CHAPTER 110 MEGAKARYOPOIESIS AND THROMBOPOIESIS
CHAPTER 110 MEGAKARYOPOIESIS AND THROMBOPOIESIS
DAVID J. KUTER
Anatomy of Megakaryocytes
Megakaryocyte Surface Membrane
Light Microscopic Appearance of Megakaryocytes
The Origin of Megakaryocytes
Early Megakaryocyte Progenitors
Late Megakaryocyte Progenitors
Effects of Thrombopoietic Growth Factors
Platelet Production and Release
The Formation of Platelets from Megakaryocytes
Regulation of Platelet Size
The Molecular Biology of Megakaryocyte Differentiation
The Regulation of Megakaryocytopoiesis and Thrombopoiesis
The Role of Thrombopoietin in Normal Physiology
Megakaryocytes in Disease
Megakaryocyte Hypoplasia Secondary to Chemotherapy
Thrombocytopenia Associated with Human Immunodeficiency Virus (HIV)
Idiopathic Thrombocytopenic Purpura
Hematopoietic Disorders Associated with Abnormal Chromosome 3Q
Agnogenic Myelofibrosis with Myeloid Metaplasia
Disorders of Thrombopoietin Production
Recombinant Thrombopoietic Growth Factors and their Clinical Uses
Other Thrombopoietic Growth Factors
Large, polyploid, bone marrow megakaryocytes shed enucleate platelets into the circulation. Most of the proteins and granules that determine the characteristics of these platelets are made in the megakaryocytes. In response to an increased need for platelets, megakaryocytes increase their number, size, and ploidy; the opposite effects occur when the demand decreases. The growth of megakaryocytes and the production of platelets are regulated almost entirely by the amount of thrombopoietin in the circulation. Upon binding to the thrombopoietin receptor, c-Mpl, thrombopoietin increases the growth of early marrow precursors of all lineages but only stimulates the maturation of late precursors of the megakaryocyte lineage and thereby increases only platelet production. Thrombopoietin is produced at a constant rate in the liver, and its level is determined primarily by the rate of clearance by c-Mpl receptors on platelets and possibly megakaryocytes. Characteristic changes in megakaryocytes occur in a wide variety of disorders of abnormal platelet production. Recombinant thrombopoietins as well as other hematopoietic growth factors such as interleukin-11 can increase platelet production and may become clinically useful.
Acronyms and abbreviations that appear in this chapter include: 5–FU, 5–fluorouracil; CHO, Chinese hamster ovary; GEMM-CFC, or mix-CFC, granulocyte-erythroid-macrophage-megakaryocyte colony-forming cells; HIV, human immunodeficiency virus; ITP, idiopathic thrombocytopenic purpura; MGDF, megakaryocyte growth and development factor; meg-BFC, megakaryocytic burst-forming cell; meg-CFC, megakaryocyte colony-forming cell; MPV, mean platelet volume; PEG-rHuMGDF, pegylated, recombinant megakaryocyte growth and development factor; PF4, platelet factor 4; rHuTPO, recombinant human thrombopoietin; vWf, von Willebrand factor.
The derivation of blood platelets from megakaryocytes has been known since 1906,1 but the process of platelet production (termed thrombopoiesis or thrombocytopoiesis) remains enigmatic. Megakaryocytes are very large, polyploid bone marrow cells whose low prevalence has made their investigation difficult. During the past two decades, in vitro cloning of megakaryocytic progenitors, production of monoclonal antibodies that specifically identify megakaryocytes and their constituents, and new molecular techniques have advanced our knowledge of megakaryocyte biology (called megakaryopoiesis or megakaryocytopoiesis). With the purification of thrombopoietin2,3,4,5,6 and 7 great strides have been made in understanding the physiology of platelet production from megakaryocytes.
ANATOMY OF MEGAKARYOCYTES
In lower vertebrate species such as fish and birds, all the circulating blood cells, including the erythrocytes and the platelets (called thrombocytes), are nucleated and are produced by diploid bone marrow precursor cells.8 However, in higher vertebrates platelets are produced by a different mechanism whose evolutionary advantage is unclear. Anucleate platelets9 are generated from unusual bone marrow cells, the megakaryocytes. Megakaryocytes normally account for approximately 0.05 to 0.1 percent of all nucleated human bone marrow cells, but their number increases as the demand for platelets rises. In contrast to the erythrocyte with a diameter of 5µm and a volume of 85 to 100 fl, megakaryocytes have average diameters of 20 to 25 µm and volumes of 4700 ± 100 fl.10 Some of the largest megakaryocytes may have diameters of 50 to 60 µm and volumes of 65,000 to 100,000 fl. These unique cells are polyploid and contain platelet-specific granules and proteins.
Mature megakaryocytes are invariably polyploid and contain from two (4N) to 32 (64N) times the normal diploid amount of DNA.11,12 Human megakaryocytes have a mean ploidy of 16N. Unlike the small percentage of hepatocytes and macrophages that have two- to four-fold the normal diploid content of DNA and whose DNA is contained in multiple separate nuclei, all of the DNA in megakaryocytes is contained within one highly lobulated nuclear envelope where each nuclear lobule represents one diploid amount (2N) of DNA. In general there is a relationship between increased ploidy and increased megakaryocyte size, but given the time needed for the cytoplasm of megakaryocytes to mature, not all small megakaryocytes are of low ploidy. In fetal life megakaryocytes are less polyploid; cultured mature megakaryocytes from fetal liver at 8 to 10 weeks of gestation are only 2N and 4N, while 8N megakaryocytes are detected at 20 weeks of gestation.13
At some ill-defined point in the megakaryocytic differentiation pathway, mitosis ceases and the unusual process of endomitosis (also called polyploidization or endoreduplication) commences.14 Endomitosis is a process in which DNA replication occurs but neither the nucleus nor the cell undergoes division (cytokinesis). Morphologically, endomitosis is associated with the dissolution of the nuclear membrane and the formation of a multipolar mitotic spindle.15 While initially it was assumed that endomitosis was simply the absence of mitosis after each round of DNA replication, studies in mice16 showed that megakaryocytes indeed enter mitosis and progress through normal prophase, prometaphase, metaphase, and up to anaphase A, but not to anaphase B, telophase, or cytokinesis. After anaphase, the nuclear membrane is reassembled about the sister chromatids as a single nucleus skipping telophase and cytokinesis, and the cells enter the next round of DNA replication.
Cessation of mitosis in the diploid megakaryocyte progenitors is apparently directly coupled to the start of endoreduplication, and it is generally assumed that most cells greater than 4N in ploidy are committed to endomitosis and rarely divide mitotically. The probability of entering the endomitotic pathway is hierarchically dependent upon the state of differentiation of the progenitor.17 Thus, the more primitive the progenitor is, the more likely it is to remain a mitotic cell. Genes with known roles in regulating the cell cycle such as the cyclin-dependent kinases and the cyclins have been studied. Although results are incomplete, during endomitosis cyclin D3 is increased,18 and the levels of cyclin B1 and cyclin B1-dependent Cdc2 kinase are reduced.19 These events may allow the megakaryocyte to abort some aspects of mitosis and reenter a phase of DNA replication without cytokinesis.20
Since platelets can produce only very small amounts of protein, their cytoplasmic characteristics are mostly determined by the megakaryocytes from which they come. Four distinct categories of granules differing in their internal constituents are produced by maturing megakaryocytes: alpha (a), dense, lysosomal, and microperoxisomal (Fig. 110-1a–e).
FIGURE 110-1 (a) Ultrastructure of the cytoplasm of a mature megakaryocyte. The majority of the granules are a granules (a-Gr) exhibiting dense nucleoid. Demarcation membranes (DM) are slightly dilated. Transverse sections of microtubules (Mt) are dispersed; at the periphery, a longitudinal microtubule runs under the cell membrane (arrows). Dense aggregates of glycogen (Gly), small cisternae of endoplasmic reticulum (ER), and free ribosomes can be recognized. ×30,320. (b) Morphology of an a granule. Dense nucleoid is located at the top; in a clear zone at the opposite pole, four transverse sections of tubular structures are seen adjacent to the granule membrane. ×37,200. (c) A dense body can be distinguished from an a granule by the black deposit when calcium is added to the fixative. ×37,200. (d) Cytochemical detection of acid phosphatase using b-glycophosphate as substrate and cerium as a trapping agent. Dense cerium-phosphate precipitates are present in lysosomal granules while a granules are unreactive. ×37,200. (e) Microperoxisome visualized using alkaline diaminobenzidine. Note the small size of a reactive granule compared to an a granule. ×37,200. (f) Distribution of a dense tracer filling the lumen of the demarcation membrane system in a maturing megakaryocyte. This system appears to outline some platelet territories (arrows). In contrast to the demarcation membrane system, which is open to the extracellular space, the endoplasmic reticulum (ER) is not labeled. ×9700. (Courtesy of Dr. Janine Breton-Gorius.)
The a granules (which are azurophilic on stained smears) are the most numerous granules seen with the electron microscope.21 The granule body itself is made early in megakaryocyte development before the demarcation membrane system and first appears in the Golgi apparatus of megakaryoblasts (stage I megakaryocytes).22 Several platelet proteins, including von Willebrand factor (vWf), platelet factor 4 (PF4), and thrombospondin have been detected in early megakaryocyte progenitors prior to the appearance of a granules.23 Figure 110-1b shows that a granules exhibit distinct zones: a diffuse granular matrix, a dense nucleoid, and a third electron-lucent compartment containing from one to six tubular structures close to the membrane.24 Immunoelectron microscopy has shown that several of the a granule proteins are compartmentalized; for example, vWf is associated with tubular structures similar to those of the vWf storage organelles of the vascular endothelium (Weibel-Palade bodies). Thrombospondin and fibrinogen are localized in the matrix,25,26 and 27 and b-thromboglobulin (b-TG) and PF4, together with mucopolysaccharides, are present in the dense nucleoid.28,29 and 30
Although some of the a granule proteins such as platelet-derived growth factor, transforming growth factor-b, PF4, and vWf are synthesized in the megakaryocyte and transported to the a granules,31 other proteins such as fibrinogen undergo GPIIb/IIIa receptor-mediated endocytosis from the plasma into the a granules of both megakaryocytes and platelets.32,33,34 and 35 Still others, such as albumin and IgG are pinocytosed from the plasma into the a granules of megakaryocytes and platelets.36 The location of megakaryocytes in close proximity to vascular sinuses may facilitate uptake of these circulating proteins.37
Several proteins have been detected on the membrane of a granules: GPIIb/IIIa,38 P-selectin (CD62P; GMP-140 or PADGEM),39 GMP-33,40 and platelet osteonectin.41
Dense granules constitute a class of granules distinguishable from a granules by their morphology (Fig. 110-1c) and their content: a nonmetabolic pool of adenine nucleotides synthesized by megakaryocytes, and calcium and serotonin.42 Dense granules of platelets are physically dense, and because of their content of serotonin and calcium, they are also electron-dense when viewed with the transmission electron microscope. The dense granule membrane bodies are made in megakaryocytes but do not acquire their content of serotonin and calcium until platelets are released into the circulation and then uptake calcium and most of the body’s circulating serotonin.43,44 This ability to accumulate and store serotonin constitutes an early marker of megakaryocytes, since even the earliest megakaryocyte progenitors are able to incorporate exogenous serotonin.44,45 The membrane of dense bodies expresses granulophysin46 and P-selectin.47
Lysosomal granules are a unique class of granules as judged by ultrastructural cytochemical localization of arylsulfatase and acid phosphatase (Fig. 110-1d), the immunolocalization of cathepsin D, and the identification of lysosome-associated membrane protein.48,49 and 50 They are formed very early during maturation, prior to the appearance of a granules.
Small granules (90 nm) that arise prior to a granule formation have been shown to contain catalase.51 These granules appear similar to the microperoxisomes of other cells (Fig. 110-1e).
MEGAKARYOCYTE SURFACE MEMBRANE
DEMARCATION MEMBRANE SYSTEM
The surface membrane of the mature megakaryocyte is deeply invaginated and highly redundant (Fig. 110-1a). It divides the cytoplasm into platelet-sized “territories” and has thus been termed the demarcation membrane system.52,53 Since the lumen of the demarcation membrane system stains with extracellular tracers (Fig. 110-1f), it is continuous with the extracellular medium.52,54 The total area of the surface and demarcation membranes may increase by 26-fold during a 72-h maturation period.52 This enormous amplification of the surface membrane is required to form platelets.52,55,56
Both the megakaryocyte cell membrane and the demarcation membrane system express GPIIb/IIIa and GPIb/IX complexes; however, these platelet glycoproteins appear first on the plasma membrane prior to the formation of the demarcation membrane system.57 One of the first signs of differentiation along the megakaryocyte lineage is the appearance on the surface of CD34+ progenitors of the platelet GPIIb/IIIa receptor,58 and its expression increases in parallel with the decline of CD34 expression during maturation.59 GPIIb has been considered to be specific for the megakaryocyte-platelet lineage.60 This is later followed by the appearance of the GPIb/IX complex57 and the collagen receptors. Expression of GPIb/IX, a receptor for vWf,61 correlates with the onset of ploidization.59 This complex has been thought to be platelet-specific, although cytokine-activated endothelial cells may also express a form of GPIb.62,63 and 64
LIGHT MICROSCOPIC APPEARANCE OF MEGAKARYOCYTES
The large, mature, polyploid megakaryocytes are readily identified by light microscopy (see Plate XXX) and are often located adjacent to bone marrow sinusoids.37 Occasionally they may actually be seen to be shedding platelets.1,65 In addition to the mature megakaryocytes, several other earlier maturational stages of megakaryocytes may be identified66 using a classification scheme based on the nuclear/cytoplasmic ratio, nuclear shape, basophilia, and granularity (Table 110-1).67 These early megakaryocytes are often distant from the sinusoid.
TABLE 110-1 MATURATION STAGES OF MEGAKARYOCYTES
Also present in the bone marrow are small, diploid megakaryocyte progenitor cells called promegakaryoblasts that are transitional cells committed to the process of endomitosis but not yet polyploid.68,69,70,71 and 72 In humans these cells have the size and morphology of lymphocytes and express GPIIb on their membrane and vWf or PF4 in their cytoplasm.23,73,74 This population is equivalent to the small acetylcholinesterase-positive marrow cells in rodents.69 (In rodents and cats, acetylcholinesterase is a specific marker for platelets and megakaryocytes69; in humans acetylcholinesterase appears to be present in immature, but not mature megakaryocytes.75) Some of these cells may retain proliferative capacity,72 but the majority of these transitional cells do not form colonies in culture. They are, however, capable of maturing into typical polyploid megakaryocytes71,76 and constitute 4 percent of all megakaryocytes.77,78 and 79 Ultrastructurally, human promegakaryoblasts are recognizable by the cytochemical demonstration of platelet peroxidase, which is present exclusively in the endoplasmic reticulum (Fig. 110-2).54 This enzyme, which is present throughout megakaryocytic maturation and is detectable in platelets, is involved in prostaglandin synthesis.80 Although several other types of marrow cells capable of transient prostaglandin synthesis exhibit a similar peroxidase activity, nonmegakaryocytes possess characteristics that distinguish them from promegakaryoblasts.81,82 Simultaneous analysis of membrane GPIIb and platelet peroxidase has shown that the latter constitutes the first marker of megakaryocyte maturation.83
FIGURE 110-2 (a) Electron micrograph of a normal human marrow promegakaryocyte treated for the detection of platelet peroxidase. This small cell (<9 µm) exhibits a dense reaction product, demonstrating the presence of platelet peroxidase in the perinuclear space and in the endoplasmic reticulum (arrows). ×12,150. (Inset) Enlargement of the Golgi zone. The Golgi saccules and vesicles are devoid of platelet peroxidase (open arrows), while the endoplasmic reticulum contains platelet peroxidase activity (closed arrow). ×25,000. (b) Maturing megakaryocyte in the marrow. The large megakaryocyte is located close to the sinus endothelium, and a small cytoplasmic bleb (arrow) is in direct contact with the blood. A neutrophil (N) is seen in the sinus. ×3600. (Courtesy of Dr. Janine Breton-Gorius.)
THE ORIGIN OF MEGAKARYOCYTES
Like all other bone marrow cells, megakaryocytes are derived from the pluripotential stem cell (Fig. 110-3). The cellular steps by which the stem cell becomes a megakaryocyte have been defined by in vitro colony studies and include early progenitors having the capacity to produce colonies consisting of cells of several lineages [such as granulocyte-erythroid-macrophage-megakaryocyte colony-forming cells (GEMM-CFC) or Mix-CFC] and later progenitors committed only to megakaryocyte differentiation.84,85
FIGURE 110-3 The origin and development of megakaryocytes. The pluripotential stem cell produces a progenitor cell committed to megakaryocyte differentiation (meg-CFC) which can undergo mitosis. Eventually the meg-CFC stops mitosis and enters endomitosis during which neither cytoplasm nor nucleus divides but DNA replication proceeds and gives rise to polyploid immature precursor cells. Upon completion of endomitosis, the immature progenitor cells become large, morphologically identifiable, mature megakaryocytes that shed platelets.
EARLY MEGAKARYOCYTE PROGENITORS
Although most CD34+/CD38– cells express the thrombopoietin receptor, c-Mpl, on their surface,86 few express other megakaryocyte-specific antigens. These cells ultimately give rise to multipotential progenitors such as the GEMM-CFC and probably also to progenitors committed to erythrocyte, megakaryocyte, and mast cell differentiation. There is no specific assay for this latter common progenitor cell, but there is a close relationship between early erythroid and megakaryocyte differentiation at the molecular, cellular, and pathological levels. For example, common transcription factors exist in these two lineages,87 and their regulatory hematopoietic cytokines, erythropoietin and thrombopoietin, are 50 percent similar.3 Both share the common cis-acting sequence, GATA-1,88,89 which is present in the promoter regions of many genes. In addition, in mice in which the thymidine kinase gene was expressed under the control of the GPIIb promoter, administration of gancyclovir resulted in eradication of both megakaryocyte and erythroid progenitors,90 suggesting that the GPIIb promoter was transcriptionally active in a bipotential progenitor cell91 and possibly in a totipotent progenitor cell.92
LATE MEGAKARYOCYTE PROGENITORS
At some point, the differentiation process produces precursor cells committed only to megakaryocyte differentiation. The transition between multipotent progenitors and megakaryocyte-restricted progenitors may involve the c-mpl oncogene, which is highly expressed in megakaryocyte cell lines and progenitor cells.93 Inhibition of c-mpl function in vitro has been reported to decrease megakaryocyte differentiation of primitive progenitors.94 A hierarchical classification system of megakaryocytic progenitors has been devised based on proliferative characteristics in culture, membrane antigen expression, and physical properties.95,96,97 and 98 The more immature progenitors have the greatest capacity for proliferation, a capacity that declines as the progenitor differentiates.99 The earliest definable committed progenitor, the megakaryocytic burst-forming cell (meg-BFC),95 gives rise to clusters of megakaryocytes (100 cells or more), is fully expressed at 21 days following initiation of culture, is resistant to 5-fluorouracil (5-FU) treatment, expresses the hematopoietic stem cell marker CD34, and is HLA-DR-negative.96,99
A later progenitor, the megakaryocyte colony-forming cell (meg-CFC),100,101 gives rise to colonies composed of smaller numbers of megakaryocytes, is expressed at 12 days of culture, is sensitive to 5-FU, expresses membrane GPIIb/IIIa102,103 and HLA-DR antigens,99 and undergoes mitosis.104 The CD34 antigen declines progressively with increasing maturity of the progenitors and is lost in the polyploid immature megakaryocyte.59
Eventually the meg-CFC stops mitosis and enters endomitosis in which polyploid precursors with scant cytoplasm are produced (Fig. 110-3). Upon the completion of endomitosis, immature megakaryocytes develop a mature cytoplasm, become morphologically identifiable, and eventually release platelets. Overall, it takes approximately 5 to 7 days to progress from the meg-CFC to the platelet. Morphologically identifiable megakaryocytes rarely divide.
EFFECTS OF THROMBOPOIETIC GROWTH FACTORS
The primary regulator of platelet production, thrombopoietin, has a major effect on almost all steps of megakaryocyte differentiation and maturation. It promotes the growth of meg-CFC, dramatically increases the rate of endomitosis, and stimulates megakaryocyte maturation.105,106 Increased megakaryocyte ploidy is seen at even very low concentrations of thrombopoietin and is the most sensitive indicator of the effect of thrombopoietin. However, the final stage of platelet release is not dependent on thrombopoietin and actually may be inhibited by large amounts of thrombopoietin.107 Other cytokines such as IL-3,108,109,110 and 111 IL-6,112,113,114,115,116,117,118,119 and 120 and IL-11121,122,123 and 124 can also promote meg-CFC growth and megakaryocyte maturation but lack much effect on endomitosis; they can all increase platelet production in vivo. Transforming growth factor b inhibits megakaryocyte growth and platelet production in vitro and in vivo.125,126 and 127 Interferon alpha and beta inhibit the growth of meg-CFC,128,129,130,131,132 and 133 but interferon gamma stimulates meg-CFC growth.134 Interferon alpha has been used clinically to reduce the platelet count in patients with essential thrombocythemia and other myeloproliferative disorders129,135,136; in patients with chronic hepatitis, interferon alpha produces a rapid decrease in the platelet count.137
PLATELET PRODUCTION AND RELEASE
THE FORMATION OF PLATELETS FROM MEGAKARYOCYTES
Although historically one of the first observations was the apparent shedding of platelets by megakaryocytes,1,65 the exact mechanism by which platelets are produced remains unclear. Incomplete data suggest that platelets are not shed from megakaryocytes with ploidy less than 8N and that larger megakaryocytes make more platelets than smaller ones. Each megakaryocyte produces an average of 1000 to 3000 platelets, and it has been estimated that 35,000 to 45,000 platelets per microliter of blood are produced per day.10,138,139 During times of marked platelet demand, normal production may increase sixfold.10 Three different mechanisms of platelet production from megakaryocytes have been proposed.
PLATELET TERRITORIES MODEL
The megakaryocyte cytoplasm is divided by the demarcation membrane system into future platelet “territories” (Fig. 110-1f), and the megakaryocyte then simply fractures into separate platelets.140 The platelets would be released into the marrow and then somehow pass into the sinusoids, leaving behind the bare megakaryocyte nucleus. Support for this mechanism comes primarily from electron microscopic data showing these platelet territories in megakaryocytes but not the more dynamic process of them fracturing into platelets. However, few bare megakaryocyte nuclei are seen in the bone marrow, and there is no evidence that platelets pass through the endothelial cells lining the sinusoids.
Megakaryocytes use the highly redundant demarcation membrane system to send pseudopodia out into the bone marrow sinusoids,52,55,141,142 and 143 and platelets and proplatelets bud off. Proplatelets are elongated strands of megakaryocyte cytoplasm (Fig. 110-4) that are larger than normal platelets141,144 and later fragment into a number of platelets. The bone marrow sinusoids are lined by very thin endothelial cells that are tightly bound to each other and may even overlap.141 The megakaryocyte pseudopodia actually pass through, not between, the endothelial cells, which may in turn play some role in regulating the process.141 This is the method initially suggested in 1906,1,65 and most subsequent data confirm many elements of this model. For example, the culture of mature megakaryocytes on basement membrane matrix leads to increased proplatelet formation.145,146 and 147 Platelets may then be liberated from these elongated processes by rupture of the links. In mice recovering from severe thrombocytopenia there are increased numbers of proplatelet processes in the sinusoids.90 Also in mice lacking the transcription factor NF-E2, the severe thrombocytopenia that is present is probably related to the inability of the megakaryocytes to form proplatelet processes.148,149 A similar defect occurs in mice with selective loss of the transcription factor GATA-1.150
FIGURE 110-4 Megakaryocyte proplatelet processes in the bone marrow sinusoid. This scanning electron micrograph shows the luminal view of the confluence of two bone marrow sinusoids with two proplatelet processes protruding through the lining endothelial cells. One of the processes has intermittent constrictions (arrows) indicating potential sites for platelet formation. Other cells depicted include lymphocytes and erythrocytes (×3000). Reproduced by permission from reference 141.
PULMONARY PLATELET PRODUCTION MODEL
Megakaryocytes or proplatelets are released from the bone marrow, travel to the lung, and are transformed into platelets therein by shear forces.151,152 and 153 This model is a further elaboration of the proplatelet model in which the locus of the production of single platelets is not in the bone marrow but in the lungs. Evidence for this mechanism is the following: (1) the demonstration that megakaryocytes can cross the bone marrow endothelial cell barrier90,154; (2) the presence of megakaryocytes and megakaryocyte nuclei in the circulation and in the pulmonary vessels151,153; (3) mathematical models suggesting that both the platelet number and the log-normal platelet size distribution can be explained by this method of platelet production.152,155 Whether these pulmonary megakaryocytes are simply occasional errant megakaryocytes that have escaped from the marrow and become trapped in the lungs or reflect a major route of cell trafficking is unclear. It has been estimated that the total amount of pulmonary megakaryocytes may account for most of the platelet production,156 but direct evidence for this is still lacking.
REGULATION OF PLATELET SIZE
Circulating platelets vary greatly in their size, density, and age.157,158 Although platelets have a log-normal distribution of sizes,152,155 the cellular mechanisms accounting for the size of platelets is poorly understood. It is unclear whether large platelets come from large, high-ploidy megakaryocytes or small, low-ploidy megakaryocytes; evidence for either exists.158,159 and 160 In general, the mean platelet volume (MPV) increases as the platelet count decreases160,161 and the larger platelets are assumed to be younger162,163,164,165 and 166 and more reactive.167 This effect is seen clinically in patients with chronic idiopathic thrombocytopenic purpura (ITP) in whom the MPV is increased due to an increased number of large platelets, called megathrombocytes; the presence of megathrombocytes is useful in distinguishing ITP from some of the other thrombocytopenic disorders.163,164,165 and 166 In reactive thrombocytosis the MPV is not increased.
The effect of thrombopoietin on platelet size is variable. In mice, administration of low doses of thrombopoietin decreased the MPV, intermediate doses produced an initial increase followed by a later decrease, and high doses gave an initial increase followed by a normalization of the MPV.168,169 In humans administered a recombinant form of thrombopoietin, the MPV usually decreased in a manner inversely proportional to the platelet count.170
THE MOLECULAR BIOLOGY OF MEGAKARYOCYTE DIFFERENTIATION
The molecular mechanisms by which cells become committed to the megakaryocyte lineage are just starting to be unraveled. Unlike hematopoietic growth factors such as thrombopoietin, which appear to prevent apoptosis and stimulate growth of cells already committed to megakaryocyte differentiation, intrinsic lineage-specific transcription factors become expressed in uncommitted precursor cells and then establish cell-specific phenotypes. The transcription factors GATA-1 and NF-E2 have been shown to be important in megakaryocyte development. The lineage-specific genes that are, in turn, controlled by these factors remain unknown.
This zinc-finger transcription factor is expressed in erythroid cells, megakaryocytes, eosinophils, and mast cells. Elimination of the entire GATA-1 gene results in embryonic death due to severe anemia.171 Since GATA-1 is found in megakaryocytes, and GATA-1 binding sites are found in many genes specific for megakaryocytes and platelets, it was anticipated that GATA-1 played a role in megakaryocyte development. When a unique portion of the GATA-1 promoter was disrupted, GATA-1 expression was eliminated in megakaryocytes but not in erythrocytes. Animals with this disrupted GATA-1 promoter were not anemic, but they had platelet counts 15 percent of normal and an increased number of small, abnormal megakaryocytes with multilobulated nuclei.150 The megakaryocytes had scant cytoplasm, few demarcation membranes, no platelet “territories,” and few platelet granules; they rarely formed proplatelets, suggesting an early defect in cytoplasmic maturation and consequently diminished platelet production. In addition these megakaryocytes had an increased proliferative capacity in vitro.87
This heterodimeric basic leucine zipper transcription factor is composed of a widely expressed p18 subunit and a p45 subunit present only in erythroid cells, megakaryocytes, and mast cells. When NF-E2 was disrupted, mice developed a mild anemia and a profound thrombocytopenia associated with a high rate of early hemorrhagic death.172,173 These mice had adequate numbers of large, abnormal megakaryocytes with hyperlobulated nuclei, rare granules, and adequate amounts of demarcation membranes, but no platelet “territories.” The mice never appear to form proplatelets.149 NF-E2 appears to affect megakaryocyte cytoplasmic differentiation and platelet production at a somewhat later step than GATA-1.174
THE REGULATION OF MEGAKARYOCYTOPOIESIS AND THROMBOPOIESIS
Over the past 50 years a number of principles of the regulation of platelet production have emerged from clinical studies175:
The platelet count in any individual remains constant throughout life unless perturbed by physiological (e.g., pregnancy) or pathological (e.g., myelodysplasia) processes.176
Among normal individuals there is a large variation in platelet counts, ranging from 150–450 × 109/liter.162 This is unlike the erythrocyte count, which is much less variable between individuals.
There is an inverse relationship between the normal platelet count and the normal mean platelet volume (MPV),177 and this produces a roughly constant circulating platelet mass.158,178 This inverse relationship extends to other species; for example, mice have a normal platelet count of 1200 × 109/liter and an MPV of 4.7 fl whereas porcupines have a normal platelet count of 30 × 109/liter and an MPV of 105 fl.179
The body “defends” the total mass of platelets, not the platelet count. Normally approximately one-third of the total platelet mass is sequestered in an exchangeable splenic pool.180 In animals181,182 or humans with enlarged spleens,180 the platelet count decreases proportionally to the increase in the size of the spleen, but the total body mass of platelets remains normal and unchanged.
The bone marrow megakaryocytes respond to changes in the demand for platelets by altering their number, size, and ploidy. In animals12,183,184 made thrombocytopenic by the injection of antibody to platelets, bone marrow megakaryocytes increase their number, size, and ploidy. In animals made thrombocytotic by platelet transfusion, the opposite changes in megakaryocytes occur (Fig. 110-5).
FIGURE 110-5 The physiological response of bone marrow megakaryocytes to changes in the platelet count. Mice were made acutely thrombocytopenic by the injection of antiplatelet antibody (a) or made thrombocytotic by the transfusion of platelets (c) and compared with untreated mice (b). The number, size, and nuclear lobulation of bone marrow megakaryocytes increased during thrombocytopenia and decreased during thrombocytosis when compared with normal animals (×16). Reproduced by permission.184
Meg-CFC do not respond acutely to the stimulus of thrombocytopenia185,186; rather, increased meg-CFC are observed subsequent to the alterations that are noted in the more differentiated megakaryocytes.185 Similarly, thrombocytosis does not result in a compensatory decrease in meg-CFC.185 These in vivo observations imply that the initial response to platelet demand is focused on the more mature nonmitotic megakaryocytes.
For almost 50 years it had been assumed that a thrombopoietin existed that regulated platelet production just as erythropoietin controlled the production of erythrocytes.187 Despite heroic efforts, it was not until 1994 that this protein was finally purified and cloned and called by several different names: thrombopoietin,5,188 c-Mpl ligand,3 megakaryocyte growth and differentiation factor (MGDF),6 or megapoietin.2 Although thrombopoietin is the historically accepted name for this protein, the term c-Mpl ligand is also appropriate given the finding that c-Mpl is the receptor for thrombopoietin. This receptor had been discovered in a murine retrovirus that caused a myeloproliferative leukemia (i.e., “mpl”) prior to the purification of thrombopoietin. The oncogene responsible for that syndrome, v-mpl, was found to encode a novel truncated hematopoietic growth factor receptor.189 When the full-length cellular homologue, c-mpl, was cloned, it was found to be a new hematopoietic growth factor receptor of unknown function that was present primarily on megakaryocytes and platelets93 and if inhibited resulted in a decrease in the growth of meg-CFC.94 This receptor was then used to help purify the c-Mpl ligand. It was subsequently demonstrated that the c-Mpl ligand was indeed thrombopoietin and that the c-Mpl receptor was the thrombopoietin receptor.190,191,192 and 193
STRUCTURE OF THROMBOPOIETIN
Thrombopoietin is produced primarily in liver parenchymal cells, while much smaller amounts are made in the kidney.194 Thrombopoietin is synthesized as a 353–amino acid precursor protein with a molecular mass of 36 kDa.3,6,195 Following the removal of the 21–amino acid signal peptide, the remaining 332 amino acids undergo glycosylation to produce an 80- to 90-kDa glycoprotein. The glycoprotein is then released into the circulation with no apparent intracellular storage in the liver or kidney.
Thrombopoietin is an unusual hematopoietic growth factor in a number of ways. First it is much larger than most other regulators of blood cell production such as G-CSF and erythropoietin. Second it has an unusual structure with an erythropoietin-like domain and a carbohydrate-rich domain. The first 153 amino acids of the mature protein are 23 percent homologous with human erythropoietin196 and probably 50 percent similar if conservative amino acid substitutions are considered. This region also contains four cysteine residues just like those in erythropoietin and is highly conserved among different species. Despite these similarities with erythropoietin, thrombopoietin does not bind to the erythropoietin receptor and erythropoietin does not bind to the thrombopoietin receptor.
Amino acids 154 to 332 comprise a novel sequence that contains six N-linked glycosylation sites; this region is less well conserved among different species. Structure-function studies have demonstrated that while the first 153 amino acids of the c-Mpl ligand are all that are required for its thrombopoietic effect in vitro,3,6,197 this truncated molecule has a markedly decreased circulatory half-life compared to the 20- to 40-h half-life of the native protein.198 Presumably, the carbohydrate-rich half of the molecule confers stability and prolongs the circulatory half-life. Similar carbohydrate sequences regulate the stability of erythropoietin.199 In addition, this part of the molecule assists in the secretion of the intact molecule from the hepatocytes by serving as a molecular chaperone or guide in protein folding; truncated muteins of this portion of the molecule have diminished secretion.200
There is a single copy of the gene for thrombopoietin on human chromosome 3q27-28.195,196,201 The gene spans approximately 7 kb with seven exons, the first two of which are noncoding. The third exon contains part of the 5′-untranslated mRNA sequence and part of the signal peptide. The erythropoietin-like region is coded for by exons 4 to 7, and all of the carbohydrate domain is encoded by exon 7. Comparison with the erythropoietin gene shows conservation of the boundaries of the coding exons except for the addition of the carbohydrate domain sequence in the final exon of the thrombopoietin gene. In addition to the functional mRNA encoded (TPO-1), two other nonfunctional m-RNA sequences (TPO-2 and TPO-3) are present due to alternative splicing.195,196
THE THROMBOPOIETIN RECEPTOR
It is now known that the thrombopoietin receptor (c-Mpl) is present on platelets and megakaryocytes, and at lesser density on most other hematopoietic precursor cells. Upon binding to thrombopoietin, the receptor undergoes dimerization, resulting in a number of signal transduction events that improve cell viability, promote growth, and possibly increase differentiation.202 In addition, receptor binding provides the major mechanism by which thrombopoietin is removed from the circulation by platelets and possibly megakaryocytes.2,203,204 and 205 Upon binding thrombopoietin, the receptor-ligand complex undergoes internalization, and the bound thrombopoietin is degraded.206 The receptor is not reexpressed on the surface.207
THE EFFECTS OF THROMBOPOIETIN IN VITRO
Binding of thrombopoietin to its receptor prevents apoptosis of megakaryocytes208 and increases their number, size, and ploidy. The rate of cellular maturation is probably also increased. These events are mediated via signal transduction pathways involving JAK, STAT, and other intracellular mediators.209,210,211,212,213,214,215,216 and 217 Addition of thrombopoietin to CD34+ cells can actually result in the majority of cells becoming megakaryocytes and then shedding platelets.218
Although thrombopoietin stimulates early precursor cells of all lineages as well as pluripotential stem cells,219 it stimulates late maturation only in megakaryocytes.
THE EFFECTS OF THROMBOPOIETIN IN VIVO
When administered to normal animals, thrombopoietin stimulates an increase in bone marrow and peripheral blood meg-CFC, an increase in bone marrow megakaryocytes, and a rise in the platelet count.220,221 and 222 Interestingly, both erythroid and multipotential precursor cells are also increased in the bone marrow and peripheral blood, but without affecting the erythrocyte or neutrophil count.
Following the daily administration of a recombinant form of thrombopoietin to normal baboons, a predictable response occurs.221,222 During the first 4 days of administration, bone marrow megakaryocyte ploidy rises to a maximum, but there is no change in the platelet count. On day 5 the platelet count begins to rise and does so at a dose-dependent rate. With continued administration of thrombopoietin, a dose-dependent plateau platelet count is attained on days 8 to 12. There is a log-linear relationship between the thrombopoietin dose and the plateau platelet count, with a maximum sixfold increase in the rate of platelet production. Upon stopping the growth factor, the platelet count returns to its baseline over 10 days without a rebound thrombocytopenia. In humans a similar time course and platelet response have been demonstrated with no acute toxicity220 but with subsequent studies indicating the potential of individuals to make antibodies to at least one of the thrombopoietin preparations under development (see Recombinant Thrombopoietic Growth Factors and Their Clinical Uses, below).
In addition to increasing the number of megakaryocytes and platelets, thrombopoietin can also affect the function of platelets. When thrombopoietin binds to its platelet receptor, it induces phosphorylation of the c-Mpl receptor and a number of other molecules in several different signal transduction pathways223,224 and 225 but does not directly cause platelet activation. However, such thrombopoietin treatment reduces by 50 percent the threshold for activation by other platelet agonists like ADP and collagen. It is unclear if this is a clinically relevant effect.
THE ROLE OF THROMBOPOIETIN IN NORMAL PHYSIOLOGY
THROMBOPOIETIN IS THE ONLY PHYSIOLOGICALLY RELEVANT REGULATOR OF PLATELET PRODUCTION
Thrombopoietin serves to “amplify” the basal production rate of megakaryocytes and platelets. When thrombopoietin or its receptor has been “knocked out” by homologous recombination in mice,226,227 and 228 the megakaryocyte and platelet mass are reduced to about 10 percent of normal, but the animals are healthy and do not spontaneously bleed. The neutrophil and erythrocyte counts are normal. In animals in which only one of the thrombopoietin genes has been deleted, the platelet count is reduced to about 65 percent of normal. However, such thrombopoietin-deficient mice can increase their platelet count if treated with other thrombopoietic growth factors such as IL-6, IL-11, or stem cell factor.229
THROMBOPOIETIN AFFECTS BONE MARROW PRECURSOR CELLS OF ALL LINEAGES
In the animals made deficient in thrombopoietin or c-Mpl, the megakaryocyte precursor cells (meg-CFC) are reduced by 90 to 95 percent, as expected. However, the myeloid and erythroid precursor cells are also reduced by 60 to 80 percent.226,229 Presumably the normal neutrophil and erythrocyte counts in these animals are maintained by the intact feedback mechanisms mediated by G-CSF and erythropoietin.
THERE IS NO “SENSOR” OF THE PLATELET MASS
Hepatic thrombopoietin production is constitutive, and the circulating levels are determined by the circulating platelet mass (Fig. 110-6). While the production of red blood cells is regulated by a cytochrome P-450 system that senses changes in oxygen delivery to tissues and alters the rate of transcription of the erythropoietin gene, thrombopoietin mRNA is produced at the same rate in normal and thrombocytopenic individuals.2,203,204 and 205,230,231 No drug or clinical condition has yet been shown to increase hepatic thrombopoietin production. Platelets and megakaryocytes contain high-affinity thrombopoietin receptors (c-Mpl) that bind and clear thrombopoietin from the circulation and thereby directly determine the circulating thrombopoietin level. When platelet production is decreased, clearance of thrombopoietin is reduced and levels rise. This type of feedback system is not unusual in hematology. Indeed, both M-CSF and G-CSF are normally regulated primarily by the amount of circulating monocytes and neutrophils, respectively.232,233 It appears that only for erythropoietin is there a true sensor of the circulating blood cell mass that in turn alters production of this hematopoietic growth factor.
FIGURE 110-6 The mechanism by which thrombopoietin (TPO) regulates platelet production from megakaryocytes. TPO (width of arrows indicates relative concentration) is produced at a constant rate by the liver and enters the circulation. Left side: When the platelet count is normal, high-affinity TPO receptors on the platelet clear most of the TPO and produce a normal plasma TPO concentration, thereby providing basal stimulation of bone marrow megakaryocytes and a normal rate of platelet production. Right side: When platelet production and the platelet count are low, the overall clearance of TPO is reduced, subsequently increasing the plasma TPO concentration and megakaryocyte and platelet production. Modified from reference 2.
MEGAKARYOCYTES IN DISEASE
As initially described in 1910,65 characteristic changes in megakaryocytes are associated with several disease processes. Most of these clinical conditions have been extensively studied with bone marrow and platelet kinetic studies10 and thrombopoietin levels.234
MEGAKARYOCYTE HYPOPLASIA SECONDARY TO CHEMOTHERAPY
The thrombocytopenia that follows chemotherapy is due to a decreased number of megakaryocytes. This results in elevated levels of thrombopoietin235 that increase the average ploidy of the remaining megakaryocytes in an effort to increase platelet production. Platelet kinetic studies have also demonstrated some element of ineffective platelet production (ineffective thrombopoiesis) in this setting.10
PERNICIOUS ANEMIA (SEE Chap. 37)
In severe pernicious anemia the low platelet count is associated with a marked increase in the number of megakaryocytes but diminished ploidy resulting in an expanded megakaryocyte mass but reduced platelet production per megakaryocyte.10 This ineffective platelet production from the megakaryocytes is comparable to the ineffective erythrocyte production also seen in this disorder.
CONGESTIVE SPLENOMEGALY (SEE Chap. 59)
As suggested above, the thrombocytopenia seen in splenomegaly secondary to liver disease has long been felt to be due to a redistribution of the normal circulating mass of platelets from the circulation to the spleen.180,236 Platelet kinetic studies have analyzed this situation further and demonstrated that the modest thrombocytopenia is not accompanied by a decrease in platelet survival but does give rise to a small increase in the number and ploidy of megakaryocytes and a small overall increase in platelet production.10 These results are at odds with data showing a reduced level of thrombopoietin and a reduction in platelet production rates in patients with cirrhosis.237
THROMBOCYTOPENIA ASSOCIATED WITH HUMAN IMMUNODEFICIENCY VIRUS (HIV) (SEE Chap. 114)
Thrombocytopenia is commonly seen in both early and late HIV infection and has been considered due to immune complex binding to platelet Fc receptors and consequent platelet removal by the spleen.238 Antiretroviral treatment is often associated with improvement. However, platelet kinetic studies suggest that platelet survival is only slightly reduced, and there is marked ineffective production of platelets from megakaryocytes.139,239 In the thrombocytopenic HIV-infected patients studied,239 megakaryocyte mass and megakaryocyte size were increased two- to threefold, but the total effective platelet production from the megakaryocytes was not increased. Administration of a recombinant thrombopoietin to these patients resulted in no change in megakaryocyte mass but did increase the effective production rate eightfold and increased the platelet count from 42 × 109/liter to 349 × 109/liter.
IDIOPATHIC THROMBOCYTOPENIC PURPURA (SEE Chap. 114)
In animal models and in humans, chronic ITP is characterized by an increase in the number, size, and ploidy of bone marrow megakaryocytes. One interpretation of these findings is that the decline in platelets in ITP results in an initial rise in thrombopoietin levels, which in turn stimulates an increase in the megakaryocytes. Support for this comes from platelet kinetic studies in which these morphological findings are associated with a sixfold increase in the rate of platelet production10,240,241 and a shortened platelet survival time. However, subsequent platelet kinetic studies242 have failed to demonstrate an increase in platelet production in ITP and suggest that the morphological findings might be related to ineffective platelet production akin to that described for HIV infection. The observation that thrombopoietin levels are normal in this patient group is consistent with either hypothesis.234,243
REACTIVE THROMBOCYTOSIS (SEE Chap. 115)
Reactive thrombocytosis occurs in association with iron deficiency, malignancy, and inflammatory states. It is associated with an increased number of megakaryocytes but with ploidy less than normal, an increased megakaryocyte mass, and an increased rate of platelet production. The increased platelet count is probably secondary to the expansion of the megakaryocyte number due to inflammatory cytokines such as IL-6,244 and ploidy is reduced due to a decreased level of thrombopoietin secondary to increased thrombopoietin clearance by the expanded platelet mass.
ESSENTIAL THROMBOCYTHEMIA (SEE Chap. 115)
Although the clonal nature of essential thrombocythemia has been challenged,245 in essential thrombocythemia and the related myeloproliferative disorders such as chronic myeloid leukemia and polycythemia vera, there is a proliferation of megakaryocytes that are of high ploidy and actively produce platelets. Whether the normal regulatory mechanism via thrombopoietin and its receptor is functioning is unclear. Platelet kinetic data suggest the proliferation is autonomous of thrombopoietin in that at increased platelet mass there is a greatly increased megakaryocyte ploidy despite a normal thrombopoietin level.234,246 Evidence suggests that platelet and possibly megakaryocyte thrombopoietin receptors are decreased tenfold in essential thrombocythemia.246
MYELODYSPLASTIC SYNDROMES (SEE Chap. 90)
Thrombocytopenia as well as thrombocytosis are found in myelodysplastic syndromes and attributed to abnormal megakaryocytes. The morphological picture is that of an increased number of small megakaryocytes of low ploidy, occasionally displaying a characteristic “pawn ball” nucleus with three lobes.247 Platelet kinetic studies10 have demonstrated a greatly expanded megakaryocyte mass (increased number of megakaryocytes of low ploidy) and ineffective platelet production from the megakaryocytes.
HEMATOPOIETIC DISORDERS ASSOCIATED WITH ABNORMAL CHROMOSOME 3q
A number of hematopoietic disorders associated with thrombocythemia or abnormal megakaryocyte formation have been associated with defects involving chromosome 3q,248 and some myeloid leukemias associated with thrombocytosis have a characteristic rearrangement of chromosome 3q21 and 3q26.201 Since the thrombopoietin gene is located on chromosome 3q27-28, it has been suggested that the thrombopoietin gene might be mediating these effects.195,196,201 However, analysis of the chromosome regions in these patients has not demonstrated involvement of the thrombopoietin gene, and blood thrombopoietin levels are normal.201,249 These results suggest that other genes close to the thrombopoietin gene may be responsible for other aspects of megakaryocyte differentiation and growth.
AGNOGENIC MYELOFIBROSIS WITH MYELOID METAPLASIA (SEE Chap. 93)
The typical finding in this myeloproliferative disorder is an increase in bone marrow megakaryocytes without dysplasia as well as a large amount of fibrosis. The fibrotic response is a polyclonal proliferation of fibroblasts which has been attributed to the release of mesenchymal growth factors such as platelet-derived growth factor or transforming growth factor–b from the abnormal megakaryocytes. Subsequent data suggest that this may not be the entire mechanism.250 Overexpression of the thrombopoietin gene using adenovectors in immunodeficient SCID mice results in thrombocytosis, increased marrow megakaryocytes, fibrosis, and extramedullary hematopoiesis that mimics the clinical disorder. However, similar overexpression of thrombopoietin in NOD-SCID mice (which have reduced monocyte and macrophage function in addition to the lymphocyte deficiency in SCID mice) produced similar thrombocytosis and megakaryocytosis but no fibrosis. These results imply that other monocyte/macrophage mediators may be involved in causing the fibrosis.
DISORDERS OF THROMBOPOIETIN PRODUCTION
The thrombopoietin physiology described above suggests possible mechanisms for clinical disorders associated with abnormalities in platelet count.231 A few of these postulated disorders have been identified.
A few families have been described that have a disorder that is clinically like the more common, sporadic cases of essential thrombocythemia.251,252 Analysis of one of these families252 identified a single point mutation in the splice donor site of intron 3 of the thrombopoietin gene that produced a new thrombopoietin mRNA with a normal protein coding region but with a shortened 5′ untranslated region that was more efficiently translated than the normal thrombopoietin transcripts. The 5′ untranslated region of thrombopoietin mRNA is unusual in that it contains numerous translation initiation sites, only one of which produces the active protein. A reduction in the number of these alternative translation initiation sites yields thrombopoietin mRNA that is more efficiently translated.253 In the families with inherited thrombocythemia, loss of these sites by mutation results in more thrombopoietin protein synthesis, higher plasma thrombopoietin levels, and chronically elevated platelet counts. In individuals with the more common, sporadic cases of essential thrombocythemia none of these mutations have been found.
Since the liver is the primary site of thrombopoietin production,254 and the thrombopoietin gene is apparently not inducible, thrombopoietin deficiency may be potentially responsible (along with splenic sequestration) for the thrombocytopenia in patients with liver failure. In animals, partial resection of the liver results in a proportional decrease in the platelet count.255 In patients with liver failure, thrombopoietin levels appear to be inappropriately low,237 leading to the suggestion that recombinant forms of thrombopoietin may be an effective therapy.
RECOMBINANT THROMBOPOIETIC GROWTH FACTORS AND THEIR CLINICAL USES
Two recombinant thrombopoietins have been extensively studied and demonstrate some clinical effect. One is a glycosylated molecule produced in Chinese hamster ovary (CHO) cells consisting of the full-length, native human amino acid sequence (recombinant thrombopoietin, rHuTPO) which has a circulatory half-life of 20 to 40 h.256,257 and 258 The other is a nonglycosylated, truncated molecule produced in E. coli composed of the first 163 amino acids of the native molecule and chemically coupled on the amino terminus to polyethylene glycol (pegylated, recombinant megakaryocyte growth and development factor, PEG-rHuMGDF).220,259,260 The half of the native thrombopoietin molecule contained in the latter drug is 50 percent similar to erythropoietin and contains all of the receptor-binding domain of thrombopoietin; it has a very short circulatory half-life and thus little biological activity in vivo. The addition of the polyethylene glycol moiety serves to stabilize the molecule in the circulation and replaces the domain that normally confers longer intravascular survival. The polyethylene glycol-thrombopoietin conjugate has a half-life of 30 to 40 h. Neither recombinant thrombopoietin product has received approval for clinical use. Some patients given PEG-rHuMGDF subcutaneously have developed antibodies to the molecule that cross-react to the endogenous thrombopoietin and cause thrombocytopenia. Intravenous administration of PEG-rHuMGDF has not been associated with antibody formation, and development of PEG-rHuMGDF by this route continues.
These recombinant thrombopoietins have demonstrated some benefit in the primary prophylaxis of thrombocytopenia associated with chemotherapy by reducing the duration, and often the depth, of the thrombocytopenia.257,259,260 In addition, they may decrease the need for platelet transfusions.257 However, when used in myeloablative chemotherapy settings such as AML261 or stem cell transplantation,262,263 and 264 neither has shown significant benefit. Both are potent mobilizers of peripheral blood progenitor cells260 and can expand cord blood progenitor cells ex vivo.265 Both can stimulate an increase in platelet count in normal platelet donors and increase the yield of plateletpheresis.266,267 Platelet counts in thrombocytopenic HIV-infected patients can also be increased.239,268
IL-3, IL-6, and IL-11 produce significant stimulation of platelet production. IL-3 and IL-6 are probably too toxic for most clinical uses, but recombinant IL-11 has modest side effects and has been approved by the FDA for use in the prevention of chemotherapy-induced thrombocytopenia.269 Recombinant human IL-11 (Neumega) stimulates megakaryocyte growth and increases platelet production with a time course similar to that of thrombopoietin. Its thrombopoietic action is not mediated through thrombopoietin release or synergism and is independent of the thrombopoietin receptor.123 Surprisingly, when the gene for the IL-11 receptor was eliminated in mice, there was no effect on the production of platelets or any other blood cell, suggesting that IL-11 is not important for normal hematopoiesis.270,271 However, in clinical studies IL-11 reduced the extent of chemotherapy-induced thrombocytopenia123,269 and in one study actually reduced the need for platelet transfusions by 27 percent.124 Its major side effects are dilutional anemia, pleural effusions, and atrial arrhythmias.
OTHER THROMBOPOIETIC GROWTH FACTORS
There is also a growing number of other molecularly designed platelet growth factors based on the structure of thrombopoietin or its receptor that are just entering preclinical testing. One of these, promegapoietin,272 is a molecular modification of thrombopoietin in which the receptor-binding region is coupled to the hematopoietic growth factor IL-3. This molecule can bind to and activate both the thrombopoietin and IL-3 receptor. Another is a thrombopoietin peptide mimetic (TPO peptide mimetic) that consists of a dimer of two identical 14–amino acid peptides with no sequence homology to thrombopoietin and that avidly binds to and activates the thrombopoietin receptor, c-Mpl.273 Neither of these molecules has entered clinical testing. These molecules define a new and growing family of molecules called the Mpl ligand family274 based on their common ability to bind and activate the receptor for thrombopoietin, c-Mpl.
Novel molecularly designed proteins275 currently under development include myelopoietin (an IL-3 receptor agonist) and progenipoietin-G (a fusion protein of flt-3 ligand and G-CSF)276; they both stimulate platelet production as well as cells of other lineages and give the promise of being “panpoietins.”
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Ernest Beutler, Marshall A. Lichtman, Barry S. Coller, Thomas J. Kipps, and Uri Seligsohn