a fellow: “Mohandas Narla” it
read. Mohan is now a great friend and colleague and I am proud to coauthor this review with him. I have found the field of hematology to be exciting, collegial, and compassionate. I look forward to many, many more years.
From www.bloodjournal.org by guest on September 30, 2016. For personal use only.
2008 112: 3939-3948
doi:10.1182/blood-2008-07-161166
Red cell membrane: past, present, and future
Narla Mohandas and Patrick G. Gallagher
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ASH 50th Anniversary Reviews (34 articles)
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