Vet. Pathol.

2 4 4 7 1-476 (1 987)

REVIEW

Erythrocyte Membrane: Structure, Function, and Pathophysiology

J. E. SMITH
Department of Pathology, College of Veterinary Medicine,
Kansas State University, Manhattan, KS

Erythrocytes are unique among mammalian cells. mechanisms responsible for the asymmetry are un-
They contain no nucleus or subcellular metabolic known, redistribution of membrane phospholipids may
have serious consequences. If the internal amino phos-
structures, yet they survive for 3 to 7 r n ~ n t h s .During
~'
their lifetime, erythrocytes travel thousands of miles pholipids become exposed to the plasma, they may
through tubes of various sizes while delivering oxygen trigger the clotting cascade. Lipid domain fluidity is
to the tissues.2oSurvival of practically all other cells determined by the molar ratio of cholesterol to phos-
in the body depends upon the proper functioning of pholipid, degree of unsaturation of phospholipid acyl
erythrocytes. They travel as individual cells, contain- chains, and phosphatidylcholine sphingomyelin ratio.
ing a concentrated solution of hemoglobin surrounded Phosphatidylcholine forms highly fluid lipid regions,
by a membrane. In spite of their apparent simplicity, while sphingomyelin induces rigid it^.^
they have limited (but necessary) metabolic capability. Proteins in the lipid domain usually extend from the
They are exposed to a high oxygen content and come inside of the erythrocyte to the outside. These mem-
in contact with a variety of endogenous and exogenous brane proteins can be divided structurally into an in-
chemicals. Their ability to survive depends on the abil- ternal hydrophilic portion, a membrane-spanning hy-
ity of the membrane to remain intact and flexible.20 drophobic portion, and an external hydrophilic portion
This paper focuses on the erythrocyte membrane struc- with attached carbohydrate^.^^ Some blood groups are
ture and function and the pathophysiological changes determined by the structure of those external carbo-
that can occur when the membrane is exposed to an hydrates.2Membrane-spanning proteins are classified
unfavorable environment. as integral proteins. Integral proteins include mem-
brane protein 3, seen in Coomasie blue-stained poly-
Erythrocyte Membrane acrylamide gels (Fig. l), and several glycophorins,
Structure and Function visualized by periodic acid-Schiff-stained gels.'* Mem-
The erythrocyte membrane consists of two domains, brane protein 3 is an anion exchange protein and allows
a lipid bilayer and the cytoskeleton. The lipid domain the erythrocyte to exchange C1- for HC03-, and to
is similar structurally to that found in most mam- transport carbon dioxide from the tissues to the
malian cells. The cytoskeleton differs from what is con- The exact function of most glycophorins is unknown.
sidered cytoskeleton in other cells because it does not
contain the structural protein tubulin and is not in- Cytoskeleton
volved in cell motility or phagocytosis. The erythrocyte cytoskeleton (Fig. I) consists of sev-
eral proteins that form a filamentous network under
Lipid domain the lipid bilayer. The network is composed of spectrin,
The lipid domain is composed of nearly equal parts ankyrin, actin, and protein 4.1. Cytoskeletal proteins
of lipid and protein. The main lipids are cholesterol interact with integral proteins and lipids of the bilayer
and phospholipids. Although cholesterol seems equally to maintain membrane integrity. The cytoskeleton has
distributed between the two halves or leaflets of the an important role in erythrocyte shape, flexibility, and
lipid bilayer, the other lipids are asymmetrically dis- lipid organization.20Spectrin is the dominant mem-
t r i b ~ t e d . ~Glycolipids,
,'~ phosphatidylcholine, and brane protein, both in number of copies per erythrocyte
sphingomyelin are located in the outer half of the bi- and in size. It consists of two proteins with molecular
layer; phosphatidylinositols, phosphatidylethanola- weights of 240,000 and 220,000 daltons. Both proteins
mine, and phosphatidylserine occur in the interior are long, flexible structures that are twisted together to
layer facing the cytoplasm. Although the forces and form heterodimers.28Tetramers, and possibly higher
47 1
412 Smith
CROSS-SECTION OF RBC MEMBRANE duces bulk viscosity in larger vessels, prevents phago-
cytosis by macro phage^,^ and aids exit from bone mar-
row. l * Erythrocyte deformability depends on cell
geometry (includingthe surface to volume ratio), mem-
brane viscoelastic properties, and intracellular viscos-
ity (primarily hemoglobin concentration).21The eryth-
rocyte’s membrane must resist mechanical stresses
applied to it while circulating through the body. Both
deformability and membrane strength are largely de-
pendent on the erythrocyte cytoskeleton.
Erythrocyte Pathophysiology
GLYCOPHORIN
Abnormalities of the erythrocyte can be divided into
those affecting primarily the lipid portion of the mem-
brane and those affecting the cytoskeleton. This divi-
sion is artificial, however, because the two structures
Fig. 1. Cross-section of the erythrocytemembrane viewed are interrelated, and drugs or chemicals that affect one
from inside the cell. The vertical graph demonstrates the structure may cause profound changes in the other.
appearance of membrane protein in polyacrylamidegel elec- Some chemicals interact with the lipid bilayer to in-
trophoresis. duce changes in erythrocyte shape; others have pro-
found oxidizing effects on the cytoskeleton and hemo-
order oligomers, are formed by a “head-to-head” in- globin.
teraction of heterodimers. Actin binds to the “tail” of
several heterodimers to generate a series of polygons Chemicals that interact with the lipid bilayer
(mostly hexagons), with spectrin tetramers as the sides. Erythrocytes can undergo two basic shape changes
Membrane protein 4.1 promotes, and may regulate, (Fig. 2). Normally, erythrocytes appear in the circu-
the actin-spectrin association. lation of many mammals as biconcave disks or dis-
The net-like structure formed by the protein-protein cocytes. The other common erythrocyte shapes are sto-
interaction of spectrin, actin, and protein 4.1, lies just matocytes, which are cup-shaped, and echinocytes,
beneath the lipid bilayer. It is attached to proteins and which have an irregular, spiculated surface. Although
lipids of the bilayer in at least three ways. Ankyrin, a wide variety of conditions can convert one shape to
another cytoskeleton protein, binds near the middle of another,’I those changes associated with drug-lipid in-
the spectrin tetramers to form a link from the network teraction are better understood than the others. When
to membrane protein 3.3 Membrane protein 4.1 links anionic amphipathic drugs are incubated with disco-
the cytoskeleton to another transmembrane protein, cytes, the drug concentrates preferentially in the outer
glycophorin.22That association may be modulated by leaflet of the lipid bilayer (Fig. 3), which expands the
polyph~sphoinositides.~~ In addition, spectrin is as- outer leaflet relative to the inner leaflet, and induces
sociated directly with phospholipids of the mem- an echinocytic change. The opposite effect is seen on
brane’s inner leaflet. The erythrocyte cytoskeleton is the lipid bilayer when cationic amphipathic drugs are
important physiologically because inherited deficien- used and erythrocytes become stomatocytic. This phe-
cies of any of its proteins cause membrane instability, nomenon is called the “bilayer couple hypothesis,” and
loss of membrane lipid, and increased tendency for was proposed by Sheetz and Singer in the 1 9 7 0 ’ ~ ~ ~
affected erythrocytes to fuse.27 Echinocytes are formed from discocytes when eryth-
The surface area of the erythrocyte skeleton appears rocyte adenosine 5‘-triphosphate (ATP) is depleted, or
to be larger than necessary to cover the inner surface the cytoplasmic Ca++is increased.l As the cytoplasmic
of the erythrocyte. Theoretical calculations indicate pool of ATP is decreased, polyphosphoinositides de-
that spectrin molecules may be 3 times longer than phosphorylate, which alters the glycophorin-mem-
required to bridge the distance between adjacent actin brane protein 4.1 interaction. When ATP is repleted,
molecule^.^ Although spectrin’s configuration under the polyphosphoinositides are rephosphorylated, and
physiological conditions is unknown, the ability of echinocytes revert back to discocytes. CytoplasmicCa++
spectrin, and thus the cytoskeleton, to expand has im- activates phospholipase C, which cleaves polyphos-
plications in erythrocyte deformability and membrane phoinositides. Because erythrocytes cannot synthesize
strength.l 3 polyphosphatidylinositol de novo, nor acquire it from
The erythrocyte’s ability to deform is necessary for plasma proteins, phospholipase C activity causes a per-
passage through capillary beds.’ Deformability also re- manent loss of inositol phospholipid and eventual, ir-
 Erythrocyte Membrane
ECHINOCYTE STOMATOCY TE
1 2
Fig. 2. At low concentrations of anionic amphipathic
drugs, normal erythrocytes or discocytes (A) are converted
to echinocytes I (B). As the concentration increases, sphero-
echinocytes I (C) then spheroechinocytes I1 (D) are formed.
Cationic amphipathic drugs produce stomatocytes I (E),
spherostomatocytes I (F),and spherostomatocytes I1 (G) as
concentration increases.
Fig. 3. Effect of amphipathic drugs on erythrocytes. a)
Anionic amphipathic drugs localize in the outer leaflet, thus
expanding it (1); cationic amphipathic drugs localize in and
expand the inner leaflet (2).
reversible echinocytic change. The exact mechanism
for the shape change is unknown.
Stomatocytes may also form when the erythrocyte
water content is increased. That phenomenon is im-
portant in the stomatocytosis seen in an inherited syn-
drome in Alaskan Malamute dogs.24
Chemicals that have oxidant effects
Because the erythrocyte carries oxygen and is ex-
posed to a wide range of chemicals dissolved in plasma,
it is particularly vulnerable to damage due to oxidants.
The effects of those oxidative stresses depend on the
compounds involved, their concentration, and the
metabolic capabilities of the erythrocyte. Many oxi-
473
G6PD 6PGD CR GPX
GLUCOSE-6-PO.
Fig. 4. Hydrogen peroxide (H,O,) detoxification path-
way, G6PD = glucose-6-phosphatedehydrogenase; 6PGD =
6-phosphogluconatedehydrogenase; GR = glutathine reduc-
tase; GSH = reduced glutathione; GSSG = oxidized gluta-
thione; NADP = nicotinamide adenine dinucleotide phos-
phate; PGX = glutathione peroxidase.
dant drugs are substituted benzene derivatives and fa-
cilitate the conversion of oxyhemoglobin to methe-
moglobin and hydrogen peroxide.* Methemoglobin
may be reduced to oxyhemoglobin with reduced nico-
tinoamide dinucleotide. Because hydrogen peroxide can
cause widespread protein denaturation and lipid per-
oxidation, the erythrocyte has efficient enzymatic ma-
chinery to convert it to water (Fig. 4). If any enzymes
or substrates in this chain of reactions become in-
adequate, either as the result of inherited deficiency,
dietary inadequacies, or drug inhibition, erythrocytes
become vulnerable to oxidant stresses.
When the oxidant stress exceeds the erythrocyte's
reductive capacity, several things can happen. The most
obvious oxidative damage is the formation of Heinz
bodies. Heinz bodies are composed of denatured
hemoglobin and some erythrocyte membrane proteins.
Although Heinz bodies may form by oxidation of in-
ternal sulf hydryl groups in hemoglobin,l5 hemichrome
formation seems more important.34
Reversible hemichromes, such as hemoglobin hy-
droxide and dihistidine femhemochrome, are formed
from methemoglobin. As the name indicates, the re-
versible hemichromes can be converted back to met-
hemoglobin, and, eventually, to reduced hemoglobin.
If irreversible hemichromes (dihistidine ferrihemo-
chrome or a mercaptide derivative) are formed, the
hemoglobin denaturation cannot be stopped, and
hemoglobin aggregates to form Heinz bodies.23Hemi-
chromes have an increased affinity for the cytoplasmic
portion of membrane protein band 3.32The resulting
membrane protein 3-hemichrome complex promotes
the polymerization of additional molecules to form a
macromolecular complex that can be recognized mi-
croscopically as Heinz bodies.
The copolymerization of membrane protein 3 and
hemichromes causes membrane protein 3 to form clus-
ters. Because membrane protein 3 is an integral protein
and spans the lipid bilayer, the cluster on the inside
also results in clustering on the outside of the eryth-
rocyte membrane. External clustering of membrane
474 Smith

6-
@-@LOWGSH
A I A-AHigh GSH

e-• Low
A-A High

1 I I I I 1 I 1

-4 0 4 8 12

0
, \,,J I I
5Or
fffCCCff
@-@LOWGSH

protein 3 creates a recognition site for auto-antibod-

ies. l9 Auto-antibody against membrane protein 3 clus-
ters may be the same auto-antibody proposed by Kay1’
to be important in recognizing senescent erythrocytes.
Erythrocytes with attached auto-antibody are trapped
and degraded by macrophages in the spleen and liver.
Alternatively, Heinz bodies can be removed by the
spleen, and the remaining portion allowed to circu-
late.33That phenomenon has been termed splenic “pit-
ting” action.
Some oxidant stresses do not result in Heinz body
formation, but do affect the erythrocyte cytoskeleton.
They may shorten erythrocyte life span by altering
erythrocyte deformability and membrane stability.
Erythrocytes from some patients with glucose-6-phos-
phate dehydrogenase deficiency have increased
amounts of high molecular weight polypeptides. l 6
Those polypeptide aggregates seem related to the de-
gree of in vivo hemolysis and the level of reduced
glutathione. Because polypeptide aggregates can be
dissociated by sulfhydryl compounds such as dithio-
threitol, they seem to be polymers of spectrin formed
by intermolecular disulfide bonds.
Similar complexes occur in sheep with an inherited
low level of glutathione, when they are challenged with
intravenous copper acetate. If the oxidant stress is con-
trolled, erythrocyte-reduced glutathione decreases (Fig.
5 ) and high molecular weight complexes appear (Fig.
6). Some high molecular weight complexes involve
hemoglobin, because treatment with sulfhydryl agents
releases hemoglobin in addition to spectrin. Those
A
‘ -A
20
lot
Fig. 6. High molecular weight complexes formed in
erythrocyte membranes of high- and low-reduced glutathione
(GSH) sheep given copper acetate intravenously (arrows).
Fig. 7. Serial changes in packed cell volume of high- and
low-reduced glutathione (GSH) sheep when copper acetate
is given intravenously (arrows).
changes cause a shortened erythrocyte life span and
decline in the packed cell volume (Fig. 7).
Other, less strilung, oxidant effects may also occur
in erythrocytes. For example, when blood is stored
under blood bank conditions, erythrocytes slowly lose
their ability to survive following t r a n s f ~ s i o n .Al-
~~
though preservation of adenosine triphosphate levels
will lengthen storage time, adenosine 5‘-triphosphate
(ATP) concentration does not seem to be the major
determinant. Erythrocyte membrane function seems
to be abnormal in stored blood. The in vitro ability of
 Erythrocyte Membrane
spectrin to bind to actin in the presence of membrane
protein 4.1 decreases during storage. Those membrane
changes in stored blood are partially reversed by di-
thiothreitol, suggesting that the change involves oxi-
dation of sulfhydryl groups during storage. The ability
of normal spectrin to bind to membrane protein 4.1
can be eliminated by the oxidation of a single disulfide
bond. Erythrocytes also lose phospholipid during stor-
age, and that loss correlates with the spectrin-actin
binding lesion.
Either oxidant stress or shape changes can shorten
erythrocyte life span. The profound effects produced
by drugs are only rarely seen in vivo, but echinocytic
and stomatocytic changes do occur physiologically and
can adversely affect erythrocyte deformability. Oxi-
dant stresses (particularly those producing Heinz bod-
ies), are seen frequently with some plant toxins and
drugs, and may cause severe anemia. Nevertheless, the
erythrocyte membrane can withstand the rigors of
chemical attack, yet remain flexible and viable.
Acknowledgements
The author acknowledges the editorial assistance of Dr.
Bradley Weeks and illustrations by Mallory R. Hoover. Re-
search was supported by NIH grant HLO 1877 and the Kansas
Agricultural Experiment Station (KAES).
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