599 Schattauer 2012

Thrombosis and Haemostasis 108.4/2012

Theme Issue Article
MicroRNAs in platelet biogenesis and function
Seema Dangwal
1
; Thomas Thum
1,2

1
Institute of Molecular and Translational Therapeutic Strategies (IMTTS), Hannover Medical School, Hannover, Germany;
2
Centre for Clinical and Basic Research, IRCCS
San Raffaele, Rome, Italy
Summary
Platelets are important to maintain primary haemostasis and play a key
role in pathology of thrombotic and occlusive vascular disorders such as
acute coronary syndrome or stroke. Despite of lacking a nucleus and ge-
nomic DNA, platelets possess diverse types of RNAs, ranging from pro-
tein coding messenger RNAs to small non-coding RNAs inherited from
their parent megakaryocytes. Indeed, platelets are capable of using
their own translational machinery to synthesise proteins upon their ac-
tivation suggesting the possibility of post-transcriptional gene regu-
lation in platelets. MicroRNAs (miRNAs) are highly conserved, tiny non-
coding RNAs exhibiting a fine-tune control of protein expression by
Correspondence to:
Thomas Thum, MD, PhD
Institute of Molecular and Translational Therapeutic Strategies (IMTTS),
Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany
Tel.: +49 511 532 5273, Fax: +49 511 532 5274
E-mail: thum.thomas@mh-hannover.de
complementary sequence recognition, binding and translational re-
pression of protein coding mRNA transcripts. Multiple functional as-
pects of miRNAs as well as their expression in platelets or megakaryo-
cytes underscore a role in platelet biology. Changes in miRNA ex-
pression patterns have been noted during platelet genesis and acti-
vation. In the present review we highlight recently identified mega-
karyocytic/platelet miRNAs and discuss their role in platelet biogenesis
and functions essential to maintain haemostasis in the body.
Keywords
Platelet reactivity, megakaryocytopoiesis, platelet miRNA, P2Y12
Received: March 31, 2012
Accepted: May 3, 2012
Prepublished online: July 10, 2012
doi:10.1160/TH12-03-0211
Thromb Haemost 2012; 108: 599604
Introduction
Circulating platelets are crucial for coagulation physiology to
maintain haemostatic balance and safeguarding vascular integrity
(1, 2). In contrast, disturbed platelet reactivity is associated with
various pathologies such as atherosclerosis, occlusive or throm-
botic cardiovascular disorders, inflammation and cancer leading
to morbidity and mortality (36). Platelets are generated from par-
ent megakaryocytes through commitment of the multipotent stem
cells to the megakaryocyte lineage, proliferation of the progenitors
and terminal differentiation of megakaryocytes in a complex
microenvironment of bone marrow (7, 8). The process of megaka-
ryocyte differentiation is characterised by a few critical steps
namely DNA endoreduplication (polyploidy up to 128 N) and ex-
pansion of cytoplasmic mass leading to increase in cell size up to
50100 micron with simultaneous acquisition of all necessary
structural and functional characteristics of platelets followed by
release of cytoplasmic fragments (2,0005,000 fragments/cell) in
blood circulation as platelets (9). In steady state, megakariocyto-
poiesis supplies 10
11
platelets per day with a new turn over every
89 days. This process is dramatically influenced by environment-
al changes and may result in more than 10 fold higher productions
under stimulating situations (10).
Although platelets lack a nucleus or genomic DNA, they are able
to translate inherited mRNAs into proteins (11, 12). Platelets in
storage continue to synthesise integrin-
3
(glycoprotein-IIIa) pro-
tein, a fibrinogen receptor involved in platelet aggregation (12, 13).
A strong correlation between platelet transcriptome and pro-
teomic profile (14), in addition to inherited functional translation
machinery, e.g. rough endoplasmic reticulum, ribosomes and
small amount of poly(A) RNAs from parent megakaryocytes sup-
ports de novo translational capabilities of platelets and suggests the
exciting possibility of post-transcriptional regulation of gene ex-
pression within platelets (15).
MicroRNAs (miRNA) are small (21 23 nucleotides) non-
coding RNAs regulating about 60% of the mammalian protein
coding genes at least in part by translational repression (16). Since
the discovery of miRNA in C. elegans, miRNAs have shown to play
crucial role in development and various pathologies (1721). Most
miRNAs expressed in multi-cellular organisms show sequence
conservation throughout the animal and plant kingdom (16). Ex-
pression of miRNAs varies from 1 10,000 copies in cellular con-
tent (22) and whereas some miRNAs are ubiquitously expressed,
many show spatiotemporal expression patterns (20, 23). Interest-
ingly, platelets inherit essential miRNA processing proteins in ad-
dition to miRNA transcripts originated in their parent megakaryo-
cytes (14). The serial analysis of gene expression (SAGE) data has
revealed the average length of platelet transcripts (2.421 kb) is
slightly greater than that of nucleated cells (1.756 kb); however, the
3UTRs of platelets mRNAs (1,047 bases) are significantly longer
than mRNAs of the nucleated cells (492 bases) suggesting possi-
bilities for post-transcriptional regulation (24). This in fact indi-
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Thrombosis and Haemostasis 108.4/2012 Schattauer 2012
600 Dangwal, Thum: MicroRNAs and platelets
(14). A year later another study by Nagalla et al. reproducibly ident-
ified the platelet miRNA expression profiles reporting the mean
miRNA levels observed in their study to be strongly correlated with
that of the previous study by Landry et al. (32). Comparative analy-
sis of these studies revealed the differential expression of miRNA
subsets among platelets, megakaryocytes and neutrophils. In addi-
tion to mature miRNAs, detection of pre-miRNA transcripts for
some abundant platelet miRNAs (miR-223, let-7c and miR-19a) as
well as presence of core components of miRNA effector complex
i.e. Dicer and Ago2 suggested that partial biogenesis of mature
miRNA from pre-miRNA templates could take place directly in
platelets (14).
The Dicer function was confirmed by the conversion of a radio-
active Dicer substrate
32
P-labelled pre-Let-7a-3 into miRNA sized
small RNA species upon incubation with platelet extracts (14). The
ability of human platelet miRNAs to mediate gene specific silenc-
ing was further examined by incubating
32
P-labeled miR-223 sen-
sor transcript with platelets or megakaryocyte extracts in separate
RISC activity assays. Either platelet or megakaryocyte extracts led
to the accumulation of a 5 end labelled 39-nt RNA species in
agreement with suspected Ago2-mediated cleavage of the sensor
sequence at 10-nt from the 5 end of the small RNA guide. Ago2 as-
sociation with endogenous mature miR-223 was confirmed in
northern blotting of platelets, Ago2 immunoprecipitation and fin-
ally the regulatory function of the Ago2-miR-223 complex was
validated by investigating the gene expression of P2Y
12
, a puriner-
gic receptor subtype expressed on platelets and many other cell
types (3335). Platelet P2Y12 receptor is highly upregulated upon
activation influencing the platelet functions, therefore it serves as a
target for new anti-platelet drugs (33, 36, 37). Disruption of
miR-223 pairing to the P2Y12 3UTR impaired its ability to repress
P2Y
12
expression in a reporter gene assay performed in a miR-223
lacking HEK293 cell-line by co-expressing pre-miR-223 with a re-
porter construct containing P2Y
12
3UTR, either with intact or
mutated miR-223 binding site (14). These studies confirm the pla-
telet miRNA system to be functionally efficient in regulating pla-
telet proteins important for platelet activation.
MiRNAs in platelet biogenesis
Megakaryocyte development and platelet production are con-
trolled by the concert action of various transcriptional regulators
to specifically activate genes of the megakaryocyte lineage and re-
press those supporting other cell types (9). Since the first report of
miRNA regulation of megakaryocytopoiesis by Garzon et al., sev-
eral studies have addressed various functional and mechanistic as-
pects of miRNAs in megakaryocytopoiesis that consequently may
affect platelet production. A pictorial summary of miRNAs in-
fluencing platelet biogenesis and functions is presented in Fi-
gure 1, and various published miRNA targets are summarised in
Table 1.
Garzon et al. studied the miRNA fingerprints of human mega-
karyocytopoiesis in bone marrow-derived CD-34 positive pro-
cates that a system progressively losing its transcriptional capabil-
ities during the biogenesis and maturation of anucleate platelets
from the nucleated megakaryocytes should perhaps balance this
deficiency by enhancing translational control and mRNA stability
(24). Moreover, miRNA-mediated gene regulation is dependent on
binding site availability and occupancy on the 3UTR of target
mRNAs; miRNAs therefore are likely to modulate platelet func-
tions via robust regulation of protein translation therein.
Biogenesis and function of miRNAs
MiRNAs are generated either as dependent transcripts co-express-
ed with the host gene transcript utilising promoters of the host
protein-coding genes or standalone primary transcripts located in
intergenic regions of the genome using their own promoters (25).
This early transcript, primary (pri)-miRNA, is processed by the ri-
bonuclease-III enzyme Drosha and the double stranded RNA-
binding protein the DiGeorge syndrome critical region-8
(DGCR-8) into a short hairpin precursor (pre)-miRNA, and there-
after exported to cytosol by exportin-5 (16, 26, 27). In the cytosol,
digestion of pre-miRNA into small RNA molecules by endonu-
clease Dicer is followed by selective incorporation of one strand of
duplex miRNA in Argonaute family protein leading its assembly in
the RNA-induced silencing complex (RISC). MiRISC renders pro-
tection and stability to mature miRNA strand against RNase and
guides miRNA to its target mRNA (28).
MiRNAs are not translated into a protein but modulate the pro-
tein synthesis by recognition of binding sites on 3UTR of protein
coding gene transcripts harbouring a sequence (28 nt) comple-
mentary to miRNA seed sequence (29). MiRNA-mRNA binding
follows the Watson-Crick base pairing but does not always require
a full complement homology (29). The complete homology
usually initiates degradation of target mRNA, whereas the partial
complementarity leads to the translational repression. MiRNA-
mediated degradation and therefore on/off switching of functional
proteins is more common in lower vertebrates than in mammals,
where the gene translation is mostly repressed by miRNA to fine-
tune control over the functional protein expression (30, 31). This
moderate control on gene expression by miRNA presumably sug-
gests a wide scope of miRNA-mRNA interactions following either
divergent (single miRNA- many targets) or convergent (many
miRNAs- single target) pathways resulting in a robust regulation of
protein synthesis in living cells (31).
Platelet miRNAs: Proof of functional
existence
A comprehensive study that investigated platelet miRNA signa-
tures was published by Landry et al. in 2009. The study reported a
huge number of miRNAs (total 219) to be expressed in platelets as
shown by locked nucleic acid (LNA) based microarray profiling
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Schattauer 2012 Thrombosis and Haemostasis 108.4/2012
601 Dangwal, Thum: MicroRNAs and platelets
genitor cells and reported a strong downregulation of 19 miRNAs
predicted to target transcription factors during megakaryocytic
differentiation using microarray, Northern blots and quantitative
real-time PCR (38). Specifically MAFB, a transcription factor, and
HOXA1 gene which were also upregulated during megakaryocyte
differentiation, were confirmed as target of miR-130a and
miR-10a, respectively (Table 1). Apparently, both miR-130a and
miR-10a were upregulated in acute megakaryoblastic leukemia
cell-line retaining the dedifferentiated state (38). Similarly
miR-34a, known to stimulate megakaryocytopoiesis by enhancing
megakaryocyte colony formation from CD-34 positive haemato-
poietic stem cells (HSCs), was increased during stimulated mega-
karyocytic differentiation in K-562 cells (39, 40).
Another miRNA, miR-155 was downregulated upon mega-
karyocytic differentiation of CD-34 positive HSCs (Fig. 1) (41,
42). A computational transcriptome analysis of HSCs obtained
from healthy human individuals predicted miR-155 mediated re-
pression of an mRNA cluster regulating myelopoiesis (41). RNA
transcripts coding transcription factors ETS-1 and MEIS-1 which
stimulate megakaryocyte gene expression are among the published
targets of miR-155 (42). Moreover, miR-155 is also reported to re-
sist megakaryocyte development in different studies (42, 43). The
overexpression of miR-155 impaired megakaryocyte proliferation
in vitro (42) and similarly, in vivo transplantation of miR-155 over-
expressing HSCs into irradiated mice showed suppressed mega-
karyocyte counts in their bone marrow (43).
Apart from inhibiting megakaryocytic differentiation, miRNAs
were also reported to stimulate megakaryocytopoiesis (44). For in-
stance, megakaryocytic erythrocytes progenitors (MEPs) show in-
creased levels of miR-150 along with megakaryocytic lineage dif-
ferentiation. Overexpression of miR-150 enhanced megakaryocyte
differentiation in vitro and in vivo with simultaneous suppression
of erythroid differentiation suggesting a differentiation switch.
MiR-150 upregulation by thrombopoietin, a primary physiologi-
Figure 1: miRNAs influencing platelet bio-
genesis and functions.
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Thrombosis and Haemostasis 108.4/2012 Schattauer 2012
602 Dangwal, Thum: MicroRNAs and platelets
cal growth factor for megakaryocyte lineage, suggests its import-
ance in megakaryocytopoiesis (45). Likewise, miR-146a was re-
portedly deregulated during megakaryocytic differentiation of
HSCs, though the direction of change remained controversial
(Fig.1). Opalinska et al. demonstrated an increase in miR-146a
levels in murine and human HSCs induced to differentiate into
megakaryocyte linage, conversely another study reported a de-
crease in its expression in human cord blood stem cells differenti-
ating into megakaryocytes (46, 47). Further, miR-146a over-
expression led to reduction in polyploid cells numbers, whereas
miR-146a inhibition by antagomiR caused an increase in poly-
ploid cells. A recent study by Starczynowsky et al. showed low lev-
els of miR-146a in MEPs in comparison to HSCs. Although a decoy
or antimiR-LNA targeted downregulation led to increased platelet
counts and decreased polyploidy of megakaryocytes, the forced ex-
pression had no effect on platelet number (48, 49). Such variations
in different studies may originate from the differences in species or
cell culture conditions or possible polymorphism in the 3 UTR of
target mRNA that necessitates defining the precise target sequence.
miRNAs in platelet function
Normal platelet functions such as activation, adhesion and aggre-
gation are essential for coagulation physiology and tissue repair.
Platelet hyper-reactivity indicates risk of atrial thrombotic con-
ditions such as myocardial infarction, stroke and peripheral artery
disease, whereas platelet hypo-reactivity indicates risk of haemor-
rhagic disorders (3). Three recent studies have addressed the pos-
sible roles of miRNAs in platelet reactivity (32, 50). The first study
by Kondkar et al. reported distinct platelet transcriptomes between
healthy individuals demonstrating hyper- or hypo-reactivity of
platelets. In comparison to hypo-reactive platelets, hyper-reactive
platelets expressed up to five-fold higher mRNA levels and 2.5-fold
higher protein levels of VAMP8, a critical component of granule
exocytosis and a predicted target of a platelet expressed miRNA-
miR-96 (Fig. 1, Table 1). The authors confirmed a dose de-
pendent decrease in VAMP8 mRNA and protein upon miR-96
overexpression in VAMP8 expressing cell-lines (50).
Likewise, another study by Nagalla et al. reported the cor-
relation between miRNA-mRNA co-expression profiles of leuko-
cyte depleted platelets and platelet reactivity. Seventy-four miR-
NAs were differentially expressed in subjects grouped according to
epinephrine-induced platelet reactivity response (32). A negative
correlation among certain differentially regulated miRNAmRNA
pairs, e.g. miR-200b:PRKAR2B, miR-495:KLHL5 and miR-
107:CLOCK, was validated by miRNA-mediated knockdown of
the respective proteins in cell-lines expressing target mRNAs
(Fig. 1). Moreover, the reduced activation in platelets lacking
PRKAR2B also supported these findings (32). Osman et al. evalu-
ated the differences in miRNA signature in resting and thrombin-
stimulated human platelets by qPCR coupled with an annotation
network for predicted target genes. Six out of 281 detected miRNA
transcripts (miR-15a, miR-3393p, miR-365, miR-495, miR-98
and miR-3613p) were differentially regulated in thrombin-acti-
vated platelets (51). These studies suggest a potential role of the
miRNA system on platelet functionality and provide a platform for
yet undiscovered facts of platelet biology.
Activated platelets release microparticles rich in growth factors
or variety of effector proteins that may exert extracellular effects
(52). A recent report of miRNA recovery from plasma micropar-
ticles indicates that platelet-derived microparticles may probably
deliver platelet miRNA at the site of action in cardiovascular sys-
tem (53). Moreover, platelets-secreted miRNAs may contribute to
the plasma miRNA pool, which has become a great attraction for
scientists searching novel biomarkers associated with various
pathological conditions (54). However the contribution of pla-
telet-secreted miRNAs remains to be clearly defined, the appli-
cation of network analysis may prove advantageous to provide a
starting platform for forthcoming experimental evidence (54).
Challenges in platelet miRNA research
Studies investigating role of platelet miRNAs in various patho-
physiological conditions are increasingly published in recent years.
However, experimentation on micron-sized anucleated cells which
are highly sensitive to the extracellular environment remains chall-
enging and requires specialised techniques to overcome those limi-
tations. Some of the initial studies therefore widely used computa-
tional prediction analysis and lacked direct mechanistic evidences
in platelets. A big challenge in platelet miRNA research is a lack of
universally optimised protocols for depletion of contaminating
nucleated blood cells such as leukocytes containing about thou-
sands fold more RNA than platelets (32). Platelet preparations
with such contaminations may therefore produce erroneous ex-
pression results. The issue has been highlighted over last years and
contamination levels up to 0.4% were considered safe for the pla-
telet gene expression profiling studies (14, 32, 55, 56). Researchers
Table 1: miRNA targets and function in megakaryocytes and pla-
telets.
miRNA Target gene Function (ref.)
miR-223 P2Y12 Platelet activation (14)
miR-200b PRKAR2B Platelet hyperreactivity (32)
miR-495 KLHL5 Platelet hyperreactivity (32)
miR-107 CLOCK Platelet hyperreactivity (32)
miR-10a HOXA1 Megakaryocytopoiesis (38)
miR-130 MAFB Megakaryocytopoiesis (38)
miR-34a MYB Megakaryocyte differentiation (40)
miR-155 ETS-1, MEIS-1 Megakaryocytopoiesis (42)
miR-150 c-MYB Megakaryocyte differentiation (45)
miR-146a CXCR4 Megakaryopoiesis (47)
miR-96a VAMP8 Platelet granule exocytosis (50)
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Schattauer 2012 Thrombosis and Haemostasis 108.4/2012
603 Dangwal, Thum: MicroRNAs and platelets
now employ improved protocols to obtain purest possible platelet
fractions from whole blood by combining different techniques to
deplete contaminating cells from platelet rich plasma preparation
such as leukocyte deprivation using leukocyte filters or magnetic
cell separation that may considerably reduce the leukocyte con-
tamination (55, 56).
Leukocyte depletion using CD-45 antibody coated magnetic
beads may produce platelet fraction as pure as up to one leukocyte
per five million platelets as reported by Nagalla et al. (32). Never-
theless, purity at the expense of sample loss may limit the suitabil-
ity of this procedure as the volume of the whole blood sample
withdrawn from patients or small animals could be limited. More-
over, the effect of high magnetic fields during the magnetic separ-
ation may result in higher platelet activation (our unpublished
data) than using other depletion techniques. Another reported
method is leukocyte filtration resulting in platelet fraction as pure
as 0.4% to 0.05% leukocyte contaminations in our hands (14). The
methods may yield optimally pure isolated platelet fraction with
minimal activation. Additionally, the use of glycophorin-a coupled
magnetic beads or erythrolysis buffer in combination with pre-
viously described leukocyte depletion methods may yield a platelet
fraction deprived of red blood cells and leukocyte.
Another challenge limiting the mechanistic experimental de-
sign in platelet research is the modulation of platelet gene ex-
pression using conventional molecular biology methods. To our
knowledge the only report demonstrating lipofectamine and elec-
troporation-based small interfering RNA-induced gene silencing
in platelets is published by Hong et al. (57), but the suboptimal
transfection efficiency (8%), unlike in the nucleated cells, ques-
tions the utility of transient transfection method in platelets. Alter-
native use of platelets derived from blood of genetically modified
animals for functional assays has been reported; however, other
possibilities such as using stably transfected platelet precursor-
megakaryocytes to yield platelets with desired gene manipulation
remains to be explored.
Conclusion
For many decades anucleate platelets were neglected in genomic
research era and thought to function by presynthesised, inherited
proteins. Since the discovery of miRNAs in platelets, differential
platelet miRNA expression patterns in healthy or diseased states
have been highlighted (58, 59). Levels of miR-340* and miR-624*
were found to be significantly high in platelets derived from blood
of patients suffering from premature coronary artery disease in
two independent small cohort studies (58). Similarly, miR-28 over-
expression was observed in platelets obtained from individuals
with myeloproliferative neoplasms (59).
Despite the unique challenges, the platelet miRNA research area
represents a great scope for novel findings to reveal yet unknown
molecular mechanisms that may regulate platelet genesis and
functions. Further advances in the platelet miRNA field may ident-
ify new therapeutic miRNA targets in platelets for thrombotic or
occlusive cardiovascular disorders as well as for certain type of
cancers, where platelets play an important role.
Acknowledgement
This work was supported by the German Ministry for EDucation
and Research (IFB-Tx to T.T., 01E00802) grant and an EFSD/Sano-
fi grant (to S.D. and T.T).
Conflicts of interest
None declared.
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