Red & White Blood Cells - Special Topics - Harper’s Illustrated Biochemistry, 29th Edition (2012)

Harper’s Illustrated Biochemistry, 29th Edition (2012)

SECTION VI. Special Topics

Chapter 52. Red & White Blood Cells

Robert K. Murray, MD, PhD

OBJECTIVES

After studying this chapter, you should be able to:

Image Understand the concept of stem cells and their importance.

Image Summarize the causes of the major disorders affecting red blood cells.

Image Discuss the general structure of the red blood cell membrane.

Image Know the biochemical bases of the ABO blood group substances.

Image Indicate the major biochemical features of neutrophils and understand the basis of chronic granulomatous disease.

Image Appreciate the importance of integrins in health and disease.

BIOMEDICAL IMPORTANCE

Blood cells have been studied intensively because they are obtained easily, because of their functional importance, and because of their involvement in many disease processes. The structure and function of hemoglobin, the porphyrias, jaundice, and aspects of iron metabolism are discussed in previous chapters. Table 52-1 summarizes the causes of a number of important diseases affecting red blood cells; some are discussed in this chapter, and the remainder are discussed elsewhere in this text. Anemia is a very prevalent condition with many causes. The discovery of the causes of certain types of anemias (eg, of pernicious anemia [a form of B12 deficient anemia] and of sickle cell anemia) has been an area where the reciprocal relationship between medicine and biochemistry referred to in Chapter 1 has been extremely beneficial. The World Health Organization (WHO) defines anemia as a hemoglobin level of <130 g/L in men and <120 g/L in females. There are many causes of anemia; only the most prevalent or biochemically relevant are mentioned here. A simplified classification of the causes of anemia is given in Table 52-2. It has been estimated that some 300,000 children are born each year with a severe inherited disorder of hemoglobin, the majority in low- or middle-income countries. Because infant mortality is decreasing, many of these children will survive to present a global health problem. Certain of the blood group systems, present on the membranes of erythrocytes and other blood cells, are of extreme importance in relation to blood transfusion and tissue transplantation. Every organ in the body can be affected by inflammation; neutrophils play a central role in acute inflammation, and other white blood cells, such as lymphocytes, play important roles in chronic inflammation. Leukemias, defined as malignant neoplasms of blood-forming tissues, can affect precursor cells of any of the major classes of white blood cells; common types are acute and chronic myelocytic leukemia, affecting precursors of the neutrophils; and acute and chronic lymphocytic leukemias. Knowledge of the molecular mechanisms involved in the causation of the leukemias is increasing rapidly, but is not discussed in any detail in this text. Combination chemotherapy, using combinations of various chemotherapeutic agents, all of which act at one or more biochemical loci, has been remarkably effective in the treatment of certain of these types of leukemias. Understanding the role of red and white cells in health and disease requires a knowledge of certain fundamental aspects of their biochemistry.

TABLE 52–1 Summary of the Causes of Some Important Disorders Affecting Red Blood Cells

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TABLE 52–2 A Brief Classification of the Causes of Anemia

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ALL BLOOD CELLS DERIVE FROM HEMATOPOIETIC STEM CELLS

Figure 52–1 summarizes the derivation of the various types of blood cells from hematopoietic stem cells. The first solid evidence for the existence of stem cells, and in particular hematopoietic stem cells, was reported from studies done in mice by Ernest McCulloch and James Till in 1963. In recent years, interest in stem cells has grown enormously, and they are now of interest to almost every area of medicine and the health sciences. A stem cell is a cell with a unique capacity to produce unaltered daughter cells (ie, self-renewal) and to generate specialized cell types (potency). Stem cells may be totipotent (capable of producing all the cells in an organism), pluripotent (able to differentiate into cells of any of the three germ layers), multipotent (produce only cells of a closely related family) or unipotent (produce only one type of cell). Stem cells are also classified as embryonic and adult; the latter are more limited in their capabilities to differentiate than the former, although genetic approaches to overcoming this restriction are becoming available.

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FIGURE 52–1 Simplified scheme of differentiation of red blood cells and other blood cells from the hematopoietic stem cell. Sites of action of interleukins (IL-7, IL-3, and IL-5), stem cell factor (SCF), thrombopoietin (TPO), FLT-3 ligand (a growth factor), granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin (EPO), monocyte colony-stimulating factor (M-CSF), and granulocyte colony-stimulating factor (G-CSF) are shown. Sites of action of important transcription factors are not shown. Various steps in the development of lymphoid cells (top part of the figure) have been omitted and abbreviated to one step. (Modified, with permission, from Scadden DT, Longo DL in Fauci AS et al (editors), Harrison’s Principles of Internal Medicine, 17th ed. McGraw-Hill, 2008. Chapter 68)

As shown in Figure 52–1, red blood cells and platelets share a common pathway of differentiation until the stage of megakaryocyte erythroid progenitors. Cells of lymphoid origin branch off at the stage of multipotent progenitors, and other white blood cells at the stage of the common myeloid progenitors. Each pathway is regulated by various factors (eg, stem cell factor, thrombopoietin, various interleukins, erythropoietin, etc), and key specific transcription factors (not indicated in the figure) are also involved at the stages indicated.

Stem cell factor is a cytokine that plays an important role in the proliferation of hematopoietic stem cells and some of their progeny. Thrombopoietin is a glycoprotein that is important in regulating the production of platelets by the bone marrow. Interleukins are cytokines produced by leukocytes; they regulate various aspects of hematopoiesis and of the immune system.

THE RED BLOOD CELL IS SIMPLE IN TERMS OF ITS STRUCTURE & FUNCTION

The major functions of the red blood cell are relatively simple, consisting of delivering oxygen to the tissues and of helping in the disposal of carbon dioxide and protons formed by tissue metabolism. Thus, it has a much simpler structure than most human cells, being essentially composed of a membrane surrounding a solution of hemoglobin (this protein forms about 95% of the intracellular protein of the red cell). There are no intracellular organelles, such as mitochondria, lysosomes, or Golgi apparatus. Human red blood cells, like most red cells of animals, are nonnucleated. However, the red cell is not metabolically inert. ATP is synthesized from glycolysis and is important in processes that help the red blood cell maintain its biconcave shape and also in the regulation of the transport of ions (eg, by the Na+-K+- ATPase and the anion exchange protein [see below]) and of water in and out of the cell. The biconcave shape increases the surface-to-volume ratio of the red blood cell, thus facilitating gas exchange. The red cell contains cytoskeletal components (see below) that play an important role in determining its shape.

About Two Million Red Blood Cells Enter the Circulation per Second

The lifespan of the normal red blood cell is 120 days; this means that slightly less than 1% of the population of red cells (~200 billion cells) is replaced daily (or ~2 million per second). The new red cells that appear in the circulation still contain ribosomes and elements of the endoplasmic reticulum. The RNA of the ribosomes can be detected by suitable stains (such as cresyl blue), and cells containing it are termed reticulocytes; they normally number about 1% of the total red blood cell count. The lifespan of the red blood cell can be dramatically shortened in a variety of hemolytic anemias. The number of reticulocytes is markedly increased in these conditions, as the bone marrow attempts to compensate for rapid breakdown of red blood cells by increasing the amount of new, young red cells in the circulation.

Erythropoietin Regulates Production of Red Blood Cells

Human erythropoietin (EPO) is a glycoprotein of 166 amino acids (molecular mass about 34 kDa). Its amount in plasma can be measured by radioimmunoassay. It is the major regulator of human erythropoiesis (Figure 52–1). As shown in the figure, earlier stages in the development of red blood cells involve stem cell factor, thrombopoietin, and interleukin-3. EPO is synthesized mainly by the kidney and is released in response to hypoxia into the bloodstream, in which it travels to the bone marrow. There it interacts with progenitors of red blood cells via a specific receptor. The receptor is a transmembrane protein consisting of two different subunits and a number of domains. It is not a tyrosine kinase, but it stimulates the activities of specific members of this class of enzymes involved in downstream signal transduction.

The availability of a cDNA for EPO has made it possible to produce substantial amounts of this hormone for analysis and for therapeutic purposes; previously the isolation of erythropoietin from human urine yielded very small amounts of the protein. The major use of recombinant EPO has been in the treatment of a small number of anemic states, such as that due to renal failure. As described in Chapter 47, attempts have been made to prolong the half-life of EPO (thus lengthening its activity) in the circulation by altering the nature of its sugar chains.

MANY GROWTH FACTORS REGULATE PRODUCTION OF WHITE BLOOD CELLS

A large number of hematopoietic growth factors have been identified in recent years in addition to erythropoietin. This area of study adds to knowledge about the differentiation of blood cells, provides factors that may be useful in treatment, and also has implications for understanding of the abnormal growth of blood cells (eg, the leukemias). Like erythropoietin, most of the growth factors isolated have been glycoproteins, are very active in vivo, and in vitro interact with their target cells via specific cell surface receptors, and ultimately (via intracellular signals) affect gene expression, thereby promoting differentiation. Many have been cloned, permitting their production in relatively large amounts. Two of particular interest are granulocyte- and granulocyte-macrophage colony-stimulating factors (G-CSF and GM-CSF, respectively). As indicated in Figure 52–1, G-CSF is relatively specific, inducing mainly granulocytes, whereas GMCSF induces a wider variety of white blood cells. When the production of neutrophils is severely depressed, this condition is referred to as neutropenia. It is particularly likely to occur in patients treated with certain chemotherapeutic regimens and after bone marrow transplantation. These patients are liable to develop overwhelming infections. G-CSF has been administered to such patients to boost production of neutrophils.

THE RED BLOOD CELL HAS A UNIQUE & RELATIVELY SIMPLE METABOLISM

Various aspects of the metabolism of the red cell, many of which are discussed in other chapters of this text, are summarized in Table 52-3.

TABLE 52–3 Summary of Important Aspects of the Metabolism of the Red Blood Cell

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The Red Blood Cell Has a Glucose Transporter in Its Membrane

The entry rate of glucose into red blood cells is far greater than would be calculated for simple diffusion. Rather, it is an example of facilitated diffusion (Chapter 40). The specific protein involved in this process is called the glucose transporter (GLUT1) or glucose permease. Some of its properties are summarized in Table 52-4. The process of entry of glucose into red blood cells is of major importance because it is the major fuel supply for these cells. About 12 different but related glucose transporters have been isolated from various human tissues; unlike the red cell transporter, some of these are insulin-dependent (eg, in muscle and adipose tissue). There is considerable interest in the latter types of transporter because defects in their recruitment from intracellular sites to the surface of skeletal muscle cells may help explain the insulin resistance displayed by patients with type 2 diabetes mellitus.

TABLE 52–4 Some Properties of the Glucose Transporter of the Membrane of the Red Blood Cell (GLUT1)

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Reticulocytes Are Active in Protein Synthesis

The mature red blood cell cannot synthesize protein. Reticulocytes are active in protein synthesis. Once reticulocytes enter the circulation, they lose their intracellular organelles (ribosomes, mitochondria, etc) within about 24 h, becoming young red blood cells and concomitantly losing their ability to synthesize protein. Extracts of rabbit reticulocytes (obtained by injecting rabbits with a chemical—phenylhydrazine—that causes a severe hemolytic anemia, so that the red cells are almost completely replaced by reticulocytes) are widely used as an in vitro system for synthesizing proteins. Endogenous mRNAs present in these reticulocytes are destroyed by use of a nuclease, whose activity can be inhibited by the addition of Ca2+. The system is then programmed by adding purified mRNAs or whole-cell extracts of mRNAs, and radioactive proteins are synthesized in the presence of 35S-labeled L-methionine or other radiolabeled amino acids. The radioactive proteins synthesized are separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and detected by radioautography.

With regard to protein synthesis, it is of interest to note that certain disorders due to genetic abnormalities cause impairment of ribosome structure and function and have been named ribosomopathies. These include some cases of Diamond-Blackfan anemia, in which mutations in a ribosomal RNA processing gene (RPS19) result in red cell hypoplasia. The 5q-syndrome presents with a similar clinical picture and is due to an insufficiency of ribosomal protein RPS 14.

Superoxide Dismutase, Catalase, & Glutathione Protect Blood Cells from Oxidative Stress & Damage

Several powerful oxidants are produced during the course of metabolism, in both blood cells and most other cells of the body. These include superoxide (Image, hydrogen peroxide (H2O2), peroxyl radicals (ROO), and hydroxyl radicals (OH) and are referred to as reactive oxygen species (ROS). Free radicals are atoms or groups of atoms that have an unpaired electron (see Chapters 15 & 45). OH is a particularly reactive molecule and can react with proteins, nucleic acids, lipids, and other molecules to alter their structure and produce tissue damage. The reactions listed in Table 52-5 play an important role in forming these oxidants and in disposing of them; each of these reactions will now be considered in turn.

TABLE 52–5 Reactions of Importance in Relation to Oxidative Stress in Blood Cells and Various Tissues

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Superoxide is formed (reaction 1) in the red blood cell by the auto-oxidation of hemoglobin to methemoglobin (approximately 3% of hemoglobin in human red blood cells has been calculated to auto-oxidize per day); in other tissues, it is formed by the action of enzymes such as cytochrome P450 reductase and xanthine oxidase. When stimulated by contact with bacteria, neutrophils exhibit a respiratory burst (see below) and produce superoxide in a reaction catalyzed by NADPH oxidase (reaction 2). Superoxide spontaneously dismutates to form H2O2 and O2; however, the rate of the same reaction is speeded up tremendously by the action of the enzyme superoxide dismutase(reaction 3). Hydrogen peroxide is subject to a number of fates. The enzyme catalase, present in many types of cells, converts it to H2O and O2 (reaction 4). Neutrophils possess a unique enzyme, myeloperoxidase, which uses H2O2 and halides to produce hypohalous acids (reaction 5); this subject is discussed further below. The selenium-containing enzyme glutathione peroxidase (Chapter 21) will also act on reduced glutathione (GSH) and H2O2 to produce oxidized glutathione (GSSG) and H2O (reaction 6); this enzyme can also use other peroxides as substrates. OH and OH can be formed from H2O2 in a nonenzymatic reaction catalyzed by Fe2+ (the Fenton reaction, reaction 7). Image and H2O2 are the substrates in the iron-catalyzed Haber-Weiss reaction (reaction 8), which also produces OH and OH. Superoxide can release iron ions from ferritin. Thus, production of OH. may be one of the mechanisms involved in tissue injury due to iron overload in hemochromatosis (see Case no. 10 Chapter 57).

Chemical compounds and reactions capable of generating potential toxic oxygen species can be referred to as prooxidants. On the other hand, compounds and reactions disposing of these species, scavenging them, suppressing their formation, or opposing their actions are antioxidants and include compounds such as NADPH, GSH, ascorbic acid, and vitamin E. In a normal cell, there is an appropriate prooxidant:antioxidant balance. However, this balance can be shifted toward the pro-oxidants when production of oxygen species is increased greatly (eg, following ingestion of certain chemicals or drugs) or when levels of antioxidants are diminished (eg, by inactivation of enzymes involved in disposal of oxygen species and by conditions that cause low levels of the antioxidants mentioned above). This state is called “oxidative stress” (see Chapter 45) and can result in serious cell damage if the stress is massive or prolonged.

ROS are now thought to play an important role in many types of cellular injury (eg, resulting from administration of various toxic chemicals or from ischemia), some of which can result in cell death. Indirect evidence supporting a role for these species in generating cell injury is provided if administration of an enzyme such as superoxide dismutase or catalase is found to protect against cell injury in the situation under study.

Deficiency of Glucose-6-Phosphate Dehydrogenase Is Frequent in Certain Areas & Is an Important Cause of Hemolytic Anemia

NADPH, produced in the reaction catalyzed by the X-linked glucose-6-phosphate dehydrogenase (Table 52-5, reaction 9) in the pentose phosphate pathway (Chapter 21), plays a key role in supplying reducing equivalents in the red cell and in other cells such as the hepatocyte. Since the pentose phosphate pathway is virtually its sole means of producing NADPH, the red blood cell is very sensitive to oxidative damage if the function of this pathway is impaired (eg, by enzyme deficiency). One function of NADPH is to reduce GSSG to GSH, a reaction catalyzed by glutathione reductase (reaction 10).

Deficiency of the activity of glucose-6-phosphate dehydrogenase, owing to mutation, is extremely frequent in some regions of the world (eg, tropical Africa, the Mediterranean, certain parts of Asia, and in North America among blacks). It is the most common of all enzymopathies (diseases caused by abnormalities of enzymes), and some 140 genetic variants of the enzyme have been distinguished; at least 400 million people are estimated to have a variant gene. It is thought that an abnormal form of this enzyme confers resistance to malaria. The disorder resulting from deficiency of glucose-6-phosphate dehydrogenase is hemolytic anemia. When an abnormal form of an enzyme causes pathology it is referred to as an enzymopathy. Consumption of broad beans (Vicia faba) by individuals deficient in activity of the enzyme can precipitate an acute attack of hemolytic anemia because they contain potential oxidants. In addition, a number of drugs (eg, the antimalarial drug primaquine [the condition caused by intake of primaquine is called primaquine-sensitive hemolytic anemia] and sulfonamides) and chemicals (eg, naphthalene) precipitate an attack, because their intake leads to generation of H2O2 or Image Normally, H2O2 is disposed of by catalase and glutathione peroxidase (Table 52-5, reactions 4 and 6), the latter causing increased production of GSSG. GSH is regenerated from GSSG by the action of the enzyme glutathione reductase, which depends on the availability of NADPH (reaction 10). The red blood cells of individuals who are deficient in the activity of glucose-6-phosphate dehydrogenase cannot generate sufficient NADPH to regenerate GSH from GSSG, which in turn impairs their ability to dispose of H2O2 and of oxygen radicals. These compounds can cause oxidation of critical SH groups in proteins and possibly peroxidation of lipids in the membrane of the red cell, causing lysis of the red cell membrane. Some of the SH groups of hemoglobin become oxidized, and the protein precipitates inside the red blood cell, forming Heinz bodies, which stain purple with cresyl violet. The presence of Heinz bodies indicates that red blood cells have been subjected to oxidative stress. Figure 52–2 summarizes the possible chain of events in hemolytic anemia due to deficiency of glucose-6-phosphate dehydrogenase.

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FIGURE 52–2 Summary of probable events causing hemolytic anemia due to deficiency of the activity of glucose-6-phosphate dehydrogenase (G6PD) (OMIM 305900).

Hemolytic Anemias Are Caused by Abnormalities Outside, Within or Inside the Red Cell Membrane

Various causes of hemolytic anemias are summarized in Figure 52–3. Causes outside the membrane (ie, extrinsic) include hypersplenism, a condition in which the spleen is enlarged from a variety of causes and red blood cells become sequestered in it. Various antibodies (eg, transfusion reactions and anti-Rh antibodies, the presence in plasma of warm, and cold antibodies that lyse red blood cells) also fall in this class, as do hemolysins released by various infectious agents, such as certain bacteria (eg, certain strains of Escherichia coli and clostridia). Some snakes release venoms that act to lyse the red cell membrane (eg, via the action of phospholipases or proteinases).

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FIGURE 52–3 Schematic diagram of some causes of hemolytic anemias. Extrinsic causes are causes outside the red cell; they include hypersplenism, various antibodies, certain bacterial hemolysins and some snake venoms. Causes intrinsic to the red cells include mutations affecting the structures of membrane proteins (eg, in hereditary spherocytosis and hereditary elliptocytosis), PNH (paroxysmal nocturnal hemoglobinuria, see Chapter 47), enzymopathies, abnormal hemoglobins, and certain parasites (eg, plasmodia causing malaria).

Causes within the membrane (intrinsic) include abnormalities of proteins. The most important conditions are hereditary spherocytosis and hereditary elliptocytosis, principally caused by abnormalities in the amount or structure of spectrin (see below). Paroxysmal nocturnal hemoglobinuria is discussed in Chapter 47.

Causes inside the red blood cell (also intrinsic) include hemoglobinopathies and enzymopathies. Sickle cell anemia and thalassemias are the most prevalent hemoglobinopathies. Abnormalities of enzymes in the pentose phosphate pathway and in glycolysis are the most frequent enzymopathies involved, particularly the former. Deficiency of glucose-6-phosphate dehydrogenase is prevalent in certain parts of the world and is a frequent cause of hemolytic anemia (see above). Deficiency of pyruvate kinase is not frequent, but it is the second commonest enzyme deficiency resulting in hemolytic anemia; the mechanism appears to be due to impairment of glycolysis, resulting in decreased formation of ATP, affecting various aspects of membrane integrity. Parasitic infections (eg, the plasmodia causing malaria) are also important causes of hemolytic anemias in certain geographic areas.

Laboratory investigations that aid in the diagnosis of hemolytic anemia are listed in Table 52-6.

TABLE 52–6 Laboratory Investigations that Assist in the Diagnosis of Hemolytic Anemia

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Methemoglobin Is Useless in Transporting Oxygen

The ferrous iron of hemoglobin is susceptible to oxidation by superoxide and other oxidizing agents, forming methemoglobin, which cannot transport oxygen. Only a very small amount of methemoglobin is present in normal blood, as the red blood cell possesses an effective system (the NADH-cytochrome b5 methemoglobin reductase system) for reducing heme Fe3+ back to the Fe2+ state. This system consists of NADH (generated by glycolysis), a flavoprotein named cytochrome b5 reductase (also known as methemoglobin reductase), and cytochrome b5. The Fe3+ of methemoglobin is reduced back to the Fe2+ state by the action of reduced cytochrome b5:

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Reduced cytochrome b5 is then regenerated by the action of cytochrome b5 reductase:

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Methemoglobinemia Is Inherited or Acquired

Methemoglobinemia can be classified as either inherited or acquired by ingestion of certain drugs and chemicals. Neither type occurs frequently, but physicians must be aware of them. The inherited form is usually due to deficient activity of cytochrome b5 reductase, but mutations can also affect the activity of cytochrome b5. Certain abnormal hemoglobins (eg, HbM) are also rare causes of methemoglobinemia. In HbM, mutation changes the amino acid residue to which heme is attached, thus altering its affinity for oxygen and favoring its oxidation. Ingestion of certain drugs (eg, sulfonamides) or chemicals (eg, aniline) can cause acquired methemoglobinemia. Cyanosis (bluish discoloration of the skin and mucous membranes due to increased amounts of deoxygenated hemoglobin in arterial blood, or in this case due to increased amounts of methemoglobin) is usually the presenting sign in both types and is evident when 10% of hemoglobin is in the “met” form. Diagnosis is made by spectroscopic analysis of blood, which reveals the characteristic absorption spectrum of methemoglobin. Additionally, a sample of blood containing methemoglobin cannot be fully reoxygenated by flushing oxygen through it, whereas normal deoxygenated blood can. Electrophoresis can be used to confirm the presence of an abnormal hemoglobin. Ingestion of methylene blue or ascorbic acid (both reducing agents) is used to treat mild methemoglobinemia due to enzyme deficiency. Acute massive methemoglobinemia (due to ingestion of chemicals) should be treated by intravenous injection of methylene blue.

MORE IS KNOWN ABOUT THE MEMBRANE OF THE HUMAN RED BLOOD CELL THAN ABOUT THE SURFACE MEMBRANE OF ANY OTHER HUMAN CELL

A variety of biochemical approaches have been used to study the membrane of the red blood cell. These include analysis of membrane proteins by SDS-PAGE, the use of specific enzymes (proteinases, glycosidases, and others) to determine the location of proteins and glycoproteins in the membrane, and various techniques to study both the lipid composition and disposition of individual lipids. Morphologic (eg, electron microscopy and freeze-fracture electron microscopy) and other techniques (eg, use of antibodies to specific components) have also been widely used. When red blood cells are lysed under specific conditions, their membranes will reseal in their original orientation to form ghosts (right-side-out ghosts). By altering the conditions, ghosts can also be made to reseal with their cytosolic aspect exposed on the exterior (inside-out ghosts). Both types of ghosts have been useful in analyzing the disposition of specific proteins and lipids in the membrane. In recent years, cDNAs for many proteins of this membrane have become available, permitting the deduction of their amino sequences and domains. All in all, more is known about the membrane of the red blood cell than about any other membrane of human cells (Table 52-7).

TABLE 52–7 Summary of Biochemical Information about the Membrane of the Human Red Blood Cell

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Analysis by SDS-PAGE Resolves the Proteins of the Membrane of the Red Blood Cell

When the membranes of red blood cells are analyzed by SDS-PAGE, about 10 major proteins are resolved (Figure 52–4), several of which have been shown to be glycoproteins. Their migration on SDS-PAGE was used to name these proteins, with the slowest migrating (and hence highest molecular mass) being designated band 1 or spectrin. All these major proteins have been isolated, most of them have been identified, and considerable insight has been obtained about their functions (Table 52-8). Many of their amino acid sequences have also been established. In addition, it has been determined which are integral or peripheral membrane proteins, which are situated on the external surface, which are on the cytosolic surface, and which span the membrane (Figure 52–5). Many minor components can also be detected in the red cell membrane by use of sensitive staining methods or two-dimensional gel electrophoresis. One of these is the glucose transporter described above.

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FIGURE 52–4 Diagrammatic representation of the major proteins of the membrane of the human red blood cell separated by SDS-PAGE. The bands detected by staining with Coomassie blue are shown in the two left-hand channels, and the glycoproteins detected by staining with periodic acid-Schiff (PAS) reagent are shown in the right-hand channel. The direction of electrophoretic migration is from top to bottom. (Reproduced, with permission, from Beck WS, Tepper RI: Hemolytic anemias III: membrane disorders. In: Hematology, 5th ed. Beck WS (editor). The MIT Press, 1991.)

TABLE 52–8 Principal Proteins of the Red Cell Membrane

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FIGURE 52–5 Diagrammatic representation of the interaction of cytoskeletal proteins with each other and with certain integral proteins of the membrane of the red blood cell. (Reproduced, with permission, from Beck WS, Tepper RI: Hemolytic anemias III: membrane disorders. In: Hematology, 5th ed. Beck WS (editor). The MIT Press, 1991.)

The Major Integral Proteins of the Red Blood Cell Membrane Are the Anion Exchange Protein & the Glycophorins

The anion exchange protein (band 3) is a transmembrane glycoprotein, with its carboxyl terminal end on the external surface of the membrane and its amino terminal end on the cytoplasmic surface. It is an example of a multipassmembrane protein, extending across the bilayer approximately 14 times. It probably exists as a dimer in the membrane, in which it forms a tunnel, permitting the exchange of chloride for bicarbonate. Carbon dioxide, formed in the tissues, enters the red cell as bicarbonate, which is exchanged for chloride in the lungs, where carbon dioxide is exhaled. The amino terminal end binds many proteins, including hemoglobin, proteins 4.1 and 4.2, ankyrin, and several glycolytic enzymes. Purified band 3 has been added to lipid vesicles in vitro and has been shown to perform its transport functions in this reconstituted system.

Glycophorins A, B, and C are also transmembrane glycoproteins but of the single-pass type, extending across the membrane only once. A is the major glycophorin, is made up of 131 amino acids, and is heavily glycosylated (about 60% of its mass). Its amino terminal end, which contains 16 oligosaccharide chains (15 of which are O-glycans), extrudes out from the surface of the red blood cell. Approximately 90% of the sialic acid of the red cell membrane is located in this protein. Its transmembrane segment (23 amino acids) is α-helical. The carboxyl terminal end extends into the cytosol and binds to protein 4.1, which in turn binds to spectrin. Polymorphism of this protein is the basis of the MN blood group system (see below). Glycophorin A contains binding sites for influenza virus and for Plasmodium falciparum, the cause of one form of malaria. Intriguingly, the function of red blood cells of individuals who lack glycophorin A does not appear to be affected.

Spectrin, Ankyrin, & Other Peripheral Membrane Proteins Help Determine the Shape & Flexibility of the Red Blood Cell

The red blood cell must be able to squeeze through some tight spots in the microcirculation during its numerous passages around the body; the sinusoids of the spleen are of special importance in this regard. For the red cell to be easily and reversibly deformable, its membrane must be both fluid and flexible; it should also preserve its biconcave shape since this facilitates gas exchange. Membrane lipids help determine membrane fluidity. Attached to the inner aspect of the membrane of the red blood cell are a number of peripheral cytoskeletal proteins (Table 52-8) that play important roles in respect to preserving shape and flexibility; these will now be described.

Spectrin is the major protein of the cytoskeleton. It is composed of two polypeptides: spectrin 1 (α chain) and spectrin 2 (β chain). These chains, measuring approximately 100 nm in length, are aligned in an antiparallel manner and are loosely intertwined, forming a dimer. Both chains are made up of segments of 106 amino acids that appear to fold into triple-stranded α-helical coils joined by nonhelical segments. One dimer interacts with another, forming a head-to-head tetramer. The overall shape confers flexibility on the protein and in turn on the membrane of the red blood cell. At least four binding sites can be defined in spectrin: (1) for self-association, (2) for ankyrin (bands 2.1, etc), (3) for actin (band 5), and (4) for protein 4.1.

Ankyrin is a pyramid-shaped protein that binds spectrin. In turn, ankyrin binds tightly to band 3, securing attachment of spectrin to the membrane. Ankyrin is sensitive to proteolysis, accounting for the appearance of bands 2.2, 2.3, and 2.6, all of which are derived from band 2.1.

Actin (band 5) exists in red blood cells as short, doublehelical filaments of F-actin. The tail end of spectrin dimers binds to actin. Actin also binds to protein 4.1.

Protein 4.1, a globular protein, binds tightly to the tail end of spectrin, near the actin-binding site of the latter, and thus is part of a protein 4.1-spectrin-actin ternary complex. Protein 4.1 also binds to the integral proteins, glycophorins A and C, thereby attaching the ternary complex to the membrane. In addition, protein 4.1 may interact with certain membrane phospholipids, thus connecting the lipid bilayer to the cytoskeleton.

Certain other proteins (4.9, adducin, and tropomyosin) also participate in cytoskeletal assembly.

Abnormalities in the Amount or Structure of Spectrin Cause Hereditary Spherocytosis & Elliptocytosis

Hereditary spherocytosis is a genetic disease, transmitted as an autosomal dominant, that affects about 1:5000 North Americans. It is characterized by the presence of spherocytes (spherical red blood cells, with a low surface-to-volume ratio) in the peripheral blood, by a hemolytic anemia (see Figure 52–3), and by splenomegaly. The spherocytes are not as deformable as are normal red blood cells, and they are subject to destruction in the spleen, thus greatly shortening their life in the circulation. Hereditary spherocytosis is curable by splenectomy because the spherocytes can persist in the circulation if the spleen is absent.

The spherocytes are much more susceptible to osmotic lysis than are normal red blood cells. This is assessed in the osmotic fragility test, in which red blood cells are exposed in vitro to decreasing concentrations of NaCl. The physiologic concentration of NaCl is 0.85 g/dL. When exposed to a concentration of NaCl of 0.5 g/dL, very few normal red blood cells are hemolyzed, whereas approximately 50% of spherocytes would lyse under these conditions. The explanation is that the spherocyte, being almost circular, has little potential extra volume to accommodate additional water and thus lyses readily when exposed to a slightly lower osmotic pressure than is normal.

One cause of hereditary spherocytosis (Figure 52–6) is a deficiency in the amount of spectrin or abnormalities of its structure, so that it no longer tightly binds the other proteins with which it normally interacts. This weakens the membrane and leads to the spherocytic shape. Abnormalities of ankyrin and of bands 3, 4.1, and 4.2 are involved in other cases.

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FIGURE 52–6 Summary of the causation of hereditary spherocytosis (OMIM 182900). Approximately 50% of cases are due to abnormalities in ankyrin and 25% to abnormalities in spectrin.

Hereditary elliptocytosis is a genetic disorder that is similar to hereditary spherocytosis except that affected red blood cells assume an elliptic, disk-like shape, recognizable by microscopy. It is also due to abnormalities in spectrin; some cases reflect abnormalities of band 4.1 or of glycophorin C.

THE BIOCHEMICAL BASES OF THE ABO BLOOD GROUP SYSTEM HAVE BEEN ESTABLISHED

Approximately 30 human blood group systems have been recognized, the best known of which are the ABO, Rh (Rhesus), and MN systems. The term “blood group” applies to a defined system of red blood cell antigens (blood group substances) controlled by a genetic locus having a variable number of alleles (eg, A, B, and O in the ABO system). The term “blood type” refers to the antigenic phenotype, usually recognized by the use of appropriate antibodies. For purposes of blood transfusion, it is particularly important to know the basics of the ABO and Rh systems. However, knowledge of blood group systems is also of biochemical, genetic, immunologic, anthropologic, obstetric, pathologic, and forensic interest. Here, we shall discuss only some key features of the ABO system. From a biochemical viewpoint, the major interests in the ABO substances have been in isolating and determining their structures, elucidating their pathways of biosynthesis, and determining the natures of the products of the A, B, and O genes.

The ABO System Is of Crucial Importance in Blood Transfusion

This system was first discovered by Landsteiner in 1900 when investigating the basis of compatible and incompatible transfusions in humans. The membranes of the red blood cells of most individuals contain one blood group substance of type A, type B, type AB, or type O. Individuals of type A have anti-B antibodies in their plasma and will thus agglutinate type B or type AB blood. Individuals of type B have anti-A antibodies and will agglutinate type A or type AB blood. Type AB blood has neither anti-A nor anti-B antibodies and has been designated the universal recipient. Type O blood has neither A nor B substances and has been designated the universal donor. The explanation of these findings is related to the fact that the body does not usually produce antibodies to its own constituents. Thus, individuals of type A do not produce antibodies to their own blood group substance, A, but do possess antibodies to the foreign blood group substance, B, possibly because similar structures are present in micro-organisms to which the body is exposed early in life. Since individuals of type O have neither A nor B substances, they possess antibodies to both these foreign substances. The above description has been simplified considerably; eg, there are two subgroups of type A: A1 and A2.

The genes responsible for production of the ABO substances are present on the long arm of chromosome 9. There are three alleles, two of which are codominant (A and B) and the third (O) recessive; these ultimately determine the four phenotypic products: the A, B, AB, and O substances.

The ABO Substances Are Glycosphingolipids & Glycoproteins Sharing Common Oligosaccharide Chains

The ABO substances are complex oligosaccharides present in most cells of the body and in certain secretions. On membranes of red blood cells, the oligosaccharides that determine the specific natures of the ABO substances appear to be mostly present in glycosphingolipids, whereas in secretions the same oligosaccharides are present in glycoproteins. Their presence in secretions is determined by a gene designated Se (for secretor), which codes for a specific fucosyl (Fuc) transferase in secretory organs, such as the exocrine glands, but which is not active in red blood cells. Individuals of SeSe or Sese genotypes secrete A or B antigens (or both), whereas individuals of the sese genotype do not secrete A or B substances, but their red blood cells can express the A and B antigens.

H Substance Is the Biosynthetic Precursor of Both the A & B Substances

The ABO substances have been isolated and their structures determined; simplified versions, showing only their nonreducing ends, are presented in Figure 52–7. It is important to first appreciate the structure of the H substancesince it is the precursor of both the A and B substances and is the blood group substance found in persons of type O. H substance itself is formed by the action of a fucosyltransferase, which catalyzes the addition of the terminal fucose in α1 → 2 linkage onto the terminal Gal residue of its precursor:

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FIGURE 52–7 Diagrammatic representation of the structures of the H, A, and B blood group substances. R represents a long complex oligosaccharide chain, joined either to ceramide where the substances are glycosphingolipids, or to the polypeptide backbone of a protein via a serine or threonine residue where the substances are glycoproteins. Note that the blood group substances are biantennary; ie, they have two arms, formed at a branch point (not indicated) between the GlcNAc—R, and only one arm of the branch is shown. Thus, the H, A, and B substances each contain two of their respective short oligosaccharide chains shown above. The AB substance contains one type A chain and one type B chain.

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The H locus codes for this fucosyltransferase. The h allele of the H locus codes for an inactive fucosyltransferase; therefore, individuals of the hh genotype cannot generate H substance, the precursor of the A and B antigens. Thus, individuals of the hh genotype will have red blood cells of type O, even though they may possess the enzymes necessary to make the A or B substances (see below). They are referred to as being Bombay phenotype (Oh).

The A Gene Encodes a GalNAc Transferase, the B Gene a Gal Transferase, & the O Gene an Inactive Product

In comparison with blood group H substance (Figure 52–7), A substance contains an additional GalNAc and B substance an additional Gal, linked as indicated. Anti-A antibodies are directed to the additional GalNAc residue found in the A substance, and anti-B antibodies are directed toward the additional Gal residue found in the B substance. Thus, GalNAc is the immunodominant sugar (ie, the one determining the specificity of the antibody formed) of blood group A substance, whereas Gal is the immunodominant sugar of the B substance. In view of the structural findings, it is not surprising that A substance can be synthesized in vitro from O substance in a reaction catalyzed by a GalNAc transferase, employing UDP-GalNAc as the sugar donor. Similarly, blood group B can be synthesized from O substance by the action of a Gal transferase, employing UDP-Gal. It is crucial to appreciate that the product of the A gene is the GalNAc transferase that adds the terminal GalNAc to the O substance. Similarly, the product of the B gene is the Gal transferase adding the Gal residue to the O substance. Individuals of type AB possess both enzymes and thus have two oligosaccharide chains (Figure 52–6), one terminated by a GalNAc and the other by a Gal. Individuals of type O apparently synthesize an inactive protein, detectable by immunologic means; thus, H substance is their ABO blood group substance.

In 1990, a study using cloning and sequencing technology described the nature of the differences between the glycosyltransferase products of the A, B, and O genes. A difference of four nucleotides is apparently responsible for the distinct specificities of the A and B glycosyltransferases. On the other hand, the O allele has a single base-pair mutation, causing a frameshift mutation resulting in a protein-lacking transferase activity.

NEUTROPHILS HAVE AN ACTIVE METABOLISM & CONTAIN SEVERAL UNIQUE ENZYMES & PROTEINS

The major biochemical features of neutrophils are summarized in Table 52-9. Prominent features are active aerobic glycolysis, active pentose phosphate pathway, moderately active oxidative phosphorylation (because mitochondria are relatively sparse), and a high content of lysosomal enzymes. Many of the enzymes listed in Table 52-5 are also of importance in the oxidative metabolism of neutrophils (see below). Table 52-10 summarizes the functions of some proteins that are relatively unique to neutrophils.

TABLE 52–9 Summary of Major Biochemical Features of Neutrophils

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TABLE 52–10 Some Important Enzymes and Proteins of Neutrophils1

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Neutrophils Are Key Players in the Body’s Defense Against Bacterial Infection

Neutrophils are motile phagocytic cells of the innate immune system that play a key role in acute inflammation. When bacteria enter tissues, a number of phenomena result that are collectively known as the “acute inflammatory response.” They include (1) the increase of vascular permeability, (2) entry of activated neutrophils into the tissues, (3) activation of platelets, and (4) spontaneous subsidence (resolution) if the invading micro-organisms have been dealt with successfully.

A variety of molecules are released from cells and plasma proteins during acute inflammation whose net overall effect is to increase vascular permeability, resulting in tissue edema (Table 52-11).

TABLE 52-11 Sources of Biomolecules with Vasoactive Properties Involved in Acute Inflammation

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In acute inflammation, neutrophils are recruited from the bloodstream into the tissues to help eliminate the foreign invaders. The neutrophils are attracted into the tissues by chemotactic factors, including complement fragment C5a, small peptides derived from bacteria (eg, N-formyl-methionyl-leucyl-phenylalanine), and a number of leukotrienes. To reach the tissues, circulating neutrophils must pass through the capillaries. To achieve this, they marginate along the vessel walls and then adhere to endothelial (lining) cells of the capillaries.

Integrins Mediate Adhesion of Neutrophils to Endothelial Cells

Adhesion of neutrophils to endothelial cells employs specific adhesive proteins (integrins) located on their surface and also specific receptor proteins in the endothelial cells. (See also the discussion of selectins in Chapter 47.)

The integrins are a superfamily of surface proteins present on a wide variety of cells. They are involved in the adhesion of cells to other cells or to specific components of the extracellular matrix. They are heterodimers, containing an α and a β subunit linked noncovalently. The subunits contain extracellular, transmembrane, and intracellular segments. The extracellular segments bind to a variety of ligands such as specific proteins of the extracellular matrix and of the surfaces of other cells. These ligands often contain ArgGly-Asp (R-G-D) sequences. The intracellular domains bind to various proteins of the cytoskeleton, such as actin and vinculin. The integrins are proteins that link the outsides of cells to their insides, thereby helping to integrate responses of cells (eg, movement and phagocytosis) to changes in the environment.

Three subfamilies of integrins were recognized initially. Members of each subfamily were distinguished by containing a common β subunit, but they differed in their subunits. However, more than three β subunits have now been identified, and the classification of integrins has become rather complex. Some integrins of specific interest with regard to neutrophils are listed in Table 52-12.

TABLE 52–12 Examples of Integrins That Are Important in the Function of Neutrophils, of Other White Blood Cells, and of Platelets1

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A deficiency of the β2 subunit (also designated CD18) of LFA-1 and of two related integrins found in neutrophils and macrophages, Mac-1 (CD11b/CD18) and p150,95 (CD11c/CD18), causes type 1 leukocyte adhesion deficiency, a disease characterized by recurrent bacterial and fungal infections. Among various results of this deficiency, the adhesion of affected white blood cells to endothelial cells is diminished, and lower numbers of neutrophils thus enter the tissues to combat infection.

Once having passed through the walls of small blood vessels, the neutrophils migrate toward the highest concentrations of the chemotactic factors, encounter the invading bacteria, and attempt to attack and destroy them. The neutrophils must be activated in order to turn on many of the metabolic processes involved in phagocytosis and killing of bacteria.

Activation of Neutrophils Is Similar to Activation of Platelets & Involves Hydrolysis of Phosphatidylinositol Bisphosphate

The mechanisms involved in platelet activation are discussed in Chapter 51 (see Figure 51–8). The process involves interaction of the stimulus (eg, thrombin) with a receptor, activation of G proteins, stimulation of phospholipase C, and liberation from phosphatidylinositol bisphosphate of inositol triphosphate and diacylglycerol. These two second messengers result in an elevation of intracellular Ca2+ and activation of protein kinase C. In addition, activation of phospholipase A2 produces arachidonic acid that can be converted to a variety of biologically active eicosanoids.

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FIGURE 52–8 Simplified scheme of the sequence of events involved in the causation of chronic granulomatous disease (OMIM 306400). Mutations in any of the genes for the four polypeptides involved (two are components of cytochrome b558 and two are derived from the cytoplasm) can cause the disease. The polypeptide of 91 kDa is encoded by a gene in the X chromosome; approximately 60% of cases of chronic granulomatous disease are X-linked, with the remainder being inherited in an autosomal recessive fashion.

The process of activation of neutrophils is essentially similar. They are activated, via specific receptors, by interaction with bacteria, binding of chemotactic factors, or antibody-antigen complexes. The resultant rise in intracellular Ca2+ affects many processes in neutrophils, such as assembly of microtubules and the actinmyosin system. These processes are respectively involved in secretion of contents of granules and in motility, which enables neutrophils to seek out the invaders. The activated neutrophils are now ready to destroy the invaders by mechanisms that include production of active derivatives of oxygen.

The Respiratory Burst of Phagocytic Cells Involves NADPH Oxidase & Helps Kill Bacteria

When neutrophils and other phagocytic cells engulf bacteria, they exhibit a rapid increase in oxygen consumption known as the respiratory burst. This phenomenon reflects the rapid utilization of oxygen (following a lag of 15-60 s) and production from it of large amounts of reactive derivatives, such as Image, H2O2, OH, and OCl (hypochlorite ion). Some of these products are potent microbicidal agents.

The electron transport chain system responsible for the respiratory burst (named NADPH oxidase) is composed of several components. One is cytochrome b558, located in the plasma membrane; it is a heterodimer, containing two polypeptides of 91 kDa and 22 kDa. When the system is activated (see below), two cytoplasmic polypeptides of 47 kDa and 67 kDa are recruited to the plasma membrane and, together with cytochrome b558, form the NADPH oxidase responsible for the respiratory burst. The reaction catalyzed by NADPH oxidase, involving formation of superoxide anion, is shown in Table 52-5 (reaction 2). This system catalyzes the one-electron reduction of oxygen to superoxide anion. The NADPH is generated mainly by the pentose phosphate cycle, whose activity increases markedly during phagocytosis.

The above reaction is followed by the spontaneous production (by spontaneous dismutation) of hydrogen peroxide from two molecules of superoxide:

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The superoxide ion is discharged to the outside of the cell or into phagolysosomes, where it encounters ingested bacteria. Killing of bacteria within phagolysosomes appears to depend on the combined action of elevated pH, superoxide ion, or further oxygen derivatives (H2O2, OH, and HOCl [hypochlorous acid; see below]) and on the action of certain bactericidal peptides (defensins) and other proteins (eg, cathepsin G and certain cationic proteins) present in phagocytic cells. Any superoxide that enters the cytosol of the phagocytic cell is converted to H2O2 by the action of superoxide dismutase, which catalyzes the same reaction as the spontaneous dismutation shown above. In turn, H2O2 is used by myeloperoxidase (see below) or disposed of by the action of glutathione peroxidase or catalase.

NADPH oxidase is inactive in resting phagocytic cells and is activated upon contact with various ligands (complement fragment C5a, chemotactic peptides, etc) with receptors in the plasma membrane. The events resulting in activation of the oxidase system have been much studied and are similar to those described above for the process of activation of neutrophils. They involve G proteins, activation of phospholipase C, and generation of inositol 1,4,5-triphosphate (IP3). The last mediates a transient increase in the level of cytosolic Ca2+, which is essential for induction of the respiratory burst. Diacylglycerol is also generated and induces the translocation of protein kinase C into the plasma membrane from the cytosol, where it catalyzes the phosphorylation of various proteins, some of which are components of the oxidase system. A second pathway of activation not involving Ca2+ also operates.

Mutations in the Genes for Components of the NADPH Oxidase System Cause Chronic Granulomatous Disease

The importance of the NADPH oxidase system was clearly shown when it was observed that the respiratory burst was defective in chronic granulomatous disease, a relatively uncommon condition characterized by recurrent infections and widespread granulomas (chronic inflammatory lesions) in the skin, lungs, and lymph nodes. The granulomas form as attempts to wall off bacteria that have not been killed, owing to genetic deficiencies in the NADPH oxidase system. The disorder is due to mutations in the genes encoding the four polypeptides that constitute the NADPH oxidase system. Some patients have responded to treatment with gamma interferon, which may increase transcription of the 91-kDa component if it is affected. Attempts are being made to develop gene therapy for this condition. The probable sequence of events involved in the causation of chronic granulomatous disease is shown in Figure 52–8.

Neutrophils Contain Myeloperoxidase, Which Catalyzes the Production of Chlorinated Oxidants

The enzyme myeloperoxidase, present in large amounts in neutrophil granules and responsible for the green color of pus, can act on H2O2 to produce hypohalous acids.

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The H2O2 used as substrate is generated by the NADPH oxidase system. Cl is the halide usually employed since it is present in relatively high concentration in plasma and body fluids. HOCl, the active ingredient of household liquid bleach, is a powerful oxidant and is highly microbicidal. When applied to normal tissues, its potential for causing damage is diminished because it reacts with primary or secondary amines present in neutrophils and tissues to produce various nitrogen-chlorine derivatives; these chloramines are also oxidants, though less powerful than HOCl, and act as microbicidal agents (eg, in sterilizing wounds) without causing tissue damage.

The Proteinases of Neutrophils Can Cause Serious Tissue Damage if Their Actions Are Not Checked

Neutrophils contain a number of proteinases (Table 52-13) that can hydrolyze elastin, various types of collagens, and other proteins present in the extracellular matrix. Such enzymatic action, if allowed to proceed unopposed, can result in serious damage to tissues. Most of these proteinases are lysosomal enzymes and exist mainly as inactive precursors in normal neutrophils. Small amounts of these enzymes are released into normal tissues, with the amounts increasing markedly during inflammation. The activities of elastase and other proteinases are normally kept in check by a number of antiproteinases (also listed in Table 52-13) present in plasma and the extracellular fluid. Each of them can combine—usually forming a noncovalent complex—with one or more specific proteinases and thus cause inhibition. In Chapter 50, it was shown that a genetic deficiency of α1-antiproteinase inhibitor (α-antitrypsin) permits elastase to act unopposed and digest pulmonary tissue, thereby participating in the causation of emphysema. α2-Macroglobulin is a plasma protein that plays an important role in the body’s defense against excessive action of proteases; it combines with and thus neutralizes the activities of a number of important proteases (Chapter 50).

TABLE 52–13 Proteinases of Neutrophils and Antiproteinases of Plasma and Tissues1

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When increased amounts of chlorinated oxidants are formed during inflammation, they affect the proteinase: antiproteinase equilibrium, tilting it in favor of the former. For instance, some of the proteinases listed in Table 52-13are activated by HOCl, whereas certain of the antiproteinases are inactivated by this compound. In addition, the tissue inhibitor of metalloproteinases and α1-antichymotrypsin can be hydrolyzed by activated elastase, and α1-antiproteinase inhibitor can be hydrolyzed by activated collagenase and gelatinase. In most circumstances, an appropriate balance of proteinases and antiproteinases is achieved. However, in certain instances, such as in the lung when α1-antiproteinase inhibitor is deficient or when large amounts of neutrophils accumulate in tissues because of inadequate drainage, considerable tissue damage can result from the unopposed action of proteinases.

RECOMBINANT DNA TECHNOLOGY HAS HAD A PROFOUND IMPACT ON HEMATOLOGY

Recombinant DNA technology has had a major impact on many aspects of hematology. The bases of the thalassemias and of many disorders of coagulation (Chapter 51) have been greatly clarified by investigations using cloning and sequencing. The study of oncogenes and chromosomal translocations has advanced understanding of the leukemias. As discussed above, cloning techniques have made available therapeutic amounts of erythropoietin and other growth factors. Deficiency of adenosine deaminase, which affects lymphocytes particularly, is the first disease to be treated by gene therapy (see Case no. 1, Chapter 57). Like many other areas of biology and medicine, hematology has been and will continue to be revolutionized by this technology. Systems biology is another approach that is beginning to be applied to normal and abnormal hematopoiesis. It depends largely on mathematical, engineering, and computational tools to further understand complex biological processes, such as hematopoiesis. Advances in genomics and proteomics will also be critical for its future development.

SUMMARY

Image Some types of anemias are very prevalent conditions. Major causes of anemia include blood loss, deficiencies of iron, folate and vitamin B12, and various factors causing hemolysis.

Image The red blood cell is simple in terms of its structure and function, consisting principally of a concentrated solution of hemoglobin surrounded by a membrane.

Image The production of red cells is regulated by erythropoietin, whereas other growth factors (eg, granulocyte and granulocytemacrophage colony-stimulating factors) regulate the production of white blood cells.

Image The red cell contains a battery of cytosolic enzymes, such as superoxide dismutase, catalase, and glutathione peroxidase, to dispose of powerful oxidants (ROS) generated during its metabolism.

Image Genetically determined deficiency of the activity of glucose-6-phosphate dehydrogenase, which produces NADPH, is an important cause of hemolytic anemia.

Image Methemoglobin is unable to transport oxygen; both genetic and acquired causes of methemoglobinemia are recognized.

Image Considerable information has accumulated concerning the proteins and lipids of the red cell membrane. A number of cytoskeletal proteins, such as spectrin, ankyrin, and actin, interact with specific integral membrane proteins to help regulate the shape and flexibility of the membrane.

Image Deficiency of spectrin results in hereditary spherocytosis and hereditary elliptocytosis, both causes of hemolytic anemia.

Image The ABO blood group substances in the red cell membrane are complex glycosphingolipids; the immunodominant sugar of A substance is N-acetyl-galactosamine, whereas that of the B substance is galactose. O substance does not contain either of these two sugar residues in the particular linkages found in the A and B substances.

Image Neutrophils play a major role in the body’s defense mechanisms. Integrins on their surface membranes determine specific interactions with various cell and tissue components.

Image Leukocytes are activated on exposure to bacteria and other stimuli; NADPH oxidase plays a key role in the process of activation (the respiratory burst). Mutations in this enzyme and associated proteins cause chronic granulomatous disease.

Image The proteinases of neutrophils can digest many tissue proteins; normally, this is kept in check by a battery of antiproteinases. However, this defense mechanism can be overcome in certain circumstances, resulting in extensive tissue damage.

Image The application of recombinant DNA technology is revolutionizing the field of hematology.

REFERENCES

Fauci AS, Braunwald E, Kasper DL, et al (editors): Harrison’s Principles of Internal Medicine, 17th ed. McGraw-Hill, 2008. (Chapters 58, 61, & 98-108 deal with various blood disorders. Chapters 66-68 deal with various aspects of hematopoietic and other stem cells).

Hofmann R, Benz Jr EJ, Shattal SJ, et al (editors): Hematology: Basic Principles and Practice, 4th ed. Elsevier Churchill Livingston, 2005.

Imlay JA: Cellular defenses against superoxide and hydrogen peroxide. Annu Rev Biochem 2008;77:755.

Israels SJ (editor): Mechanisms in Hematology, 4th 3rd ed. Core Health Sciences Inc, 2011.

Naria A, Ebert BL: Ribosomopathies: human disorders of ribosome dysfunction. Blood 2010;115:3196.

Orkin SH, Higgs DR: Sickle cell disease at 100 years. Science 2010;329:291.

Scriver CR, Beaudet AL, Valle D, et al (editors): The Molecular Bases of Inherited Disease, 8th ed. McGraw-Hill, 2001. (This text is now available online and updated as The Online Metabolic & Molecular Bases of Inherited Disease at www.ommbid.com Subscription is required, although access may be available via university and hospital libraries and other sources). A number of the chapters concern topics described in this chapter.

van den Berg JM, van Koppen E, Ahlin A, et al: Chronic granulomatous disease: the European experience. PLoS ONE 2009;4:e5234.

Weatherall DJ: The inherited diseases of hemoglobin are an emerging global health problem. Blood 2010;115:4331.

Whichard ZL, Sarkar CA, Kimmel M, Corey SJ: Hematopoiesis and its disorders: a systems biology approach. Blood 2010;115:2339.

Yonekawa K, Harlan JM: Targeting leukocyte integrins in human diseases. J Leukoc Biol 2005;77:129.