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Blood cells can be studied histologically in smears prepared by spreading a drop of blood in a thin layer on a microscope slide (Figure 12–3). In such films the cells are clearly visible and distinct from one another, facilitating observation of their nuclei and cytoplasmic characteristics. Blood smears are routinely stained with mixtures of acidic (eosin) and basic (methylene blue) dyes. These mixtures may also contain dyes called azures that are more useful in staining cytoplasmic granules containing charged proteins and proteoglycans. Azurophilic granules produce metachromasia in stained leukocytes like that seen with mast cells in connective tissue. Some of these special stains, such as Giemsa and Wright stain, are named after hematologists who introduced their own modifications into the original mixtures.
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Erythrocytes (red blood cells [RBCs]) are terminally differentiated structures lacking nuclei and completely filled with the O2-carrying protein hemoglobin. RBCs are the only blood cells whose function does not require them to leave the vasculature.
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MEDICAL APPLICATION
Anemia is the condition of having a concentration of erythrocytes below the normal range. With fewer RBCs per milliliter of blood, tissues are unable to receive adequate O2. Symptoms of anemia include lethargy, shortness of breath, fatigue, skin pallor, and heart palpitations. Sickle cell anemia is caused by a homozygous mutation causing an amino acid substitution in hemoglobin, which renders the mature RBCs deformed and slightly rigid (Figure 12–5) and can lead to capillary blockage.
An increased concentration of erythrocytes in blood (erythrocytosis or polycythemia) may be a physiologic adaptation found, for example, in individuals who live at high altitudes, where O2 tension is low. Elevated hematocrit increases blood viscosity, putting strain on the heart, and, if severe, can impair circulation through the capillaries.
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Human erythrocytes suspended in an isotonic medium are flexible biconcave discs (Figure 12–4). They are approximately 7.5 μm in diameter, 2.6-μm thick at the rim, but only 0.75-μm thick in the center. Because of their uniform dimensions and their presence in most tissue sections, RBCs can often be used by histologists as an internal standard to estimate the size of other nearby cells or structures.
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The biconcave shape provides a large surface-to-volume ratio and facilitates gas exchange. The normal concentration of erythrocytes in blood is approximately 3.9-5.5 million per microliter (μL, or mm3) in women and 4.1-6.0 million/μL in men.
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Erythrocytes are normally quite flexible, which permits them to bend and adapt to the small diameters and irregular turns of capillaries. Observations in vivo show that at the angles of capillary bifurcations, erythrocytes with normal adult hemoglobin frequently assume a cuplike shape. In larger blood vessels RBCs may adhere to one another loosely in stacks called rouleaux (Figure 12–4c).
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The erythrocyte plasmalemma, because of its ready availability, is the best-known membrane of any cell. It consists of about 40% lipid, 10% carbohydrate, and 50% protein. Most of the latter are integral membrane proteins (see Chapter 2), including ion channels, the anion transporter called band 3 protein, and glycophorin A. The glycosylated extracellular domains of the latter proteins include antigenic sites that form the basis for the ABO blood typing system. Several peripheral proteins are associated with the inner surface of the membrane, including spectrin, dimers of which form a lattice bound to underlying actin filaments, and ankyrin, which anchors the spectrin lattice to the glycophorins and band 3 proteins. This submembranous meshwork stabilizes the membrane, maintains the cell shape, and provides the cell elasticity required for passage through capillaries.
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Erythrocyte cytoplasm lacks all organelles but is densely filled with hemoglobin, the tetrameric O2-carrying protein that accounts for the cells’ uniform acidophilia. When combined with O2 or CO2, hemoglobin forms oxyhemoglobin or carbaminohemoglobin, respectively. The reversibility of these combinations is the basis for the protein’s gas-transporting capacity.
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Erythrocytes undergo terminal differentiation (discussed in Chapter 13) which includes loss of the nucleus and organelles shortly before the cells are released by bone marrow into the circulation. Lacking mitochondria, erythrocytes rely on anaerobic glycolysis for their minimal energy needs. Lacking nuclei, they cannot replace defective proteins.
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Human erythrocytes normally survive in the circulation for about 120 days. By this time defects in the membrane’s cytoskeletal lattice or ion transport systems begin to produce swelling or other shape abnormalities, as well as changes in the cells’ surface oligosaccharide complexes. Senescent or worn-out RBCs displaying such changes are recognized and removed from circulation, mainly by macrophages of the spleen, liver, and bone marrow.
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Leukocytes (WBCs) leave the blood and migrate to the tissues where they become functional and perform various activities related to immunity. Leukocytes are divided into two major groups, granulocytes and agranulocytes, based on the density of their cytoplasmic granules (Table 12–2). All are rather spherical while suspended in blood plasma, but they become amoeboid and motile after leaving the blood vessels and invading the tissues. Their estimated sizes mentioned here refer to observations in blood smears in which the cells are spread and appear slightly larger than they are in the circulation.
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Granulocytes possess two major types of abundant cytoplasmic granules: lysosomes (often called azurophilic granules in blood cells) and specific granules that bind neutral, basic, or acidic stains and have specific functions.
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Granulocytes also have polymorphic nuclei with two or more distinct (almost separated) lobes and include the neutrophils, eosinophils, and basophils (Figure 12–1 and Table 12–2). All granulocytes are also terminally differentiated cells with a life span of only a few days. Their Golgi complexes and rough ER are poorly developed, and with few mitochondria they depend largely on glycolysis for their energy needs. Most granulocytes undergo apoptosis in the connective tissue and billions of neutrophils alone die each day in adults. The resulting cellular debris is removed by macrophages and, like all apoptotic cell death, does not itself elicit an inflammatory response.
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Agranulocytes lack specific granules, but do contain some azurophilic granules (lysosomes). The nucleus is spherical or indented but not lobulated. This group includes the lymphocytes and monocytes (Figure 12–1 and Table 12–2). The differential count (percentage of all leukocytes) for each type of leukocyte is also presented in Table 12–2.
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All leukocytes are key players in the constant defense against invading microorganisms and in the repair of injured tissues, specifically leaving the microvasculature in injured or infected tissues. At such sites factors termed cytokines are released from various sources and these trigger loosening of intercellular junctions in the endothelial cells of local postcapillary venules (Figure 12–6). Simultaneously the cell adhesion protein P-selectin appears on the endothelial cells’ luminal surfaces following exocytosis from cytoplasmic Weibel-Palade bodies. The surfaces of neutrophils and other leukocytes display glycosylated ligands for P-selectin, and their interactions cause cells flowing through the affected venules to slow down, like rolling tennis balls arriving at a patch of velcro. Other cytokines stimulate the now slowly rolling leukocytes to express integrins and other adhesion factors that produce firm attachment to the endothelium (see Figure 11–21d). In a process called diapedesis (Gr. dia, through + pedesis, to leap), the leukocytes send extensions through the openings between the endothelial cells, migrate out of the venules into the surrounding tissue space, and head directly for the site of injury or invasion. The attraction of neutrophils to bacteria involves chemical mediators in a process of chemotaxis, which causes leukocytes to rapidly accumulate where their defensive actions are specifically needed.
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The number of leukocytes in the blood varies according to age, sex, and physiologic conditions. Healthy adults have 4500-11,000 leukocytes per microliter of blood.
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Neutrophils (Polymorphonuclear Leukocytes)
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Mature neutrophils constitute 50%-70% of circulating leukocytes, a figure that includes slightly immature forms released to the circulation. Neutrophils are 12-15 μm in diameter in blood smears, with nuclei having two to five lobes linked by thin nuclear extensions (see Table 12–2; Figure 12–7). In females, the inactive X chromosome may appear as a drumstick-like appendage on one of the lobes of the nucleus (Figure 12–7c) although this characteristic is not always seen. Neutrophils are inactive and spherical while circulating but become amoeboid and highly active during diapedesis and upon adhering to ECM substrates such as collagen.
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Neutrophils are usually the first leukocytes to arrive at sites of infection where they actively pursue bacterial cells using chemotaxis and remove the invaders or their debris by phagocytosis.
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The cytoplasmic granules of neutrophils provide the cells’ functional activities and are of two main types (Figure 12–8). Azurophilic primary granules or lysosomes are large, dense vesicles with a major role in both killing and degrading engulfed microorganisms. They contain proteases and antibacterial proteins, including the following:
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Myeloperoxidase (MPO), which generates hypochlorite and other agents toxic to bacteria
Lysozyme, which degrades components of bacterial cell walls
Defensins, small cysteine-rich proteins that bind and disrupt the cell membranes of many types of bacteria and other microorganisms.
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MEDICAL APPLICATION
Several kinds of neutrophil defects, often genetic in origin, can affect function of these cells, for example, by decreasing adhesion to the wall of venules, by causing the absence of specific granules, or with deficits in certain factors of the azurophilic granules. Individuals with such disorders typically experience more frequent and more persistent bacterial infections, although macrophages and other leukocytes may substitute for certain neutrophil functions.
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Specific secondary granules are smaller and less dense, stain faintly pink, and have diverse functions, including secretion of various ECM-degrading enzymes such as collagenases, delivery of additional bactericidal proteins to phagolysosomes, and insertion of new cell membrane components.
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Activated neutrophils at infected or injured sites also have important roles in the inflammatory response, which begins the process of restoring the normal tissue microenvironment. They release many polypeptide chemokines that attract other leukocytes and cytokines, which direct activities of these and local cells of the tissue. Important lipid mediators of inflammation are also released from neutrophils.
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Neutrophils contain glycogen, which is broken down into glucose to yield energy via the glycolytic pathway. The citric acid cycle is less important, as might be expected in view of the paucity of mitochondria in these cells. The ability of neutrophils to survive in an anaerobic environment is highly advantageous, because they can kill bacteria and help clean up debris in poorly oxygenated regions, for example, damaged or necrotic tissue lacking normal microvasculature.
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Neutrophils are short-lived cells with a half-life of 6-8 hours in blood and a life span of 1-4 days in connective tissues before dying by apoptosis.
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MEDICAL APPLICATION
Neutrophils look for bacteria to engulf by pseudopodia and internalize them in vacuoles called phagosomes. Immediately thereafter, specific granules fuse with and discharge their contents into the phagosomes that are then acidified by proton pumps. Azurophilic granules then discharge their enzymes into this acidified vesicle, killing and digesting the engulfed microorganisms.
During phagocytosis, a burst of O2 consumption leads to the formation of superoxide anions (O2–) and hydrogen peroxide (H2O2). O2– is a short-lived, highly reactive free radical that, together with MPO and halide ions, forms a powerful microbial killing system inside the neutrophils. Besides the activity of lysozyme cleaving cell wall peptidoglycans to kill certain bacteria, the protein lactoferrin avidly binds iron, a crucial element in bacterial nutrition whose lack of availability then causes bacteria to die. A combination of these mechanisms will kill most microorganisms, which are then digested by lysosomal enzymes. Apoptotic neutrophils, bacteria, semidigested material, and tissue-fluid form a viscous, usually yellow collection of fluid called pus.
Several neutrophil hereditary dysfunctions have been described. In one of them, actin does not polymerize normally, reducing neutrophil motility. With an NADPH oxidase deficiency, there is a failure to produce H2O2 and hypochlorite, reducing the cells’ microbial killing power. Children with such dysfunctions can experience more persistent bacterial infections.
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Eosinophils are far less numerous than neutrophils, constituting only 1%-4% of leukocytes. In blood smears, this cell is about the same size as a neutrophil or slightly larger, but with a characteristic bilobed nucleus (Table 12–2; Figure 12–9). The main identifying characteristic is the abundance of large, acidophilic specific granules typically staining pink or red.
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Ultrastructurally the eosinophilic-specific granules are seen to be oval in shape, with flattened crystalloid cores (Figure 12–9c) containing major basic proteins (MBP), an arginine-rich factor that accounts for the granule’s acidophilia and constitutes up to 50% of the total granule protein. MBPs, along with eosinophilic peroxidase, other enzymes and toxins, act to kill parasitic worms or helminths. Eosinophils also modulate inflammatory responses by releasing chemokines, cytokines, and lipid mediators, with an important role in the inflammatory response triggered by allergies. The number of circulating eosinophils increases during helminthic infections and allergic reactions. These leukocytes also remove antigen-antibody complexes from interstitial fluid by phagocytosis.
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Eosinophils are particularly abundant in connective tissue of the intestinal lining and at sites of chronic inflammation, such as lung tissues of asthma patients.
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MEDICAL APPLICATION
An increase in the number of eosinophils in blood (eosinophilia) is associated with allergic reactions and helminthic infections. In patients with such conditions, eosinophils are found in the connective tissues underlying epithelia of the bronchi, gastrointestinal tract, uterus, and vagina, and surrounding any parasitic worms present. In addition, these cells produce substances that modulate inflammation by inactivating the leukotrienes and histamine produced by other cells. Corticosteroids (hormones from the adrenal cortex) produce a rapid decrease in the number of blood eosinophils, probably by interfering with their release from the bone marrow into the bloodstream.
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Basophils are also 12-15 μm in diameter but make up less than 1% of circulating leukocytes and are therefore difficult to find in normal blood smears. The nucleus is divided into two irregular lobes, but the large specific granules overlying the nucleus usually obscure its shape.
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The specific granules (0.5 μm in diameter) typically stain purple with the basic dye of blood smear stains and are fewer, larger, and more irregularly shaped than the granules of other granulocytes (see Table 12–2; Figure 12–10). The strong basophilia of the granules is due to the presence of heparin and other sulfated GAGs. Basophilic-specific granules also contain much histamine and various other mediators of inflammation, including platelet activating factor, eosinophil chemotactic factor, and the enzyme phospholipase A that catalyzes an initial step in producing lipid-derived proinflammatory factors called leukotrienes.
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By migrating into connective tissues, basophils appear to supplement the functions of mast cells, which are described in Chapter 5. Both basophils and mast cells have metachromatic granules containing heparin and histamine, have surface receptors for immunoglobulin E (IgE), and secrete their granular components in response to certain antigens and allergens.
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MEDICAL APPLICATION
In some individuals a second exposure to a strong allergen, such as that delivered in a bee sting, may produce an intense, adverse systemic response. Basophils and mast cells may rapidly degranulate, producing vasodilation in many organs, a sudden drop in blood pressure, and other effects comprising a potentially lethal condition called anaphylaxis or anaphylactic shock.
Basophils and mast cells also are central to immediate or type 1 hypersensitivity. In some individuals substances such as certain pollen proteins or specific proteins in food are allergenic, that is, elicit production of specific IgE antibodies, which then bind to receptors on mast cells and immigrating basophils. Upon subsequent exposure, the allergen combines with the receptor-bound IgE molecules, causing them to cross-link and aggregate on the cell surfaces and triggering rapid exocytosis of the cytoplasmic granules. Release of the inflammatory mediators in this manner can result in bronchial asthma, cutaneous hives, rhinitis, conjunctivitis, or allergic gastroenteritis.
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By far the most numerous type of agranulocyte in normal blood smears, lymphocytes constitute a family of leukocytes with spherical nuclei (Table 12–2; Figure 12–11). Lymphocytes are typically the smallest leukocytes and constitute approximately one-third of these cells. Although they are morphologically similar, mature lymphocytes can be subdivided into functional groups by distinctive surface molecules (called “cluster of differentiation” or CD markers) that can be distinguished using antibodies with immunocytochemistry or flow cytometry. Major classes include B lymphocytes, helper and cytotoxic T lymphocytes (CD4+ and CD8+, respectively), and natural killer (NK) cells. These and other types of lymphocytes have diverse roles in immune defenses against invading microorganisms and certain parasites or abnormal cells. T lymphocytes, unlike B cells and all other circulating leukocytes, differentiate outside the bone marrow in the thymus. Functions and formation of lymphocytes are discussed with the immune system in Chapter 14.
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Although generally small, circulating lymphocytes have a wider range of sizes than most leukocytes. Small, newly released lymphocytes have diameters similar to those of RBCs; medium and large lymphocytes are 9-18 μm in diameter, with the latter representing activated lymphocytes or NK cells. The small lymphocytes are characterized by spherical nuclei with highly condensed chromatin and only a thin surrounding rim of scant cytoplasm, making them easily distinguishable from granulocytes. Larger lymphocytes have larger, slightly indented nuclei and more cytoplasm that is slightly basophilic, with a few azurophilic granules, mitochondria, free polysomes, and other organelles (Figure 12–11d).
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Lymphocytes vary in life span according to their specific functions; some live only a few days and others survive in the circulating blood or other tissues for many years.
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MEDICAL APPLICATION
Given their central roles in immunity, lymphocytes are obviously important in many diseases. Lymphomas are a group of disorders involving neoplastic proliferation of lymphocytes or the failure of these cells to undergo apoptosis. Although often slow-growing, all lymphomas are considered malignant because they can very easily become widely spread throughout the body.
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Monocytes are precursor cells of macrophages, osteoclasts, microglia, and other cells of the mononuclear phagocyte system in connective tissue of nearly all organs (see Chapter 5). Circulating monocytes have diameters of 12-15 μm and have nuclei that are large and usually distinctly indented or C-shaped (Figure 12–12). The chromatin is less condensed than in lymphocytes and typically stains lighter than that of large lymphocytes. Cells of the mononuclear phagocyte system arise in developing organs from monocytes formed in the embryonic yolk sac and are supplemented throughout life by monocytes from the bone marrow. All monocyte-derived cells are antigen-presenting cells with important roles in immune defense as well as tissue repair.
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The cytoplasm of the monocyte is basophilic and contains many small lysosomal azurophilic granules, some of which are at the limit of the light microscope’s resolution. These granules are distributed through the cytoplasm, giving it a bluish-gray color in stained smears. Mitochondria and small areas of rough ER are present, along with a Golgi apparatus involved in the formation of lysosomes (Figure 12–12e).
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MEDICAL APPLICATION
Extravasation or the accumulation of immigrating monocytes occurs in the early phase of inflammation following tissue injury. Acute inflammation is usually short-lived as macrophages undergo apoptosis or leave the site, but chronic inflammation usually involves the continued recruitment of monocytes. The resulting continuous presence of macrophages can lead to excessive tissue damage that is typical of chronic inflammation.
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Blood platelets (or thrombocytes) are very small non-nucleated, membrane-bound cell fragments only 2-4 μm in diameter (Figure 12–13a). As described in Chapter 13, platelets originate by separation from the ends of cytoplasmic processes extending from giant polyploid bone marrow cells called megakaryocytes. Platelets promote blood clotting and help repair minor tears or leaks in the walls of small blood vessels, preventing loss of blood from the microvasculature. Normal platelet counts range from 150,000 to 400,000/μL (mm3) of blood. Circulating platelets have a life span of about 10 days.
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In stained blood smears, platelets often appear in clumps. Each individual platelet is generally discoid, with a very lightly stained peripheral zone, the hyalomere, and a darker-staining central zone rich in granules, called the granulomere. A sparse glycocalyx surrounding the platelet plasmalemma is involved in adhesion and activation during blood coagulation.
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Ultrastructural analysis (Figure 12–13b) reveals a peripheral marginal bundle of microtubules and microfilaments, which helps to maintain the platelet’s shape. Also in the hyalomere are two systems of membrane channels. An open canalicular system of vesicles is connected to invaginations of the plasma membrane, which may facilitate platelets’ uptake of factors from plasma. A much less prominent set of irregular tubular vesicles comprising the dense tubular system is derived from the ER and stores Ca2+ ions. Together, these two membranous systems facilitate the extremely rapid exocytosis of proteins from platelets (degranulation) upon adhesion to collagen or other substrates outside the vascular endothelium.
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Besides specific granules, the central granulomere has a sparse population of mitochondria and glycogen particles (Figure 12–13b). Electron-dense delta granules (δG), 250-300 nm in diameter, contain ADP, ATP, and serotonin (5-hydroxytryptamine) taken up from plasma. Alpha granules (αG) are larger (300-500 nm in diameter) and contain platelet-derived growth factor (PDGF), platelet factor 4, and several other platelet-specific proteins. Most of the stained granules seen in platelets with the light microscope are alpha granules.
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The role of platelets in controlling blood loss (hemorrhage) and in wound healing can be summarized as follows:
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Primary aggregation: Disruptions in the microvascular endothelium, which are very common, allow the platelet glycocalyx to adhere to collagen in the vascular basal lamina or wall. Thus, a platelet plug is formed as a first step to stop bleeding (Figure 12–14).
Secondary aggregation: Platelets in the plug release a specific adhesive glycoprotein and ADP, which induce further platelet aggregation and increase the size of the platelet plug.
Blood coagulation: During platelet aggregation, fibrinogen from plasma, von Willebrand factor and other proteins released from the damaged endothelium, and platelet factor 4 from platelet granules promote the sequential interaction (cascade) of plasma proteins, giving rise to a fibrin polymer that forms a three-dimensional network of fibers trapping RBCs and more platelets to form a blood clot, or thrombus (Figure 12–14). Platelet factor 4 is a chemokine for monocytes, neutrophils, and fibroblasts, and proliferation of the fibroblasts is stimulated by PDGF.
Clot retraction: The clot initially bulges into the blood vessel lumen, but soon contracts slightly due to the activity of platelet-derived actin and myosin.
Clot removal: Protected by the clot, the endothelium and surrounding tunic are restored by new tissue, and the clot is then removed, mainly dissolved by the proteolytic enzyme plasmin, which is formed continuously through the local action of plasminogen activators from the endothelium on plasminogen from plasma.
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MEDICAL APPLICATION
Aspirin and other nonsteroidal anti-inflammatory agents have an inhibitory effect on platelet function and blood coagulation because they block the local prostaglandin synthesis, which is needed for platelet aggregation, contraction, and exocytosis at sites of injury. Bleeding disorders result from abnormally slow blood clotting. One such disease directly related to a defect in the platelets is a rare autosomal recessive glycoprotein Ib deficiency, involving a factor on the platelet surface needed to bind subendothelial collagen and begin the cascade of events leading to clot formation.
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SUMMARY OF KEY POINTS
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Blood SUMMARY OF KEY POINTS
The liquid portion of circulating blood is plasma, while the cells and platelets comprise the formed elements; upon clotting, some proteins are removed from plasma and others are released from platelets, forming a new liquid termed serum.
Important protein components of plasma include albumin, diverse α- and β-globulins, proteins of the complement system, and fibrinogen, all of which are secreted within the liver, as well as the immunoglobulins.
RBCs or erythrocytes, which make up the hematocrit portion (~45%) of a blood sample, are enucleated, biconcave discs 7.5 μm in diameter, filled with hemoglobin for the uptake, transport, and release of O2, and with a normal life span of about 120 days.
WBCs or leukocytes are broadly grouped as granulocytes (neutrophils, eosinophils, basophils) or agranulocytes (lymphocytes, monocytes).
All leukocytes become active outside the circulation, specifically leaving the microvasculature in a process involving cytokines, selective adhesion, changes in the endothelium, and transendothelial migration or diapedesis.
All granulocytes have specialized lysosomes called azurophilic granules and smaller specific granules with proteins for various cell-specific functions.
Neutrophils, the most abundant type of leukocyte, have polymorphic, multilobed nuclei, and faint pink cytoplasmic granules that contain many factors for highly efficient phagolysosomal killing and removal of bacteria.
Eosinophils have bilobed nuclei and eosinophilic-specific granules containing factors for destruction of helminthic parasites and for modulating inflammation.
Basophils, the rarest type of circulating leukocyte, have irregular bilobed nuclei and resemble mast cells with strongly basophilic specific granules containing factors important in allergies and chronic inflammatory conditions, including histamine, heparin, chemokines, and various hydrolases.
Lymphocytes, agranulocytes with many functions as T- and B-cell subtypes in the immune system, range widely in size, depending on their activation state, and have roughly spherical nuclei with little cytoplasm and few organelles.
Monocytes are larger agranulocytes with distinctly indented or C-shaped nuclei, which circulate as precursors of macrophages and other cells of the mononuclear phagocyte system.
Platelets are small (2-4 μm) cell fragments derived from megakaryocytes in bone marrow, with a marginal bundle of actin filaments, alpha granules and delta granules, and an open canalicular system of membranous vesicles; rapid degranulation on contact with collagen triggers blood clotting.