Blood

Whole blood is a suspension of cellular elements in plasma

If you spin down a sample of blood containing an anticoagulant for ~5 minutes at 10,000 g, the bottom fraction contains formed elements — RBCs (or erythrocytes), WBCs (leukocytes, which include granulocytes, lymphocytes, and monocytes), and platelets (thrombocytes); the top fraction is blood plasma ( Fig. 18-1 ). The RBCs having the highest density are at the bottom of the tube, whereas most of the WBCs and platelets form a whitish gray layer—the buffy coat —between the RBCs and plasma. Only a small amount of WBCs, platelets, and plasma is trapped in the bottom column of RBCs.

The hematocrit (see p. 102 ) is the fraction of the total column occupied by RBCs. The normal hematocrit is ~40% for adult women and ~45% for adult men. The hematocrit in the newborn is ~55% and falls to ~35% at 2 months of age, from which time it rises during development to reach adult values at puberty. The hematocrit is a measure of concentration of RBCs, not of total body red cell mass. Expansion of plasma volume in a pregnant woman reduces the hematocrit, whereas her total red cell volume also increases but less than plasma volume (see p. 1142 ). Immediately after hemorrhage, the hematocrit may be normal despite the loss of blood volume (see pp. 585–586 ). Total RBC volume is ~28 mL/kg body weight in the adult woman and ~36 mL/kg body weight in the adult man.

Plasma is a pale-white watery solution of electrolytes, plasma proteins, carbohydrates, and lipids. Pink-colored plasma suggests the presence of hemoglobin caused by hemolysis (lysis of RBCs) and release of hemoglobin into the plasma. A brown-green color may reflect elevated bilirubin levels (see Box 46-1 ). Plasma can also be cloudy in cryoglobulinemias (see pp. 438–439 ). The electrolyte composition of plasma differs only slightly from that of interstitial fluid on account of the volume occupied by proteins and their electrical charge (see Table 5-2 ).

Plasma proteins at a normal concentration of ~7.0 g/dL account for a colloid osmotic pressure or oncotic pressure of ~25 mm Hg (see p. 470 ). Principal plasma proteins are albumin, fibrinogen, globulins, and other coagulation factors. The molecular weights of plasma proteins range up to 970 kDa ( Table 18-1 ). The plasma concentration of albumin ranges from 3.5 to 5.5 g/dL, which provides the body with a total plasma albumin pool of ~135 g. Albumin is synthesized by the liver at a rate of ~120 mg/kg body weight per day and, due to catabolism, has a half-life in the circulation of ~20 days. Urinary losses of albumin are normally negligible (<20 mg/day; see p. 470 ). Plasma concentration of albumin is typically decreased in hepatic cirrhosis (see p. 569 ). Hepatic synthesis of albumin is strongly enhanced by a low plasma colloid osmotic pressure.

Many plasma proteins are involved in blood coagulation through coagulation cascades, the end point of which is the cleavage of fibrinogen into fibrin monomers that further assemble into a fibrin polymer. The fibrinogen molecule is a dimer of identical heterotrimers, each composed of Aα-, Bβ-, and γ chains. Fibrinogen is synthesized only by the liver (see Table 46-3 ) and circulates in plasma at concentrations of 150 to 300 mg/dL. The acute-phase response ( Box 18-1 ) greatly enhances fibrinogen synthesis. During clotting, the cross-linked polymers of fibrin form strands that trap red and white cells, platelets, and plasma inside the thrombus (i.e., blood clot). Subsequent interaction of myosin and actin in the platelets of the clot allows the clot to shrink to a plug that expels a slightly yellow-tinged fluid. This residual fluid is serum, which differs principally from plasma by the absence of fibrinogen and other coagulation factor. However, serum still contains albumin, antibodies, and other proteins. Note that plasma can also form a clot, but a plasma clot does not retract because it lacks platelets.

Subtraction of the albumin and fibrinogen moiety from total protein concentration yields the concentration of all the proteins grouped as globulins. Electrophoresis can be used to fractionate plasma proteins. The electrophoretic mobility of a protein depends on its molecular weight (size and shape) as well as its electrical charge. Plasma proteins comprise the following in decreasing order of electrophoretic mobility ( Fig. 18-2 A ): albumin, α ₁ -globulins, α ₂ -globulins, β-globulins, fibrinogen, and γ-globulins. The three most abundant peaks are albumin, fibrinogen, and γ-globulins. The γ-globulins include the immunoglobulins or antibodies, which can be separated into IgA, IgD, IgE, IgG, and IgM. Immunoglobulins are synthesized by B lymphocytes and plasma cells.

Clinical laboratories most often perform electrophoresis of blood proteins on serum instead of plasma (see Fig. 18-2 B ). Table 18-1 shows the major protein components that are readily resolved by electrophoresis. Proteins present in plasma at low concentrations are identified by immunological techniques, such as radioimmunoassay (see p. 976 ) or enzyme-linked immunosorbent assay. Not listed in Table 18-1 are several important carrier proteins present in plasma: ceruloplasmin (see p. 970 ), transcobalamin (see p. 937 ), corticosteroid-binding globulin (CBG; see p. 1021 ), insulin-like growth factor (IGF)–binding proteins (see p. 996 ), sex hormone–binding globulin (SHBG or TeBG; see pp. 1119–1120 ), thyroid-binding globulin (see pp. 1008–1009 ), and vitamin D–binding protein (see p. 1064 ). The liver synthesizes most of the globulins and coagulation factors. N18-1

Bone marrow is the source of most blood cells

If you spread a drop of anticoagulated blood thinly on a glass slide, you can detect under the microscope the cellular elements of blood. In such a peripheral blood smear, the following mature cell types are easily recognized: erythrocytes; granulocytes divided in neutrophils, eosinophils, and basophils; lymphocytes; monocytes; and platelets ( Fig. 18-3 ).

Hematopoiesis is the process of generation of all the cell types present in blood. Because of the diversity of cell types generated, hematopoiesis serves multiple roles ranging from the carriage of gases to immune responses and hemostasis. Pluripotent long-term hematopoietic stem cells (LT-HSCs) constitute a population of adult stem cells found in bone marrow that are multipotent and able to self-renew. The short-term hematopoietic stem cells (ST-HSCs) give rise to committed stem cells or progenitors, which after proliferation are able to differentiate into lineages that in turn give rise to burst-forming units (BFUs) or colony-forming units (CFUs), each of which ultimately will produce one or a limited number of mature cell types: erythrocytes, the megakaryocytes that give rise to platelets, eosinophils, basophils, neutrophils, monocytes-macrophages/dendritic cells, and B or T lymphocytes and natural killer cells ( Fig. 18-4 ). Soluble factors known as cytokines guide the development of each lineage. The research of Donald Metcalf demonstrated the importance of a family of hematopoietic cytokines that stimulate colony formation by progenitor cells, the colony-stimulating factors. The main colony-stimulating factors are granulocyte-macrophage colony-stimulating factor (GM-CSF; see p. 70 ), granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), interleukin-3 (IL-3) and IL-5 (see p. 70 ), thrombopoietin (TPO), and erythropoietin (EPO; see pp. 431–433 ).

GM-CSF is a glycoprotein that stimulates proliferation of a common myeloid progenitor and promotes the production of neutrophils, eosinophils, and monocytes-macrophages. Recombinant GM-CSF (sargramostim [Leukine]) is used clinically after bone marrow transplantation and in certain acute leukemias.

G-CSF and M-CSF are glycoproteins that guide the ultimate development of granulocytes and monocytes-macrophages/dendritic cells, respectively. Recombinant G-CSF (filgrastim [Neupogen]) is used therapeutically in neutropenia (e.g., after chemotherapy). M-CSF is also required for osteoclast development (see p. 1057 and Fig. 52-4 ).

IL-3 (also known as multi-CSF) has a broad effect on multiple lineages. The liver and the kidney constitutively produce this glycoprotein. IL-5 (colony-stimulating factor, eosinophil), a homodimeric glycoprotein, sustains the terminal differentiation of eosinophilic precursors.

TPO binds to a TPO receptor called c-Mpl, which is the cellular homolog of the viral oncogene v- mpl (murine myeloproliferative leukemia virus). On stimulation by TPO, the Mpl receptor induces an increase in the number and size of megakaryocytes—the cells that produce platelets—which thereby greatly augments the number of circulating platelets.

EPO, N18-2 which is homologous to TPO, is produced by the kidney and to a lesser extent by the liver. This cytokine supports erythropoiesis or red cell development ( Fig. 18-5 ). As described on pages 93–94 , hypoxia increases the abundance of the α subunit of hypoxia-inducible factor 1 (HIF-1α), which enhances production of EPO messenger RNA. Although EPO is not absolutely required for early commitment of progenitor cells to the erythroid lineage, it is essential for the differentiation of burst-forming unit–erythroid cells (BFU-Es) to colony-forming unit–erythroid cells (CFU-Es) or proerythroblasts (also known as pronormoblasts), which still lack hemoglobin. The further maturation of cells downstream of proerythroblasts does not require EPO. Recombinant EPO has proved effective in the treatment of anemia. Hemoglobin first appears at the stage of polychromatic erythroblasts and is clearly evident in orthochromatic erythroblasts. The subsequent exocytosis of the nucleus produces reticulocytes (see Fig. 18-5 ), whereas the loss of ribosomes and mitochondria yields mature erythrocytes, which enter the circulation. The mature erythrocyte has a life span of ~120 days. Immature reticulocytes may also appear in the circulation when erythropoiesis is heavily activated.

RBCs are mainly composed of hemoglobin

As evidenced by the magnitude of the hematocrit, RBCs are the most abundant elements in blood. RBCs are non-nucleated biconcave cells with a diameter of ~7.5 µm and a volume of ~90 fL (90 × 10 ⁻¹⁵ L). Maintaining the shape of the RBC is a cytoskeleton that is anchored to the plasma membrane by glycophorin and the “band 3” Cl-HCO ₃ exchanger (see Fig. 2-9 ). The distinctive shape of the RBC provides a much larger surface-to-volume ratio than that of a spherical cell; this maximizes diffusion area and minimizes intracellular diffusion distances for gas exchange. The RBC performs three major tasks: (1) carrying O ₂ from the lungs to the systemic tissues, (2) carrying CO ₂ from tissues to the lungs, and (3) assisting in the buffering of acids and bases.

Table 18-2 lists the properties of RBCs that are routinely determined in the clinical laboratory. The most important constituent of the RBC is hemoglobin. Globin synthesis begins in the proerythroblast (see Fig. 18-5 ). By the end of the orthochromatic-erythroblast stage, the cell has synthesized all the hemoglobin it will carry. Normal blood hemoglobin content is ~14.0 g/dL in the adult female and ~15.5 g/dL in the adult male. The hemoglobin concentration in red cell cytosol is extremely high, ~5.5 mM. The mean cell hemoglobin concentration is ~35 g/dL RBCs, or about five times the concentration of proteins in plasma. Enclosure of hemoglobin in red cells has the advantage of minimizing the loss of hemoglobin from the plasma through filtration across the blood capillary walls. The structure and various chemical forms of hemoglobin, as well as the carriage of O ₂ and CO ₂ by hemoglobin, are discussed in Chapter 29 .

Because the mature RBCs contain no nucleus or other organelles, they can neither synthesize proteins nor engage in oxidative metabolism. The RBC can engage in two metabolic pathways: glycolysis, which consumes 90% of glucose uptake; and the pentose shunt (see Fig. 58-1 ), which consumes the remaining 10% of glucose. The cell generates its ATP exclusively by glycolysis. An important constituent of the RBC is 2,3-diphosphoglycerate (2,3-DPG). RBCs use DPG mutase to convert 1,3-diphosphoglycerate (1,3-DPG), part of the normal glycolytic pathway (see Fig. 58-6 A ), into 2,3-DPG. In RBCs, the cytosolic concentration of 2,3-DPG is normally 4 to 5 mM, about the same as the concentration of hemoglobin. 2,3-DPG acts on hemoglobin by reducing the O ₂ affinity of hemoglobin (see pp. 654–655 ).

Erythrocytes contain glutathione at ~2 mM, more than any other cell of the body outside the hepatocyte (see p. 955 ). A high ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) protects the RBC against oxidant damage. Glutathione reductase regenerates GSH from GSSG in a reaction that consumes reduced nicotinamide adenine dinucleotide phosphate (NADPH). The RBC generates all its NADPH from the glycolytic intermediate glucose-6-phosphate through the pentose-shunt pathway (see Fig. 58-1 ).

RBCs carry two cytoplasmic isoforms of carbonic anhydrase (see p. 656 ), N18-3 CA I and CA II. These enzymes, which rapidly interconvert CO ₂ and , play a critical role in carrying metabolically produced CO ₂ from the systemic tissues to the pulmonary capillaries for elimination in the exhaled air (see pp. 655–657 ). CA II has one of the fastest known enzymatic turnover rates.

N18-3

Carbonic Anhydrases

Overview

The carbonic anhydrases (CAs) are a family of zinc-containing enzymes with at least 16 members among mammals; eTable 18-1 lists some of these isoforms. Physiologically, the CAs catalyze the interconversion of CO ₂ and although they also can cleave aliphatic and aromatic ester linkages. CA I is present mainly in the cytoplasm of erythrocytes. CA II is a ubiquitous cytoplasmic enzyme. CA IV is a GPI-linked enzyme (see p. 13 ) found, for example, on the outer surface of the apical membrane of the renal proximal tubule (see pp. 828–829 ). A hallmark of many CAs is their inhibition by sulfonamides (e.g., acetazolamide).

Reaction Catalyzed by CAs

Before we consider the action of CA, it is instructive to examine the interconversion of CO ₂ and in the absence of enzyme. When [CO ₂ ] increases,

${\text{CO}}_2+H_{2}O\underrightarrow{\text{slow}}{\text{H}}_2{\text{CO}}_3\underrightarrow{\text{fast}}{H}^{+}+{\text{HCO}}_3^{-}$

CO ₂ can also form by directly combining with OH ⁻ , a reaction that becomes important at high pH values, when [OH ⁻ ] is also high:

Because the dissociation of H ₂ O replenishes the consumed OH ⁻ , the two mechanistically distinct pathways for formation are functionally equivalent. Of course, both reaction sequences are reversible. However, in the absence of CA, the overall speed of the interconversion between CO ₂ and is slow at physiological pH. In fact, it is possible to exploit this slowness experimentally to generate solutions that are temporarily “out of equilibrium” N28-4 . Unlike normal (i.e., equilibrated) solutions, such out-of-equilibrium solutions can have virtually any combination of [CO ₂ ], [ ], and pH in the physiological range.

Structural biologists have solved the crystal structures of several CAs. At the reaction site, three histidines coordinate a zinc atom that, along with a threonine, plays a critical role in binding CO ₂ and . In CA II, the fastest of the CAs, a fourth histidine acts as a proton acceptor/donor. Extensive site-directed mutagenesis studies have provided considerable insight into the mechanism of the CA reaction. The CAs have the effect of catalyzing the slow CO ₂ hydration in Equation NE 18-1 . Actually, these enzymes catalyze the top reaction in Equation NE 18-2 , the direct combination of CO ₂ with OH ⁻ to form . CA II catalyzes both reactions in Equation NE 18-2 :

$\begin{array}{c}\left(\text{His}\right)\text{Enzyme}-\text{Zn}-{\text{OH}}^{-}+{\text{CO}}_2\rightleftarrows \left(\text{His}\right)\text{Enzyme}-\text{Zn}-{\text{HCO}}_3^{-}\\ \left(\text{His}\right)\text{Enzyme}-\text{Zn}-{\text{HCO}}_3^{-}+H_{2}O\rightleftarrows \left(\text{His}\right)\text{Enzyme}-\text{Zn}-H_{2}O+{\text{HCO}}_3^{-}\\ \left(\text{His}\right)\text{Enzyme}-\text{Zn}-H_{2}O\rightleftarrows (H^{+}-\text{His})\text{Enzyme}-\text{Zn}-{\text{OH}}^{-}\\ (H^{+}-\text{His})\text{Enzyme}-\text{Zn}-{\text{OH}}^{-}\rightleftarrows \left(\text{His}\right)\text{Enzyme}-\text{Zn}-{\text{OH}}^{-}+H^{+}\end{array}$

CA II has one of the highest turnover numbers of any known enzyme: Each second, one CA II molecule can convert >1 million CO ₂ molecules to ions. In the erythrocyte, this rapid reaction is important for the carriage of CO ₂ from the peripheral blood vessels to the lungs (see pp. 655–657 ). In the average cell, CAs are important for allowing the rapid buffering of H ⁺ by the buffer pair.

Another role of CAs may be in minimizing the pH changes that occur on membrane surfaces that contain transporters that move H ⁺ or . For example, the extrusion of H ⁺ from the cell by an Na-H exchanger would generate a low pH at the outer surface of the cell. CA IV, with its extracellular catalytic domain, would consume much of the extruded H ⁺

$H^{+}+{\text{HCO}}_3^{-}\to {\text{CO}}_2+H_{2}O$

and thereby minimize the fall in surface pH.

Preliminary data suggest that the electrogenic Na/HCO ₃ cotransporters (NBCe1 and NBCe2; see p. 122 ), and perhaps also the other Na ⁺ -coupled “ ” transporters (the electroneutral NBCs NBCn1 and NBCn2, the Na ⁺ -driven Cl-HCO ₃ exchanger NDCBE; see p. 124 ), actually move . As enters the cell, the following reaction would not only replenish the but would also generate H ⁺ on the outer surface of the cell:

${\text{HCO}}_3^{-}\to H^{+}+{\text{CO}}_3^{2-}$

The consequence would be a buildup of H ⁺ on the extracellular surface, just as in the case of an Na-H exchanger. An extracellular CA like CA IV would consume this newly generated H ⁺ :

$H^{+}+{\text{HCO}}_3^{-}\to {\text{CO}}_2+H_{2}O$

The net reaction (summing Equation NE 18-5 and Equation NE 18-6 ) would be as follows:

$2{\text{HCO}}_3^{-}\to {\text{CO}}_3^{2-}+{\text{CO}}_2+H_{2}O$

Thus, when a transporter like NBCe1 appears to transport 2 into the cell, what is really happening is that 1 is being transported by the NBCe1, and 1 CO ₂ and 1 H ₂ O are entering the cell by another mechanism. CA enzymes on both the extra- and intracellular surfaces of the cell minimize the pH changes (by consuming or generating H ⁺ , as necessary) generated by NBCs and H ⁺ transporters.

CA Deficiency in Humans

For a discussion of genetic deficiencies of CAs in humans, see Box 39-1 . The homozygous absence of normal CA II causes CA II deficiency syndrome, characterized by osteopetrosis, renal tubular acidosis, and cerebral calcification. At least seven different mutations can cause genetic defects. The mutation that is common in patients of Arabic descent causes mental retardation but less severe osteopetrosis. Other patients may carry two different mutations. Indeed, the first three patients described with CA II deficiency syndrome, sisters in the same family, were compound heterozygotes, having received one mutation from their mother and a second from their father.

Although CA I deficiency exists, the homozygous condition has no obvious consequences because CA I and CA II normally contribute about equally to the CA activity in RBCs.

Nontraditional CAs

Three soluble CAs—VIII, X, and XI—are unusual in that they lack one or more of the three homologous histidine residues that enzymatically active CAs use to coordinate Zn ²⁺ . In addition, two receptor tyrosine phosphatases—RPTPβ and RPTPγ—have nontraditional CA domains at the location of the ligand-binding site. It is likely that these RPTPs are CO ₂ and/or sensors.

eTABLE 18-1

Some Human CAs *

ISOFORM	MOLECULAR MASS (kDa)	CELLULAR LOCATION	TISSUE DISTRIBUTION	RELATIVE ACTIVITY	SENSITIVITY TO SULFONAMIDES (e.g., ACZ)
I	29	Cytosol	RBCs and GI tract	15%	High
II	29	Cytosol	Nearly ubiquitous	100%	High
III	29	Cytosol	8% of soluble protein in slow-twitch (type I) muscle	1%	Low
IV	35	Extracellular surface of membrane (GPI linked)	Widely distributed, including acid-transporting epithelia	~100%	Moderate
VA, VB	35, 36	Mitochondria (soluble)	Mitochondria	~20%, ~70%	Moderate
VI	39-42	Secreted	Saliva, milk	~25%	High
VII	29	Cytosol	Cytosol	~70%	Very high
IX	54/58	Catalytic domain on extracellular surface †	Certain cancers	~100%	High
XII	44	Catalytic domain on extracellular surface †	Certain cancers	~30%	(Binds ACZ with unknown affinity)
XIII	29	Cytosol	Widely distributed	~10%	High
XIV	54	Catalytic domain on extracellular surface †	Kidney, heart, skeletal muscle, brain, retinal pigment epithelium, certain cancers	~100%	Moderate

ACZ, acetazolamide; GI, gastrointestinal.

* Several additional CAs have been cloned. In some cases they have not been functionally characterized.

† Integral membrane protein with one membrane-spanning segment.

References

• Purkerson JM, Schwartz GJ: The role of carbonic anhydrases in renal physiology. Kidney Int 2007; 71: pp. 103-115.
• Sly WS, Hu PY: Human carbonic anhydrases and carbonic anhydrase deficiency. Annu Rev Biochem 1995; 64: pp. 375-401.
• Wykoff CC, Beasley N, Watson PH, et. al.: Expression of the hypoxia-inducible and tumor-associated carbonic anhydrases in ductal carcinoma in situ of the breast. Am J Pathol 2001; 158: pp. 1011-1019.
• https://en.wikipedia.org/wiki/Carbonic_anhydrase (last accessed August 25, 2015).

CO ₂ carriage also depends critically on the Cl-HCO ₃ exchanger AE1 (see pp. 124–125 and 656 ) in the RBC membrane. The transporter was originally known as band 3 protein because of its position on a sodium dodecyl sulfate–polyacrylamide gel of RBC membrane proteins. AE1 is the most abundant membrane protein in RBCs, with ~1 million copies per cell. One AE1 molecule can transport as many as 50,000 ions per second—it is one of the fastest known transporters. AE1 and most other members of the SLC4 family of HCO ₃ transporters are blocked by a disulfonic stilbene known as DIDS.

The water channel AQP1 (see p. 110 ) is the second most abundant membrane protein in RBCs, with ~200,000 copies per cell. AQP1 appears to contribute more than half of the CO ₂ permeability of the RBC membrane (see pp. 655–657 ).

Leukocytes defend against infections

Table 18-3 summarizes the relative abundance of various leukocytes in the blood. These WBCs are in two major groups: the granulocytes, on the one hand, and the lymphocytes and monocytes, on the other. Granulocytes are so named because of their cytoplasmic granules, which on a blood smear stained with Giemsa stain or Wright stain appear red (eosinophils), blue (basophils), or intermediate (neutrophils). The nuclear material is irregularly shaped in the form of the letter S or Z. The name polymorphonuclear leukocytes applies to all three types of granulocytes, but often is used to refer specifically to neutrophils. The average diameter of a neutrophil is ~12 µm, smaller than that of a monocyte (14 to 20 µm) or eosinophil (~13 µm), somewhat larger than that of a basophil (~11 µm), and much larger than that of a lymphocyte (6 to 10 µm). Granulocytes have a brief life span in the blood (<12 hours), but on activation can migrate into the tissues.