Int J Physiol Pathophysiol Pharmacol 2016;8(1):28-34

www.ijppp.org /ISSN:1944-8171/IJPPP0027847

Review Article
Role of autophagy in megakaryocyte differentiation
and platelet formation
Tao You1,3,4, Qi Wang1,2, Li Zhu1,2,3
1
Cyrus Tang Hematology Center, 2Collaborative Innovation Center of Hematology, 3MOH Key Lab of Thrombosis
and Hemostasis, 4Jiangsu Institute of Hematology of The First Affiliated Hospital, Soochow University, Suzhou,
Jiangsu, P.R. China
Received March 30, 2016; Accepted April 8, 2016; Epub April 25, 2016; Published April 30, 2016

Abstract: Autophagy is a conserved biological process for digestion and recycling of cytoplasmic constituents in
eukaryotic cells. Autophagy may trigger cell death or promote cell survival following various forms of stress. The
emerging roles of autophagy in megakaryopoiesis, thrombopoiesis, and platelet function have been uncovered us-
ing not only in vitro and in vivo genetic models, but also in clinical observations of autophagic structure in patients
with thrombocytopenic disorders. Inhibition of autophagy in early stage of megakaryocyte differentiation appears to
impede megakaryocyte maturation, reduce platelet formation, and affect platelet function, whereas autophagic de-
ficiency in mature megakaryocytes gives rise to abnormal platelet activation and function without changing platelet
size and number. On the other hand, induction of autophagy by rapamycin in megakaryocytes exhibited substantial
therapeutic benefits in patients with immune thrombocytopenic purpura (ITP). This mini-review is to highlight recent
progresses in understanding the regulation of autophagy in megakaryopoiesis, thrombopoiesis and platelet func-
tion to bridge the gap between autophagy and megakaryocyte/platelet pathophysiology.

Keywords: Autophagy, megakaryopoiesis, thrombopoiesis, platelets

Introduction stress response by removing aggregated pro-

tein and recycling degraded products [6]. On
Autophagy is a conserved catabolic process in the flip side of the coin, autophagy may trigger
eukaryotic cells. During autophagy, targeted programmed cell death under certain condi-
cytoplasmic components undergo sequestra- tions [7]. The complete process of autophagy,
tion by double membrane vesicles called which includes activation, cargo identification,
autophagosomes, which then deliver their con- autophagosome formation, lysosome fusion
tents to lysosomes for degradation or recycle and degradation, is mediated by a series of
[1]. Since its discovery in 1960s, autophagy autophagy-related genes (ATGs) [8]. Over the
has been flourishingly investigated and com- past decades, the pleiotropic roles of autopha-
prehensively linked to various biological pro- gy in the development of hemocytes have been
cesses and pathological conditions. According characterized in erythropoiesis, as well as in
to different transportation and substrates, au- blood cancers [9, 10]. Recently, autophagy has
tophagy can be divided into three distinct forms been demonstrated to be indispensable for nor-
termed macroautophagy, microautophagy and mal megakaryopoiesis and platelet function
chaperone-mediated autophagy, of which mac- using animal models with lineage specific dele-
roautophagy consists of pexophagy, mitophagy tion of ATG [11, 12]. In addition, accumulating
and non-selective autophagy [2]. evidence of autophagy has hitherto been noted
in ITP, myelodysplastic syndromes, and chronic
The crucial roles of autophagy have been impli- myelogenous leukemia since the initial discov-
cated in nutrient starvation, infection, cell ery of putative autophagic vacuoles from mega-
death and repair [3-5]. Autophagy is consid- karyocytes in 1970s [13-15]. In the present
ered to play a mainly protective role in cellular review, we will first discuss the molecular basis
 Autophagy in megakaryocyte and platelet

of the autophagic machinery briefly and then dylinositol 3-kinase VPS34 (Vsp34), to initiate
move on to more specific regulation of mega- the formation of autophagosomes [28]. Once
karyopoiesis and thrombopoiesis by autopha- Vsp34 is activated, the generation of phospha-
gic modulators, followed by touching upon the tidylinositol 3-phosphate (PtdInS(3)P) will re-
clinical relevance of autophagic regulation and cruit WD repeat domain phosphoinositide-in-
thrombocytopenic disorders. teracting protein 2 (WIPI2) to the surface of the
phagophore through binding with ATG16L [29].
Autophagic signaling Concurrently, ATG12 and ATG5 bind to ATG16L
thus forming an E3-like complex, which then
The process of autophagy was initially discov- binds to ATG3 and promotes autophagosome
ered by Porter et al in 1962 from glucagon nucleation. On the other hand, activated ATG3
treated liver cells, in which lysosomes contain- covalently binds to LC3, which is lipidated by
ing other organelles were observed [16]. ATG16L and conjugates with PE on the mem-
Further discoveries in injury response and recy- brane of autophagosomes [30]. During this
cling/degradation of cellular constituents led to step, p62 pinpoints specific organelles as a
the invention of the term “autophagy” by de cargo docking receptor, as well as guiding LC3
Duve [17]. Amongst all the three types of auto- into autophagosomes [31]. Eventually, the
phagy identified, macroautophagy is the canon- autophagosomes fuses with the acidic lyso-
ical pathway that has been most extensively somes to form autolysosome, from which the
studied [18]. Damaged organelles are subject- outside LC3 molecules are conversed to the
ed to sequestration by double layered autopha- cleaved form by ATG4, while the inner LC3 and
gosomes that subsequently deliver their con- cargos undergo degradation [32].
tents for acidic hydrolysis and degradation by
fusing with lysosomes [2]. Microautophagy, to Recently, the roles of autophagy in hematopoi-
some extent, resembles macroautophagy in esis have been recognized. Several studies
spite of direct engulfment of intracellular con- demonstrate that autophagy is involved in both
stituents by lysosomes [19]. On the other hand, megakaryopoiesis and erythropoiesis, for ins-
chaperone-mediated autophagy operated in a tance, in differentiation of reticulocytes to red
quite different manner involving the hsc70-con- blood cells, as well as in thrombopoiesis [11,
taining complex with high selectivity for sub- 33, 34]. Induction of autophagy is associated
strates [20]. with cell death and differentiation in chronic
myeloid leukemia cell line, when the efficacy of
Autophagy-related genes (ATGs) are the key induction of autophagy has been tested in th-
regulators of autophagic signaling. Originally rombocytopenic disorders [35, 36].
cloned from the yeast Saccharomyces cerevisi-
ae, the homologues of ATGs in mammalian Megakaryopoiesis and thrombopoiesis
cells have been identified, and their functions
unveiled [21, 22]. In mammals, nutritional depri- Megakaryopoiesis is a complicated process
vation, growth factors, and oxidative stress can mediated by different hematopoietic cells and
regulate autophagy through AMP-activated pro- extracellular factors. Hematopoietic stem cells
tein kinase (AMPK) and mammalian target of are committed to megakaryocyte lineage, inc-
rapamycin (mTOR) [23, 24]. In the setting of luding mixed progenitor colony (CFU-Mix), or
starvation, increased ratio of AMP/ATP acti- common myeloid progenitor (CMP), colony-
vates AMPK, which then induces cellular pro- forming unit-granulocyte-erythrocyte-monocy-
tective autophagy through inhibition of mTORC1 te-megakaryocyte (CFU-GEMM), mixed MK/ery-
[25]. UNC-51 like kinase 1 (ULK1), focal adhe- throid progenitor cell (MEP), colony-forming
sion kinase family interacting protein of 200 kD unit-megakaryocyte (CFU-MK), burst-forming
(FIP200), ATG101 and ATG13 form a protein unit-megakaryocyte (BFU-MK), and eventually
complex with mTORC1 that is an inhibitor of differentiated mature megakaryocytes [37].
autophagy [26]. Increased formation of this The maturation of MKs is characterized by en-
complex following dephosphorylation of ATG13 domitosis, cytoplasmic maturation, and assem-
induced by metabolic stress or rapamycin pro- bling of all constituents required for the produc-
motes autophagy [27]. Similarly, activated tion of functional platelets. Mature megakaryo-
UKL1 phosphorylates Beclin-1, which forms a cytes can be identified by specific cell sur-
complex with ATG14L, P150, and phosphati- face markers including CD41, CD61 (integrin

29 Int J Physiol Pathophysiol Pharmacol 2016;8(1):28-34

 Autophagy in megakaryocyte and platelet

to the vascular niches and

increases platelet produc-
tion [48]. Recently, the
gradients of reactive oxy-
gen species are also impli-
cated in megakaryocyte
maturation [49].

Upon maturation, mega-

Figure 1. Inhibition of autophagy in
hematopoietic stem cell impairs mega-
karyocytes acquire all nec-
karyopoiesis and platelet function. Inhi- essary cellular constitu-
bition of autophagy by genetic deletion ents for thrombopoiesis,
of Atg7 in mice at different stages of which are demarcated in-
lineage development impairs mega- to micro anucleated parti-
karyocyte differentiation, as well as
platelet function. MK-PPF, Proplatelet-
cles prior to shedding [50].
forming megakaryocyte. Atg7 KO, Atg7 Meanwhile, the cell mem-
knockout. brane and cell skeleton
including tubulin and actin
undergo extensive rear-
αIIbβ3), CD42 (glycoprotein Ib) and glycopro- rangement, which allows the protrusion of mas-
tein V [38]. The engagement of transcription sive pseudopodial projections termed proplate-
factors, cytokines and extracellular stress syn- lets [51, 52]. Eventually, these proplatelets
ergically promotes the maturation of megakary- dispatch from megakaryocytes with their con-
ocytes [39]. Transcription factors, such as SCL, stituents, thus giving rise to circulating plate-
GATA1, GATA2, NF-E2, allows the development lets. The dispatching of platelets from mature
of megakaryocyte/erythroid progenitor cells megakaryocytes is termed thrombopoiesis. Of
[40]. FOG1 (ZFPM1) regulates the transcription- note, apoptosis has been shown to be involved
al activity of GATA-1 and contributes to early in the eventual stage of platelet release [53].
megakaryocyte differentiation, when NFE2 and
SCL-1 regulate the later differentiation of mega- Roles of autophagy in megakaryocyte matura-
karyocytes and production of platelets [41, 42]. tion and platelet function
Moreover, PU.1 and Fli-1 also guide the differ-
There is substantial evidence supporting the
entiation of megakaryocytes from progenitor
roles of autophagy in megakaryopoiesis. In
cells [43]. In contrast, C-myb (MYB) balances
1970s, TEM (Transmission Electron Micros-
with GATA1 by playing an inhibitory role in
cope) studies from patients with carcinoid syn-
megakaryopoiesis [44]. Besides transcription
drome identified autophagosome-like struc-
factors, the differentiation of megakaryocytes
tures inside platelets but not megakaryocytes,
is also fine-tuned by hormones, especially th-
indicating the possible involvement of increased
rombopoietin. By interaction with its receptor
autophagy [13]. A later study in dogs with burn
c-MPL, thrombopoietin acts as the canonical
injury revealed the presence of autophagocyto-
potentiator of differentiation of megakaryo-
sis in megakaryocytes, which was considered
cytes and thrombopoiesis [45]. Activation of
as potential cell recycling process [54]. Evi-
downstream signals consisting of MAPK, PI3K, dence of activated autophagy is also noticed in
and STATs, works in concert to promote mega- ITP, which exhibits extensive cytoplasmic vacu-
karyopoiesis [46]. Bone marrow niches provide oles representing programmed cell death. Ne-
platforms for megakaryocytes and platelet vertheless, these vacuoles observed appear to
development by supplying gradients of oxygen, be with single membrane rather than with dou-
chemokines, and infrastructures for megakary- ble membranes resembling autophagosomes
ocyte migration [47]. Additionally, megakaryo- [15].
cyte migration is regulated by stromal cell-
derived factor-1α (SDF-1), angiopoietin 1 (Ang- The inhibitor of autophagy, mTORC1, has been
1), and TPO, etc. SDF-1α navigates terminal reported to regulate both early and late steps
megakaryocytes towards the vascular endothe- of megakaryocyte development [55]. Additio-
lium, when activation of VEGFR1 promotes nally, inhibition of mTORC1 with rapamycin
SDF-1 mediated migration of megakaryocytes induces autophagy, decreases the size and

30 Int J Physiol Pathophysiol Pharmacol 2016;8(1):28-34

 Autophagy in megakaryocyte and platelet

ploidy of megakaryocytes, and impedes the study may also suppress P70S6K and 4E-BP
maturation of megakaryocytes through a p21 pathways. In addition to megakaryocytes, fur-
and cyclin D3 dependent manner [56]. These ther studies in platelets confirmed the pres-
findings suggest an important role of autopha- ence of autophagic proteins and showed that
gy in the regulation of megakaryocyte develop- class III PtdIns3K dependent autophagy was
ment. Of note, both mTORC1 containing ra- required for normal platelet function [60].
pamycin-associated TOR protein (Raptor) and
mTORC2 containing rapamycin-insensitive com- Closing remarks
panion of Raptor (Rictor) are involved in the
regulation of cell cycle [57]. However, it can be Autophagy, as a conserved biological process,
difficult to conclude that autophagy directly has been well studied and associated with can-
regulates megakaryopoiesis due to the non- cer, metabolic disorders, autoimmune disease,
specificity of pharmacological approach such and radiation damage. Altered autophagy is
as rapamycin. In that case, genetic approaches implicated in hematopoietic and blood cells
using gene knockout mice were developed to indicated by morphological studies. Given that
underscore the role of autophagy. Consequently, most evidence of autophagy hitherto is from
a recent study demonstrated that abrogation of cancer cells, the explicit function of autophagy
autophagy from stem cell stage by hematopoi- in megakaryocytes and platelets remains to be
etic knockout of ATG7 lead to impaired mega- elucidated. Fortunately, recent genetic studies
karyopoiesis, thrombopoiesis and hemostasis, uncovered the indispensable role of autophagy
producing larger but fewer dysfunctional plate- in both megakaryopoiesis and platelet func-
lets [11]. However, deletion of ATG7 in mature tion. In addition, results from a clinical trial on
megakaryocytes and platelets using PF4-driven ITP suggest that rapamycin is effective for
crew method only result in abnormal hemosta- treating immune-induced thrombocytopenia.
sis while platelet number and size remain Thus, targeting autophagy may yield a promis-
unchanged. Further investigation demonstrat- ing approach for thrombocytopenic disease, for
ed abnormal aggregation and cargo granule example, in MDS or secondary to chemo-/
packing in these platelets [12]. In light of these radiation-therapy.
findings, autophagy is likely to be indispensable
for the early stage of megakaryocyte develop- Acknowledgements
ment, and is required for normal platelet func-
tion as well (Figure 1). This work was supported by the grants from
Natural Science Foundation of China [grant
Being a chronic myelogenous leukemia (CML) 81370373, 81170132, 91439112 to LZ]; the
cell line, K562 retains the capability of mega- Priority Academic Program Development of
karyocytic differentiation, thus providing a Jiangsu Higher Education Institutions (PAPD) of
favorable tool to study megakaryopoiesis. One China [to LZ].
group reported that inhibition of autophagy in
K562 cells by knockdown of autophagic genes Disclosure of conflict of interest
substantially impedes megakaryopoiesis [58].
Consistently, Lapatinib treatment induces auto- None.
phagic cell death and megakaryocytic differen-
Address correspondence to: Li Zhu, Cyrus Tang
tiation in K562 cells, both of which can be
inhibited by knockdown of ATG7 or application Hematology Center, Soochow University, Rm 509,
of 3-MA [35]. In contrast, another group showed Bldg 703, 199 Ren’ai Road, Suzhou, Jiangsu
that although autophagy was readily observed 215123, P.R. China. Tel: 86 15195682930; Fax: 86
during induction of megakaryocytic differentia- 512 65882122; E-mail: [email protected]
tion by 12-O-tetradecanoyl-phorbol-13-acetate
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