MicroRNAs in Platelet Physiology and Function

Cory R. Lindsay, PhD1 Leonard C. Edelstein, PhD1

1 The Cardeza Foundation for Hematologic Research and the
Department of Medicine, Sidney Kimmel Medical College, Thomas
Jefferson University, Philadelphia, Pennsylvania
Semin Thromb Hemost
Address for correspondence Leonard C. Edelstein, PhD, The Cardeza
Foundation for Hematologic Research and the Department of
Medicine, Sidney Kimmel Medical College, Thomas Jefferson
University, Jeff Alumni Hall, Room 394, 1020 Locust St., Philadelphia,
PA 19107 (e-mail: leonard.edelstein@jefferson.edu).

Abstract Platelets are anucleate blood cells that are best known for their role in hemostasis and
thrombosis. Perhaps due to the necessity of maintaining a proteome over an 8- to 9-day
lifespan or the need to adapt to environmental situations, platelets retain many of the
RNA metabolic processes of nucleated cells such as the ability to splice, translate, and
regulate RNA levels through posttranscriptional mechanisms. In fact, in the absence of
transcription, the dependence on posttranscriptional mechanisms to regulate gene
expression may have resulted in microRNAs (miRNAs) making up a greater proportion of

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the platelet transcriptome than observed in other cells. miRNAs are
22 nucleotide RNA
molecules that regulate gene expression through messenger RNA (mRNA) degradation
or inhibition of translation. miRNAs regulate differentiation of the platelet precursor,
the megakaryocyte. Identification of miRNA:mRNA pairs that are associated with
platelet phenotypes has led to the discovery of novel regulators of platelet function
Keywords in healthy and diseased subjects. Circulating miRNAs may originate from platelets and
► platelets can serve as biomarkers for platelet function. Platelet microparticles have been
► megakaryocytes demonstrated to have the ability to deliver miRNAs of extracellular targets and alter
► microRNA gene expression in those targets. This review summarizes the current state of
► microparticles knowledge of miRNAs in megakaryocytes, platelets, and platelet microparticles.

Estimates of the proportion of the human genome that is
transcribed into RNA range up to 85% but only 3% is translated
into protein.1 It is now understood that noncoding RNA regulates
the expression of the protein-coding fraction of the genome at
multiple levels including transcription, processing, and transla-
tion.2 Platelets, anucleate cells primarily associated with their
role in thrombosis and hemostasis, have been found to be rich in
many classes of noncoding RNA including microRNAs (miRNAs)
and long noncoding RNAs.3,4 While platelets lack nuclei and thus
transcription, there is evidence that platelets possess most
posttranscriptional features of RNA metabolism including
splicing, translation, and miRNA-mediated regulation.5–7
MicroRNA Function
miRNAs are
22 nucleotide regulatory RNAs expressed in
multicellular organisms.8 The latest version of miRBase
(version 21; http://www.mirbase.org/) lists 2,588 mature
human miRNAs, while the GENCODE reference set (v22) of
all evidence-based features in the human genome lists 4,093
(http://www.gencodegenes.org/). However, a recent report
by Londin et al suggests that these repositories may be
incomplete.9 MiRNAs regulate most (>60%) mammalian
protein-coding genes.10 Some miRNAs are expressed ubiqui-
tously, but many are tissue and/or developmental stage
specific.11 Cell miRNA content is highly variable and ranges
from 1 to 10,000 copies.12
MiRNA biogenesis has been reviewed in depth else-
where.13 The canonical miRNA pathway consists of pri-
miRNAs transcribed from DNA genes by RNA polymerase II,
which are then processed into pre-miRNAs in the nucleus by a
type III RNase Drosha–containing complex. After transloca-
tion to the cytoplasm, a different type III RNAse, Dicer,
shortens the pre-miRNA into a
22 nucleotide double-
stranded RNA molecule. One strand of this molecule is loaded
into the RNA-induced silencing complex (RISC) that contains

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Thrombosis and Hemostasis; Guest Publishers, Inc., 333 Seventh Avenue, 10.1055/s-0035-1570077.
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Tel: +1(212) 584-4662.
mRNA in Platelets Lindsay, Edelstein
Argonaute proteins. Guided by the miRNA sequence, the RISC
then causes translational inhibition followed by messenger
RNA (mRNA) degradation, which is the primary effect of
miRNAs.14–16 The impact of miRNAs on gene expression is
to fine-tune and reduce noise in protein expression.17–19
Importantly, small differences (as little as a 20% change) in
miRNA levels have been shown to cause autoimmune disease
and predispose to malignancy.20
miRNAs target multiple mRNAs, and most mRNAs are
targeted by multiple miRNAs. Target prediction is a critical
aspect of miRNA research, because it is through the targets
that miRNA function is realized. Different miRNA target
prediction algorithms are publicly available, including
TargetScan, Miranda, PicTar, RNA22, and Microcosm. The
algorithms utilize homology to the seed region of the miRNA
(nucleotides 2–8), free energy of binding between the miRNA
and target, conservation of the target sequences, and/or co-
expression of miRNA and target in the same cell to predict
mRNA targets. However, there is a lack of consensus on the
optimal prediction method.21 Unbiased, transcriptome-wide,
cell-based approaches have been used to begin to clarify the
situation. High-throughput sequencing of cross-linking
immunoprecipitation (HITS-CLIP) can be used to identify
miRNA targets in vivo. In T-cells,
40% of the miR-155
mRNA targets identified by HITS-CLIP did not have
canonical seed matches used in most in silico prediction
algorithms, underscoring the caution required when utilizing
these algorithms.22
MicroRNAs and Megakaryocytopoiesis
Through the use of megakaryocyte (MK)-like cell lines and
murine or human CD34þ hematopoietic stem cells, miR-150,
miR-155, miR-146a, miR-34a, and others have been identified
as regulating megakaryopoiesis (reviewed by Edelstein et al23
and Edelstein and Bray24,25). Here we will summarize the
most recent finding on the role of miRNAs in MK
differentiation.
miR-146b and miR-105 Promote Megakaryopoiesis
Zhai et al26 reported miR-146b levels increased during mega-
karyocytic differentiation of cord blood (CB)–derived CD34þ
cells and K562 erythroleukemia cells treated with the phorbol
ester phorbol 12-myristate 13-acetate. miR-146b functioned
as a positive regulator of megakaryopoiesis through down-
regulation of PDGFRA, an inhibitor of megakaryopoiesis. The
levels of CD61-positive cells were consistently higher in
thrombopoietin (TPO)-induced CD34þ cells overexpressing
miR-146b, while overexpression of miR-146b in K562 was
observed to increase the ploidy and lobulation of nuclei
characteristic of MKs. Conversely, reduction of miR-146b
levels also repressed megakaryocytic maturation of K562
cells, but was rescued by silencing PDGFRA.
Kamat et al27 screened a total of 466 human miRNAs for
their effect on common myeloid progenitor differentiation
from CD34þ cells derived from human embryonic stem cells.
Overexpression of miR-105 resulted in the strongest effect on
differentiation of MKs, measured by the fold change in surface
Seminars in Thrombosis & Hemostasis
level expression of CD41 and CD42 in developing MKs com-
pared with control. miR-513a, miR-571, and miR-195 were
also shown to enhance MK maturation, although not to the
extent of miR-105.
miR-142 and miR-486–3p Inhibit Megakaryopoiesis
miR-142 is a hematopoietic-specific miRNA that was
previously implicated in the differentiation of lymphocytes
and other cell lineages. In miR-142, null mice platelet counts
are reduced and multiple facets of MK differentiation are
altered. This includes smaller MKs (measured by cell area), a
reduction in ploidy, and diminished proplatelet network
formation.28
c-Myb is a transcription factor with established roles in
hematopoietic differentiation.29 Comparing the expression
profiles of CB-derived CD34þ cells lacking c-Myb expres-
sion to controls, Bianchi et al identified miR-486–3p as a
c-Myb target.30 They found that miR-486 helps control MK
versus erythroid cell lineage fate through regulation of the
transcription factor c-Maf. miR-486 overexpression
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restrained differentiation of MKs—denoted by the reduc-
tion of more mature MKs (CD34–CD41 þ , CD41 þ CD42b
and CD41 þ CD42b þ ), while erythroid differentiation
was enhanced. Morphological characteristics of these over-
expressing cell lines included markedly smaller cells with
less lobulated nuclei. miR-486 knockdown, in contrast,
showed an increase in MK maturation in both early and
late stages of development.
MicroRNA Control of Megakaryocyte Maturation in
Malignancies and Other Disorders
NPM1 mutations are the most frequent genetic alteration in
acute myeloid leukemia. Using a conditional mouse model of
the most common NPM1 mutation in leukemogenesis,
Sportoletti et al31 reported a reduced platelet count and
upregulation of miR-10a, miR-10b, and miR-20a, which had
previously been reported to downregulated during in vitro
differentiation of MKs.32
Immature MKs are frequently found in cases of immune
thrombocytopenia (ITP), which is often preceded by viral
infection. After adding plasma samples from patients with ITP
to CB mononuclear cells differentiating into MKs, Wang et al
found the yield and maturity of MKs was reduced and the
expression levels of 14 different viral miRNAs were signifi-
cantly upregulated in these cells.33 These results suggest a
functional link between viral infection and megakaryocytic
defects.
Primary myelofibrosis (PMF) is a myeloproliferative
neoplasm that is characterized by MK hyperplasia and
bone marrow fibrosis. miRNA and mRNA profiles of
CD34þ cells isolated from PMF patients identified miR-
155–5p as a negative regulator of JARID2 expression. The
reduction of JARID2 expression resulted in diminished in
vitro expansion of MKs from CD34þ cells. This was con-
firmed with a reduced percentage of CD41þ cells and a
reduction in megakaryocytic cells based on morphological
analysis in both JARID2-siRNA-expressing and miR-155-
overexpressing cells.34

Platelet MicroRNA Content and Function
Platelets are more accessible than MKs, and more data
characterizing platelet miRNA and its transcriptome have
been obtained. Many reports over the past several years
suggest platelet miRNAs are biologically and clinically rele-
vant as: (1) tools to understand platelet physiology and MK
gene expression; (2) potential regulators of platelet RNA and
protein expression levels; and (3) a source for circulating
miRNAs. Despite the absence of a nucleus, platelets have
mRNA and mRNA splicing machinery, and translate mRNA
into proteins relevant to hemostasis and inflammation.6,35,36
Notably, platelets continue to translate proteins from mRNA
under blood bank storage conditions.37 The Provost labora-
tory has demonstrated that human platelets contain func-
tional miRNA processing machinery.7 In addition, they
recently reported that mRNAs that undergo translation in
the platelet, such as SERPINE1, are found associated with
Ago2:miRNA complexes. Upon platelet activation, these
mRNAs are released from these complexes, allowing them
to undergo translation.38 RNA-Seq analysis of 10 platelet
samples from healthy subjects indicates that 2,244 known
and novel miRNAs are present in at least half the samples.9,39
MicroRNAs in Platelet Physiology and Gene
Expression
Our group has utilized platelet RNA expression profiles to
understand the mechanisms that underlie interindividual
variation in platelet function. This variation can fall along
demographic lines or according to physiological state. In
some cases, validated miRNA:mRNA regulatory pairs
in platelets have been identified. While RNA profiling can
identify novel regulators of platelet function in health and
disease conditions, there are a few precautions to take when
interpreting the results. (1) Leukocytes can contain 60 to 200
times as much miRNA as platelets, and proper removal of
them is necessary.4 Magnetic immunodepletion of leukocytes
and erythrocytes can result in a greater than 1:106 leukocyte:
platelet ratio, allowing for a more accurate representation of
the platelet transcriptome. (2) Because there are differences
in platelet miRNA profiles by age, self-identified race, and
gender, controls must be well matched to the experimental
group.40,41 (3) Transformed cell lines are often used as a
substitute for primary cells. However, unsupervised hierar-
chical clustering of miRNA expression data indicates that cell
lines resemble each other rather than their postulated cell of
origin (►Fig. 1A).
Kondkar et al first reported that VAMP8 mRNA is more
highly expressed in platelets hyperresponsive to epineph-
rine signaling. Through a single nucleotide polymorphism
that was associated with epinephrine activity, they also
identified miR-96 as a regulator of VAMP8 that could down-
regulate VAMP8 mRNA and protein levels.42 We expanded
upon this result by demonstrating that miRNA expression
profiles could be used to identify platelet samples with
differing aggregation responses to epinephrine. 43 Using a
bioinformatics approach, we identified three miRNA:
mRNA in Platelets Lindsay, Edelstein
mRNA regulatory pairs—miR-200b:PRKAR2B, miR-495:
KLHL5, and miR-107:CLOCK — that were validated experi-
mentally. Furthermore, PRKAR2B was confirmed as a newly
identified regulator of platelet function.43 Simon et al41
identified networks of miRNA:mRNA pairs that were
associated with age and gender in the Platelet RNA and
eXpression 1 (PRAX1) study.
In PRAX1, we discovered that platelets from black subjects
were more responsive to activation through the thrombin
receptor, protease-activated receptor 4 (PAR4). Concordantly,
we were able to identify a large number of miRNAs and
mRNAs that are differentially expressed by both self-
identified race and PAR4 reactivity.40 Among the miRNAs
differentially expressed by race was the DLK1-DIO3 cluster of
54 miRNAs. One member of this cluster, miR-376c, was found
to regulate phosphatidylcholine transfer protein (PCTP),
which itself is differentially expressed by race. PCTP was
then confirmed as a regulator of platelet PAR4 function.40
These data suggested the hypothesis that differentially
expressed miRNAs could contribute to the greater thrombotic
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risk in blacks as compared with whites.44,45
Platelet MicroRNA in Disease
Alterations in platelet miRNA expression have also been
observed in a variety of clinical conditions. Polycythemia
vera (PV) and essential thrombocythemia (ET) are BCR-
ABL–negative myeloproliferative neoplasms that are diffi-
cult to distinguish from nonclonal conditions due to the
variety of causative mutations.46 A study with PV patients
demonstrated that miR-26b was upregulated in the
platelets of patients with PV but not in the control subjects,
suggesting molecular diagnosis may be possible.46 Xu and
colleagues47 delineated a three-miRNA (miR-10a, miR-
148a, and miR-490–5p) “fingerprint” that discriminated
ET from normal platelets.
Generally recognized as the most abundant miRNA in
platelets, miR-223 has been investigated by many groups
for its role in disease. While Stratz et al reported that there
was no overall change in the platelet miRNA profile of
patients with type 2 diabetes, Elgheznawy and colleagues
have reported that miR-223 is lower in diabetic subjects.48,49
This discrepancy may be due to differences in the presence of
antiplatelet medication or experimental approach (profiling
vs. candidate gene). Elgheznawy et al also report that in
diabetic platelets, calpain cleaves Dicer, resulting in lower
levels of miR-223, miR-142, miR-143, and miR-155. Coagula-
tion factor XIII-A was identified using proteomic and molec-
ular techniques as a miR-223 target. Mice deficient in miR-223
exhibited shorter bleeding times, larger FeCl3-induced
thrombi, greater sensitivity to low doses of thrombin, and
impaired clot retraction.48 These results differed from that of
Leierseder et al, who reported that miR-223-deficient mice
had no defects in platelet number or function. However, these
dissimilarities may be due to differences in assay type and
agonist concentration.50 Platelet miR-223 expression has
also been reported to be lower in subjects with increased
on-clopidogrel activity.51
Seminars in Thrombosis & Hemostasis
mRNA in Platelets Lindsay, Edelstein

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Fig. 1 Comparison of primary cell, cell line, and circulating miRNA profiles. Unsupervised hierarchical clustering (average linkage) of miRNA
expression profiles of (A) cell lines (CL) (from left to right: Meg-01, K562, Raji, Jurkat, U937), 4 or (B) plasma and serum (healthy samples from
Williams et al65 or NCBI GEO Accession GSE25609) compared with platelets (PLT), T-cells, B-cells, granulocytes (Gran), and erythrocytes (Ery).4
Only miRNAs found in both samples were used. Color bar indicates log2 expression relative to mean expression for all miRNAs in each sample
individually.

MicroRNAs in Stored Platelets Circulating MicroRNAs

Platelets undergo a gradual loss in quality when stored either
at room temperature or refrigerated.52 Attempts to limit
bacterial contamination in room temperature–stored
platelets include the use of nucleic acid cross-linking agents.
Osman et al studied the effect of these pathogen reduction
systems on miRNA content and found that one of them,
Intercept, reduced the levels of 6 of the 11 tested miRNAs.
However, neither miRNA cross-linking nor alterations in
miRNA synthesis or function were observed.53 Yu et al
measured miRNA levels in platelets 1, 3, and 5 days after
apheresis and found 10 had altered expression levels.54 Of
those 10, miR-326 increased over time and was found to
downregulate expression of the antiapoptotic Bcl-xL,
resulting in apoptosis of the platelets.55 Deep sequencing of
platelets stored for 1 to 5 days revealed a gradual loss of the
most abundant miRNAs and suggests the use of miRNA
profiles as biomarkers for stored platelet quality.56
miRNAs from platelets are found in the circulation and have
the potential to serve as biomarkers for platelet-mediated
diseases. In general, miRNAs are very biochemically stable
and compared with mRNAs have superior performance char-
acteristics as biomarkers for disease activity.57–59 This
stability is thought to result from the miRNAs being
packaged inside extracellular vesicles such as microparticles,
liposomes, or complexed with RNA-binding proteins or high-
density lipoproteins (reviewed by Creemers et al60). Because
platelets contain a significant proportion of the cellular
fraction of miRNA in blood and because platelet micropar-
ticles (PMPs) are the most abundant microparticle, it is
thought that a substantial fraction of circulating miRNAs
originate from platelets.4,61 For example, circulating levels
of miRNAs highly expressed in platelets have been found to be
associated with myocardial infarction risk, diabetes, and
smoking.62–64 However, the most abundant platelet miRNA,

Seminars in Thrombosis & Hemostasis


miR-223, actually makes up a greater fraction of granulocyte
miRNA content than of platelets4 and unsupervised hierar-
chical clustering of miRNA profiles suggests that plasma and
serum miRNA profiles more closely resemble erythrocytes
rather than platelets, perhaps because they contribute the
most cellular miRNA mass per volume of blood (►Fig. 1B).4,65
Other studies have identified an association between
circulating miRNAs and platelet function. Willeit and
colleagues found that plasma miRNA levels were lower in
healthy subjects taking antiplatelet medication.66 Zhang et al
found that reduced circulating miR-223 levels predicted high
platelet function in troponin-negative non-ST elevation acute
coronary syndrome patients.67 It was also reported that lower
plasma miR-223 was associated with high platelet reactivity
to adenosine diphosphate (ADP)-signaling in patients with
coronary artery disease, possibly due to miR-223 regulation of
the P2Y12 ADP receptor.7,68 Finally, circulating miR-126 was
found to correlate with circulating P-selectin levels in diabetic
patients and this level was sensitive to aspirin treatment,
suggesting a platelet origin.69
MicroRNA Transfer by Platelet Microparticles
Microparticles are small (0.1–1 μm) vesicles shed by many cell
types including platelets upon activation.70 In addition
to their protein contents, microparticles also contain a
substantial quantity of miRNAs.71 Notably, microparticles
and platelet-like particles are able to transfer miRNAs to
other cells types,71–76 raising the possibility that miRNAs
released upon platelet activation could regulate gene
expression in other cells, such as endothelial, monocytic,
neutrophilic, or smooth muscle. PMP engulfment and miRNA
delivery to neutrophils has recently been reported to be
dependent on the presence of platelet 12-lipoxygenase and
secreted phospholipase A2-IIA.77
When we compared the expression level of PMP mRNAs
and miRNAs reported by Duchez et al with the expression
level of platelet mRNAs and miRNAs from the PRAX1
Fig. 2 Correlation between human platelet and megakaryocyte miRNAs. (A) Log2 mRNA and (B) miRNA expression levels were determined in
platelets 40,41 and compared with expression level in PMPs.77 Statistical significance was calculated by Pearson rank correlation. Line indicates
linear regression, dashed lines are 95% confidence interval.
mRNA in Platelets Lindsay, Edelstein
dataset,77 we found an extremely high degree of correlation
between platelet and PMP mRNA and miRNA levels (►Fig. 2).
However, some miRNAs did not correlate well—raising the
possibility of selective packaging or different stability of RNA
species during MK–platelet–PMP transitions.
Laffont et al were the first to report transfer of miRNA to
endothelial cells by PMPs.75 They demonstrated that PMPs
contain functional Ago2:miR-223 complexes which can
regulate FBXW7 and EFNA1 expression in endothelial cells.
Consistent with the data that the PMP miRNA profile reflects
the parental platelet, thrombopoietin was reported to
increase miR-223 maturation in platelets, resulting in higher
levels of miR-223 in PMPs. This increase in miR-223 lead to
decreased IGF1-R expression in cultured endothelial cells
resulting in apoptosis.76 Following up on this work, Shan et
al reported that miR-223 levels were increased in patients
with atherosclerosis. Injection of a locked nucleic acid miR-
223 inhibitor into an atherosclerotic mouse model resulted in
increased aortic lesion size and increased intimal:medial
ratio in a model of carotid artery ligation injury, suggesting
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miR-223 has antiatherogenic properties.78 When Gidlöf and
colleagues profiled the miRNA content of platelets from
STEMI patients, they found miR-22, miR-185, miR-320b, and
miR-425–5p were reduced in the platelets of patients
compared with controls. These miRNAs were even lower in
the thrombus as compared with circulating platelets, suggest-
ing platelet activation had led to miRNA release. Finally, they
demonstrated that PMP-delivered miR-320b could repress
ICAM-1 expression in cultured endothelial cells.74
Summary and Future Directions
Given that the average platelet lifespan is 8 to 9 days and the
average half-life of a cellular protein is 46 hours, it is reason-
able to assume that the platelet must maintain its tran-
scriptome to preserve its proteome. In addition, several
reports of signal-dependent splicing and translation suggest
that the platelet can actively alter its gene expression
Seminars in Thrombosis & Hemostasis
mRNA in Platelets Lindsay, Edelstein
according to environmental conditions.5,6,79–81 The lack of
transcription obligates the platelet to utilize posttranscrip-
tional mechanisms to regulate gene expression, a fact that
may account for the large proportion of the transcriptome
consisting of miRNAs and the number of extended 3′UTRs
observed in platelets.4,82
There are many benefits to studying platelet miRNA
content: (1) differential mRNA and miRNA expression has
been associated with platelet function and can be used
to identify novel regulators of platelet function; (2)
understanding the mechanisms behind variation in platelet
gene expression can lead to knowledge of interindividual
variation in platelet function; (3) expression levels of miRNAs
in platelets and in circulation have been used as biomarkers to
identify differences in platelet function in both health and
pathological conditions; (4) gathering evidence that PMPs
can deliver miRNAs to extracellular targets and affect their
gene expression suggests that the platelet transcriptome has
functional consequences outside of the platelet; and (5) given
the relative accessibility of platelets versus MKs and the
relatedness of their transcriptomes, platelet miRNA can
lead to insights regarding MK biology.25
Several critical questions remain to be answered about
platelet miRNAs. Do miRNAs function inside platelets? What
proportion of the circulating miRNA content derives from
platelets? Are changes in miRNA expression the cause or
result of a pathological condition? Do PMPs deliver miRNAs in
vivo? With the increasing evidence for the role noncoding
RNAs play in biology, it is likely that miRNAs are important
regulators of platelet physiology.
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