Rodak's Hematology Clinical Principles and Applications

PART II

Blood Cell Production, Structure, and Function

CHAPTER 9

Erythrocyte metabolism and membrane structure and function

George A. Fritsma*

OUTLINE

Energy Production—Anaerobic Glycolysis

Glycolysis Diversion Pathways (Shunts)

Hexose Monophosphate Pathway

Methemoglobin Reductase Pathway

Rapoport-Luebering Pathway

RBC Membrane

RBC Deformability

RBC Membrane Lipids

RBC Membrane Proteins

Osmotic Balance and Permeability

Objectives

After completion of this chapter, the reader will be able to:

1. List the erythrocyte metabolic processes that require energy.
2. Diagram the Embden-Meyerhof anaerobic glycolytic pathway (EMP) in the red blood cell (RBC), highlighting adenosine triphosphate (ATP) consumption and generation.
3. Name the components of the hexose-monophosphate pathway that detoxify peroxide and the process that accomplishes detoxification.
4. Describe the RBC metabolic pathway that generates 2,3-BPG, state the effect of its formation on ATP production, and explain its importance in oxygen transport.
5. Diagram the methemoglobin reductase pathway and explain its importance in maintaining functional hemoglobin.
6. Explain the importance of semipermeability of biological membranes.
7. Describe the arrangement and function of lipids in the RBC membrane.
8. Explain cholesterol exchange between the RBC membrane and plasma, including factors that affect the exchange.
9. Define, locate, and explain the role of RBC transmembrane proteins in maintaining membrane stability and provide examples.
10. Cite the relative concentrations of RBC cytoplasmic potassium, sodium, and calcium, and name the structures that maintain those concentrations.
11. Discuss how ankyrin, protein 4.1, actin, adducin, tropomodulin, dematin, and band 3 interact with α- and β-spectrin and the lipid bilayer.
12. Name conditions caused by abnormalities of vertical linkages and horizontal (lateral) linkages in RBC transmembrane and cytoskeletal proteins.

CASE STUDY

After studying the material in this chapter, the reader should be able to respond to the following case study:

Cyanosis is blue skin coloration, visible in Caucasians, that occurs when the blood does not deliver enough oxygen to the tissues. It is a common sign of heart or lung disease, in which the blood fails to become oxygenated or is distributed improperly throughout the body. In the 1940s, Dr. James Deeny, an Irish physician, was experimenting with the use of vitamin C (ascorbic acid), a potent reducing agent, as a treatment for heart disease.¹ To his disappointment, it was ineffective for nearly all patients. However, he discovered two brothers with the distinction of being truly blue men. When he treated them with vitamin C, each turned a healthy pink. Neither man was determined to have either heart or lung disease.

1. What does it mean to say that vitamin C is a reducing agent?
2. What must be happening if vitamin C was able to cure the cyanosis?
3. What is the significance of finding this condition in brothers?

The erythrocyte (red blood cell, RBC) is the primary blood cell, circulating at 5 million RBCs per microliter of blood on average. It is anucleate and biconcave and has an average volume of 90 fL. The cytoplasm provides abundant hemoglobin, a complex of globin, protoporphyrin, and iron that transports elemental oxygen (O₂) from high partial pressure to low partial pressure environments, that is, from lung capillaries to the capillaries of organs and tissues. Hemoglobin, plasma proteins, and additional RBC proteins also transport molecular carbon dioxide (CO₂) and bicarbonate (HCO₃⁻) from the tissues to the lungs. Hemoglobin is composed of four globin molecules, each supporting one heme molecule; each heme molecule contains a molecule of iron (Chapters 10 and 11). The biconcave RBC shape supports deformation, enabling the circulating cell to pass smoothly through capillaries, where it readily exchanges O₂ and CO₂ while contacting the vessel wall.^{2, 3}

RBCs are produced through erythrocytic (normoblastic) maturation in bone marrow tissue (Chapter 8). The nucleus, while present in maturing marrow normoblasts, becomes extruded as the cell passes from the bone marrow to peripheral blood. Cytoplasmic ribosomes and mitochondria disappear 24 to 48 hours after bone marrow release, eliminating the cells’ ability to produce proteins or support oxidative metabolism. Adenosine triphosphate (ATP) is produced within the cytoplasm through anaerobic glycoglycocalyx. 22 They serve aslysis (Embden-Meyerhof pathway, EMP) for the lifetime of the cell. ATP drives mechanisms that slow the destruction of protein and iron by environmental peroxides and superoxide anions, maintaining hemoglobin’s function and membrane integrity. Oxidation, however, eventually takes a toll, limiting the RBC circulating life span to 120 days, whereupon the cell becomes disassembled into its reusable components globin, iron, and the phospholipids and proteins of the cell membrane, while the protoporphyrin ring is excreted as bilirubin (Chapters 8 and23).

This chapter is one of a series of four that present the physiology of normal RBC production, structure, function, and senescence. These include Chapter 8, Erythrocyte Production and Destruction; Chapter 10, Hemoglobin Metabolism; and Chapter 11, Iron Kinetics and Laboratory Assessment. This chapter describes RBC energy production, the protective mechanisms that preserve structure and function, and the structure, function, deformability, and maintenance of the cell membrane. Taken as a unit, these four chapters form the basis for understanding RBC disorders (anemias), as described in Chapters 19 through 28 [Chapter 19 Chapter 20 Chapter 21 Chapter 22 Chapter 23 Chapter 24 Chapter 25 Chapter 26 Chapter 27Chapter 28].

Energy production—anaerobic glycolysis

Lacking mitochondria, the RBC relies on anaerobic glycolysis for its energy.⁴ The hemoglobin exchange of O₂ and CO₂ is a passive function, however the cells’ metabolic processes listed inBox 9-1 require energy. As energy production slows, the RBC grows senescent and is removed from the circulation (Chapter 8). Hematologists have identified hereditary deficiencies of nearly every glycolytic enzyme, and their common result is shortened RBC survival, known collectively as hereditary nonspherocytic hemolytic anemia (Chapter 24).

BOX 9-1

Erythrocyte Metabolic Processes Requiring Energy

Intracellular cationic gradient maintenance

Maintenance of membrane phospholipid distribution

Maintenance of skeletal protein deformability

Maintenance of functional hemoglobin with ferrous iron

Protecting cell proteins from oxidative denaturation

Glycolysis initiation and maintenance

Glutathione synthesis

Nucleotide salvage reactions

Glucose enters the RBC without energy expenditure via the transmembrane protein Glut-1.⁵ Anaerobic glycolysis, the EMP (Figure 9-1), requires glucose to generate ATP, a high-energy phosphate source. With no cytoplasmic glycogen organelles, RBCs lack internal energy stores and rely on plasma glucose for glycolysis-generated ATP. Through the EMP, glucose is catabolized to pyruvate (pyruvic acid), consuming two molecules of ATP per molecule of glucose and maximally generating four molecules of ATP per molecule of glucose, for a net gain of two molecules of ATP.

FIGURE 9-1 Glucose metabolism in the erythrocyte. ADP, Adenosine diphosphate; ATP, adenosine triphosphate; G6PD, glucose-6-phosphate dehydrogenase; NAD, nicotinamide adenine dinucleotide (oxidized form);NADH, nicotinamide adenine dinucleotide (reduced form); NADP, nicotinamide adenine dinucleotide phosphate (oxidized form); NADPH, nicotinamide adenine dinucleotide phosphate (reduced form).

The sequential list of biochemical intermediates involved in glucose catabolism, with corresponding enzymes, is given in Figure 9-1. organize glycolysis into three phases.

The first phase of glycolysis employs glucose phosphorylation, isomerization, and diphosphorylation to yield fructose 1,6-bisphosphate (F1,6-BP). Fructose-bisphosphate aldolase cleaves F1,6-BP to produce glyceraldehyde-3-phosphate (G3P; Figure 9-1 and Table 9-1). Intermediate stages employ, in order, the enzymes hexokinase, glucose-6-phosphate isomerase, and 6-phosphofructokinase. The initial hexokinase and 6-phosphofructokinase steps consume a total of 2 ATP molecules and limit the rate of glycolysis.

TABLE 9-1

Glucose Catabolism: First Phase

Substrates	Enzyme	Products
Glucose, ATP	Hexokinase	G6P, ADP
G6P	Glucose-6-phosphate isomerase	F6P
F6P, ATP	6-Phosphofructokinase	F1,6-BP, ADP
F1,6-BP	Fructose-bisphosphate adolase	DHAP, G3P

ADP, Adenosine diphosphate; ATP, adenosine triphosphate; DHAP, dihydroxyacetone phosphate; F1,6-BP, fructose-1,6-bisphosphate; F6P, fructose-6-phosphate; G3P, glyceraldehyde-3-phosphate; G6P, glucose-6-phosphate.

The second phase of glucose catabolism converts G3P to 3-phosphoglycerate (3-PG). The substrates, enzymes, and products for this phase of glycolytic metabolism are summarized inTable 9-2. In the first step, G3P is oxidized to 1,3-bisphosphoglycerate (1,3-BPG) through the action of glyceraldehyde-3-phosphate dehydrogenase (G3PD). 1,3-BPG is dephosphorylated byphosphoglycerate kinase, which generates 2 ATP molecules and 3-PG.

TABLE 9-2

Glucose Catabolism: Second Phase

Substrates	Enzyme	Product
G3P	Glyceraldehyde-3-phosphate dehydrogenase	1,3-BPG
1,3-BPG, ADP	Phosphoglycerate kinase	3PG, ATP
1,3-BPG	Bisphosphoglycerate mutase	2,3-BPG
2,3-BPG	Bisphosphoglycerate phosphatase	3-PG

1,3-BPG, 1,3-bisphosphoglycerate; 2,3-BPG, 2,3-bisphosphoglycerate; 3-PG, 3-phosphoglycerate; ADP, adenosine diphosphate; ATP, adenosine triphosphate; G3P, glyceraldehyde-3-phosphate.

The third phase of glycolysis converts 3-PG to pyruvate and generates ATP. Substrates, enzymes, and products are listed in Table 9-3. The product 3-PG is isomerized byphosphoglycerate mutase to 2-phosphoglycerate (2-PG). Enolase (phosphopyruvate hydratase) then converts 2-PG to phosphoenolpyruvate (PEP). Pyruvate kinase (PK) splits off the phosphates, forming 2 ATP molecules and pyruvate. PK activity is allosterically modulated by increased concentrations of F1,6-BP, which enhances the affinity of PK for PEP.⁵ Thus, when the F1,6-BP is plentiful, increased activity of PK favors pyruvate production. Pyruvate may diffuse from the erythrocyte or may become a substrate for lactate dehydrogenase with regeneration of the oxidized form of nicotinamide adenine dinucleotide (NAD⁺). The ratio of NAD⁺ to the reduced form (NADH) modulates the activity of this enzyme.

TABLE 9-3

Glucose Catabolism: Third Phase

Substrates	Enzyme	Product
3-PG	Phosphoglycerate mutase	2-PG
2-PG	Enolase (phosphopyruvate hydratase)	PEP
PEP, ADP	Pyruvate kinase	Pyruvate, ATP

2-PG, 2-phosphoglycerate; 3-PG, 3-phosphoglycerate; ADP, adenosine diphosphate; ATP, adenosine triphosphate; PEP, phosphoenolpyruvate.

Glycolysis diversion pathways (shunts)

Three alternate pathways, called diversions or shunts, branch from the glycolytic pathway. The three diversions are the hexose monophosphate pathway (HMP) or aerobic glycolysis, themethemoglobin reductase pathway, and the Rapoport-Luebering pathway.

Hexose monophosphate pathway

Aerobic or oxidative glycolysis occurs through a diversion of glucose catabolism into the HMP, also known as the pentose phosphate shunt (Figure 9-1). The HMP detoxifies peroxide (H₂O₂), which arises from O₂ reduction in the cell’s aqueous environment, where it oxidizes and destroys heme iron, proteins, and lipids, especially lipids containing thiol groups.⁵ By detoxifying peroxide, the HMP extends the functional life span of the RBC.

The HMP diverts glucose-6-phosphate (G6P) to ribulose 5-phosphate by the action of glucose-6-phosphate dehydrogenase (G6PD). In the process, oxidized nicotinamide adenine dinucleotide phosphate (NADP) is converted to its reduced form (NADPH). NADPH is then available to reduce oxidized glutathione (GSSG) to reduced glutathione (GSH) in the presence of glutathione reductase. Glutathione is a cysteine-containing tripeptide, and the designation GSH highlights the sulfur in the cysteine moiety. Reduced glutathione becomes oxidized as it reduces peroxide to water and oxygen via glutathione peroxidase.

During steady-state glycolysis, 5% to 10% of G6P is diverted to the HMP. After oxidative challenge, HMP activity may increase up to thirtyfold.⁶ The HMP catabolizes G6P to ribulose 5-phosphate and carbon dioxide by oxidizing G6P at carbon 1. The substrates, enzymes, and products of the HMP are listed in Table 9-4.

TABLE 9-4

Glucose Catabolism: Hexose Monophosphate Pathway

Substrates	Enzyme	Product
G6P	Glucose-6-phosphate dehydrogenase and 6-Phosphogluconolactonase	6-PG
6-PG	6-Phosphogluconate dehydrogenase	R5P

6-PG, 6-phosphogluconate; G6P, glucose-6-phosphate; R5P, ribulose 5-phosphate.

G6PD provides the only means of generating NADPH for glutathione reduction, and in its absence erythrocytes are particularly vulnerable to oxidative damage (Chapter 24).⁷ With normal G6PD activity, the HMP detoxifies oxidative compounds and safeguards hemoglobin, sulfhydryl-containing enzymes, and membrane thiols, allowing RBCs to safely carry O₂. However, in G6PD deficiency, the most common inherited RBC enzyme deficiency worldwide, the ability to detoxify is hampered, resulting in hereditary nonspherocytic anemia.

Methemoglobin reductase pathway

Heme iron is constantly exposed to oxygen and peroxide.⁸ Peroxide oxidizes heme iron from the ferrous (+2) to the ferric (+3) state. The affected hemoglobin molecule is calledmethemoglobin. Although the HMP prevents hemoglobin oxidation by reducing peroxide, it is not able to reduce methemoglobin once it forms. NADPH is able to do so, but only slowly. The reduction of methemoglobin by NADPH is rendered more efficient in the presence of methemoglobin reductase, also called cytochrome b₅ reductase. Using H⁺ from NADH formed when G3P is converted to 1,3-BPG, cytochrome b₅ reductase acts as an intermediate electron carrier, returning the oxidized ferric iron to its ferrous, oxygen-carrying state. This enzyme accounts for more than 65% of the methemoglobin-reducing capacity within the RBC.⁸

Rapoport-luebering pathway

A third metabolic shunt generates 2,3-bisphosphoglycerate (2,3-BPG; also called 2,3-diphosphoglycerate or 2,3-DPG). 1,3-BPG is diverted by bisphosphoglycerate mutase to form 2,3-BPG. 2,3-BPG regulates oxygen delivery to tissues by competing with oxygen for the oxygen-binding site of hemoglobin (Chapter 10). When 2,3-BPG binds heme, oxygen is released, which enhances delivery of oxygen to the tissues.

2,3-BPG forms 3-PG by the action of bisphosphoglycerate phosphatase. This diversion of 1,3-BPG to form 2,3-BPG sacrifices the production of two ATP molecules. There is further loss of two ATP molecules at the level of PK, because fewer molecules of PEP are formed. Because two ATP molecules were used to generate 1,3-BPG and production of 2,3-BPG eliminates the production of four molecules, the cell is put into ATP deficit by this diversion. There is a delicate balance between ATP generation to support the energy requirements of cell metabolism and the need to maintain the appropriate oxygenation and deoxygenation status of hemoglobin. Acidic pH and low concentrations of 3-PG and 2-PG inhibit the activity ofbisphosphoglycerate mutase, thus inhibiting the shunt and retaining 1,3-BPG in the EMP. These conditions and decreased ATP activate bisphosphoglycerate phosphatase, which returns 2,3-BPG to the glycolysis mainstream. In summary, these conditions favor generation of ATP by causing the conversion of 1,3-BPG directly to 3-PG and returning 2,3-BPG to 3-PG for ATP generation downstream by PK.

Rbc membrane

Rbc membrane deformability

RBCs are biconcave and average 90 fL in volume.⁹ Their average surface area is 140 μm², a 40% excess of surface area compared with a 90-fL sphere. This excess surface-to-volume ratio enables RBCs to stretch undamaged up to 2.5 times their resting diameter as they pass through narrow capillaries and through splenic pores 2 μm in diameter; this property is called RBCdeformability.¹⁰ The RBC plasma membrane, which is 5 μm thick, is 100 times more elastic than a comparable latex membrane, yet it has tensile (lateral) strength greater than that of steel. The deformable RBC membrane provides the broad surface area and close tissue contact necessary to support the delivery of O₂ from lungs to body tissue and CO₂ from body tissue to lungs.

RBC deformability depends not only on RBC geometry but also on relative cytoplasmic (hemoglobin) viscosity. The normal mean cell hemoglobin concentration (MCHC) ranges from 32% to 36% (Chapter 14 and inside front cover), and as MCHC rises, internal viscosity rises.¹¹ MCHCs above 36% compromise deformability and shorten the RBC life span because viscous cells become damaged as they stretch to pass through narrow capillaries or splenic pores. As RBCs age, they lose membrane surface area, while retaining hemoglobin. As the MCHC rises, the RBC, unable to pass through the splenic pores, is destroyed by splenic macrophages (Chapter 8).

Rbc membrane lipids

Besides geometry and viscosity, membrane elasticity (pliancy) also contributes to deformability. The RBC membrane consists of approximately 8% carbohydrates, 52% proteins, and 40% lipids.¹² The lipid portion, equal parts of cholesterol and phospholipids, forms a bilayer universal to all animal cells (Figure 13-10). Phospholipids form an impenetrable fluid barrier as their hydrophilic polar head groups are arrayed upon the membrane’s surfaces, oriented toward both the aqueous plasma and the cytoplasm, respectively.¹³ Their hydrophobic nonpolar acyl tails arrange themselves to form a central layer dynamically sequestered (hidden) from the aqueous plasma and cytoplasm. The membrane maintains extreme differences in osmotic pressure, cation concentrations, and gas concentrations between external plasma and the cytoplasm.¹⁴ Phospholipids reseal rapidly when the membrane is torn.

Cholesterol, esterified and largely hydrophobic, resides parallel to the acyl tails of the phospholipids, equally distributed between the outer and inner layers, and evenly dispersed within each layer, approximately one cholesterol molecule per phospholipid molecule. Cholesterol’s β-hydroxyl group, the only hydrophilic portion of the molecule, anchors within the polar head groups, while the rest of the molecule becomes intercalated among and parallel to the acyl tails. Cholesterol confers tensile strength to the lipid bilayer.¹⁵

The ratio of cholesterol to phospholipids remains relatively constant and balances the need for deformability and strength. Membrane enzymes maintain the cholesterol concentration by regularly exchanging membrane and plasma cholesterol. Deficiencies in these enzymes are associated with membrane abnormalities such as acanthocytosis, as the membrane loses tensile strength (Chapter 24). Conversely, as cholesterol concentration rises, the membrane gains strength but loses elasticity.

The phospholipids are asymmetrically distributed. Phosphatidylcholine and sphingomyelin predominate in the outer layer; phosphatidylserine (PS) and phosphatidylethanolamine form most of the inner layer. Distribution of these four phospholipids is energy dependent, relying on a number of membrane-associated enzymes, whimsically termed flippases, floppases, andscramblases, for their positions.¹⁶ When phospholipid distribution is disrupted, as in sickle cell anemia and thalassemia (Chapters 27 and 28) or in RBCs that have reached their 120-day life span, PS, the only negatively charged phospholipid, redistributes (flips) to the outer layer. Splenic macrophages possess receptors that bind PS and destroy senescent and damaged RBCs.

Membrane phospholipids and cholesterol may also redistribute laterally so that the RBC membrane may respond to stresses and deform within 100 milliseconds of being challenged by the presence of a narrow passage, such as when arriving at a capillary. Redistribution becomes limited as the proportion of cholesterol increases. Plasma bile salt concentration also affects cholesterol exchange.¹⁷ In liver disease with low bile salt concentration, membrane cholesterol concentration becomes reduced. As a result, the more elastic cell membrane shows a “target cell” appearance when the RBCs are layered on a glass slide (Figure 19-1).

Glycolipids (sugar-bearing lipids) make up 5% of the external half of the RBC membrane.¹⁸ They associate in clumps or rafts and support carbohydrate side chains that extend into the aqueous plasma to anchor the glycocalyx. The glycocalyx is a layer of carbohydrates whose net negative charge prevents microbial attack and protects the RBC from mechanical damage caused by adhesion to neighboring RBCs or to the endothelium. Glycolipids may bear copies of carbohydrate-based blood group antigens, for example, antigens of the ABH and the Lewis blood group systems.

Rbc membrane proteins

Although cholesterol and phospholipids constitute the principal RBC membrane structure, transmembrane (integral) and cytoskeletal (skeletal, peripheral) proteins make up 52% of the membrane structure by mass.¹⁹ A proteomic study reveals there are at least 300 RBC membrane proteins, including 105 transmembrane proteins. Of the purported 300 membrane proteins, about 50 have been characterized and named, some with a few hundred copies per cell, and others with over a million copies per cell.²⁰

Transmembrane proteins

The transmembrane proteins serve a number of RBC functions, as listed in Table 9-5.²¹ Through glycosylation they support surface carbohydrates, which join with glycolipids to make up the protective glycocalyx.²² They serve as transport and adhesion sites and signaling receptors. Any disruption in transport protein function changes the osmotic tension of the cytoplasm, which leads to a rise in viscosity and loss of deformability. Any change affecting adhesion proteins permits RBCs to adhere to one another and to the vessel walls, promoting fragmentation (vesiculation), reducing membrane flexibility, and shortening the RBC life span. Signaling receptors bind plasma ligands and trigger activation of intracellular signaling proteins, which then initiate various energy-dependent cellular activities, a process called signal transduction.

TABLE 9-5

Names and Properties of Selected Transmembrane RBC Proteins

Transmembrane Protein	Band	Molecular Weight (D)	Copies per Cell (×103)	% of Total Protein	Function
Aquaporin 1					Water transporter
Band 3 (anion exchanger 1)	3	90,000–102,000	1200	27%	Anion transporter, supports ABH antigens
Ca2+-ATPase					Ca2+ transporter
Duffy		35,000–43,000			G protein-coupled receptor, supports Duffy antigens
Glut-1	4.5	45,000–75,000		5%	Glucose transporter, supports ABH antigens
Glycophorin A	PAS-1	36,000	500–1000	85% of glycophorins	Transports negatively charged sialic acid, supports MN determinants
Glycophorin B	PAS-4	20,000	100–300	10% of glycophorins	Transports negatively charged sialic acid, supports Ss determinants
Glycophorin C	PAS-2	14,000–32,000	50–100	4% of glycophorins	Transports negatively charged sialic acid, supports Gerbich system determinants
ICAM-4					Integrin adhesion
K+-Cl– cotransporter
Kell		93,000			Zn2+-binding endopeptidase, Kell antigens
Kidd		46,000–50,000			Urea transporter
Na+,K+-ATPase
Na+-K+-2Cl− cotransporter
Na+-Cl− cotransporter
Na+-K+ cotransporter
Rh		30,000–45,500			D and CcEe antigens
RhAG		50,000			Necessary for expression of D and CcEe antigens; gas transporter, probably of CO2

ATPase, Adenosine triphosphatase; Duffy, Duffy blood group system protein; ICAM, intracellular adhesion molecule; Kell, Kell blood group system protein; PAS, periodic acid–Schiff dye; RBC, red blood cell; Rh, Rh blood group system protein;RhAG, Rh antigen expression protein.

The transmembrane proteins assemble into one of two macromolecular complexes named by their respective cytoskeletal anchorages: ankyrin or protein 4.1 (Figure 9-2). These complexes and their anchorages provide RBC membrane structural integrity, because the membrane relies on the cytoskeletal proteins positioned immediately within (underneath) the lipid bilayer for its ability to retain (and return to) its biconcave shape despite deformability. The transmembrane proteins provide vertical membrane structure.

FIGURE 9-2 Representation of the human red blood cell membrane. The transmembrane proteins assemble in one of two complexes defined by their anchorage to skeletal protein ankyrin and skeletal protein 4.1. Band 3 is the most abundant transmembrane protein. In the ankyrin complex band 3 and protein 4.2 anchor to ankyrin, which is bound to the spectrin backbone. In the 4.1 complex, band 3, Rh, and other transmembrane proteins bind the complex of dematin, adducin, actin, tropomyosin, and tropomodulin through protein 4.1. CD47, Signaling receptor; Duffy, Duffy blood group system protein; GPA, glycophorin A; GPC, glycophorin C; Kell,Kell blood group system protein; LW, Landsteiner-Weiner blood group system protein; Rh, Rh blood group system protein; RhAG, Rh antigen expression protein; XK, X-linked Kell antigen expression protein.

Blood group antigens.

Transmembrane proteins support carbohydrate-defined blood group antigens.²³ For instance, band 3 (anion transport) and Glut-1 (glucose transport) support the majority of ABH system carbohydrate determinants by virtue of their high copy numbers.²⁴ ABH system determinants are also found on several low copy number transmembrane proteins. Certain transmembrane proteins provide peptide epitopes. For instance, glycophorin A carries the peptide-defined M and N determinants, and glycophorin B carries the Ss determinants, which together comprise the MNSs system.^{25, 26}

The Rh system employs two multipass transmembrane lipoproteins and a multipass glycoprotein, each of which crosses the membrane 12 times.^27-30 The two lipoproteins present the D and CcEe epitopes, respectively, but expression of the D and CcEe antigens requires the separately inherited glycoprotein RhAG, which localizes near the Rh lipoproteins in the ankyrin complex. Loss of the RhAG glycoprotein prevents expression of both the D and CcEe antigens (Rh-null) and is associated with RBC morphologic abnormalities (Chapter 24). Additional blood group antigens localize to the 4.1 complex or specialized proteins.

The gpi anchor and paroxysmal nocturnal hemoglobinuria.

A few copies of the phospholipid phosphatidylinositol (PI), not mentioned in the RBC Membrane Lipids section, reside in the outer, plasma-side layer of the membrane. PI serves as a base upon which a glycan core of sugar molecules is synthesized, forming the glycosylphosphatidylinositol (GPI) anchor. Over 30 surface proteins bind to the GPI anchor including decay-accelerating factor (DAF, or CD55) and membrane inhibitor of reactive lysis (MIRL, or CD59).^{31, 32} These proteins appear to float on the surface of the membrane as they link to the GPI anchor. The phosphatidylinositol glycan anchor biosynthesis, class A (PIGA) gene codes for a glycosyltransferase required to add N-acetylglucosamine to the PI base early in the biosynthesis of the GPI anchor on the membrane. In paroxysmal nocturnal hemoglobinuria (Chapter 24), an acquired mutation in the PIGA gene affects the cells’ ability to synthesize the GPI anchor. Without the GPI anchor on the membrane, CD55 and CD59 become deficient, and the cells are susceptible to complement-mediated hemolysis.

Nomenclature.

Numerical naming, for instance, band 3, protein 4.1, and protein 4.2, derives from historical (pre-proteomics) protein identification techniques that distinguished 15 membrane proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), as illustrated in Figure 9-3.³³ Bands migrate through the gel, with their velocity a property of their molecular weight and net charge, and are identified using Coomassie blue dye. The glycophorins, with abundant carbohydrate side chains, are stained using periodic acid–Schiff (PAS) dye.

FIGURE 9-3 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of RBC membrane proteins stained with Coomassie blue dye. Lane A illustrates numerical band naming based on migration. Lane B names and illustrates the positions of some of the major proteins. Source: (Adapted from Costa FF, Agre P, Watkins PC, et al: Linkage of dominant hereditary spherocytosis to the gene for the erythrocyte membrane-skeleton protein ankyrin, N Engl J Med 323:1046, 1990.)

Band 3, protein 4.2, and RhAG, members of the ankyrin complex, link their associated proteins and the bilayer membrane to the cytoskeletal proteins through ankyrin. Likewise, glycophorin C, Rh, and blood group Duffy link the 4.1 complex through protein 4.1.³⁷ The 4.1 anchorage also includes the more recently defined proteins adducin and dematin, which link with band 3 and Glut-1, respectively.³⁸

Cytoskeletal proteins

The principal cytoskeletal proteins are the filamentous α-spectrin and β-spectrin (Table 9-6), which assemble to form an antiparallel heterodimer held together with a series of lateral bonds.³⁹ Antiparallel means that the carboxyl (COOH) end of one strand associates with the amino (NH₃) end of the other, and the two heterodimers associate head-to-head to form a tetramer (Figure 9-2). The spectrins form a hexagonal lattice (Figure 9-4) that is immediately adjacent to the cytoplasmic membrane lipid layer and provides lateral or horizontal membrane stability.⁴⁰ Because the skeletal proteins do not penetrate the bilayer, they are also called peripheral proteins.

FIGURE 9-4 Spectrin-based cytoskeleton on the cytoplasmic side of the human red blood cell membrane. A, Junctional complex composed of actin filaments containing 14 to 16 actin monomers, band 4.1, adducin, and tropomyosin, which probably determines the length of the actin filaments. B, Spectrin dimers form a lattice that binds band 3 and protein 4.2 (not shown) via ankyrin and band 3, Glut-1 and Duffy (not shown), and glycophorin via protein 4.1.

TABLE 9-6

Names and Properties of Selected Skeletal RBC Proteins

Skeletal Protein	Band	Molecular Weight (D)	Copies per Cell (×103)	% of Total Protein	Function
α-spectrin β-spectrin	1 2	240,000–280,000 220,000–246,000	240 240	16% 14%	Filamentous antiparallel heterodimer, primary cytoskeletal proteins
Adducin	2.9	80,000–103,000	60	4%	Caps actin filament
Ankyrin	2.1	206,000–210,000	120	4.5%	Anchors band 3 and protein 4.2
Dematin	4.9	43,000–52,000	40	1%	Actin bundling protein
F-actin	5	42,000–43,000	400–500	5.5%	Binds β-spectrin
G3PD	6	35,000–37,000	500	3.5%
Protein 4.1	4.1	66,000–80,000	200	5%	Anchors 4.1 complex
Protein 4.2 (protein kinase)	4.2	72,000–77,000	200	5%	Anchors ankyrin complex
Tropomodulin	5	41,000–43,000	30	—	Caps actin filament
Tropomyosin	7	27,000–38,000	80	1%	Regulates actin polymerization

G3PD, Glucose-3-phosphate dehydrogenase glyceraldehyde; RBC, red blood cell.

Spectrin stabilization.

The secondary structure of both α- and β-spectrin features triple-helical repeats of 106 amino acids each; 20 such repeats make up α-spectrin, and 16 make up β-spectrin.⁴¹ Essential to the cytoskeleton are the previously mentioned ankyrin, protein 4.1, adducin and dematin, and, in addition, actin, tropomyosin, and tropomodulin (Figure 9-4).³⁵ A single helix at the amino terminus of α-spectrin consistently binds a pair of helices at the carboxyl terminus of the β-spectrin chain, forming a stable triple helix that holds together the ends of the heterodimers.⁴²Joining these ends are actin and protein 4.1. Actin forms short filaments of 14 to 16 monomers whose length is regulated by tropomyosin. Adducin and tropomodulin cap the ends of actin, and dematin appears to stabilize actin in a manner that is the subject of current investigation.⁴³

Membrane deformation.

Spectrin dimer bonds that appear along the length of the molecules disassociate and reassociate (open and close) during RBC deformation.⁴⁴ Likewise, the 20 α-spectrin and 16 β-spectrin repeated helices unfold and refold. These flexible interactions plus the spectrin-actin-protein 4.1 junctions between the tetramers are key regulators of membrane elasticity and mechanical stability, and abnormalities in any of these proteins result in deformation-induced membrane fragmentation. For instance, hereditary elliptocytosis (ovalocytosis) arises from one of several autosomal dominant mutations affecting spectrin dimer-to-dimer lateral bonds or the spectrin–ankyrin–protein 4.1 junction.⁴⁵ In hereditary elliptocytosis, the membrane fails to rebound from deformation, and RBCs progressively elongate to form visible elliptocytes, which causes a mild to severe hemolytic anemia.⁴⁶ Conversely, autosomal dominant mutations that affect the integrity of band 3, ankyrin, protein 4.2, or α- or β-spectrin are associated with hereditary spherocytosis (Chapter 24).^{47, 48} In these cases there are too few vertical anchorages to maintain membrane stability. The lipid membrane peels off in small fragments called “ blebs,” or vesicles, whereas the cytoplasmic volume remains intact. This generates spherocytes with a reduced membrane-to-cytoplasm ratio.

Osmotic balance and permeability

The RBC membrane is impermeable to cations Na⁺, K⁺, and Ca²⁺. It is permeable to water and the anions bicarbonate (HCO₃⁻) and chloride (Cl⁻), which freely exchange between plasma and RBC cytoplasm.⁴⁹ Aquaporin 1 (Table 9-5) is a transmembrane protein that forms pores or channels whose surface charges create inward water flow in response to internal osmotic changes.

The ATP–dependent cation pumps Na⁺ -ATPase and K⁺ -ATPase (Table 9-5) regulate the concentrations of Na⁺ and K⁺, maintaining intracellular-to-extracellular ratios of 1:12 and 25:1, respectively.^{50, 51} Ca²⁺ -ATPase extrudes calcium, maintaining low intracellular levels of 5 to 10 μmol/L. Calmodulin, a cytoplasmic Ca²⁺-binding protein, controls the function of Ca²⁺-ATPase.⁵² These enzymes, in addition to aquaporin, maintain osmotic balance.

The cation pumps consume 15% of RBC ATP production. ATP loss or pump damage permits Ca²⁺ and Na⁺ influx, with water following osmotically. The cell swells, becomes spheroid, and eventually ruptures. This phenomenon is called colloid osmotic hemolysis.

Sickle cell disease provides an example of increased cation permeability. When crystallized sickle hemoglobin deforms the cell membranes, internal levels of Na⁺, K⁺, and especially Ca²⁺rise, which results in hemolysis.⁵²

Summary

• Glucose enters the RBC with no energy expenditure via the transmembrane protein Glut-1.
• The anaerobic Embden-Meyerhof pathway (EMP) metabolizes glucose to pyruvate, consuming two ATP molecules. The EMP subsequently generates four ATP molecules per glucose molecule, a net gain of two.
• The hexose-monophosphate pathway (HMP) pathway aerobically converts glucose to pentose and generates NADPH. NADPH reduces glutathione. Reduced glutathione reduces peroxides and protects proteins, lipids, and heme iron from oxidation.
• The methemoglobin reductase pathway converts ferric heme iron (valence 3⁺iron, methemoglobin) to reduced ferrous (valence 2⁺ form), which binds O₂.
• The Rapoport-Luebering pathway generates 2,3-BPG and enhances O₂delivery to tissues.
• The RBC membrane is a lipid bilayer whose hydrophobic components are sequestered from aqueous plasma and cytoplasm. The membrane provides a semipermeable barrier separating plasma from cytoplasm and maintaining an osmotic differential.
• RBC membrane phospholipids are asymmetrically distributed. Phosphatidylcholine and sphingomyelin predominate in the outer layer; phosphatidylserine and phosphatidylethanolamine form most of the inner layer.
• Enzymatic plasma to membrane exchange maintains RBC membrane cholesterol.
• Acanthocytosis and target cells are associated with abnormalities in the concentration or distribution of membrane cholesterol and phospholipids.
• RBC transmembrane proteins channel ions, water, and glucose and anchor cell membrane receptors. They also provide the vertical support connecting the lipid bilayer to the underlying cytoskeleton to maintain membrane integrity.
• RBC cytoplasm K⁺concentration is higher than plasma K⁺, whereas Na⁺ and Ca²⁺ concentrations are lower. Disequilibria are maintained by membrane enzymes K⁺-ATPase, Na⁺-ATPase, and Ca²⁺-ATPase. Pump failure leads to Na⁺ and water influx, cell swelling, and lysis.
• The shape and flexibility of the RBC, which are essential to its function, depend on the cytoskeleton. The cytoskeleton is derived from a group of peripheral proteins on the interior of the lipid membrane. The major structural proteins are α- and β-spectrin, which are bound together and connected to transmembrane proteins by ankyrin, actin, protein 4.1, adducin, tropomodulin, dematin, and band 3. Cytoskeletal proteins provide the horizontal or lateral support for the membrane.
• Hereditary spherocytosis arises from defects in proteins that provide vertical support for the membrane. Hereditary elliptocytosis is due to defects in cytoskeletal proteins that provide horizontal support for the membrane.
• Membrane proteins are extracted using sodium dodecyl sulfate, separated using polyacrylamide gel electrophoresis, and stained with Coomassie blue. They are numbered from the point of application; lower numbers correlate to high protein molecular weight and lower net charge.

Now that you have completed this chapter, go back and read again the case study at the beginning and respond to the questions presented.

Review questions

Answers can be found in the Appendix.

1. Which RBC process does notrequire energy?
2. Oxygen transport
3. Cytoskeletal protein deformability
4. Preventing the peroxidation of proteins and lipids
5. Maintaining cytoplasm cationic electrochemical gradients
6. What pathway anaerobically generates energy in the form of ATP?
7. Hexose monophosphate pathway
8. Rapoport-Luebering pathway
9. Embden-Meyerhof pathway
10. 2,3-BPG pathway
11. Which is true concerning 2,3-BPG?
12. The least abundant of RBC organophosphates
13. Enhances O₂release from hemoglobin
14. Source of RBC glucose
15. Source of RBC ATP
16. To survive, the RBC must detoxify peroxides. What hexose-monophosphate shunt product(s) accomplishes detoxification?
17. ATP
18. 2,3-BPG
19. Pyruvic and lactic acid
20. NADPH and reduced glutathione
21. Which of the following helps maintain RBC shape?
22. Membrane phospholipids
23. Cytoskeletal proteins
24. GPI anchor
25. Glycocalyx
26. The glycolipids of the RBC membrane:
27. Provide flexibility.
28. Carry RBC antigens.
29. Constitute ion channels.
30. Attach the cytoskeleton to the lipid layer.
31. RBC membranes block passage of most large molecules such as proteins, but allow passage of small molecules such as the cations Na+, K+, and Ca++. What is the term for this membrane property?
32. Semipermeable
33. Deformable
34. Intangible
35. Flexible
36. RBC membrane phospholipids are arranged:
37. In a hexagonal lattice.
38. In chains beneath a protein exoskeleton.
39. In two layers whose composition is asymmetric.
40. So that hydrophobic portions are facing the plasma.
41. RBC membrane cholesterol is replenished from the:
42. Plasma.
43. Mitochondria.
44. Cytoplasm.
45. EMB pathway.
46. The hemoglobin iron ion may become oxidized to the +3 valence state by several pathological mechanisms. What portion of the Embden-Meyerhof pathway reduces iron to the physiologic +2 valence state?
47. Methemoglobin reductase pathway
48. Hexose monophosphate pathway
49. Rapoport-Luebering pathway
50. The 2,3-BPG shunt
51. Which of the following is an example of a transmembrane or integral membrane protein?
52. Glycophorin A
53. Ankyrin
54. Spectrin
55. Actin
56. Abnormalities in the horizontal and vertical linkages of the transmembrane and cytoskeletal RBC membrane proteins may be seen as:
57. Shape changes.
58. Methemoglobin increase.
59. Reduced hemoglobin content.
60. Enzyme pathway deficiencies.

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^*The author acknowledges the contribution of Kathryn Doig, who is the previous author of this chapter.