STRUCTURE AND FUNCTION OF THE HEMATOLOGIC SYSTEM

Plasma and Plasma Proteins

In adults, plasma accounts for 50% to 55% of blood volume. Plasma is a complex aqueous liquid containing a variety of organic and inorganic elements (Table 25-1). The concentration of these elements varies depending on diet, metabolic demand, hormones, and vitamins. Plasma differs from serum in that serum is plasma that has been allowed to clot in the laboratory in order to remove fibrinogen and other clotting factors that may interfere with some diagnostic tests.

The plasma contains a large number of proteins (plasma proteins) that constitute about 7% of the total plasma weight. These vary in structure and function and can be classified into two major groups, albumin and globulins. Most plasma proteins are produced by the liver. The major exception is antibodies (immunoglobulins), which are produced by plasma cells in the lymph nodes and other lymphoid tissues (see Chapter 7).

Albumin (about 60% of total plasma protein at a concentration of about 4 g/dl) serves as a carrier molecule for normal components of blood as well as drugs that have low solubility in water (e.g., free fatty acids, lipid-soluble hormones, thyroid hormones, bile salts). Its most essential role is regulation of the passage of water and solutes through the capillaries. Albumin molecules are large and do not diffuse freely through the vascular endothelium, and thus they maintain the critical colloidal osmotic pressure (or oncotic pressure) that regulates the passage of water and solutes into the surrounding tissues (see Chapters 1 and 3). Water and solute particles tend to diffuse out of the arterial portions of the capillaries because the blood pressure is greater in arterial than in venous blood vessels (see Chapter 3). Water and solutes move from tissue cells into the venous portions of the capillaries where the pressures are reversed, oncotic pressure being greater than intravascular pressure or hydrostatic pressure. In the case of decreased production (e.g., cirrhosis, other diffuse liver diseases, protein malnutrition) or excessive loss of albumin (e.g., certain kidney diseases, extensive burns), the reduced oncotic pressure leads to excessive movement of fluid and solutes into the tissue and decreased blood volume.¹

The remaining plasma proteins, or globulins, are often classified by their properties in an electric field (serum electrophoresis). Under the normal conditions used to perform serum electrophoresis, albumin is the most rapidly moving protein. The globulins are classified by their movement relative to albumin: alpha (α) globulins (those moving most closely to albumin), beta (β) globulins, and gamma (γ) globulins (those with the least movement). Depending on the electrophoretic procedure the alpha and beta globulins may be subdivided into subregions (α₁, α₂, β₁, or β₂-globulins). Fibrinogen is a major plasma protein (about 4% of total plasma protein) that would move between the beta and gamma regions but is removed during the formation of serum. The gamma globulin region consists primarily of immunoglobulin G (IgG) (see Chapter 7).

Plasma proteins can also be classified into groups by function: clotting, defense, transport, or regulation. The clotting factors promote coagulation and stop bleeding from damaged blood vessels. Fibrinogen is the most plentiful of the clotting factors and is the precursor of the fibrin clot. Proteins involved in defense, or protection, against infection include antibodies and complement proteins (see Chapters 6 and 7). Transport proteins specifically bind and carry a variety of inorganic and organic molecules, including iron (transferrin), copper (ceruloplasmin), steroid hormones, and vitamins (e.g., retinol-binding protein). The plasma lipids, triglycerides, phospholipids, cholesterol, and fatty acids are carried through the blood as complexes with plasma proteins; they are known as lipoproteins (see Chapters 1 and 30). Regulatory proteins include a variety of enzymatic inhibitors (e.g., α₁-antitrypsin) that protect the tissues from damage, precursor molecules (e.g., kininogen) that are converted into active biologic molecules when needed, and protein hormones (e.g., cytokines) that communicate between cells.

Plasma also contains several charged inorganic ions (electrolytes) that regulate cell function, osmotic pressure, and blood pH. (Electrolytes are described in Chapters 1 and 3.)

Cellular Components of the Blood

The cellular elements of the blood are broadly classified as red blood cells (RBCs) (i.e., erythrocytes), white blood cells (WBCs) (i.e., leukocytes), and platelets (thrombocytes). The components of the blood are listed in Table 25-2.

Spleen

The spleen is the largest of the secondary lymphoid organs. It is a site of fetal hematopoiesis; its mononuclear phagocytes filter and cleanse the blood; its lymphocytes mount an immune response to blood-borne microorganisms; and it serves as a blood reservoir (see Chapter 27).

The spleen is a concave, encapsulated organ that weighs about 150 g and is about the size of a fist. It is located in the left upper abdominal cavity, curved around a portion of the stomach (see Figure 7-3). Strands of connective tissue (trabeculae) extend from the capsule, dividing the spleen into compartments (Figure 25-5). The compartments contain masses of lymphoid tissue called splenic pulp. The spleen is interlaced with many blood vessels, some of which are capable of distending to store blood. Blood that circulates through the spleen comes from the splenic artery, which branches from the descending aorta and reenters the circulatory system through the splenic vein and into the portal vein.

The portion of arterial blood that enters the spleen first encounters the white splenic pulp, which consists of masses of lymphoid tissue containing macrophages and lymphocytes, primarily T lymphocytes in proximity to the arterioles (see Figure 25-5, B and E). Cellular clumps (lymphoid follicles) are formed in the white pulp around the splenic arterioles. The lymphoid follicles consist primarily of B lymphocytes. These are the chief sites of immune function within the spleen. Here blood-borne antigens encounter lymphocytes, initiating the immune response and the conversion of lymphoid follicles into germinal centers (see Chapter 7).⁷

Some of the blood that enters the terminal capillaries continues through the microcirculation and enters highly distensible storage areas called venous sinuses in the red pulp of the spleen. The venous sinuses are capable of storing more than 300 ml of blood. Passive dilation of the venous sinuses enables the spleen to increase its storage capacity as needed by the body. Sudden reductions in blood pressure cause the sympathetic nervous system to stimulate constriction of the sinuses, resulting in expulsion of as much as 200 ml of blood into the venous circulation, which helps restore blood volume and increases the hematocrit by as much as 4%.

The endothelial lining of the venous sinuses is discontinuous (having gaps between endothelial cells) and therefore extremely permeable so that blood cells are allowed to exit the circulation (Figure 25-6). The red pulp contains a system of loosely interconnected resident macrophages that provide the principal site of splenic filtration. Because of the slow circulation in the sinuses, the macrophages easily phagocytose old, damaged, or dead blood cells of all kinds (but chiefly erythrocytes), microorganisms, macromolecules, and particles of debris. Hemoglobin from phagocytosed erythrocytes is catabolized, and heme (iron) is stored in the cytoplasm of the macrophages or released back into the blood (see Figure 25-16). The macrophages also can remove particulate inclusions containing denatured hemoglobin (Heinz bodies) from erythrocytes without harming the cells themselves. Blood that filters through the red pulp also finds its way into the venous sinuses and hence into the portal circulation.

The spleen is not absolutely necessary for life or for adequate hematologic function. However, splenic absence from any cause (atrophy, traumatic injury, or removal because of disease) has several secondary effects on the body. For example, leukocytosis (high levels of circulating leukocytes) often occurs after splenectomy, suggesting that the spleen exerts some control over the rate of proliferation of leukocyte stem cells in the bone marrow or their release into the bloodstream. Circulating levels of iron may also decrease, reflecting the spleen’s role in the iron cycle. The immune response to encapsulated bacteria (e.g., Streptococcus pneumoniae [pneumococcus], Neisseria meningitidis [meningococcus], Haemophilus influenzae), which is primarily an IgM response, may be severely diminished resulting in increased susceptibility to disseminated infections. Loss of the spleen results in an increase in morphologically defective blood cells in the circulation, confirming the spleen’s role in removing old or damaged cells.

Lymph Nodes

Structurally, lymph nodes are part of the lymphatic system. Lymphatic vessels collect interstitial fluid from the tissues and transport it, as lymph, through vessels of increasing size to the thoracic duct, which drains into the superior vena cava returning the lymph to the circulation. Lymph nodes are distributed throughout the body and provide filtration of the lymph during its journey through the lymphatics. Each lymph node is enclosed in a fibrous capsule, branches of which (trabeculae) extend inward to partition the node into several compartments (Figure 25-7). Reticular fibers of connective tissue divide the compartments into a meshwork throughout the lymph node. The node consists of outer (cortex) and inner (paracortex) cortical areas and an inner medulla. Lymph enters through multiple small afferent lymphatic vessels into the subcapsular sinus, just beneath the capsule, drains into the cortical sinuses to the medullary sinuses, from which the lymph is collected and leaves the node by way of the efferent lymphatic vessel. Blood flows into the lymph nodes through the lymphatic artery, which ends in groups of postcapillary venules disturbed throughout the outer cortex. The blood is drained through the lymphatic vein.

Functionally, however, lymph nodes are part of the hematologic and immune systems and are the primary site for the first encounter between antigen and lymphocytes. Lymphocytes enter the lymph node from the blood through the postcapillary venules by means of diapedesis across the endothelial lining. B lymphocytes tend to migrate preferentially to nodes in the cortex and medulla, whereas T lymphocytes predominantly migrate to the paracortex (see Figure 25-7). Macrophages reside in the lymph node, help filter the lymph of debris, foreign substances, and microorganisms, and provide antigen-processing functions. The dendritic cells encounter and process antigens and microorganisms in other tissues, enter the lymph node through the afferent lymph vessels, and migrate throughout the nodes. The reticular network provides adhesive surfaces for trapping large numbers of phagocytes and lymphocytes and facilitating their organization into follicles or primary nodules.⁸ The presence of antigen, either removed from the lymph by macrophages or presented on the surface of dendritic cells, results in the production of secondary nodules containing germinal centers. In the germinal centers lymphocytes, particularly B cells, respond to antigenic stimulation by undergoing proliferation and further differentiation, including class-switch, into memory cells and plasma cells (see Chapter 7). Plasma cells migrate to the medullary cords. The B lymphocyte proliferation in response to a great deal of antigen (e.g., during infection) may result in lymph node enlargement and tenderness (reactive lymph node).

Bone Marrow

Bone marrow is confined to the cavities of bone and is the primary site of residence of hematopoietic stem cells (Figure 25-8). Adults have two kinds of bone marrow: red, or active (hematopoietic), marrow (also called myeloid tissue) and yellow, or inactive, marrow. The large quantity of fat in inactive marrow gives its characteristic yellow color. Not all bones contain active marrow. In adults, active marrow is found primarily in the flat bones of the pelvis (34%), vertebrae (28%), cranium and mandible (13%), sternum and ribs (10%), and in the extreme proximal portions of the humerus and femur (4% to 8%). Inactive marrow predominates in cavities of other bones. (Bones are discussed further in Chapter 41.)

Hematopoietic marrow is vascularized by the primary arteries of the bones, which terminate in a capillary network forming large venous sinuses. Hematopoietic marrow and fat fill the spaces surrounding the network of venous sinuses. Newly produced blood cells traverse narrow openings between endothelial cells in the venous sinus walls and thus enter the circulation. Normally, immature cells have not developed the appropriate surface receptors to interact with the endothelium and enter the circulation.

The stromal compartment of the bone marrow contains a variety of cell types, including mesenchymal stem cells, macrophages/osteoclasts, and endothelial-like cells.⁹ The mesenchymal stem cells can differentiate into fibroblasts, osteoblasts, or adipocytes. Bone marrow fibroblasts secrete a large variety of cytokines (e.g., macrophage colony-stimulating factor [M-CSF], granulocyte-macrophage colony-stimulating factor [GM-CSF], IL-6) that are necessary for hematopoiesis (see Table 25-4). Osteoblasts are responsible for the formation of new bone.¹⁰ Additionally, osteoclasts secrete cytokines that drive hematopoietic cell differentiation. Adipocytes are fat cells containing large depositions of lipid. Adipocytes secrete several growth factors, as well as leptin. Leptin preferentially stimulates mesenchymal stem cells to differentiate into osteoblasts.

Macrophages and osteoclasts have common monocytic precursors. Bone marrow macrophages secrete cytokines and chemokines that regulate proliferation of hematopoietic progenitor cells. Other monocytic cells differentiate under the direction of stromal cells and osteoblasts and undergo intercellular fusion into large multinucleate osteoclasts. Osteoclasts remodel bone by resorption and can produce cytokines that affect proliferation of hematopoietic cells.

The hematologic compartment of the bone marrow consists of a variety of cellular niches that favor differentiation of various hematopoietic progenitor cells.¹¹ The niches are distinguished by a variety of locally produced cytokines and growth factors, so that one niche may be more likely to support differentiation of erythroid precursors into erythrocytes, whereas granulocytes may differentiate in a different site.

Cell Differentiation

Each type of blood cell originates from common hematopoietic stem cells that proliferate and differentiate under control of a variety of cytokines and growth factors (Figure 25-9 and see Table 25-4). During this process some hematopoietic stem cells undergo different paths of differentiation into more differentiated stem cells that are committed to a particular line of blood cells.

All humans originate from a single cell (the fertilized egg) that has the capacity to proliferate and eventually differentiate into the huge diversity of cells of the human body. After fertilization, the egg divides over a 5-day period to form a hollow ball (blastocyst) that implants on the uterus. Until about 3 days after fertilization, each cell (blastomere) is undifferentiated and retains the capacity to differentiate into any cell type. In the 5-day blastocyst, the outer layer cells have undergone differentiation and commitment to become the placenta. Cells of the inner cell mass (embryonic stem cells), however, continue to have unlimited differentiation potential (currently referred to as being pluripotent) and can grow into different kinds of tissue—blood, nerves, heart, bone, and so forth. After implantation, cells of the inner cell mass begin differentiation into other cell types. Differentiation is a multistep process and results in intermediate groups of stem cells (multipotent stem cells) with more limited, but still impressive, abilities to differentiate into many different types of cells (see Figure 25-9).¹²

The hematopoietic organs contain a population of hematopoietic stem cells that have partially differentiated.¹³ They have the capacity to differentiate easily into any of the hematologic cell populations but are very difficult to differentiate into other cell types, like nerve or muscle cells.¹² The challenge of getting any partially committed multipotent stem cells to differentiate reliably involves coaxing them with identical chemical signals that the body uses naturally for differentiation. This is a daunting task with potentially astonishing clinical implications. For example, bone marrow might become the reservoir from which stem cells are harvested and then stimulated to produce nerve cells to help with the treatments of spinal cord injuries.

As with all stem cells, the hematopoietic stem cells are self-renewing (they have the ability to proliferate without further differentiation) so that a relatively constant population of stem cells is available.¹² Some hematopoietic stem cells will continue differentiation into hematopoietic progenitor cells. Progenitor cells retain proliferative capacity but are committed to possible further differentiation into particular types of hematologic cells: lymphoid (lymphocytes, NK cells), granulocyte-monocyte (granulocytes, monocytes, macrophages), and megakaryocyte-erythroid (platelets, erythrocytes) progenitor cells (see Figure 25-9).

As with all other forms of cellular differentiation, successful hematopoiesis requires that progenitor cells interact with neighboring cells (e.g., stromal cells of the bone marrow) through a variety of adhesion molecules and are exposed to particular signaling molecules (e.g., cytokines) (see Table 25-4).¹⁴ Stromal cells apparently express steel factor, a stem cell factor, which activates stem cells to develop. Several cytokines participate in hematopoiesis, particularly colony-stimulating factors (CSFs or hematopoietic growth factors), which stimulate the proliferation of progenitor cells and their progeny and initiate the maturation events necessary to produce fully mature cells (see Figure 25-9).¹⁵ Multiple cell types in the hematopoietic organs, including endothelial cells, fibroblasts, and lymphocytes, produce CSFs.

Hematopoiesis in the bone marrow occurs in two separate pools, the stem cell pool and the bone marrow pool (Figure 25-10). The stem cell pool is responsible for maintaining the number of pluripotent stem cells and partially committed progenitor cells. The bone marrow pool contains cells that are proliferating and maturing in preparation for release into the circulation and mature cells that are stored for later release into the peripheral blood. The peripheral blood also contains two pools of cells; those in the circulation and those stored around the walls of the blood vessels (often called the marginating storage pool). The marginating storage pool primarily consists of neutrophils that adhere to the endothelium in vessels where the blood flow is relatively slow. These cells can rapidly move into tissues and mucous membranes when needed in an inflammatory response. The infiltrating cells are replenished from the circulating pool.

Under certain conditions of rapid depletion of the circulating pool, the circulating hematologic cells need to be rapidly replenished. Medullary hematopoiesis can be accelerated by any or all of three mechanisms: (1) conversion of yellow bone marrow, which does not produce blood cells, to hematopoietic red marrow by the actions of erythropoietin (a hormone that stimulates erythrocyte production); (2) faster differentiation of progenitor cells; and (3) faster proliferation of stem cells into progenitor cells (see Table 25-4).

Clinical Uses of Colony-Stimulating Factors

Neutrophils are normally present in the blood in the range of 4000 to 6000 cells/μL, and in response to a bacterial infection, numbers usually increase to 10,000 to 20,000 cells/μL. Susceptibility to infection develops when normal levels drop below 1000 cells/μL, such as during congenital neutropenia or as a consequence of cytotoxic therapy for cancer. Similarly normal levels of other hematologic cells may be suppressed, e.g., congenital or acquired immune deficiencies and anemia (see Chapters 8 and 9).

The numbers of circulating hematologic cells are under the control of CSFs (see Table 25-4). Administration of CSFs can raise white cell numbers to extremely high levels in healthy individuals. These excessive levels of white blood cells may result in production of toxic products and tissue damage. Therapy with CSFs has been tested in individuals with subnormal levels of circulating blood cells, such as acquired immunodeficiency syndrome (AIDS), aplastic anemia, or congenital neutropenia or as a consequence of cytotoxic therapy lymphoma or leukemia (Figure 25-11). CSF therapy can stimulate increases in circulating granulocyte-monocyte populations, but the degree of response depends on the available numbers of stem and progenitor cells that have survived chemotherapy or the effects of disease. CSF treatment has corrected some cases of congenital neutropenia and resulted in reconstitution of hematopoiesis after bone marrow transplantation.¹⁶ CSF treatment can result in shorter periods of intensive nursing and hospitalization. Recombinant CSFs (e.g., granulocyte colony-stimulating factor [G-CSF], GM-CSF, erythropoietin) are being mass-produced for therapeutic use.

Erythropoiesis

In the confines of the bone marrow erythroid progenitor cells proliferate and differentiate into large, nucleated proerythroblasts, which are committed into producing cells of the erythroid series (Figure 25-12). Erythroid development from the proerythroblast onward is contained in a compartment referred to as the erythron.¹⁷ The proerythroblast, which has ribosomes and can produce protein, differentiates through several intermediate forms of erythroblast while synthesizing hemoglobin and progressively eliminating most intracellular structures, including the nucleus. Thus the maturing erythroblast becomes more compact and progressively assumes the shape and characteristics of an erythrocyte. Hemoglobin is readily apparent and increases in quantity as nuclear size shrinks throughout the basophilic and polychromatophilic stages. The orthochromatic erythroblast (normoblast) is the smallest of the nucleated erythrocyte precursors.

The last immature form of erythroblast is the reticulocyte, which is anucleate and contains a meshlike (reticular) network of ribosomal ribonucleic acid (RNA) that is visible microscopically after staining with certain dyes. The reticulocyte contains polyribosomes (for globin synthesis) and mitochondria (for oxidative metabolism and heme synthesis). The reticulocyte matures into an erythrocyte within 24 to 48 hours. During this period, mitochondria and ribosomes disappear and the cell becomes smaller and more disk like. With these final changes, the erythrocyte loses its capacity for hemoglobin synthesis and oxidative metabolism. Reticulocytes remain in the marrow approximately 1 day and are released into the venous sinuses. They continue to mature in the bloodstream and may travel to the spleen for several days of additional maturation. The normal reticulocyte count is 1% of the total red blood cell count. Approximately 1% of the body’s circulating erythrocyte mass normally is generated every 24 hours. Therefore, the reticulocyte count is a useful clinical index of erythropoietic activity and indicates whether new red cells are being produced.

Regulation of Erythropoiesis

In healthy individuals, the total volume of circulating erythrocytes remains surprisingly constant. Most steps of erythropoiesis are primarily under the control of a feedback loop involving the glycoprotein erythropoietin (see Table 25-4). In conditions of tissue hypoxia, erythropoietin is secreted by the liver and, primarily, by the peritubular cells of the kidney (Figure 25-13). Rising levels of circulating erythropoietin cause a compensatory increase in proliferation and differentiation of proerythroblasts in the bone marrow. The density of cellular erythropoietin receptor decreases progressively during erythroid maturation to almost undetectable levels on reticulocytes. The normal steady-state rate of production of approximately 2.5 million erythrocytes per second can increase to 17 million per second during anemia or under conditions of low oxygen, such as high-altitude or pulmonary disease. Thus the body responds to reduced oxygenation of blood in two ways: (1) stimulation of chemoreceptors of the carotid body and aortic arch that signal the brain to increase respiration and (2) stimulation of receptors on the kidney peritubular cells to increase erythropoietin synthesis and release.

One of the most significant advances in the study of hematopoietic growth factors has been the development of erythropoietin for use in individuals with chronic renal failure. In 1986 large amounts of recombinant human erythropoietin (r-HuEPO) became widely available for clinical research. Erythropoietin is administered intravenously or subcutaneously for the treatment of anemia caused by decreased production of erythropoietin. An immediate effect of increased endogenous or exogenous erythropoietin is an increase in the blood reticulocyte count, followed by increasing levels of erythrocytes. The most significant side effect associated with r-HuEPO is increased blood pressure.

Hemoglobin Synthesis

Hemoglobin (Hb), the oxygen-carrying protein of the erythrocyte, constitutes approximately 90% of the cell’s dry weight. Hemoglobin-packed blood cells take up oxygen in the lungs and exchange it for carbon dioxide in the tissues. A single erythrocyte can contain as many as 300 hemoglobin molecules. Hemoglobin increases the oxygen carrying capacity of blood by 100-fold. Each hemoglobin molecule is composed of two pairs of polypeptide chains (the globins) and four colorful complexes of iron plus protoporphyrin (the hemes) (Figure 25-14).¹⁸ Hemoglobin is responsible for blood’s ruby-red color.

Several variants of hemoglobin exist, but they differ only slightly in primary structure based on the use of different polypeptide chains; alpha, beta, gamma, delta, epsilon, or zeta (α, β, γ, δ, ε, or ζ) (Table 25-5). Each polypeptide chain contains approximately 150 amino acids and is arranged in the knotted-sausage configuration shown in Figure 25-14. The chains assemble to form a tetrahedron containing two pairs of identical chains. Hemoglobin A, the most common type in adults, is composed of two α- and two β-polypeptide chains (α2β2). A normal variant, fetal hemoglobin (hemoglobin F) is a complex of two α- and two γ-polypeptide chains (α2γ2) that binds oxygen with a much greater affinity than adult hemoglobin.

Heme is a large, flat, iron-protoporphyrin disk that is synthesized in the mitochondria and can carry one molecule of oxygen (O₂). Thus an individual hemoglobin molecule with its four hemes can carry four oxygen molecules. If all four oxygen-binding sites are occupied by oxygen, the molecule is said to be saturated. Through a series of complex biochemical reactions, protoporphyrin, a complex four-ringed molecule, is produced and bound with ferrous iron. It is crucial that the iron be correctly charged; reduced ferrous iron (Fe²⁺) can bind oxygen in the lungs and release it in the tissues, where oxygen concentration is less, whereas ferric iron (Fe³⁺) cannot. Binding of oxygen to ferrous iron (oxyhemoglobin) temporally oxidizes Fe²⁺ to Fe³⁺, but after the release of oxygen the body reduces the iron to Fe²⁺ (deoxyhemoglobin [reduced hemoglobin]) and reactivates the hemoglobin’s capacity to bind oxygen. Without reactivation by methemoglobin reductase, the Fe³⁺-containing hemoglobin (methemoglobin) cannot bind oxygen.

Several other molecules can competitively bind to deoxyhemoglobin. Carbon monoxide (CO) directly competes with oxygen for binding to ferrous ion with an affinity that is about 200-fold greater than oxygen. Thus even a small amount of CO can dramatically decrease the ability of hemoglobin to bind and transport oxygen. Hemoglobin also binds carbon dioxide (CO₂), but at a binding site separate from where oxygen binds. In the lungs, CO₂ is released allowing hemoglobin to bind oxygen.

Erythrocytes may play a role in the maintenance of vascular relaxation. Nitric oxide (NO) produced by blood vessels is a major mediator of relaxation and dilation of the vessel walls. In the lungs, hemoglobin can concurrently bind oxygen to the ferrous ion and NO to cysteine residues in the globins. As hemoglobin transfers its oxygen to tissue, it may also shed small amounts of nitric oxide, contributing to dilation of the blood vessels and helping get the oxygen into tissues.

Nutritional Requirements for Erythropoiesis

Normal development of erythrocytes and synthesis of hemoglobin depends on an optimal biochemical milieu and adequate supplies of the necessary building blocks, including protein, vitamins, and minerals (Table 25-6). If these components are lacking for a prolonged time, erythrocyte production slows and anemia (insufficient numbers of functional erythrocytes) may result (see Chapter 26).

Erythropoiesis cannot proceed in the absence of vitamins, especially B₁₂, folate (folic acid), B₆, riboflavin, pantothenic acid, niacin, ascorbic acid, and vitamin E. Dietary vitamin B₁₂ is a large molecule that requires a protein secreted by parietal cells into the stomach (intrinsic factor [IF]) for transport across the ileum. Once absorbed, vitamin B₁₂ is stored in the liver and used as needed in erythropoiesis. Defects in IF production lead to decreased B₁₂ absorption and pernicious anemia.

Folate is the second most important vitamin for erythrocyte production and maturation. Folate is necessary for DNA synthesis, being a component of three of the four DNA bases (thymine, adenine, and guanine), and RNA synthesis. Folate absorption occurs principally in the upper small intestine and is stored in the liver. Folate deficiency is more common than vitamin B₁₂ deficiency and occurs more rapidly. Folate stores can be depleted within a few months, whereas vitamin B₁₂ depletion can take years. Folate supplements are prescribed for pregnant women because pregnancy increases the demand for folate and may cause anemia.

Normal Destruction of Senescent Erythrocytes

After about 100 to 120 days in the circulation, old erythrocytes are removed by tissue macrophages, primarily in the spleen. Although mature erythrocytes lack nuclei, mitochondria, and endoplasmic reticulum, they do have cytoplasmic enzymes capable of glycolysis (anaerobic glucose metabolism) and production of small quantities of ATP, which provides the energy needed to maintain cell function and membrane pliability. Metabolic processes diminish as the erythrocyte ages, so less ATP is available to maintain plasma membrane function. The senescent red cell becomes increasingly fragile and loses its reversible deformability, becoming susceptible to rupture while passing through narrowed regions of the microcirculation.

Additionally, the plasma membrane of senescent red cells undergoes phospholipid rearrangement that is recognized by receptors on macrophages (primarily in the spleen) that selectively remove and sequester the red cells. If the spleen is dysfunctional or absent, macrophages in the liver (Kupffer cells) take over.

The erythrocytes are digested by proteolytic and lipolytic enzymes in the phagolysosomes (digestive vacuoles) of the macrophage. The heme and globin of methemoglobin dissociate easily, and the globin is broken down into its component amino acids. The iron in hemoglobin is oxidized, forming Fe⁺³ (methemoglobin), and recycled (see following section).

Porphyrin is reduced to bilirubin, which is transported to the liver, conjugated, and finally excreted in the bile as glucuronide (Figure 25-15). Approximately 6 g of hemoglobin is catabolized daily, producing 200 mg of bilirubin. Bacteria in the intestinal lumen transform conjugated bilirubin into urobilinogen. Although a small portion is reabsorbed to be either metabolized further by the liver or excreted by the kidney into the urine, most urobilinogen is excreted in feces. Conditions causing accelerated erythrocyte destruction increase the load of bilirubin for hepatic clearance, leading to increased serum levels of unconjugated bilirubin and increased urinary excretion of urobilinogen. Gallstones (cholelithiasis) can result from a chronically elevated rate of bilirubin excretion.

Iron Cycle

Approximately 67% of total body iron is bound to heme in erythrocytes (hemoglobin) and muscle cells (myoglobin), and approximately 30% is stored in mononuclear phagocytes (i.e., macrophages) and hepatic parenchymal cells as either ferritin or hemosiderin. The remaining 3% (less than 1 mg) is lost daily in urine, sweat, bile, and epithelial cells in the gut.

Iron is continually recycled.¹⁹ The methemoglobin released from the breakdown of senescent or damaged erythrocytes (see preceding section) is dissociated by the enzyme heme oxygenase, and the iron released into the bloodstream, where it is free to bind again to transferrin, or stored in the macrophage’s cytoplasm as ferritin or hemosiderin (Figure 25-16). A minute amount of iron is stored in muscle cells by the heme-containing protein myoglobin. Unavailable stores of iron are present in cytochromes, catalases, and peroxidase enzymes.

The protein ferritin is the major intracellular iron storage protein. Apoferritin, which is ferritin without attached iron, can store thousands of atoms of iron. Several (24) apoferritin complexes combine to form the micelle ferritin. Large aggregates of micelles (if a large amount of iron is present) produce numerous ferritin micelles, known as hemosiderin. Hemosiderin is visible as an iron-based pigment under a light microscope as cell inclusions. The iron within deposits of hemosiderin is poorly available to supply iron when needed. Conditions leading to large amounts of iron include hemolysis, severe congestion, unusual increases in dietary iron consumption, increased absorption, or decreased loss. The most common cause of hemosiderin deposition is simple bruising. Hemosiderin in small amounts within iron-rich tissues (i.e., spleen, liver, bone marrow) is considered normal. Large aggregates or its presence in tissue such as the lungs or subcutaneous tissue suggest a pathologic condition.

Iron balance is maintained through controlled absorption rather than excretion. Dietary iron (primarily as Fe²⁺) is transported by a divalent metal ion transporter directly across the membranes of epithelial cells in the duodenum and proximal jejunum.²⁰ (Transport mechanisms are described in Chapter 1.) Regulation of iron transport across the plasma membrane of gastrointestinal epithelial cells is related to the cells’ iron content and the overall rate of erythropoiesis. If the body’s iron stores are low or the demand for erythropoiesis increases, iron is transported rapidly through the epithelial cell and into the plasma. If body stores are high and erythropoiesis is not increased, iron transport is shut down, although iron can cross the epithelial cells’ plasma membrane passively and is stored as ferritin. Excretion of iron occurs when the epithelial cells of the intestinal mucosa slough off.

Iron from either dietary sources or erythrocyte catabolism is transported in the blood bound to apotransferrin, which is then called transferrin; under normal conditions, only one third of the iron-binding sites on transferrin molecules are occupied. Apotransferrin is a glycoprotein synthesized primarily by hepatocytes in the liver but also produced in small quantities by tissue macrophages, submaxillary and mammary glands, and ovaries or testes. Iron for hemoglobin production is carried by transferrin to the bone marrow, where it binds to transferrin receptors on erythroblasts. Transferrin receptors are on the plasma membrane of all nucleated cells, although at particularly high levels on erythroid precursors and rapidly proliferating cells (e.g., lymphocytes), and are thought to be the only route of cellular entry for transferrin-attached iron. Transferrin is recycled (transferrin cycle) in the following manner:

The iron is transported to the erythroblast’s mitochondria (the site of hemoglobin production), where the enzyme heme synthetase inserts ferrous iron into protoporphyrin to form heme. Heme then is bound to globin to form hemoglobin. Iron not used in erythropoiesis is stored temporarily as ferritin or hemosiderin and later excreted.

Adhesion

Normally, platelets are generally observed “rolling” along the margins of vessels. At sites of vessel injury, however, platelets become adherent to the site of endothelial damage where the subendothelial matrix is exposed and endothelial cells have released vWF and decreased their antithrombotic activities (Figure 25-19).²⁵ Platelet adhesion is mostly mediated by the binding of platelet surface receptor glycoprotein-Ib (GPIb) (in a complex with clotting factors IX and V) to von Willebrand factor (vWF) (Figure 25-20).²⁷ The vWF protein is found in the subendothelial matrix and is released by endothelial cells and platelets. Deficiencies in GPIb (Bernard-Soulier syndrome) or of vWF (von Willebrand disease) lead to highly defective hemostasis and congenital bleeding disorders. Platelet adhesion narrows the diameter of the blood vessel resulting in increasing shear forces that could strip platelets off the vessel surface. However, those same forces induce conformational changes in the vWF molecule that result in increased affinity with GPIb, thus stabilizing the adherent platelet.²⁸

Platelet adhesion is also facilitated by other interactions between platelet receptors and exposed molecules of the subendothelial matrix. For instance, adhesion is increased through binding of the platelet collagen receptor GPVI to exposed collagen in the matrix.

Activation

As a result of interactions with the endothelium or the subendothelial matrix, as well as exposure to inflammatory mediators produced by the endothelium and other cells, the platelets are activated. Activation results in dynamic changes in platelet shape from smooth spheres to those with spiny projections and degranulation (also called the platelet-release reaction) resulting in the release of various potent biochemicals (Figure 25-21).²⁹

Platelets contain three types of granules: lysosomes, dense bodies, and alpha granules.³⁰ The contents of the dense bodies and alpha granules are particularly important in hemostasis. The dense bodies contain ADP, serotonin, and calcium. ADP recruits and activates other platelets through specific receptors. During activation the platelet plasma membrane undergoes several important changes, including becoming ruffled and sticky, cellular spreading to make tight contacts between neighboring platelets causing the platelet plug to seal the injured endothelium, and externalization of the phospholipid phosphatidylserine, which provides a matrix for activation of clotting factors (see Figure 25-20). Serotonin is a vasoactive amine that functions like histamine and increases vasodilation and vascular permeability. Calcium is necessary for many of the adhesive interactions as well as intracellular signaling mechanisms that control platelet activation.

Alpha granules contain a large number of clotting factors (e.g., fibrinogen, factor V), growth factors (e.g., PDGF), and heparin-binding proteins (e.g., platelet factor 4). Many of these mediators either promote or inhibit platelet activity and the eventual process of clot formation. PDGF stimulates smooth muscle cells and promotes tissue repair. Heparin-binding proteins enhance clot formation at the site of injury.

Platelets also initiate production of the prostaglandin derivative thromboxane-A₂ (TXA₂), which counters the effects of PGI₂ that is produced by endothelial cells (see Figure 25-20). TXA₂ promotes the degranulation of platelets, increases expression of platelet fibrinogen receptors, and stimulates platelet aggregation. The balance between TXA₂ and PGI₂ affects platelet aggregation, which is favored by TXA₂ excess and inhibited by PGI₂ excess. An isoform of cyclooxygenase (COX-1) converts arachidonic acid to TXA₂ in platelets. Aspirin at low doses specifically and irreversibly inactivates COX-1, decreasing production of TXA₂ and decreasing platelet activation.³¹ A few days of low doses of aspirin lead to more than 95% inhibition of TXA₂.

Other stimuli of platelet activation include epinephrine, thrombin, and collagen. Thrombin and collagen are particularly strong stimuli. Thrombin cleaves the extracellular domain of G-protein–coupled protease-activated receptors (PARs), thereby initiating transmembrane signaling.

Aggregation

Platelet aggregation is stimulated primarily by TXA₂ and ADP, which induce functional fibrinogen receptors on the platelet. The GPIIb-IIIa complex (also called integrin αIIbβ3) undergoes a conformational change during activation to become a calcium-dependent receptor for fibrinogen (see Figure 25-20). It is a member of the integrin receptor family and binds other matrix proteins (e.g., fibronectin, fibrinogen, thrombospondin). Although the GPIIb-IIIa complex is the most abundant aggregation receptor on the platelet, receptors for vWF (GPIb) and collagen (GPVI) also contribute to the process. Interplatelet aggregation and clot retraction that form the primary hemostatic plug are facilitated by fibrinogen bridges between receptors on the platelets. The GPIIb-IIIa–fibrinogen pathway is essential for the formation of a thrombus and as such is an important therapeutic target for blockage by antiplatelet drugs.³¹ In addition, fibrin strands within the clot shorten, becoming denser and stronger, helping the clot to approximate the edges of the injured vessel wall and sealing the site of injury. Contraction of myosin and actin filaments in the platelet cytoskeleton mediates platelet contraction and fusing of the platelet mass into a secondary hemostatic plug. Contraction expels serum from the fibrin meshwork, resulting in greater packing and increased strength. This process usually begins within a few minutes after a clot has formed, and most of the serum is expelled within 20 to 60 minutes.³²

If blood vessel injury is minor, primary hemostasis is achieved by formation of the platelet plug within 3 to 5 minutes of injury. Platelet plugs seal the many minute ruptures that occur daily in the microcirculation, particularly in capillaries. With too few platelets, numerous small hemorrhagic areas called purpuras develop under the skin and throughout the tissues (see Chapter 27). If primary hemostasis is inadequate to prevent further bleeding, the process proceeds through secondary hemostasis to create a larger complex of more tightly interactive platelets within a matrix created by activation of the clotting system.

KEY TERMS

Agranulocyte 955, 956

Albumin 953

Antithrombin III (AT-III) 978

Apoferritin 969

Apotransferrin 971

Basophil 956

Blood clot 976

Bone marrow 962

Clotting (coagulation) system 976

Clotting factor 954

Colony-stimulating factor (CSF, hematopoietic growth factor) 963

Cyclooxygenase (COX-1) 976

D-dimer 980

Dendritic cell 957

Deoxyhemoglobin 968

Endomitosis 971

Eosinophil 956

Erythroblast 966

Erythrocyte (red blood cell [RBC]) 954

Erythropoiesis 965

Erythropoietin 965

Fibrin degradation product (FDP) 980

Fibrinolysis 972

Fibrinolytic system (plasminogen-plasmin system) 979

Globin 967

Globulin 953

GPIIb-IIIa complex 976

Granulocyte 955

Hematopoiesis 961

Hematopoietic stem cell 962

Heme 967

Hemoglobin (Hb) 967

Hemosiderin 969

Hemostasis 972

Immunocyte 955

Leukocyte (white blood cell [WBC]) 955

Lymphocyte 956

Macrophage 957

Marginating storage pool 963

Mast cell 956

Megakaryocyte 957

Mesenchymal stem cell 961

Methemoglobin 968

Monocyte 957

Mononuclear phagocyte system (MPS) 957

Myeloid tissue 962

Myoglobin 969

Natural killer (NK) cell 957

Neutrophil (polymorphonuclear neutrophil [PMN]) 955

Nitric oxide (NO) 972

Normoblast 966

Oxyhemoglobin 968

Phagocyte 955

Plasma 952

Plasma protein 952

Plasmin 979

Plasminogen 979

Platelet (thrombocyte) 957

Platelet adhesion 973

Platelet aggregation 976

Platelet-release reaction 973

Proerythroblast 965

Prostacyclin (PGI) 972

Protein C 979

Protein S 979

Protoporphyrin 968

Reticulocyte 966

Serum 952

Steel factor 963

Stromal cell 963

Thrombomodulin 979

Thromboxane A₂ (TXA₂) 976

Tissue factor (TF, tissue thromboplastin) 977

Tissue factor pathway inhibitor (TFPI) 979

Tissue plasminogen activator (t-PA) 980

Transferrin 971

Urokinase-like plasminogen activator (u-PA) 980

von Willebrand factor (vWF) 973