Red blood cell membrane: Structure and dynamics

INTRODUCTION — Erythropoiesis is a tightly regulated and complex process that generates two million enucleated red blood cells (RBCs) every second. These RBCs ultimately have to deform from their normal 7- to 8-micron diameter discoid shape to pass through the 3-micron diameter capillaries and the 1- to 2-micron-wide endothelial slits in the red pulp of the spleen (picture 1).

Estimates based on the 120-day typical RBC lifespan suggest that RBCs make approximately 500,000 passages through the circulation during their lifespan. This topic reviews the membrane characteristics (structural proteins, organization, and dynamic properties) that allow RBCs to circulate without being destroyed or obstructing blood flow in capillaries or splenic sinusoids.

Conditions with abnormalities of RBC membrane are discussed separately.

●Stomatocytosis – (See "Hereditary stomatocytosis (HSt) and hereditary xerocytosis (HX)".)

●Projections and target cells – (See "Burr cells, acanthocytes, and target cells: Disorders of red blood cell membrane".)

●Spherocytosis – (See "Hereditary spherocytosis".)

●Elliptocytosis – (See "Hereditary elliptocytosis and related disorders".)

●Bite cells and Heinz bodies – (See "Unstable hemoglobin variants" and "Drug-induced hemolytic anemia".)

RBC SHAPE AND DEFORMABILITY

RBC shape/geometry — The RBC differs from most eukaryotic cells in that its shape is created by the geometry of its membrane and submembrane cytoskeleton, rather than any internal or external structures. In contrast to most other cells, RBCs have no cytoplasmic structures or organelles, no nucleus, and no cell-cell junctions that contribute to their shape.

Normal RBCs are biconcave discs (picture 2). In a two-dimensional view, such as on a peripheral blood smear, the biconcave disc appears as a round cell with an area of central pallor (picture 3). The basis for this shape is the highly regulated ratio of membrane surface area to cytoplasmic volume, designated "SA/V." The advantageous SA/V ratio of RBCs gives them a remarkable ability to deform and alter their shape to pass through narrow channels.

Membrane deformability is a property that reflects the extent of the membrane deformation that can be induced by a defined level of applied force. Most deformations do not involve an increase in surface area; rather, the membrane geometry is altered to allow a change in shape. Normal RBCs can undergo large linear extensions (of up to 230 percent of their original diameter) while maintaining a constant surface area. However, an increase of even 3 to 4 percent in surface area results in cell lysis.

The more deformable the membrane, the less force required for the cell to deform. A high degree of deformability is needed for RBCs to traverse small capillaries (as small as 3 microns) and splenic sinusoids (as small as 1 to 2 microns) that are smaller than the diameter of the RBCs (7 to 8 microns). The ability of the red cell to undergo extensive deformation is essential for both its function and its survival [1-4].

Red cell deformability is a function of three major attributes [5,6]:

●Shape/geometry (ie, the SA/V ratio) (see 'Surface area to volume ratio (SA/V)' below)

●Viscosity of the cytoplasm (see 'Cytoplasmic viscosity' below)

●Material/mechanical properties of the membrane related to protein-protein, lipid-lipid, and protein-lipid interactions (see 'Composition of the membrane/cytoskeleton' below)

In contrast to mature RBCs, reticulocytes, generated by enucleation of orthochromatic erythroblasts, are the last stage of terminal erythroid differentiation; their deformability is only approximately 10 percent that of mature RBCs [7,8]. The deformability and mechanical stability of reticulocytes both improve during maturation [7].

Major reorganizations of membrane phospholipids, cytoskeletal proteins, and integral proteins accompany acquisition of the discoid shape and the enhanced deformability. These changes include:

●Loss of membrane lipids

●Loss of integral proteins such as transferrin receptors, cell adhesion molecules such as integrins, insulin receptors, and fibronectin receptors

●A major reorganization of the cytoskeletal protein network

●Altered phosphorylation of membrane proteins

●Progressive loss of volume and cell water, resulting in an increased SA/V ratio

Surface area to volume ratio (SA/V) — Normal RBCs have a volume of 90 fL and a surface area of 140 square microns. If the RBC were a sphere of identical volume, its surface area would be only 98 square microns. Thus, the biconcave discoid shape provides approximately 40 square microns of excess surface area (an additional 43 percent).

Reductions in the SA/V ratio (less plasma membrane for a given amount of cytoplasm) lead to changes in RBC shape (typically, stomatocytes and spherocytosis) that translate into reduced deformability and often an increased rate of hemolysis. On the peripheral blood smear, spherocytes appear smaller than normal RBCs and lack central pallor. Increased osmotic fragility is a characteristic feature of spherocytes. In vitro studies have demonstrated that reduction of SA/V by approximately 25 percent leads to rapid and complete entrapment in the spleen [9].

Reduced SA/V can be documented by osmotic gradient ektacytometry, which documents decreased membrane surface area and increased osmotic fragility, and by documentation of decreased membrane content of band 3 by labeling with eosin-5-maleimide (EMA) [10,11]. The figure illustrates examples of these techniques as used in the diagnosis of RBC membrane disorders (figure 1).

There are two mechanisms by which the SA/V ratio can be decreased:

●Decreased plasma membrane surface area – A relative decrease in the amount of membrane area for the same amount of cell volume decreases the SA/V ratio. This occurs in disorders affecting RBC membrane homeostasis. Examples include:

•Autoimmune hemolytic anemia (AIHA), in which pieces of the RBC membrane coated with immunoglobulins on the surface are removed by splenic macrophages.

•Hereditary spherocytosis, in which defects in membrane cytoskeletal proteins such as ankyrin, spectrin, or band 3 (anion exchanger) lead to decreased cohesion between the lipid bilayer and spectrin-based membrane cytoskeleton and resultant membrane loss [10,12].

•Hemolytic hereditary elliptocytosis, in which RBC fragmentation occurs as a consequence of decreased membrane mechanical stability [12].

●Increased cell water – A relative increase in the amount of cell water decreases the SA/V ratio. This occurs in hemolysis due to inappropriate use of water as a diluent for RBC transfusion and in certain hereditary anemias with genetic defects that affect RBC hydration (eg, hereditary stomatocytosis [HSt]). In HSt, the increased SA/V results in a uni-concave cup, which appears as a stomatocyte on the peripheral blood smear. These disorders were discussed in a 2017 review [13]. They are discussed in detail separately. (See "Hereditary stomatocytosis (HSt) and hereditary xerocytosis (HX)".)

These disorders are discussed in more detail and in separate topic links provided below. (See 'Clinical consequences' below.)

Loss of membrane at a slower rate is a function of normal RBC aging. During their normal lifespan, RBCs become increasingly spherical (with a progressive reduction in SA/V) due to loss of surface area; this occurs as reticuloendothelial macrophages remove small pieces of membrane. This is the dominant mechanism for the removal of senescent RBCs from the circulation.

In contrast to conditions with decreased SA/V ratios, target cells are RBCs that have an increased SA/V ratio (more membrane for a given amount of cell volume). On the peripheral blood smear, target cells may be larger than normal RBCs and often have an area of central density within the region of central pallor (picture 4). Spur cells (picture 5) are also produced by an increased SA/V ratio; these are contrasted with burr cells (picture 6).

Target cells and spur cells can form with increased insertion of membrane cholesterol, as occurs in liver disease, or with decreased cytoplasmic volume, as occurs in thalassemia and hereditary xerocytosis. In contrast to spherocytes, target cells have a near-normal lifespan, and individuals with target cells have minimal hemolysis and generally are not anemic. Target cells and spur cells have decreased, rather than increased, osmotic fragility. (See "Burr cells, acanthocytes, and target cells: Disorders of red blood cell membrane".)

Cytoplasmic viscosity — Cytoplasmic viscosity (resistance to flow) also impacts RBC deformability. The major contributing factor to viscosity in the RBC is the mean corpuscular hemoglobin concentration (MCHC), which is determined by the ratio of hemoglobin to cell water [5]. This is because hemoglobin is by far the most abundant protein in the RBC.

It is important to note that while the cell volume of mammalian RBCs varies over 10-fold, the MCHC is relatively invariant across species at approximately 33 g/dL (range, 27 to 35 g/dL). This MCHC produces a viscosity of 5 to 15 centipoise (cP), which is 5 to 15 times greater than the viscosity of water. Above an MCHC of 35 g/dL, further increases in MCHC lead to exponential increases in viscosity (eg, 45 cP at 40 g/dL; 170 cP at 45 g/dL; and 650 cP at 50 g/dL), and viscosity may become the primary determinant of cellular deformability. This relationship between MCHC and viscosity is illustrated in the figure (figure 2).

The lipid components and integral proteins of the RBC membrane involved in transport function play a crucial role in volume homeostasis, maintaining the MCHC at levels that do not unduly influence the ability of the cell to deform. (See 'Composition of the membrane/cytoskeleton' below.)

An abnormally high MCHC generally occurs when cell water is decreased (ie, cellular dehydration), which may be due to abnormalities in membrane channels or other alterations to membrane permeability. Examples include:

●Hereditary disorders affecting cellular channels (hereditary xerocytosis with pathogenic variants in PIEZO1, which encodes a mechanically activated stretch channel, or in KCNN4, which encodes the Gardos channel). (See "Hereditary stomatocytosis (HSt) and hereditary xerocytosis (HX)".)

●Hereditary disorders in which abnormal hemoglobins precipitate and interact with the RBC membrane (sickle cell disease, hemoglobin C disease, thalassemia) [3,5,14,15]. (See "Pathophysiology of thalassemia", section on 'Effects on the RBC' and "Pathophysiology of sickle cell disease", section on 'Effects on the RBC'.)

Reduced deformability may impair oxygen delivery in these disorders.

Membrane stability — The mechanical behavior of the RBC membrane is complex and depends upon the magnitude and duration of applied stresses [5,6,16-18]. The property of mechanical membrane stability is defined as the maximum extent of deformation that a membrane can undergo and still recover its initial shape. Any further increase in deformation results in membrane failure and cell fragmentation.

Membrane deformability and mechanical stability are regulated by multiple membrane properties such as elastic shear modulus, bending modulus, and yield stress.

●Elastic shear modulus is a measure of the magnitude of force needed to induce uni-axial deformation at a constant surface area. The higher the value, the greater the force needed to induce deformation. Shear modulus is regulated by both cytoskeletal and membrane proteins.

●Bending modulus is a measure of the contribution of bending effects to the total work of deformation. Bending modulus becomes important when there are large changes in membrane curvature, such as with spicule formation. Bending modulus is regulated primarily by the lipid bilayer.

●Yield stress is the value of applied force at which the membrane begins to flow; it is a measure of the cohesion between the lipid bilayer and the underlying membrane skeleton.

At small values of applied force for short duration, the red cell membrane behaves as a viscoelastic solid since it is capable of undergoing large elastic extensions and completely recovers its initial shape. Studies of biochemically perturbed RBC membranes and RBC membranes from various disease states have shown that deformability and stability can change with no fixed relationship to each other [19]. This observation implies that these two properties are regulated by different cytoskeletal proteins and their interactions.

Normal membrane stability allows RBCs to circulate for 120 days without fragmenting. Decreased stability leads to RBC fragmentation under normal circulating stresses, and increased stresses in the circulation (such as those caused by microthrombi or vascular anomalies) lead to RBC fragmentation even when membrane stability and deformability are optimized. (See 'Clinical consequences' below.)

COMPOSITION OF THE MEMBRANE/CYTOSKELETON

Structural organization and dynamic regulation — The plasma membrane of the RBC is a continuous sheet-like membrane that envelops the cellular contents (table 1). It consists of a complex, ordered array of lipids (approximately one billion molecules) and proteins (approximately 10 million molecules) in an extraordinarily thin (6 to 10 nm) lipid bilayer punctuated by penetrating or attached proteins (figure 3). Approximately 40 percent of the membrane mass is lipid, 52 percent is protein, and 8 percent is carbohydrate [20,21].

Increasingly sophisticated methods of analysis continue to identify proteins present in the RBC membrane, some of which remain uncharacterized. By mass spectrometry, there appear to be at least 1000 membrane proteins [22]. The filamentous network of associated proteins and glycoproteins, also referred to as the membrane skeleton or cytoskeleton, is important for maintaining cell shape and regulating membrane properties to produce a flexible yet mechanically resilient and stable membrane [5,6]. The membrane proteins are divided into two general groups: integral proteins and peripheral proteins (figure 3).

Besides mechanical deformability and stability, the RBC membrane has numerous other properties that arise in part from specialized interactions between specific membrane proteins or lipids, or both [12]:

●Self-assemble and self-seal

●Maintain asymmetry between inner and outer leaflets

●Provide physical continuity and solute impermeability

●Provide biconcave disc shape with maximum deformability

●Allow certain ions to enter the cell, some against a concentration gradient

●Allow certain nutrients to enter the cell

●Transmit signals between the cytoplasm and the extracellular environment

●Anchor proteins of the submembrane cytoskeleton

The RBC membrane is both extremely elastic (soft) and structurally resistant (strong). It is 100-fold softer than a latex membrane of equivalent thickness, and it is stronger than steel [12]. This is likely due to the triple helical structure of spectrin proteins and the dynamic interactions among proteins at the junctional complex. (See 'Spectrin' below.)

The interactions among membrane proteins must be regulated because the RBC undergoes regular cycles of deformation and relaxation as it circulates, requiring the membrane to accommodate to extensive and dynamic changes in cell shape.

Only limited information is available regarding these regulatory pathways.

Potential regulatory mechanisms include phosphorylation, variations in intracellular magnesium and 2,3-bisphosphoglycerate (2,3-BPG, also called 2,3 diphosphoglycerate [2,3-DPG]) during the oxygenation/deoxygenation cycle, and calmodulin effects due to elevated intracellular calcium [8,19,23]. (See "Structure and function of normal hemoglobins", section on 'Cooperativity'.)

Most of the available evidence suggests that phosphorylation tends to lower the affinity of protein-protein interactions. As examples [24]:

●Phosphorylation of protein 4.1 results in a fivefold decrease in its affinity for spectrin and its ability to promote spectrin-actin association.

●Phosphorylation of beta-spectrin reduces membrane mechanical stability.

●Increased levels of intracellular 2,3-DPG and elevated cytoplasmic concentrations of calcium, in association with calmodulin, decrease membrane mechanical stability by destabilizing spectrin-actin-protein 4.1 and spectrin-actin-adducin interactions [19,25]. The calcium concentrations inside RBCs are not sufficient to stimulate proteases.

Lipid bilayer — Like other plasma membranes, the plasma membrane of the RBC is a lipid bilayer with inner and outer leaflets. The major lipid components of the RBC membrane are unesterified cholesterol and phospholipids, which are present in nearly equimolar quantities, along with small amounts of free fatty acids and glycolipids [20]. Plasma membranes are fluid structures, yet they are highly ordered with respect to the distribution of molecules both across the bilayer and within the plane of the bilayer. Cholesterol appears to be evenly distributed between the inner and outer leaflets, but the distribution of four major membrane phospholipids is highly asymmetric [12].

The primary membrane phospholipids include the uncharged phospholipids phosphatidylcholine (PC; 30 percent) and sphingomyelin (SM; 25 percent) and the charged phospholipids phosphatidylethanolamine (PE; 28 percent) and phosphatidylserine (PS; 14 percent).

The distribution of these phospholipids in the membrane is highly asymmetrical [26-28]:

●The outer monolayer contains more than 75 percent of the choline-containing uncharged phospholipids PC and SM

●The inner monolayer contains most of the negatively charged phospholipids (approximately 80 percent of the PE and all of the PS)

The mature RBC is unable to synthesize lipids de novo. However, lipid renewal pathways produce considerable turnover of the various phospholipids with no net change in lipid composition. In addition, the cholesterol content of the membrane is regulated by exchange between plasma cholesterol and membrane cholesterol; membrane cholesterol content in turn regulates phospholipid "scramblase" activity, which maintains the asymmetry of phospholipids in the membrane [29,30]. Scramblase activity is ATP-independent. Other contributions to the maintenance of asymmetric phospholipid distribution in the normal RBC membrane appear to be a function of the following processes:

●A slow and symmetric rate of passive diffusion of choline-containing phospholipids (PC and SM) across the two lipid monolayers, with a time scale on the order of hours [27]. By contrast, cholesterol diffuses across the membrane on a physiologic time scale on the order of seconds (half-time of seconds or less) [31].

●A direct interaction between charged phospholipids and membrane skeletal proteins [32].

●Active transport of the aminophospholipids (PS and PE) from the outer to the inner monolayer by an aminophospholipid translocase (also called "flippase") [27,30,33]. Flippase is the opposing enzyme to scramblase. Flippase activity requires ATP.

Loss of this asymmetric organization occurs in several disease states, including beta thalassemia and sickle cell disease [34,35]. In patients with these diseases, the membrane phospholipid bilayer is scrambled in such a way that some PS moves to the outer leaflet [36,37]. This abnormality is thought to be due to an oxidative injury caused by aggregates of unpaired globin chains (eg, excess alpha globin chains in the beta thalassemia syndromes) or oxidant membrane damage induced by sickle hemoglobin. The clinical significance of this abnormality is not fully defined, but it is very likely that PS on the outer leaflet is recognized by reticuloendothelial macrophages as a recognition signal for phagocytosis [38]. This abnormality may also contribute to thrombogenesis, endothelial adhesion, and reduced RBC survival, which is seen in these disorders. (See "Pathophysiology of sickle cell disease", section on 'Vaso-occlusion'.)

The shape of the lipid bilayer can be altered by slight variations in the surface area of either the inner or outer leaflet [39,40]. Increasing the surface area of the inner leaflet produces a stomatocytic shape (picture 7), while increasing that of the outer leaflet promotes outward curvature and transforms the cell into an echinocyte (burr cell) (picture 6) [39,40]. (See "Hereditary stomatocytosis (HSt) and hereditary xerocytosis (HX)" and "Burr cells, acanthocytes, and target cells: Disorders of red blood cell membrane".)

Integral membrane proteins — Integral membrane proteins are proteins that are tightly embedded in the membrane through hydrophobic interactions with the lipids in the bilayer. Band 3 (the anion exchanger), aquaporin-1 (the water channel), glut-1 (glucose transporter), and glycophorins are the most abundant proteins of this class. These proteins span the membrane and have distinct structural and functional domains, both within the bilayer and on either side of the membrane. There are as many as 50 additional integral membrane proteins, many of which are present at low abundance [12].

Many of the blood group proteins (perhaps as many as half) define various blood group antigens [12]. As an example, the Rh blood group system antigens are carried by a family of palmitoylated integral membrane proteins that include Rh30 proteins (RhD and RhCE) and the Rh50 glycoprotein [41]. The function of this protein complex remains undefined, although evidence suggests that it may be a gas transporter for ammonia and carbon dioxide [42].

Band 3 — Band 3, also called the anion exchanger (AE1) or solute carrier family 4, is the major integral membrane protein in the RBC, constituting approximately one-fourth of total membrane protein. Each RBC contains approximately 1.2 million copies of band 3.

Band 3 is a 911 amino acid glycoprotein. It is the product of a single gene on chromosome 17 (SLC4A1) [43]. The gene is transcribed by two promoters: The upstream promoter produces erythroid band 3 while the downstream promoter produces renal band 3 [44]. Defects in the production of renal band 3 account for some cases of familial distal (type 1) renal tubular acidosis. (See "Overview and pathophysiology of renal tubular acidosis and the effect on potassium balance".)

Band 3 is composed of three functionally distinct domains:

●The hydrophilic cytoplasmic domain (residues 1 to 403) interacts with a variety of peripheral membrane and cytoplasmic proteins including ankyrin, band 4.2 (see 'Ankyrin and band 4.2' below), protein 4.1, adducin, hemoglobin, and a number of glycolytic enzymes (figure 4). These interactions are thought to provide cohesion between the plasma membrane and the underlying spectrin-based membrane skeleton and to in turn prevent loss of membrane surface [44,45]. Abnormalities affecting this domain are seen in hereditary spherocytosis (accounting for 15 to 25 percent of cases) and Southeast Asian ovalocytosis (SAO; a form of hereditary elliptocytosis in which nine amino acids [codons 400 to 408] are absent). (See "Hereditary spherocytosis", section on 'Pathophysiology' and "Hereditary elliptocytosis and related disorders".)

●The hydrophobic transmembrane domain (residues 404 to 882) forms the anion transporter, which exchanges one bicarbonate ion for one chloride ion. Abnormalities affecting ion transport can cause cryohydrocytosis, a form of hereditary stomatocytosis. (See "Hereditary stomatocytosis (HSt) and hereditary xerocytosis (HX)".)

●The acidic carboxyl-terminal domain (residues 883 to 911) has no known function, although this domain binds carbonic anhydrase.

Band 3 also contains a single N-glycosidically linked oligosaccharide that carries several blood groups including I and Diego [46]. (See "Red blood cell antigens and antibodies".)

Aquaporin-1 — Aquaporin-1 belongs to a family of membrane channel proteins that serve as selective pores for water transport [47]. In the RBC, aquaporins allow the cell to remain in osmotic equilibrium with the extracellular fluid. In addition, the Colton blood group antigens represent a polymorphism on the aquaporin-1 protein [47,48]. Aquaporin-1 null deficiency is a rare disorder with a very mild hemolytic anemia [49].

Glut-1 — Glut-1 is a glucose transporter in the RBC. Rare cases of GLUT-1 mutations have been described; these manifest with mild RBC changes and significant non-hematologic abnormalities [50].

Piezo-1 — Piezo-1 is a mechanosensitive nonselective cation channel. Disease-causing variants in the PIEZO1 gene lead to RBC dehydration and mild to moderate compensated hemolytic anemia in hereditary xerocytosis [51].

Glycophorins — Four sialic acid-rich glycoproteins (glycophorin A, B, C, and D) comprise a class of integral proteins that constitute approximately 2 percent of RBC membrane proteins [52]. Glycophorins A, B, and C are distinct proteins encoded by three distinct genes, two located on chromosome 4 (glycophorins A and B) and one on chromosome 2 (glycophorin C). Glycophorin D is produced from the glycophorin C gene using a downstream initiation site [52].

Glycophorins consist of three domains:

●A cytoplasmic domain containing a cluster of basic residues positioned near the plasma membrane

●A hydrophobic domain that forms a single alpha-helix spanning the bilayer

●A heavily glycosylated extracellular domain

Glycophorin carbohydrates impart a strong negative charge to the RBC surface that is thought to be important in reducing RBC interactions with each other and with the vascular endothelium. Glycophorin C binds to submembrane proteins p55 and protein 4.1 and regulates their abundance in the membrane.

Glycophorins carry a number of blood group systems [52]:

●Glycophorin A – MN blood group

●Glycophorin B – S blood group

●Glycophorin C – Gerbich blood group

Glycophorins have also been shown to play a role in malaria infection or resistance [53,54]. (See "Protection against malaria by variants in red blood cell (RBC) genes", section on 'MNS blood group system (glycophorins A and B)'.)

Peripheral membrane proteins — Peripheral membrane proteins are proteins located on the cytoplasmic surface of the lipid bilayer; they are attached to the integral membrane proteins rather than to the membrane itself. As a result, peripheral membrane proteins can be readily released from the membrane (eg, by manipulation of ionic strength). Their principal function is thought to be related to structure, elasticity, and mechanical stability of the membrane and the geometry of the cell rather than transport of solutes or other molecules across the membrane [5,6].

Major protein components of the membrane cytoskeleton include spectrin, ankyrin, band 4.2, actin, tropomyosin, tropomodulin, protein 4.1, adducin, and dematin (figure 3).

High-resolution electron micrographs of extended membrane skeletons show a highly repeated and remarkably regular organization of spectrin/actin/protein 4.1 complexes in which each complex is linked to adjacent complexes by multiple spectrin tetramers [55]. This creates an irregular network in the resting RBC membrane in which the basic unit is composed of a hexagonal lattice with six spectrin molecules [23,56].

Ankyrin provides a critical physical link between the lipid bilayer and the membrane cytoskeleton via its interactions with the integral membrane protein band 3 [23,57].

Spectrin — Spectrin is composed of two subunits, alpha and beta, encoded on chromosomes 1 and 14, respectively [58]. Synthesis of beta-spectrin is rate-limiting for the assembly of the spectrin tetramer, while alpha-spectrin is synthesized in excess.

Spectrin is a flexible, rod-like protein present at approximately 200,000 copies per RBC. The alpha and beta subunits are intertwined side-to-side and are joined to other spectrin molecules by flexible joining regions like beads on a string. Spectrin heterodimers associate head-to-head to form (alpha-beta)₂ tetramers. The tetramers appear to predominate in the membrane cytoskeleton. Oligomers larger than the tetramers are also observed; they appear to be formed by spectrin dimers joined by head-to-head linkages into a central ring [55].

Cryo-electron microscopy studies have demonstrated that the native spectrin filaments are approximately 46 nm long [59]. When fully extended, spectrin heterotetramers can be as long as 190 nm (ie, they can stretch over four times their resting length). The surface density of spectrin varies across the RBC membrane, perhaps related to the amount of membrane stress.

Functions of spectrin include maintaining the membrane composition of other proteins and lipids as well as the biconcave disc shape of RBCs. In the nondeformed state, spectrin molecules exist in a folded conformation. During reversible deformation, the change in geometric shape occurs at a constant surface area. This process is characterized by rearrangement of the skeletal network in which some spectrin molecules become uncoiled and extended, while others are more compressed and folded [18,19].

Spectrin deficiency can cause hereditary spherocytosis. Because beta-spectrin is rate-limiting for assembly of the spectrin tetramer, variants affecting a single beta-spectrin allele are sufficient to cause spherocytosis, whereas variants affecting alpha-spectrin must be present at both alleles to cause spherocytosis (ie, autosomal recessive inheritance).

Beta-spectrin is the spectrin subunit that interacts with other cytoskeletal proteins:

●The carboxyl-terminal region has a binding site for ankyrin. Ankyrin in turn binds to the cytoplasmic tail of the integral membrane protein band 3, and this provides the major linkage between the lipid bilayer and the membrane cytoskeleton [60].

●The amino-terminal region has a binding site protein 4.1. Protein 4.1 in turn binds to the integral membrane protein glycophorin C, and this provides a second linkage between the lipid bilayer and the membrane cytoskeleton [61].

●The amino-terminal region has binding sites for multiple short actin filaments. A junctional complex is formed between beta-spectrin, actin, and protein 4.1 [62]. Adducin also stabilizes the spectrin/actin/protein 4.1 junctional complex.

Ankyrin and band 4.2 — Ankyrin (previously called band 2.1) is an 1879 amino acid protein encoded by a single gene on chromosome 8. Ankyrin is present at approximately 100,000 copies per RBC [57].

As noted above, ankyrin provides the major contribution to the mechanical coupling between the lipid bilayer and the submembrane cytoskeleton. This is achieved by binding to the integral membrane protein Band 3 and submembrane beta-spectrin [23,57,60]. Protein 4.2 binds to both band 3 and ankyrin and strengthens this linkage [63].

Actin, tropomyosin, and tropomodulin — Actin in RBCs is assembled in an unusual configuration involving short filaments with a highly uniform length (approximately 35 nm). The proteins tropomyosin and tropomodulin may regulate the length of these actin filaments [62].

Actin oligomers are critical for the organization of the junctional complex; they bind to spectrin tetramers in the complex. An average of six spectrin tail regions form a complex with each actin oligomer, creating an irregular network with an approximately hexagonal lattice [23,56]. Each spectrin-actin junction is stabilized by the formation of a ternary complex with protein 4.1 (and, to a lesser degree, adducin).

Protein 4.1 — Protein 4.1 is a 622 amino acid protein encoded by a single gene on chromosome 1. Protein 4.1 is present at approximately 200,000 copies per cell. A major function of protein 4.1 is to stabilize the spectrin-actin interaction, which is inherently weak [62].

Adducin — Adducin is less abundant than some of the other cytoskeletal proteins in the RBC; it is present at only approximately 30,000 copies per cell. Adducin binds spectrin and the integral membrane protein band 3, and it stabilizes two spectrin interactions:

●The spectrin-actin interaction

●The spectrin/actin/protein 4.1 junctional complex interaction with band 3 in the lipid bilayer [64]

Adducin is a target for the calcium-dependent regulatory protein calmodulin; as a result, its ability to promote spectrin-actin interactions is regulated by the intracellular calcium concentration.

Dematin — Dematin stabilizes the interaction of spectrin with actin. Phosphorylation of dematin modulates its ability to stabilize the interaction. While dematin deficiency has not been identified in human RBCs, its deficiency in murine RBCs leads to marked cell fragmentation and severe anemia.

CLINICAL CONSEQUENCES — Conditions that interfere with normal membrane deformability and/or stability can reduce the normal surface area to volume (SA/V) ratio, alter RBC shape, and reduce the lifespan of the RBC (ie, cause hemolysis) [65].

An abnormal vasculature can also cause the RBC membrane to fail despite its optimized SA/V ratio. This is of particular relevance in microangiopathic anemias such as thrombotic thrombocytopenic purpura (TTP) and disseminated intravascular coagulation (DIC), and in vascular anomalies, in which significant mechanically induced RBC fragmentation is seen. (See 'Thrombotic microangiopathies' below.)

Inherited hemolytic anemias — Some inherited hemolytic anemias are due to mutations affecting cytoskeletal or integral membrane proteins (figure 3).

Examples of inherited hemolytic anemias with decreased membrane stability include the following:

●Hereditary spherocytosis (HS) syndromes may be due to mutations affecting one of the peripheral membrane proteins (eg, spectrin, ankyrin, band 3, band 4.2, Rh-associated glycoprotein [RhAG]). Loss of membrane causes a decreased SA/V ratio, decreased deformability, and decreased membrane stability. Hemolysis with spherocytes on the peripheral blood smear is typical of these disorders. (See "Hereditary spherocytosis", section on 'Genetics'.)

●Hereditary elliptocytosis (HE) syndromes may be due to weakened spectrin-spectrin association (mutations affecting alpha- or beta-spectrin), weakened spectrin-actin-protein 4.1 association (mutations affecting protein 4.1), or weakened interactions between band 3 and the peripheral membrane proteins (mutation affecting the membrane-spanning domain and cytoplasmic tail of band 3) [5,12]. Of note, not all HE syndromes (non-hemolytic HE) are associated with hemolysis; some individuals come to medical attention due to an incidental finding of elliptocytes on the peripheral blood smear. (See "Hereditary elliptocytosis and related disorders".)

●Hereditary stomatocytosis (HSt) syndromes may have mild hemolysis at baseline, but in some of these syndromes, increased hemolysis is observed in settings of increased mechanical stress [13]. In some of the overhydrated forms of HSt, the membrane defects result in increased cell volume due to impaired channel function. In these cases, the SA/V ratio is decreased by increasing volume rather than by decreasing membrane area. Hereditary xerocytosis due to pathogenic variants in PIEZO1 and in KCNN4 has increased cytoplasmic viscosity in addition to membrane abnormalities due to cell dehydration and consequent decreased osmotic fragility. (See "Hereditary stomatocytosis (HSt) and hereditary xerocytosis (HX)".)

While normal RBCs completely recover their shape following repeated cycles of deformation in the circulation, RBCs in these conditions that have weakened or abnormal junctions between skeletal proteins fail to recover their initial shape and undergo plastic deformation (figure 4). This explains why splenectomy is effective in treating hemolytic anemia in some of these conditions, because many of these misshapen cells are sequestered and removed from circulation by the spleen [65]. However, splenectomy is avoided in some of the other conditions given the risk of thromboembolism. (See "Hereditary stomatocytosis (HSt) and hereditary xerocytosis (HX)", section on 'Splenectomy'.)

Inherited hemoglobinopathies — In hemoglobinopathies, there is no specific abnormality in the membrane or the cytoskeletal proteins. However, abnormal hemoglobins may precipitate and bind to the membrane, inducing deformation of the membrane. Repeated cycles of this process may cause the cells to become irreversibly damaged and unable to return to the normal biconcave disc shape. In some cases, precipitated hemoglobin at the membrane may induce membrane phagocytosis by reticuloendothelial macrophages. This is the case in sickle cell disease and more severe forms of thalassemia, especially alpha thalassemias. (See "Pathophysiology of sickle cell disease" and "Pathophysiology of thalassemia", section on 'Effects on the RBC'.)

Acquired hemolytic anemias — In acquired hemolytic anemias due to autoimmune mechanisms (eg, warm autoimmune hemolytic anemia [AIHA]), RBCs become coated by antibodies, which leads to partial phagocytosis by reticuloendothelial macrophages and reduced membrane surface area. The resulting spherocytes have a reduced SA/V ratio and reduced cellular deformability. (See "Warm autoimmune hemolytic anemia (AIHA) in adults", section on 'Red cell destruction'.)

By contrast, in liver disease, target cells or spur cells are formed when increased membrane cholesterol content increases the cell surface area; these cells have an increased SA/V ratio and are not prone to hemolyze. (See "Burr cells, acanthocytes, and target cells: Disorders of red blood cell membrane".)

Thrombotic microangiopathies — Membrane failure can also occur with a structurally normal RBC that is subjected to increasing degrees of deformation in which the membrane becomes more extended and some of the major structural molecules (see 'Spectrin' above) attain their maximal linear extension. This point is the limit of reversible deformability. A continued application of force would require an increase in surface area and the breaking of junctional complexes; this occurs at the weakest of the lateral protein-protein associations (spectrin-spectrin junction or spectrin-actin-protein 4.1R junction), leading to loss of membrane stability and membrane fragmentation.

A good example of this is the thrombotic microangiopathies (TMAs), in which small vessel microthrombi create extremely tortuous passages. Although RBCs have an optimized SA/V ratio and deformability that is adequate for normal circulation, they are unable to deform adequately to traverse these microthrombi. As a result, they become mechanically sheared, producing schistocytes (picture 8 and picture 9) on the peripheral blood smear. (See "Diagnostic approach to suspected TTP, HUS, or other thrombotic microangiopathy (TMA)", section on 'Microangiopathic hemolytic anemia (MAHA)'.)

SUMMARY

●Importance of membrane deformability – Red blood cells (RBCs) must undergo extensive passive deformation as they traverse the circulation and squeeze through capillaries and splenic sinusoids that are smaller than one-half the diameter of the RBC (picture 1). The biconcave disc shape (picture 2) and cytoplasmic viscosity of the RBC optimize deformability and stability of the RBC membrane to prevent hemolysis during these deformations. The three main determinants of deformability are the ratio of membrane (surface area) to cytoplasm (volume), cytoplasmic viscosity (mainly determined by mean corpuscular hemoglobin concentration [MCHC]), and membrane properties. (See 'RBC shape and deformability' above.)

●Membrane properties – The plasma membrane of the RBC is a continuous sheet-like membrane that envelops the cellular contents. It consists of an ordered array of lipids (approximately one billion molecules) and proteins (approximately 10 million molecules) (table 1) in an extraordinarily thin (6 to 10 nm) lipid bilayer punctuated by penetrating or attached proteins (referred to as integral membrane proteins and peripheral membrane proteins, respectively) (figure 3). These lipids and proteins maintain a striking asymmetry that allows ions, nutrients, and regulatory signals to enter the cell, at times against a concentration gradient. (See 'Structural organization and dynamic regulation' above.)

●Membrane composition – The RBC plasma membrane is a lipid bilayer with inner and outer leaflets. Its major components are unesterified cholesterol and phospholipids. The outer leaflet contains mostly uncharged phospholipids (phosphatidylcholine [PC] and sphingomyelin [SM]), and the inner leaflet contains mostly charged phospholipids (phosphatidylethanolamine [PE] and phosphatidylserine [PS]). Plasma cholesterol exchanges with membrane cholesterol, and membrane cholesterol regulates the activity of a "flippase" that maintains the asymmetry of the phospholipids. (See 'Lipid bilayer' above.)

●Protein-protein and protein-carbohydrate interactions – Integral membrane proteins are tightly embedded in the membrane through hydrophobic interactions with lipids in the bilayer. Band 3 (the anion exchanger), aquaporin-1 (the water channel), glut-1 (glucose transporter), and glycophorins are the most abundant. Carbohydrates in the glycophorins impart a strong net negative charge to the cell surface, which helps reduce interaction of RBCs with each other and with the vascular endothelium. (See 'Integral membrane proteins' above.)

●Structural membrane proteins – Peripheral membrane proteins are attached to the integral membrane proteins rather than to the membrane itself. Some of the most abundant and best characterized include spectrin, ankyrin, band 4.2, actin, tropomyosin, tropomodulin, protein 4.1, adducin, and dematin. Their principal function is related to structure, elasticity, and mechanical stability of the membrane and the geometry of the cell. They form an irregular network in which the basic unit is a hexagonal lattice of spectrin molecules. Ankyrin provides the major connection to the integral membrane proteins via interaction with band 3. (See 'Peripheral membrane proteins' above.)

●Effects on RBC shape and lifespan – Conditions that interfere with normal membrane deformability and/or stability can reduce the normal surface area to volume (SA/V) ratio, alter RBC shape, and cause hemolysis. Inherited hemolytic anemias with decreased membrane stability include hereditary spherocytosis (HS), some forms of hereditary elliptocytosis (HE), and some forms of hereditary stomatocytosis (HSt). Autoimmune hemolysis and membrane damage due to precipitated hemoglobins may also reduce the SA/V ratio and/or alter membrane stability and cause hemolysis. In thrombotic microangiopathies, RBC membrane stability is normal but the constraints of the vasculature result in membrane failure. (See 'Clinical consequences' above.)

ACKNOWLEDGMENT — UpToDate gratefully acknowledges Stanley L Schrier, MD (deceased), who contributed as Section Editor on earlier versions of this topic and was a founding Editor-in-Chief for UpToDate in Hematology.