Control of Erythropoiesis

Alterations in the concentration of hemoglobin in the blood lead to changes in tissue oxygen
tension within the kidney. In response to hypoxia, the kidney secretes a hormone called EPO.
This hormone induces erythroid progenitor cells to differentiate into pronormoblasts, thereby
bringing about expansion of the erythroid marrow and an increase in red cell production. This,
in turn, leads to an increase in the size of the erythron and an increase in tissue oxygen levels.
Each of the major steps in this process is discussed in greater detail in the sections that follow.
Tissue Oxygen
Tissue oxygen tension depends on the relative rates of oxygen supply and demand. Oxygen
supply is a complex function of interacting but semi-independent variables, including (a) blood
flow, (b) blood hemoglobin concentration, (c) hemoglobin oxygen saturation, and (d)
hemoglobin oxygen affinity. Each of these functions may be altered to compensate for a
deficiency in one of the others. For example, in severe anemia, cardiac output and respiratory
rate may increase, and hemoglobin oxygen affinity may be reduced through the 2,3-
diphosphoglycerate effect. Conversely, in respiratory insufficiency, secondary polycythemia
occurs.
Despite the influence of cardiovascular and respiratory adjustments, tissue oxygen tension
decreases roughly in proportion to the degree of anemia. Conversely, induced polycythemia of
moderate degree leads to normal or increased tissue oxygen tension and to increased tolerance
to hypoxia. These changes occur despite the increase in blood viscosity that accompanies
polycythemia, suggesting that peripheral vascular resistance decreases to compensate for
increased viscosity. However, with advanced degrees of polycythemia, the increase in viscosity
may be great enough to negate the advantages of increased oxygen-carrying capacity.
Tissue hypoxia is the fundamental stimulus to erythropoiesis, as was first suggested by
Miescher in 1893. This concept has been amply confirmed (1). However, it now seems clear
that hypoxia does not exert its effects by a direct action on the marrow, as Miescher believed,
but instead acts by inducing the elaboration of a hormone, EPO. The nature of the tissue
oxygen receptors (or oxygen sensor) has only recently been understood. These sensors are
located within the kidney because production of EPO can be induced by renal artery
constriction or by hypoxic perfusion of the isolated kidney.
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Erythropoietin
Structure of Erythropoietin
EPO is a glycoprotein hormone produced by the kidney, and it is the major humoral regulator of
red cell production. EPO was originally purified from the urine of patients with aplastic anemia
(192). It has an MW of 34,000 daltons as determined electrophoretically and contains 30%
carbohydrate, of which 11% consists of sialic acid, 11% total hexose, and 8% N-
acetylglucosamine (193). The potency of EPO is expressed in units, with one unit being equal to
the amount of EPO present in one tenth of the International Reference Preparation (194). This
unit had been originally defined as the amount of EPO that produced in the starved rat the
same erythropoietic response (increase in serum EPO level) as 5 μmol of cobalt (1). The potency
of the purified human urinary EPO has been determined to be 70,400 U/mg of protein or
50,000 U/mg of total weight (192).
Purification of human urinary EPO has allowed the isolation and cloning of the human genomic
DNA encoding for human EPO (195). The gene encoding EPO has been localized on human
chromosome 7 (7 pter-q22) (196). The EPO gene exists as a single copy in a 5.4-kb DNA
fragment and contains four introns and five exons for the 193–amino acid polypeptide (195).
The gene also includes a 27–amino acid signal peptide (leader sequence), which is cleared
during EPO secretion, and a 166–amino acid peptide with an MW of 18,398 (195). The C-
terminal arginine is absent from both recombinant and natural hormone, presumably because
of posttranslational modification by a carboxypeptidase (197). The human EPO contains four
cysteine residues linked by disulfide bonds, which, when reduced or alkylated, lead to
significant loss of activity (199,200).
Recombinant EPO synthesized by mammalian cells is highly glycosylated, and the carbohydrate
structure of the recombinant hormone is similar to but distinct from that of the natural
hormone (197). Recombinant EPO has an MW of 30,000 by velocity sedimentation (200) and
contains approximately 39% carbohydrate (201); however, recombinant EPO migrates slightly
slower on sodium dodecyl sulfate–electrophoresis than urinary EPO, which suggests a slightly
higher apparent MW of 40,000 compared to an MW of 34,000 of the native urinary EPO (202).
The discrepancy between the electrophoretically obtained apparent MWs of natural and
recombinant hormone is almost certainly due to distinctly different glycosylation of
recombinant EPO in the Chinese hamster ovary cell line compared to the glycosylation of native
EPO in kidney cells. Recombinant EPO must have additional carbohydrate, less negatively
charged carbohydrates, or both compared to urinary EPO to result in reduced electrophoretic
mobility.
Glycosylation of the hormonal peptide is absolutely necessary for its in vivo activity. The bulk of
glycosylation of EPO is at a single site of N-linked carbohydrate. Asialated EPO and nonglyco-
sylated recombinant EPO produced in bacteria have no activity in vivo, which can be at least
partially attributed to rapid clearance of the hormone by the liver via the galactose receptors of
hepatic cells (200,203).
The importance of glycosylation of EPO to EPO’s in vivo activity and half-life led to modification
of the EPO gene/protein to make a more effective pharmaceutical. The gene coding for EPO
was modified by adding a second site of N-linked glycosylation, such that when the gene is
expressed in Chinese hamster ovary cells, the amount of carbohydrate attached to the modified
EPO peptide is almost doubled. The new product was called darbepoetin or novel erythroid-
stimulating protein. Darbepoetin is highly related to EPO, such that it binds to the same
receptor, but it has a longer in vivo half-life than recombinant EPO so that fewer injections per
week than recombinant EPO are required for therapeutic use (204).
Studies on the amino acid sequences of human and murine EPO have shown a very high degree
of conservation of the molecule structure in these two species (205,206). Which part of the EPO
molecule is primarily responsible for its function is not known, although results of studies with
monoclonal antibodies have indicated that the amino acids in positions 99 to 129 may play an
important role in the binding of EPO to cell membrane (207).
Site and Regulation of Erythropoietin Production
Almost 50 years ago, Jacobson et al. established that the kidney is the major organ of EPO
production in adult rats (208). Also, humans with end-stage renal failure were found to have
low serum EPO concentrations, which were restored to normal after successful renal
transplantation (210). The cloning of the murine EPO gene has allowed studies on the
production of EPO-specific mRNA in anemic mice. Induction of anemia leads, within 1 hour, to
the appearance of EPO-encoding mRNA in the kidney and liver of anemic mice and rats
(210,212). After bleeding, the EPO-mRNA in the kidney increases 500 to 1,000 times compared
to normal kidney, whereas the liver produces only 7% of the total EPO-mRNA (211). In no tissue
other than the kidneys and the liver is EPO-mRNA detectable, even in the presence of severe
anemia (213). These changes in EPO-mRNA were followed by parallel changes in serum EPO
concentration determined by radioimmunoassay, indicating that EPO production in response to
anemia represents de novo synthesis rather than release of preformed hormone (212). More
recent evidence indicates that the anemia-induced increase in EPO-mRNA is to a great extent
due to an increased transcription (213). Murine EPO-mRNA was detected by ribonuclease
protection assay at 14 days of gestation in the liver and almost a week later in the kidneys,
which assume a major role in EPO production after birth (214). In cases of paraneoplastic
erythrocytosis, EPO-mRNA has been detected in the neoplastic cells (215,216).
Specialized cells producing EPO have been identified in renal and hepatic parenchyma by the
technique of in situ hybridization using radioactive probes specific for EPO-mRNA (Fig. 6.12)
(217,218,219). These rare EPO-producing cells are found in the interstitium of the renal
parenchyma, outside the tubular basement membrane, mostly in the inner cortex and outer
medulla. The nature of these interstitial peritubular EPO-producing cells remains controversial;
however, the bulk of experimental evidence indicates that these cells are fibroblastlike type I
interstitial cells (220,221). In liver, mRNA is detected in hepatocytes. The number of the
interstitial renal EPO-producing cells increases in response to anemia, indicating that increased
demands for EPO are met by an increase in the number of EPO-producing cells rather than by
an increased synthesis of EPO by a preset number of cells (212). Thus, it appears that EPO is
synthesized de novo in response to hypoxia and that there is no detectable storage of the
hormone.
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Increased levels of circulating EPO do not seem to exert any negative effect on EPO production.

Figure 6.12. In situ hybridization of murine kidney cells using a probe specific for murine
erythropoietin (EPO)-mRNA. EPO-producing cells are found in the interstitium, covered heavily
with grains. Arrows indicate peritubular interstitial cells not producing EPO. (Courtesy of Dr.
Stephen Koury.)
The mechanism by which hypoxia leads to EPO synthesis has only recently been understood.
Results of several studies have shown that the EPO gene contains sequences that are oxygen
sensitive and are involved in the regulation of EPO gene expression (222). By the use of plasmid
constructs, transgenic mice, and cell transfection, it has been shown that these oxygen-
sensitive sequences, located at the region flanking the 3′ end of the EPO gene, can confer to
cells the ability to respond to hypoxia by an increase of the protein encoded by the reporter
gene (223). In the same area of the EPO gene, an enhancer has been identified with sequences
suggesting that it belongs to the family of the nuclear hormone receptor superfamily (224). The
ligand for this oxygen-sensitive enhancer was identified as a protein of 120 kd termed hypoxia-
inducible factor 1 (HIF-1) (225,226,227). This DNA-binding protein is tightly regulated by the
oxygen tension and is considered to be the physiologic regulator of EPO transcription (228).
HIFs are heterodimeric, helix-loop-helix, transcription factors consisting of two subunits, HIF-1α
and HIF-1β. The concentration and transcriptional activity of HIF-1α increase in a geometric
fashion on exposure to hypoxia. In addition to EPO, a large number of genes (glucose
transporters, glycolytic enzymes, vascular endothelial growth factors, and many others) are
activated during hypoxia to aid the cell in adapting to hypoxic conditions (229). HIF-1α is
constitutively expressed under normoxic conditions, but it is rapidly degraded via the ubiquitin
proteosome complex after it is tagged with the protein of von Hippel-Lindau. Binding of the
protein of von Hippel-Lindau to HIF-1α requires hydroxylation of the latter by a proline
hydroxylase (230,231,232,233). Prolylhydroxylase is an oxygen and iron-dependent enzyme.
Under hypoxic condition, little or no proline hydroxylation takes place; thus, the protein of von
Hippel-Lindau does not bind to HIF-1α, which accumulates, heterodimerizes with HIF-1β, and
recruits the p300/CREB-binding protein transcriptional coactivator, and the whole complex
binds to EPO enhancer to promote EPO gene transcription. Recruitment of p300 is inhibited by
asparagine hydroxylation that is catalyzed by an oxygen-sensitive asparaginyl-hydroxylase
(234). It seems that these two amino acid hydroxylases, by their dependence on normal
intracellular oxygen for their function, act as the oxygen sensor in the EPO-producing interstitial
cells in the kidney, and by regulating the function of HIF-1 at least at two distinct points (233),
they ultimately control EPO synthesis and production.
Action of Erythropoietin
Erythropoietin Receptors
EPO binds to specific molecules on the cell surface, the EPORs. Expression of both EPO and
EPORs is necessary for adult life. Deletion of either the genes that code for EPO or the EPOR in
mice results in identical phenotypes of fetal death at day 12 to 13 as a result of severe anemia
(235). Arguably, the most important control point of erythropoiesis is the interaction of EPO
with the receptor for EPO. The activation of the EPOR generates an intracellular signal in
immature erythroid cells that promotes the survival of these cells that would otherwise
undergo apoptosis. EPO may also promote proliferation. A direct instructive effect of EPO on
either primitive hematopoietic cells or mature erythroid cells to direct differentiation is not
supported by most data.
Specific EPORs are expressed on hematopoietic cells that respond to EPO and have been
identified on human (23) and murine erythroid cells (236), on erythroleukemic cell lines (236),
in fetal liver tissue rich in erythroid elements, in mouse and rat placenta (215,238), and in
megakaryocytes (238,239). The density of EPORs on erythroid cells is relatively small
(approximately 1,000 molecules per cell) and varies correlating with the cell’s responsiveness to
and dependence on EPO (239). EPORs are detectable by autoradiography on human BFUs-E;
their density increases as the BFU-E matures to CFU-E (29). Erythroid cells at a stage between
CFU-E and proerythroblast seem to have the highest density of EPORs, which decrease as the
proerythroblast matures and eventually disappear at the stage of orthochromatic erythroblast
(29,238). EPORs are not expressed on reticulocytes or red cells.
Whereas functional EPORs were detected in mouse and rat placenta (215,238), these placental
EPORs apparently cannot function to transport significant amounts of EPO from the maternal
circulation to the circulation of the fetus. The deletion of the EPO gene in a mouse fetus results
in severe anemia and death at days 12 to 13 postinception (235). If sufficient EPO from the
mother were transported across the placenta to the fetus, one would expect normal birth of
the mice missing the EPO gene (EPO-/-) but death by anemia shortly afterward. Thus, the fetal
death of EPO-/- mice proves that fetal EPO production is necessary for survival. The detection of
receptors for EPO in megakaryocytes (238,239) is understandable because EPO concentration
can affect platelet levels. In contrast, the physiologic significance of the presence of receptors
for EPO in neurons, the kidneys, endothelial cells, embryonic muscles, and breast cancer cells is
not yet established. It is doubtful that expression of receptors for EPO in nonerythroid tissues is
required for normal embryonic development because tissue-specific expression of the EPO
receptor under control of an erythroid promoter in EPOR-/- embryonic stem cells results in
apparently normal mice. This likely proves that EPOR expression is only required in erythroid
cells for normal development (240).
Cloning of the murine erythroleukemia EPOR gene revealed a DNA sequence encoding a 507–
amino acid peptide of 62 kd with a single membrane–spanning domain in a direction such that
the amino-terminal site is located extracellularly and the carboxy-terminal site is located
intracytoplasmically (241). The EPOR undergoes glycosylation and phosphorylation before being
incorporated into the cell membrane as a 70- to 78-kd protein (241). Cells transfected with this
gene express both high- and low-affinity EPORs (241). The human EPOR gene has been localized
in the long arm of chromosome 19 (242). After the cloning of the EPOR gene, other receptor
genes were cloned that were similar in sequence, such that it is now recognized that the EPOR
is related to a large family of receptors, the hematopoietic receptor superfamily, that includes
receptors for interleukins-2 through -9, granulocyte colony-stimulating factor, and granulocyte-
macrophage colony-stimulating factor, and receptors for other factors such as growth hormone
and prolactin that do not modulate hematopoietic cells (243).
After binding of EPO to its receptor, the hormone is rapidly endocytosed and degraded
(239,244). Whether the receptor is also degraded or recycles to the membrane is not known,
although experimental evidence favors degradation (236). Crystallographic studies confirm
that, as is the case for other members of the hematopoietic superfamily of receptors, one
molecule of EPO may simultaneously bind to two receptors for EPO. It is clear that activation of
the receptors by the hormone leads to formation of receptor homodimers (245); however,
evidence suggests that EPOR dimers likely exist even before the binding of EPO. EPO binding
shifts and stabilizes an active receptor conformation that brings the two EPORs in closer contact
(246,247).
Tyrosine phosphorylation of the EPOR (248) is the first observable event after EPO binding.
Because the EPOR lacks a kinase domain, a tyrosine protein kinase must associate with the
receptor. The tyrosine protein kinase JAK2, a member of the Janus family of kinases, is the
primary EPOR-associated kinase being able to bind to conserved sequence of amino acids found
in cytoplasmic domains of the EPOR (249,250) and other receptors related in a sequence to the
receptors for EPO. JAK2 is also implicated as the only significant EPOR-interacting kinase
because deletion of the JAK2 kinase gene in mice results in fetal death on day 12 to 13
associated with severe anemia that mirrors the phenotype after either deletion of the EPO gene
or the gene coding the receptor for EPO (251). A single amino acid mutation (Val617Phe)
located in
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the regulatory pseudokinase domain of this kinase results in constitutive activation of JAK2.
Such a somatic mutation has been detected in the majority of patients with polycythemia vera
and in a lower frequency in patients with other myeloproliferative disorders (252,253).
To summarize the current view of this research, two EPOR molecules bind to one EPO molecule.
JAK2 kinase molecules associated with each receptor are activated by the physical process of
bringing the inactive (or low-activity) kinases in close proximity by a shift in receptor
conformation, such that these low-activity kinases cross-phosphorylate each other to gain full
activity. The activated JAK2 kinase then phosphorylates all eight tyrosine residues of the EPOR
cytoplasmic domain. These phosphorylated tyrosine residues of the receptor become docking
sites for signaling molecules that may be phosphorylated by JAK2 to become active, such as the
signal-transducing activators of transcription (STAT) proteins. STAT1 and STAT5 become
phosphorylated, dimerize, and are transported to the nucleus to mediate gene transcription.
Alternatively, the translocation of active signaling molecules from the cytoplasm to the plasma
membrane via docking to the phosphorylated EPOR activates a signaling cascade. Examples
include the translocation of phosphatidylinositol-3-kinase (PI3-kinase) from the cytoplasm to
phosphorylate plasma membrane lipids at the cell surface (254).
Domains of the receptor for EPO, the specific tyrosine residues of the receptor, or both are
apparently important in activation of distinctive signaling pathways. For example, tyrosine
residue 343 is required for EPO-dependent activation of STAT5 signaling (256), whereas the
distal end and tyrosine residue 479 of the EPOR are required for activation of both PI3-kinase
and MAP kinase cascade. The importance of EPOR tyrosine phosphorylation is difficult to
reconcile with a study that showed that transgenic mice expressing receptors for EPO without
any cytoplasmic tyrosine residues are essentially normal regarding erythropoiesis and
hematocrit (257). Thus, the tyrosine residues of the receptor for EPO are likely important in
more subtle ways than previously believed. This is suggested also by the fact that all eight
tyrosines are conserved in both number and alignment between the murine and human EPOR
genes. If these residues served no important purpose, one would expect differences in either
tyrosine numbers or alignment between the mouse and human genes.
The distal end of the EPOR acts as a negative regulatory domain to which tyrosine protein
phosphatases dock to phosphorylated tyrosine residues to attenuate EPO signaling. This is
suggested by the findings in a transgenic mouse created to express a truncated human receptor
EPO, replacing the murine EPOR. This mouse was found to have a severe erythrocytosis,
mimicking the disease state noted in humans with polycythemia vera who have elevated red
cell mass due to mutations in the negative regulatory domain of the EPOR gene (258).
The role of the transcription factor, STAT5, in signaling after phosphorylation and activation by
binding to the receptor for EPO is controversial. STAT5 is expressed from two very similar
genes, STAT5a and STAT5b. In transgenic mice in which both genes for STAT5a and STAT5b
were deleted or “knocked out” (STAT5a/b-/- mice), STAT5a/b was found to be neither essential
nor significant for erythropoiesis (259). However, other investigators obtained and studied the
same STAT5a/b-/- mice and reported a marked fetal anemia in the STAT5-/- mice and lesser but
significant anemia in newborn and adult STAT5-/- mice (260). Erythropoiesis in STAT5a/b-/- mice
was then re-examined by the initial investigators, who reported that the hematocrit was slightly
reduced at fetal and newborn stages, but comparison of photographs of the STAT5 -/- fetus did
not appear to be significantly different from the wild-type fetus (258). Thus, STAT5 seems to
have some role in erythropoiesis, different from the initial conclusion that STAT5 has no effect,
but it also plays a less significant role in fetal erythropoiesis than was initially claimed (260).
EPO is known to weakly activate STAT1 and to strongly activate STAT5 in primary erythroid
cells, such that STAT1 activation may partially compensate for STAT5 and explain why deletion
of STAT5 in mice results in a relatively mild inhibition of erythropoiesis (261). A potential target
of STAT5 is the Bcl-Xl gene. Bcl-Xl is an antiapoptotic protein believed to be essential for EPO-
dependent survival. Two recent studies with Bcl-Xl–deficient mice confirmed that expression of
Bcl-Xl is required for normal erythropoiesis, but they showed that Bcl-Xl promotes the survival
of mature erythroid cells that no loner depend on EPO for survival (262,263). Thus, Bcl-Xl must
be regulated by factors other than EPO-activated STAT5.
A large body of evidence now shows that both the MAP kinase cascade and PI3-kinase cascade
are activated after EPO activation of the EPOR. Although EPO may act to (a) promote the
survival of immature progenitors by preventing apoptosis or programmed cell death, (b) drive
the proliferation of progenitor cells, and (c) direct the differentiation of progenitor cells along
erythroid maturation, it is not clear if some intracellular signaling pathways distinctly activate
only one of these events. Some data suggest that the EPO-dependent activation of the MAP
kinase pathway is more important in directing proliferation, whereas the PI3-kinase pathway is
equally important in survival and proliferation (254). The central role of PI3 kinase in signaling
downstream of the receptor for EPO is suggested from the surprising ability of a constitutively
active Akt (a kinase directly downstream of PI3 kinase) to partially rescue erythroid
development (CFU-E) when expressed in JAK2-/- fetal liver cells (255).
Mechanism of Action
EPO is a hormone that promotes erythroid differentiation (1). The role of EPO during the very
early stages of erythropoiesis is still undefined. Cell lines with features of multipotent
hematopoietic progenitor cells (264,265) and purified human blood BFUs-E (29) express a small
number of EPORs, suggesting a possible role of EPO for their survival and differentiation.
Although, in vivo, the BFU-E pool is unaffected by acute changes of the level of serum EPO and
BFUs-E are EPO independent for their survival, they can respond to EPO by increasing their
cycling, which is part of the process of erythroid differentiation (40). Chronic administration of
recombinant EPO (rEPO) to humans with end-stage renal disease results in global stimulation of
the bone marrow with an increase in the concentration and cycling of all types of
hematopoietic progenitors, but this effect is most likely indirect (41).
The erythroid cell that is the most sensitive to EPO is a cell between the CFU-E and the
proerythroblast (29,238), and this erythroid cell can be considered the primary target of EPO
action. These cells express the highest density of EPORs on their membrane and are absolutely
dependent on EPO for their survival (29,266,267). Studies on murine splenic erythroid cells
infected with the anemia strain of Friend virus (268) have shown that binding of EPO is followed
by a series of biochemical events, including increase in Ca2+ uptake (60), internalization of the
hormone (244), increase in total RNA synthesis (269), glucose and iron uptake (239), rate of
transcription of the α- and β-globin gene (211,270), expression of transferrin receptors (59),
and eventually increase of hemoglobin synthesis as well as of membrane bands 3 and 4.1
(267,271). All of these changes result in an increased rate of erythroid differentiation, ending
with an increase in the reticulocyte production and an eventual increase in the red cell mass.
One of the most impressive effects of EPO on the erythroid cells is the ability of the hormone to
maintain the viability of these cells irrespective of any effect on cycling and differentiation
(266,267). In the absence of EPO, erythroid cells die. It has been shown that EPO retards the
cleavage of DNA that occurs normally in CFU-E (272). In the absence of EPO, DNA cleavage is
rapid and proceeds to cell death. The pattern of rapid DNA cleavage occurring in erythroid cells
deprived of EPO is characteristic of cells undergoing apoptosis (programmed cell death) (272).
In the presence of EPO, cell death is avoided and the erythroid cells are allowed to differentiate
and form red cells. These findings suggest that the
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hormone promotes erythroid differentiation simply by allowing cell survival, which is a

prerequisite for both cell proliferation and maturation. This model also suggests that, under
normal conditions, a large number of generated CFU-E are not surviving, but, under conditions
of high EPO levels, expansion of the erythroid marrow is seen simply by allowing survival of
more CFU-E, resulting in an increased rate of red cell production. Within the same context,
once the red cell mass is restored to normal, the ensuing decrease of EPO levels leads to a rapid
turn-off of erythropoiesis by allowing programmed cell death to occur (272).
The observation that relatively immature erythroid progenitor cells continue to develop in fetal
mice in which either the gene for EPO or the gene coding for the EPOR has been deleted (235)
supports the model that EPO acts primarily by promoting survival of more mature erythroid
cells and that EPO has no or a less significant role in proliferation of erythroid precursors or in
directing erythroid differentiation of immature hematopoietic cells. The abundant erythroid
cells (proerythroblasts near the CFU-E stage of erythroid development) from the spleens of
either EPO-/- or EPOR-/- mice undergo apoptosis or programmed death unless these spleen cells
are either cultured in vitro in EPO (EPO-/- mice) or forced by transfection with complementary
DNA to express the EPOR (EPOR-/- mice) and then cultured in the presence of EPO. Thus,
neither EPO nor receptors for EPO are necessary for the proliferation and differentiation of
stem cells and early progenitor cells into relatively mature erythroid cells. Both EPO and the
receptors for EPO, however, are absolutely required for erythroid cells to survive the transition
from CFU-E/proerythroblasts to mature erythro-blasts, suggesting a clear role of EPO in
directing the survival of these cells.
In addition to erythroid cells, EPO affects megakaryocytes and their progenitors CFUs-MK. EPO
acts as a colony-stimulating factor for murine CFU-MK (273), whereas in humans, it potentates
the effect of megakaryocyte colony-stimulating factors present in lymphocyte-conditioned
medium (274). It also promotes the differentiation of murine megakaryocytes (275), which
express EPORs (276), and, when injected at high doses into mice, it increases platelet
production (277). In patients with end-stage renal disease treated with EPO, a minor increase in
the platelet count, averaging approximately 30,000/μl, has been noted (278). EPO does not
seem to contribute in a significant way to the regulation of platelet counts because anephric
patients can maintain a normal platelet count (278). Its various effects on megakaryopoiesis
and thrombopoiesis may be explained by the extensive homology between EPO and
thrombopoietin, the major humoral regulator of platelet mass (279).