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1 The Cardeza Foundation for Hematologic Research and the Address for correspondence Leonard C. Edelstein, PhD, The Cardeza
Department of Medicine, Sidney Kimmel Medical College, Thomas Foundation for Hematologic Research and the Department of
Jefferson University, Philadelphia, Pennsylvania Medicine, Sidney Kimmel Medical College, Thomas Jefferson
University, Jeff Alumni Hall, Room 394, 1020 Locust St., Philadelphia,
Semin Thromb Hemost 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
Estimates of the proportion of the human genome that is human miRNAs, while the GENCODE reference set (v22) of
transcribed into RNA range up to 85% but only 3% is translated all evidence-based features in the human genome lists 4,093
into protein.1 It is now understood that noncoding RNA regulates (http://www.gencodegenes.org/). However, a recent report
the expression of the protein-coding fraction of the genome at by Londin et al suggests that these repositories may be
multiple levels including transcription, processing, and transla- incomplete.9 MiRNAs regulate most (>60%) mammalian
tion.2 Platelets, anucleate cells primarily associated with their protein-coding genes.10 Some miRNAs are expressed ubiqui-
role in thrombosis and hemostasis, have been found to be rich in tously, but many are tissue and/or developmental stage
many classes of noncoding RNA including microRNAs (miRNAs) specific.11 Cell miRNA content is highly variable and ranges
and long noncoding RNAs.3,4 While platelets lack nuclei and thus from 1 to 10,000 copies.12
transcription, there is evidence that platelets possess most MiRNA biogenesis has been reviewed in depth else-
posttranscriptional features of RNA metabolism including where.13 The canonical miRNA pathway consists of pri-
splicing, translation, and miRNA-mediated regulation.5–7 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-
MicroRNA Function
tion to the cytoplasm, a different type III RNAse, Dicer,
miRNAs are 22 nucleotide regulatory RNAs expressed in shortens the pre-miRNA into a 22 nucleotide double-
multicellular organisms.8 The latest version of miRBase stranded RNA molecule. One strand of this molecule is loaded
(version 21; http://www.mirbase.org/) lists 2,588 mature into the RNA-induced silencing complex (RISC) that contains
Argonaute proteins. Guided by the miRNA sequence, the RISC level expression of CD41 and CD42 in developing MKs com-
then causes translational inhibition followed by messenger pared with control. miR-513a, miR-571, and miR-195 were
RNA (mRNA) degradation, which is the primary effect of also shown to enhance MK maturation, although not to the
miRNAs.14–16 The impact of miRNAs on gene expression is extent of miR-105.
to fine-tune and reduce noise in protein expression.17–19
Importantly, small differences (as little as a 20% change) in miR-142 and miR-486–3p Inhibit Megakaryopoiesis
miRNA levels have been shown to cause autoimmune disease miR-142 is a hematopoietic-specific miRNA that was
and predispose to malignancy.20 previously implicated in the differentiation of lymphocytes
miRNAs target multiple mRNAs, and most mRNAs are and other cell lineages. In miR-142, null mice platelet counts
targeted by multiple miRNAs. Target prediction is a critical are reduced and multiple facets of MK differentiation are
aspect of miRNA research, because it is through the targets altered. This includes smaller MKs (measured by cell area), a
that miRNA function is realized. Different miRNA target reduction in ploidy, and diminished proplatelet network
prediction algorithms are publicly available, including formation.28
TargetScan, Miranda, PicTar, RNA22, and Microcosm. The c-Myb is a transcription factor with established roles in
algorithms utilize homology to the seed region of the miRNA hematopoietic differentiation.29 Comparing the expression
(nucleotides 2–8), free energy of binding between the miRNA profiles of CB-derived CD34þ cells lacking c-Myb expres-
and target, conservation of the target sequences, and/or co- sion to controls, Bianchi et al identified miR-486–3p as a
expression of miRNA and target in the same cell to predict c-Myb target.30 They found that miR-486 helps control MK
mRNA targets. However, there is a lack of consensus on the versus erythroid cell lineage fate through regulation of the
optimal prediction method.21 Unbiased, transcriptome-wide, transcription factor c-Maf. miR-486 overexpression
miR-223, actually makes up a greater fraction of granulocyte dataset,77 we found an extremely high degree of correlation
miRNA content than of platelets4 and unsupervised hierar- between platelet and PMP mRNA and miRNA levels (►Fig. 2).
chical clustering of miRNA profiles suggests that plasma and However, some miRNAs did not correlate well—raising the
serum miRNA profiles more closely resemble erythrocytes possibility of selective packaging or different stability of RNA
rather than platelets, perhaps because they contribute the species during MK–platelet–PMP transitions.
most cellular miRNA mass per volume of blood (►Fig. 1B).4,65 Laffont et al were the first to report transfer of miRNA to
Other studies have identified an association between endothelial cells by PMPs.75 They demonstrated that PMPs
circulating miRNAs and platelet function. Willeit and contain functional Ago2:miR-223 complexes which can
colleagues found that plasma miRNA levels were lower in regulate FBXW7 and EFNA1 expression in endothelial cells.
healthy subjects taking antiplatelet medication.66 Zhang et al Consistent with the data that the PMP miRNA profile reflects
found that reduced circulating miR-223 levels predicted high the parental platelet, thrombopoietin was reported to
platelet function in troponin-negative non-ST elevation acute increase miR-223 maturation in platelets, resulting in higher
coronary syndrome patients.67 It was also reported that lower levels of miR-223 in PMPs. This increase in miR-223 lead to
plasma miR-223 was associated with high platelet reactivity decreased IGF1-R expression in cultured endothelial cells
to adenosine diphosphate (ADP)-signaling in patients with resulting in apoptosis.76 Following up on this work, Shan et
coronary artery disease, possibly due to miR-223 regulation of al reported that miR-223 levels were increased in patients
the P2Y12 ADP receptor.7,68 Finally, circulating miR-126 was with atherosclerosis. Injection of a locked nucleic acid miR-
found to correlate with circulating P-selectin levels in diabetic 223 inhibitor into an atherosclerotic mouse model resulted in
patients and this level was sensitive to aspirin treatment, increased aortic lesion size and increased intimal:medial
suggesting a platelet origin.69 ratio in a model of carotid artery ligation injury, suggesting
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.
according to environmental conditions.5,6,79–81 The lack of 10 Friedman RC, Farh KK, Burge CB, Bartel DP. Most mammalian
transcription obligates the platelet to utilize posttranscrip- mRNAs are conserved targets of microRNAs. Genome Res 2009;
tional mechanisms to regulate gene expression, a fact that 19(1):92–105
11 Wienholds E, Kloosterman WP, Miska E, et al. MicroRNA
may account for the large proportion of the transcriptome
expression in zebrafish embryonic development. Science 2005;
consisting of miRNAs and the number of extended 3′UTRs 309(5732):310–311
observed in platelets.4,82 12 Chen C, Ridzon DA, Broomer AJ, et al. Real-time quantification of
There are many benefits to studying platelet miRNA microRNAs by stem-loop RT-PCR. Nucleic Acids Res 2005;33(20):
content: (1) differential mRNA and miRNA expression has e179
13 Krol J, Loedige I, Filipowicz W. The widespread regulation of
been associated with platelet function and can be used
microRNA biogenesis, function and decay. Nat Rev Genet 2010;
to identify novel regulators of platelet function; (2)
11(9):597–610
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gene expression can lead to knowledge of interindividual predominantly act to decrease target mRNA levels. Nature 2010;
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miR-430 reduces translation before causing mRNA decay in zebra-
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