Thrombopoiesis

Kenneth Kaushansky

The production of platelets is a complex process that involves hematopoietic stem cells (HSCs), their
differentiated progeny, the marrow microenvironment and hematopoietic cytokines. Much has been learned
in the 110 years since James Homer Wright postulated that marrow megakaryocytes were responsible for
blood platelet production, at a time when platelets were termed the “dust of the blood”. In the 1980s a
number of in vitro culture systems were developed that could produce megakaryocytes, followed by the
identification of several cytokines that could stimulate the process in vitro. However, none of these cytokines
produced a substantial thrombocytosis when injected into animals or people, nor were blood levels inversely
related to platelet count, the sine qua non of a physiological regulator. A major milestone in our
understanding of thrombopoiesis occurred in 1994 when thrombopoietin, the primary regulator of platelet
production was cloned and initially characterized. Since that time many of the molecular mechanisms of
thrombopoiesis have been identified, including the effects of thrombopoietin on the survival, proliferation,
and differentiation of hematopoietic stem and progenitor cells, the development of polyploidy and proplatelet
formation, the final fragmentation of megakaryocyte cytoplasm to yield blood platelets, and the regulation of
this process. While much progress has been made, several outstanding questions remain, such as the nature of
the signals for final platelet formation, the molecular nature of the regulation of marrow stromal
thrombopoietin production, and the role of these physiological processes in malignant hematopoiesis.
Semin Hematol 52:4–11. C 2014 Elsevier Inc. All rights reserved.

THE HISTORY OF THROMBOPOIESIS facilitating additional purification attempts; however,

while some claims were made of its biological activities,
Carnot, Wright, and others in the early 20th century
attempts to produce a cDNA for thrombopoietin failed.
defined the critical role of platelets in blood coagulation,
Occasionally in science, a finding from one field,
and their origin from the marrow megakaryocyte based on
although in itself important, can have a catalytic and
elegant camera lucida images. Based on an evolving
profound effect on a seemingly unrelated area of research.
understanding of erythropoiesis, particularly the identifi-
The discovery and characterization of the murine myelo-
cation of erythropoietin as the humoral regulator of
proliferative leukemia virus (MPLV) had such an impact on
erythrocyte production, Keleman coined the term “throm-
the search for thrombopoietin. MPLV causes an acute
bopoietin” in 1958 to describe the humoral substance
myeloproliferative neoplasm in infected mice2; in 1990,
responsible for platelet production.1
the responsible oncogene (v-mpl) was cloned, and the proto-
In the mid-1960s and 1970s, several groups attempted
oncogene (c-Mpl) obtained 2 years later.3,4 Based on the
to purify thrombopoietin from the plasma of thrombocy-
predicted structure of the encoded protein it was immedi-
topenic animals; these early efforts were severely handi-
ately evident that c-Mpl encodes a member of the
capped by inconvenient and insensitive assays for the
hematopoietic cytokine receptor family, which includes the
hormone, the in vivo incorporation of radiolabeled
receptors for erythropoietin, granulocyte colony-stimulating
methionine into newly formed platelets, and the attempts
factor, granulocyte-macrophage colony-stimulating factor,
failed to produce unequivocal proof of the existence of
growth hormone, prolactin, and several interleukins (ILs).
thrombopoietin. In the 1980s a number of in vitro
However, when “c-Mpl receptor” was cloned, the corre-
megakaryocyte differentiation assays were developed,
sponding “c-Mpl ligand” was unknown. Based on the cell
from which the receptor was cloned, the bipotent erythroid/
School of Medicine, Stony Brook University, Stony Brook, NY. megakaryocytic cell line HEL, we, and others, postulated
Some of the work mentioned in this paper was supported by the
National Institutes of Health Grant No. R01 DK49855. that the c-Mpl ligand might be thrombopoietin.
Conflicts of interest: none.
Address correspondence to Kenneth Kaushansky, MD, MACP, Stony
Brook University, School of Medicine, Health Sciences Center
4-225, Stony Brook, NY, 11794-8430. E-mail: kenneth. THE CLONING AND CHARACTERIZATION OF
kaushansky@stonybrookmedicine.edu THROMBOPOIETIN
0037-1963/$ - see front matter
& 2014 Elsevier Inc. All rights reserved. Three distinct approaches yielded cDNA for thrombo-
http://dx.doi.org/10.1053/j.seminhematol.2014.10.003 poietin. Using an in vitro megakaryocyte-based assay, and

4 Seminars in Hematology, Vol 52, No 1, January 2015, pp 4–11

Thrombopoiesis 5

the plasma from 1,100 thrombocytopenic rats, scientists at And culture of marrow megakaryocytes in thrombopoietin
Kirin Pharmaceuticals employed a 12-step conventional leads to pronounced polyploidy.15 However, the final
purification scheme to obtain sufficient purified thrombo- stages of platelet formation and release appear to be
poietin to obtain amino acid sequence, and then cloned thrombopoietin-independent, as withdrawal of the hor-
cDNA for rat and then multiple species of thrombopoie- mone from late-stage megakaryocyte cultures does not
tin, including the human hormone.5 Using the c-Mpl eliminate proplatelet formation; in fact, thrombopoietin
proto-oncogene product coupled to an affinity matrix, withdrawal is reported to stimulate it.16
scientists at Genentech obtained sufficient purified porcine Also at odds with the prevailing conventional wisdom on
Mpl ligand to allow amino acid sequencing and cDNA thrombopoietin, in addition to its effects on megakaryocytic
cloning6 of ovine and human hormones. In contrast to the progenitors and mature cells, thrombopoietin affects
biochemical purifications utilized by these groups, an hematopoietic stem cells (HSC) in vitro, especially when
expression cloning strategy using a chemically mutated used in combination with IL-3 or SCF17,18. Numerous
c-Mpl-bearing cell line was used by Lok and Kaushansky studies reported the expression of c-Mpl on the surface of
to obtain cDNA for murine and then human thrombo- HSCs19,20, indicating the stem cell effects of thrombopoie-
poietin.7,8 Initial in vitro experiments using the corre- tin are direct. And based on these results, thrombopoietin
sponding recombinant proteins demonstrated the effect of has been included in many ex vivo cytokine cocktails
thrombopoietin on megakaryocyte maturation, and injec- designed to expand HSCs for therapeutic use.21,22
tions into normal mice resulted in impressive increases in More recently, an intriguing paracrine role for throm-
marrow megakaryocytes and peripheral blood platelet bopoietin/c-Mpl in maintaining quiescent Tie2þ HSCs at
counts.8 the osteoblastic niche has been identified23. Osteoblasts
The cloned human thrombopoietin cDNA encodes a were found to release thrombopoietin, supporting the
polypeptide of 353 amino acids, including the 21 amino survival and quiescence of HSCs; inhibition of this
acid secretory leader sequence7; the mature protein con- interaction reduced the number of HSCs at the osteo-
sists of two domains. The amino-terminal 154 residues are blastic niche. Another mechanism by which thrombopoie-
homologous to erythropoietin, which like other members tin affects HSCs is by promoting DNA repair,24 a finding
of the hematopoietic cytokine family displays a four helix that could eventually be clinically translated to HSC
bundle fold,9 and binds to the c-Mpl receptor. The “protection” from genotypic damage during ionizing
carboxyl-terminal domain of thrombopoietin bears no radiation or chemotherapy.
resemblance to any known proteins, and acts to prolong
the circulatory half-life of the hormone10; it also serves as
In Vivo Thrombopoietic Effects of
an intramolecular chaperone, aiding in the proper folding
of the polypeptide into the mature hormone.11 Hematopoietic Cytokines
Once recombinant thrombopoietin was available puri-
fied protein was tested in a range of experimental animals.
THE BIOLOGICAL ACTIVITIES OF
The initial results were remarkable; within 5 days of daily
THROMBOPOIETIN
administration of subnanogram quantities of recombinant
The availability of the recombinant protein allowed the murine “c-Mpl ligand”, mouse platelet counts were
first detailed studies of the biological properties of throm- quintupled.8 These initial experiments made obvious the
bopoietin. Previous conjecture was that the hormone was fact that the c-Mpl ligand obtained by the multiple groups
solely a megakaryocyte differentiation factor, driving the was thrombopoietin, as prior studies of the administration
maturation of megakaryocytes and platelet formation, but of other megakaryocytic factors (e.g. IL-6, IL-11) would
had no effect on immature cells of the lineage or other result in, at most, a 50% increase in blood platelet counts.
hematopoietic cell types. Initial studies with recombinant That thrombopoietin is the primary physiological regu-
thrombopoietin dispelled many of these incorrect assump- lator of thrombopoiesis was made clear by genetic studies;
tions. Thrombopoietin alone is able to stimulate the the generation of mice engineered to lack either Thpo or
proliferation of nearly all marrow megakaryocytic progenitor c-Mpl resulted in a 85-90% reduction (although not
cells (colony-forming unit, megakaryocyte [CFU-MK]) complete elimination) of platelet counts, and its blood
in vitro, and acts in synergy with other hematopoietic levels were inversely related to platelet count25,26. That the
cytokines, such as IL-3, IL-11, and stem cell factor in vitro HSC effects of thrombopoietin were physiological
(SCF)12 to promote the growth of CFU-MK. In vitro, was then demonstrated when competitive repopulation
thrombopoietin acts to increase megakaryocyte size and assays were performed on the c-Mpl27, revealing an
expression of lineage-specific megakaryocyte surface pro- approximate 8-fold reduction in HSC activity of marrow
teins, such as glycoprotein (GP)Ib and GPIIb/IIIa.8,13 cells when compared to normal mouse marrow. Likewise,
Studies of megakaryocyte ultrastructure show increased transplantation of normal HSCs into lethally irradiated
demarcation membrane and platelet granule formation normal recipient mice resulted in a 15-20 fold greater
following culture with thrombopoietin, indicating that the increase in post-transplant stem cell expansion compared
hormone primes megakaryocytes for platelet production.14 to transplantation into Thpo-/- recipients.28 And the final
6 K. Kaushansky

proof of the critical, non-redundant role of thrombopoie- microenvironmental “niches”, the osteoblastic niche35 and
tin in HSC biology came from “experiments of nature”; the vascular niche.36 Hematopoietic stem and progenitor
children born with congenital amegakaryocytic thrombo- cells localize to both, but the functional effects on cells at
cytopenia, who nearly always progress to aplastic anemia these distinct locations differ. Megakaryocyte maturation
(stem cell depletion), almost universally display homozy- and platelet formation are dependent on cellular migration
gous, or compound heterozygous inactivating mutations of from the osteoblastic to the vascular niche. At this latter
c-Mpl,29 or far more rarely, Thpo. site, once adequately mature, megakaryocytes extend
As noted above, elimination of c-Mpl or Thpo reduces proplatelet processes through or between cells of the
thrombopoiesis to about 10-15% of normal. A number of sinusoidal endothelial layer and shed platelets into the
investigators have attempted to determine the origins of the bloodstream.37
remaining platelet production. Given a modest effect of Marrow stromal cells are responsible for crafting these
IL-3, GM-CSF, oncostatin-M, IL-6 and IL-11 on mega- local microeenvironments, through their expression of
karyopoiesis in vitro and in vivo, a number of studies soluble and surface bound cytokines, counter-receptors
creating double knock-outs of c-Mpl and these other for integrins and other adhesion molecules on the surface
cytokines or their receptors have been performed. The of hematopoietic cells, and through their secretion of
combined reductions failed to reduce platelet levels below extracellular macromolecules. A number of marrow cell
that seen with c-Mpl deficiency alone. Based on the synergy types elaborate matrix molecules, including osteoblasts,
shown between thrombopoietin and erythropoietin in vitro, endothelial cells, fibroblasts, adipocytes, CXCL12-
we also genetically combined c-Mpl deficiency and Epo abundant reticular (CAR) cells (which surround sinusoidal
receptor deficiency; we also found that the combined endothelial cells), and even hematopoietic cells such as
elimination failed to further reduce thrombopoiesis over macrophages and megakaryocytes.38 In specific reference
that seen in c-Mpl deficient mice. However, the combination to megakaryocyte development, marrow stromal cells have
of CXCL12 (previously termed stromal derived factor 1) and been shown to secrete thrombopoietin,39,40 the primary
fibroblast growth factor (FGF)-4, two cytokines that promote regulator of thrombopoiesis, CXCL12,41 which is a
megakaryocyte homing to the vascular niche in marrow, were primary chemokine attracting megakaryocytes and other
found to restore thrombopoiesis in c-Mpl or Thpo null mice, hematopoietic cells to the marrow microenvironment, and
unlike IL-6 or IL-1130. The investigators proposed that cells acts to stimulate megakaryocyte growth,42 to express cell
or substances in the vascular niche was responsible for the surface-bound SCF,43,44 which acts in synergy with
favorable effects of the two cytokines. thrombopoietin to promote megakaryocyte growth,45
Despite significant advances in understanding the role of and to express VCAM-1 and fibronectin, which bind to mega-
thrombopoietin in HSCs, progenitors and megakaryocytes, karyocyte integrin α4β1 and promote cell growth.46,47
the physiologically relevant effects of thrombopoietin and Different fragments of microenvironmental fibronectin
c-Mpl on platelet function remains somewhat elusive. have been shown to differentially support erythroid, or
Platelets express c-Mpl and the molecular machinery required megakaryocytic progenitors47,48, potentially providing a
for thrombopoietin signal transduction, including; JAK2, mechanism for enhancing or suppressing thrombopoiesis
STAT3, STAT5, Akt and Ras (see below). Superphysiolog- depending on the animal’s blood cell needs. Platelet-
ical amounts of thrombopoietin (4100ng/ml) directly trigger endothelial cell adhesion molecule (PECAM)-1 plays a
platelet aggregation in vitro (reviewed in31), whilst more critical role in megakaryocyte migration, as its genetic
physiological concentrations of the hormone prime platelets elimination blunts cell migration in response to
for stimulation with other agonists, possibly by increasing CXCL12.49 And the interaction of microenvironmental
activity of Ras32. Thrombopoietin also has significant effects VWF and its megakaryocyte receptor glycoprotein Ib/V/
on platelet adhesion under flow. Low thrombopoietin IX appears particularly important for proplatelet forma-
concentrations (0.01-1ng/ml) accelerate firm platelet adhe- tion. In contrast, type I collagen, which localizes to the
sion to von Willebrand factor and prevent de-attachment at osteoblastic niche, prevents platelet formation, where
higher flow rates, suggesting that thrombopoietin may be megakaryocyte proliferation is favored over maturation
important in thrombus formation33. However, despite many into platelets.50
years of clinical trials, and current clinical use, only a single
publication reports an excess of thrombosis in patients treated
with thrombopoietin or thrombopoietin receptor agonists, Regulation of Thrombopoiesis
and that was found in patients with severe liver disease While a number of hematopoietic cytokines can
undergoing invasive and vascular procedures.34 stimulate megakaryocyte growth in vitro (IL-3, GM-
CSF, SCF), and when injected in vivo some produce
modest increases in blood platelet counts (e.g. IL-6, IL-11),
The Thrombopoietic Marrow
the blood levels of these proteins do not vary with platelet
Microenvironment count except for IL-6 (see below on inflammation). In
Evidence from many sources has established the exis- contrast, plasma concentrations of thrombopoietin vary
tence of two distinct anatomical and functional marrow inversely with the platelet count in patients with reduced
Thrombopoiesis 7

platelet production.51 Multiple organs display RNA tran- intrinsic kinase activity; instead it recruits and directly
scripts for thrombopoietin, being present at highest levels associates with the cytoplasmic kinase JAK2 to mediate
in the liver in normal animals.6,7 One model that accounts phosphorylation and activation of downstream signaling
for the regulation of blood thrombopoietin levels is based proteins. JAK2 associates with c-Mpl prior to the receptor
on the capacity of platelets to adsorb thrombopoietin from being trafficked to the membrane; in fact, the c-Mpl/JAK2
solution, internalize and destroy it52; in patients with association stabilizes expression of the receptor, increasing
thrombocytosis, the steady-state level of thrombopoietin the presence of c-Mpl at the membrane.60
production is overwhelmed by platelet-mediated destruc- Extensive work using both c-Mpl-expressing cell lines
tion, and so levels are low, and thrombopoiesis is low; in and primary marrow cells has identified a variety of
contrast, in thrombocytopenic patients, with little of the different intracellular proteins activated and squelched
hepatic thrombopoietin production removed by platelets, following thrombopoietin engagement of c-Mpl. Due to
blood levels of the hormone to rise, driving increased the relatively close homology between c-Mpl and the
thrombopoiesis. Additional support for this model comes erythropoietin receptor, initial studies focused on the JAK/
from Thpoþ/- mice;26 loss of one allele of Thpo leads to a STAT pathway and identified JAK2 and TYK2 as the
40% reduction in platelet counts. immediate kinases that bind to c-Mpl and become
In addition to this “autoregulation” model, a growing activated following thrombopoietin binding,61,62 leading
body of evidence suggests other mechanisms regulate to the activation of the transcription factors STAT3 and
thrombopoietin production. At baseline, it is very difficult STAT5.63 Thrombopoietin also stimulates the phosphor-
to detect specific mRNA in marrow stromal cells. How- ylation and formation of the Shc-Grb2-SOS adaptor
ever, transcript levels are substantially increased in marrow protein complex,64,65 activates the phosphatases SHIP
stromal cells in response to thrombocytopenia.39,53 The and SHPTP-2, and both the phosphoinositide-3-kinase
mechanism underlying this response is only beginning to (PI3K)/Akt66,67 and Raf-1/MAP kinase pathways.68
be understood. It is known that platelet derived growth The activation of both PI3K and MAPK are instru-
factor (PDGF)-BB and fibroblast growth factor (FGF)-2 mental in mediating many effects of thrombopoietin on c-
stimulate, and platelet factor 4, thrombospondin and Mpl bearing cells. A number of additional transcription
transforming growth factor (TGF)-β inhibit thrombopoie- factors are activated in stem cells and megakaryocytes in
tin production from cultures of marrow stromal cells;54 on response to thrombopoietin. The Hox genes were first
balance, whole platelet extracts suppresses thrombopoietin recognized for their effects on body pattern development,
production. but were subsequently shown important in a number of
In addition to marrow stromal cell thrombopoietin pro- mature cell settings. HoxB4 and HoxA9 were shown to
duction, a number of inflammatory states (e.g. ulcerative influence the levels of HSCs.69,70
colitis, rheumatoid arthritis) are associated with thrombo- Thrombopoietin induced PI3K and MAPK lead to the
cytosis, and increased thrombopoietin levels.55,56 The synthesis of HoxB4,71 and the nuclear translocation of
inflammation-induced increase in thrombopoietin expres- HoxA9,72 helping to explain the effect of the hormone on
sion is mediated by IL-6, which stimulates hepatocyte HSCs. The cytokine can also stimulate expression of
thrombopoietin production both in vitro and in vivo.57,58 hypoxia inducible factor,73 a transcription factor critical
A final new model that helps explain the regulation of for the expression of vascular endothelial cell growth
blood thrombopoietin levels, and hence thrombopoiesis, is factor, which also influences stem cell expansion.74 In
platelet binding to the hepatic Ashwell-Morrell receptor, addition to these molecular pathways that underlie the
which triggers enhanced hepatic thrombopoietin pro- favorable effects of the hormone on HSC expansion and/
duction (see: http://www.thsna.org/Presentation_Upload/ or survival, thrombopoietin also influences stem/progeni-
presentation_uploads/86_57_86_57_Hoffmeister_2014_ tor cell lineage fate determination. The relative level of
03_THSNA_CHICAGO.pdf). expression of the transcription factor c-Myb influences the
lineage choice of bipotent erythroid/megakaryocytic pro-
genitors.75 By influencing the level of microRNA (miR)-
The Molecular Mechanisms of Thrombopoiesis 150, which influences the stability of c-Myb mRNA and
c-Mpl is a member of the type I cytokine receptor its translational efficiency, thrombopoietin acts to favor
family, along with receptors for a number of interleukins, the megakaryocytic lineage.76
colony stimulating factors, growth hormone and erythro- One of the most obvious effects of thrombopoietin on
poietin. The receptors of this family are multimeric; either megakaryocytic progenitors is the induction and advance-
homo- or heterodimeric, or heterotrimeric59. The c-Mpl ment of endomitosis, resulting in a highly (32-128N)
receptor is a homodimer. The binding of cognate ligand to polyploid cell. Detailed videomicroscopy revealed that
these receptors induces a conformational change in the megakaryocytes replicate their DNA but abort mitosis in
multimeric receptor, which triggers a number of phos- mid anaphase,77 prior to cellular or nuclear division, and
phorylation events, including that of both the cytoplasmic do so over and again resulting in a highly polyploid cell.
domain of the receptor and its associated proteins. How- The small G protein RhoA is expressed by many cells
ever, type I cytokine receptors, including c-Mpl, lacks types, and is distributed throughout the cytoplasm during
8 K. Kaushansky

midphase of the cell cycle. During mitosis, RhoA becomes prevented, suggesting other E3 ubiquitin ligases may also
highly localized to the mitotic spindle, by virtue of it’s be involved.
activation by the guanine nucleotide exchange factors
GEF-H1 and ECT2. Endomitotic megakaryocytes express
abundant RhoA, but it fails to localize to the mitotic Remaining Questions
spindle77,78 as occurs in normal diploid cells. Recent Much has been learned about thrombopoiesis in the
studies have shown that endomitosis is critically dependent 100þ years since the origins of platelets from the marrow
on inactivation of GEF-H1 and ECT2, and hence, their megakaryocyte was first postulated, particularly in the
inactivation of RhoA.79 Consistent with this finding, 20 years since the cloning and initial characterization of
when RhoA was genetically eliminated in megakaryocytes thrombopoietin. However, many questions remain. For
and platelets; the resultant mice displayed enhanced example, are there other physiologically relevant “throm-
polyploidy.80 However, a direct link between thrombo- bopoietic substances”, or cytokines exclusively restricted to
poietin signaling and RhoA (in)activation has not yet been promoting the formation and release of platelets from
established. Obviously, additional studies will be required large, highly polyploid megakaryocytes? Do we now know
to fully understand the molecular mechanisms underlying all of the relevant signaling pathways employed by c-Mpl
megakaryocyte endomitosis. in transducing the myriad of signals sent to a cell by
Given its importance to hematopoiesis and the growth thrombopoietin? What is the molecular link between
promoting intracellular signaling pathways it activates, RhoA inactivation during megakaryocyte endomitosis
stringent regulatory mechanisms are required to ensure and thrombopoietin, if any? What are all the clinical roles
thrombopoietin signaling is tightly controlled. Two main that thrombopoietin receptor agonists are likely to play,
mechanisms exist by which thrombopoietin regulates its and are they truly thrombogenic in certain circumstances?
own activity; activation of negative regulators, and inter- And while the clear pathogenetic role of c-Mpl and its
nalization and degradation of its activated receptor. Of the downstream singling kinase, JAK2 in patients with mye-
proteins activated or upregulated in response to thrombo- loproliferative neoplasms was not discussed above, does
poietin, Lyn, Lnk and suppressors of cytokine signaling thrombopoietin contribute to mutant c-Mpl or mutant
(SOCS) have all been identified as mediating important JAK2 mediated disease? Only additional research will
negative feedback mechanisms. Inhibition of the Src definitively address these and other important physiolog-
family kinase Lyn, enhanced thrombopoietin -mediated ical, pathological and therapeutic questions.
ERK1/2 activation and proliferation in c-Mpl bearing cell
lines, and promoted megakaryocyte differentiation in bone
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