Platelet MicroRNAs

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Platelet MicroRNAs Patrick Provost*,† *

CHUQ Research Center/CHUL Pavilion, Quebec City, QC, Canada, †Department of Microbiology, Infectious Diseases and Immunology, Faculty of Medicine, Universit
e Laval, Quebec City, QC, Canada

THE MICRORNA PATHWAY OF HUMAN CELLS 127 Biogenesis and Function of MicroRNAs 127 The Repertoire of Human MicroRNAs 127 Role of MicroRNAs 128 Identification of mRNAs Regulated by MicroRNAs 129 MicroRNAs in Health and Disease 129 PLATELETS 130 The Platelet Proteome 130 The Platelet Transcriptome 130 Platelet mRNA 3’UTRs 131 THE MICRORNA PATHWAY OF PLATELETS 131 Platelet MicroRNAs 131 Detection of Platelet MicroRNAs 131 Biogenesis of Platelet MicroRNAs 131 The Repertoire of Platelet MicroRNA Sequences 131 Function of MicroRNAs in Platelets 133 MicroRNAs as Possible Regulators of mRNA Translation in Platelets 133 A Role for MicroRNAs in Platelet Function? 133 A Role for Platelet MicroRNAs in Other Cell Types 134 Platelet MicroRNA Data: Between Mice and Men 134 USE OF PLATELET MICRORNAs AS BIOMARKERS 134 Platelet MicroRNAs and Reactivity 134 Platelet MicroRNAs and Cancer 134 Platelet MicroRNAs and Diabetes 135 Platelet MicroRNAs and Chronic Kidney Disease 135 Clinical Monitoring of Platelet MicroRNAs 135 THERAPEUTIC APPLICATIONS 135 CONCLUSION AND PERSPECTIVES 136 ACKNOWLEDGMENTS 136 REFERENCES 136

THE MICRORNA PATHWAY OF HUMAN CELLS The microRNA-guided RNA silencing pathway, or microRNA pathway, is a central gene regulatory process based on small RNA species known as microRNAs (Fig. 6.1).2,3 Key regulators of gene expression, microRNAs are noncoding RNA species expressed in the vast majority of eukaryotes, including humans. Representing a family of unexpectedly small endogenous RNAs of
19–24 nucleotides (nt) in length, microRNAs are diverse in sequence and expression patterns, evolutionarily widespread, and involved in sequence-specific, post-transcriptional regulation of gene expression.4–6 MicroRNAs are generated upon the sequential processing of microRNA precursor molecules by the ribonucleases III (RNases III) Drosha and Dicer (Fig. 6.2).

Platelets. https://doi.org/10.1016/B978-0-12-813456-6.00006-0 Copyright © 2019 Elsevier Inc. All rights reserved.

Predicted to regulate
60% of the genes in humans,2,7,8 it may prove difficult to find a biological function or cellular process that is not influenced, at least to some degree, by microRNAs.

Biogenesis and Function of MicroRNAs Our current knowledge on the biogenesis and function of microRNAs in humans, which form a compartmentalized process, is summarized in Fig. 6.1. Encoded in the genome of nucleated cells, microRNA genes, either of intronic or intergenic origin, are transcribed mainly by RNA polymerase II9 into long, highly structured and sometimes polycistronic primary microRNAs (pri-microRNAs). These pri-microRNAs form hairpin loop structures that are cleaved into
70-nt microRNA precursors (pre-microRNAs) by the nuclear Microprocessor complex. This complex is composed of the RNase III Drosha10 and its partner protein DiGeorge syndrome Critical Region gene 8 (DGCR8)11–13 (Fig. 6.1), with heme acting as a cofactor. Critical for Microprocessor to process pri-microRNAs with high fidelity, heme converts a DGCR8 dimer into an active conformation capable of recognizing the terminal loop of pri-microRNAs.14 The pre-microRNA products accumulate in the nucleus and are exported via Exportin-515,16 to the cytoplasm, where the RNase III Dicer recognizes them.17–19 Dicer processes the stem and removes the loop of pre-microRNA substrates to generate microRNA:microRNA* duplexes. For that process, Dicer is assisted by TAR RNA-binding protein 2 (TRBP2),20 an integral cofactor that acts as a gatekeeper to ensure efficient Dicer binding and processing of pre-microRNAs in RNA-crowded environment.21 Following the recruitment of Argonaute 2 (Ago2) protein,20 a microRNA strand selection and separation step, based on the relative thermodynamic stability of the duplex extremities, leads to the formation of a microRNA-containing ribonucleoprotein (miRNP) complex. Guided by its associated microRNA component, this miRNP recognizes and regulates translation of a specific messenger RNA (mRNA) in a sequence-specific manner.2 The targeted mRNAs are then translocated to the P-bodies, likely through a process that requires dynamic phosphorylation of Ago2 on serine 798,22 where they are eventually cleaved and degraded.23 Alternatively, the mRNA targets may be “recycled” and returned to the translational machinery for expression upon a specific cellular signal.24,25

The Repertoire of Human MicroRNAs The nomenclature and detailed structure of the different microRNA species, which are generated upon the successive processing activity of the RNases III Drosha and Dicer, are illustrated in Fig. 6.2. It is important to note that both strands of a microRNA:microRNA* duplex may be incorporated into effector miRNP complexes and exert mRNA regulatory effects,26,27 sometimes on a family of related genes, enzymatic or signaling pathways.

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Fig. 6.1 The human microRNA-guided RNA silencing pathway. MicroRNAs can regulate translation of specific mRNAs. (Adapted from Ouellet et al.,1 with permission from Hindawi).

Fig. 6.2 MicroRNA species of the microRNA-guided RNA silencing pathway. Sequential processing of microRNA precursor species by Drosha and Dicer governs microRNA biogenesis.

As many as 2654 different human microRNA sequences, generated from 1917 pre-microRNAs, have been reported to date in miRBase (Release 22; http://www.mirbase.org), the primary searchable online database of published microRNA sequences and annotations.28–30 Although this number appears to be relatively small compared to protein-coding mRNAs, the facts that (i) each microRNA may recognize on average
200–300 different mRNAs, (ii) each mRNA target may be recognized by tens of different microRNAs, and (iii)

microRNAs act in concert underlie the ability of microRNAs to regulate up to
60% of the genes in humans.2,7,8

Role of MicroRNAs MicroRNA species confers to miRNP complexes the ability to recognize cellular mRNAs through specific binding sites, usually located in their 30 untranslated region (UTR).2 As depicted

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Fig. 6.3 MicroRNA recognition of specific mRNAs. Determinants of microRNA:mRNA interactions—importance of the microRNA seed region and cooperativity of contiguous microRNA binding sites. (Adapted from Ouellet et al.,1 with permission from Hindawi).

in Fig. 6.3, the main determinants of a typical microRNA: mRNA interaction are as follows: (i)

perfect base pairing of the microRNA seed region (nucleotides 2–8 from the 50 end), which is critical to microRNA function; (ii) a central mispaired region, which prevents Ago2mediated cleavage of the mRNA target and allow translational repressive control; and. (iii) complementary base pairing of the 30 region, which stabilizes further the microRNA:mRNA interaction. The contiguous microRNA binding sites may allow cooperation between mRNA-associated miRNP complexes (not shown in Fig. 6.3) for an enhanced, synergistic control of mRNA translation. The miRNP complex initiate mRNA target binding through the microRNA seed region, resulting in four distinct reaction pathways: target cleavage, transient binding, stable binding, and Argonaute unloading.31 The target cleavage requires extensive sequence complementarity and dramatically accelerates miRNP recycling for further target cleavage. The stable binding of miRNP is efficiently established with the seed match only, which may explain the seed-match rule of microRNA target selection. Although microRNAs are known mainly as repressors of gene expression, they have also been shown to enhance mRNA translation, when acting within Fragile X mental retardation syndrome-related protein 1a (FXR1a)-containing miRNP complexes in nondividing, cell cycle arrested or quiescent cells.32,33 The gene regulatory effects of microRNAs at the transcriptional level are beyond the scope of this chapter and will not be discussed here.

Identification of mRNAs Regulated by MicroRNAs Recognition of mRNA targets by microRNAs is mediated through imperfect base pairing, which renders mRNA targets difficult to predict. However, several microRNA target prediction tools are available on the web, such as: (i)

the predicted targets section of specific microRNA entries in miRBase (http://www.mirbase.org);28–30 (ii) miRDB (http://mirdb.org);34 (iii) RNA22 (https://cm.jefferson.edu/rna22);7 (iv) TargetScan (http://www.targetscan.org/vert_71);35 (v) miRTar (http://mirtar.mbc.nctu.edu.tw);

(vi) miRWalk, a comprehensive atlas of predicted and validated miRNA-target interactions (http://zmf.umm.uniheidelberg.de/apps/zmf/mirwalk2);36 and. (vii) miRTarBase, an experimentally validated microRNAtarget interaction database (http://mirtarbase.mbc.nctu. edu.tw/php/index.php).37 DIANA Tools (http://diana.imis.athena-innovation.gr/ DianaTools/index.php) offer a target prediction algorithm, called microT-CDS (trained on a positive and a negative set of miRNA Recognition Elements (MREs) located in both the 3’-UTR and CDS regions; http://www.microrna.gr/microTCDS).38 They also provide a database of experimentally supported mRNA targets of microRNAs (TarBase v8; http:// carolina.imis.athena-innovation.gr/diana_tools/web/index. php?r¼tarbasev8/index).39 All these computational tools are based on general rules elaborated from the different aspects and features of experimentally validated microRNA:mRNA target interactions, and provide rather exhaustive lists of potential mRNA targets that could be regulated by microRNAs of interest, or vice-versa (i.e., possible regulatory microRNAs for mRNA of interest). These predictive tools are thus very helpful in guiding investigators in prioritizing and performing the experimental validation of microRNA:mRNA pairs of interest.40

MicroRNAs in Health and Disease Considering their role and importance in regulating gene expression in humans, microRNAs are expected to play a significant role in health and disease, as reviewed previously in the context of diabetes, neurological diseases, and viral infections.3,41–46 As illustrated in Fig. 6.4, a functional microRNA pathway is required for a tightly regulated expression of the cellular proteins, which is required to maintain cellular/ tissue homeostasis and to remain healthy (1). Involved in the fine-tuning of gene expression, a dysfunctional microRNA biogenesis (e.g., when the function of a core protein component of the microRNA pathway is deleted, mutated, or misexpressed) (2) and/or an altered microRNA function (e.g., deletion, mutation or mis-expression of a microRNA) (3) may deregulate gene expression, compromise cellular/ tissue homeostasis and lead to human genetic diseases. The latter situation may also be encountered when the corresponding microRNA binding sites are affected by single nucleotide polymorphisms (SNPs).47 The same concepts and

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Fig. 6.4 MicroRNAs and human diseases. Deregulation of microRNA biogenesis and/or function may lead to human genetic diseases. (1) A functional microRNA pathway is required for the fine regulation of gene/protein expression, which is required to maintain cellular/tissue homeostasis and to remain healthy. Cellular/tissue homeostasis may be compromised and lead to human diseases upon deregulation of gene expression caused by (2) a dysfunctional microRNA biogenesis and/or (3) an altered microRNA function.

As reviewed in this book, platelets play myriad roles far beyond their involvement in the maintenance of hemostasis as well as in thrombosis and vessel occlusion that underlies stroke and acute coronary syndromes. Most platelet functions are mediated by a repertoire of proteins inherited from their megakaryocytic precursor cells and/or synthesized de novo from megakaryocyte-derived mRNAs.

synthesis of specific proteins, such as cyclooxygenase 2 (COX2),58 has also been documented in human platelets. Using RNA-Seq and ribosome profiling of primary human platelets, Mills et al.59 confirmed that platelet mRNA transcripts are broadly occupied by ribosomes and that the decay of platelet mRNAs is slowed by the natural loss of the mRNA surveillance and ribosome rescue factor Pelota. Together, these studies suggest that platelet mRNAs can be translated into proteins and contribute to the platelet proteome along processes that can be induced and regulated by appropriate stimuli. As discussed further in Chapter 7, the platelet proteome is much more dynamic than previously believed.

The Platelet Proteome

The Platelet Transcriptome

Despite lacking genomic DNA, and thereby incapable of transcribing nuclear genes into mRNAs, platelets contain rough endoplasmic reticulum and ribosomes49 that can use mRNAs as a template for de novo protein synthesis.50–52 Indeed, after Newman et al.53 reported the amplification of platelet-specific messenger RNA using polymerase chain reaction (PCR), a study by Roth et al.54 subsequently confirmed that circulating human blood platelets retain a small amount of poly(A) + RNA from their megakaryocytic precursor cells; these observations on platelets paralleled their ability for protein biosynthesis.50 Later, Weyrich et al.55 showed that thrombin-activated, but not resting, platelets synthesize Bcl-3 along a process blocked by mRNA translation inhibitors. These investigators also demonstrated that platelets synthesize numerous proteins after they adhere to fibrinogen, which mediates outside-in signaling by engagement of αIIb/ß3 integrins.55 Similarly, Evangelista et al.56 reported the de novo cyclooxygenase-1 synthesis, which is involved in thromboxane A2 formation in platelets, in response to thrombin and fibrinogen. More recently, the cytokine interleukin-18 (IL18) was shown to be synthesized de novo upon activation, in contrast to the IL-18-binding protein, which is present in premade form in human platelets.57 The constitutive

It is the advent of most advanced technologies, such as RNASeq, that unveiled the great complexity of the transcriptional landscape of anucleate human platelets.60,61 Whereas previous serial analysis of gene expression (SAGE) and microarray profiling studies estimated the number of protein-coding transcripts present in human platelets to
6000,62–67 RNA-Seq analyses brought that number to
9500.60,61 A strong correlation between transcript abundance and protein expression was established,66–68 further supporting the functionality of these transcripts in platelets. Notably, platelet factor 4 (PF4), CCL5 (RANTES), Tubulin beta-1 (TUBB-1), Glycoprotein Ib (GPIb; GP1BB), ITGA2b (GPIIb) and ITGB3 (GPIIIa) are among the top transcripts detected in human platelets.67 Platelet genomics is discussed in more detail in Chapter 5. Perhaps one of the most surprising observations was that of the existence mRNA splicing in human platelets,69 a process usually encountered in nucleated cells. In response to surface receptor activation, platelets excise introns from IL-1ß premRNA, yielding a mature mRNA that is translated into protein. Suggesting that the regulation of platelet mRNA translation is more complex than previously thought, these findings support the existence of post-transcriptional control of gene expression in human platelets.

principles may hold true and be applicable to platelets in the context of the cardiovascular system and related diseases.48

PLATELETS

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Platelet mRNA 3’UTRs

Detection of Platelet MicroRNAs

The 3’UTR of mRNAs, involved in the post-transcriptional control of gene expression, harbor regulatory elements that play an important role in the modulation of mRNA translation. Interestingly, SAGE analyses unveiled that the 3’UTR of platelet mRNAs is significantly longer (1047 nt) than in nucleated cells (492 nt), whereas the average length of platelet mRNA 5’UTRs does not differ significantly from nucleated cells (platelets: 151 nt; nucleated cells: 120 nt). Indeed, the 3’UTR of platelet mRNAs warrants special considerations. For instance, the annotated 3’UTR of P2RY1 is nearly 1 kb, whereas the actual 3’UTR in platelets is considerably longer.70 According to microRNA target prediction algorithms, this additional 2.5 kb harbors additional microRNA binding sites. This indicates that actual transcript data, not predicted annotation data, should be used for microRNA target site predictions in platelets.70 It is also relevant to note that the regulatory element Brd box (bearded box), whose regulation likely involves complementary sequences found at the 50 end of certain microRNAs,71 has been found to be enriched in platelet transcripts.72 It may be speculated that, by harboring longer 3’UTRs, platelet mRNAs may contain more regulatory elements than their nucleated cell counterparts, including binding sites for specific microRNAs. An attractive explanation would be that a system, i.e., platelets, that loses its ability for transcriptional regulation may compensate for this deficiency by enhancing its capacity for mRNA translational control, such as those involving microRNAs.

Several experimental approaches have been developed and/or used to detect microRNAs, such as Northern blotting,76 RNase protection assay (RPA),81 reverse transcription and quantitative real-time PCR (qRT-PCR),82 micro-array profiling,76 and highthroughput sequencing (also known as next-generation RNA sequencing, or RNA-seq).60,80 Whereas the first three, hybridization-based methods aim to detect specific, known, individual microRNA sequences, the latter two are large-scale approaches designed to quantify either all the known microRNAs (micro-array) or any RNA sequences within the range of sizes under study. A study based on qPCR and performed by Osman and F€alker allowed the detection of 281 microRNA sequences, of which 6 microRNAs (miR-15 a, miR-339-3 p, miR-365, miR495, miR-98, and miR-361-3 p) were up- or down-regulated in thrombin-activated human platelets.82 Affirming the widespread existence of microRNAs in human platelets and revealing an alteration in the level of specific microRNAs upon thrombin stimulation, this work suggested a potential link between microRNA regulation of gene expression and the ability of platelets to respond to specific conditions and/or stimuli. Complemented by appropriate bioinformatics tools and analyses, next-generation RNA-seq represents a very powerful, gene discovery tool, as demonstrated by its instrumental use in discovering mutations in the NBEAL gene that cause the gray platelet syndrome83 (Chapter 48).

THE MICRORNA PATHWAY OF PLATELETS

Biogenesis of Platelet MicroRNAs

Platelet MicroRNAs

Not only do human platelets contain microRNAs, but they also harbor the main cytoplasmic protein components of the microRNA pathway, including Dicer,76 TRBP2,76 FMRP,84 and Ago2.76 As expected, the nuclear components Drosha and DGCR8 could not be detected in platelets,76 which is consistent with their anucleate nature (Fig. 6.5). Mediating microRNA biogenesis in nucleated cells, the Dicer•TRBP2 complex also seems responsible for platelet microRNA formation. Indeed, whereas both proteins could be coimmunoprecipitated, supporting the existence of a Dicer•TRBP2 complex, either Dicer or TRBP2 immunoprecipitate prepared from human platelets could convert a premiRNA substrate into a mature microRNA.76 The detection of pre-microRNA species in human platelets76 supports a scenario in which pre-microRNAs would serve as a template for the synthesis of platelet microRNAs. Their relative abundance,76 however, militates against a major contribution of de novo biogenesis of microRNAs within platelets and rather suggests that most of the mature microRNAs detected in circulating platelets may be inherited from megakaryocytes.

Studying the aberrant microRNA expression profile of peripheral blood cells of patients with polycythemia vera73 and the differential expression of microRNAs in hematopoietic cell lineages,74 Bruchova and coworkers were the first to suggest the presence of microRNAs in platelets. However, the authors used a simplified platelet isolation protocol, based solely on lowspeed centrifugation of platelet-rich plasma, and did not characterize the purity of their platelet preparations, likely prone to contamination by other blood cells, such as leukocytes. Atreya and colleagues subsequently reported the membrane arraybased differential profiling of microRNAs using platelet concentrates containing
0.01% (or 1/10,000) of contaminating white blood cells,75 whose contribution could not be excluded since leukocytes contain as much as 12,500-fold more RNA than platelets. Using a more stringent platelet purification protocol, yielding <1 contaminating leukocyte per
3 million platelets and a level of leukocyte RNA contamination estimated to <0.4%, Landry et al.76 elucidated the existence in anucleate human platelets of a functional microRNA pathway devoid of its habitual nuclear initiation step (Fig. 6.5). Human platelets were found to harbor an abundant and diverse array of microRNAs, which are known as key regulators of mRNA translation in human nucleated cells. Covering a range greater than
2.5 log in expression levels, the platelet microRNA profile was markedly different from that of human neutrophils, supporting further the lack of leukocyte contribution to the platelet data.76 One of the most abundant microRNA family represented in human platelets is let-7,76 which is known to be involved in cell differentiation processes.77–79 Representing 48% of the platelet microRNA content,80 the presence of let-7 microRNAs in terminally differentiated platelets was expected. What is surprising, though, is the variety and relative abundance of their microRNA content, which makes of platelets one of the richest sources of human microRNAs reported to date.76,80

The Repertoire of Platelet MicroRNA Sequences The complete repertoire of platelet microRNA sequences goes far beyond the microRNAs annotated in miRBase. RNA-Seq analyses revealed that, as in nucleated cells, platelet microRNAs bear signs of post-transcriptional modifications, mainly terminal adenylation and uridylation.80 Poly-uridylation of pre-microRNAs by 30 terminal uridylyl transferase (TUTase) 4 (TUT4)85 has been shown to block Dicer processing and decrease microRNA levels, whereas mono-uridylation of mature microRNAs by TUT4 and TUT7, which is pervasive and physiologically significant, disrupt target mRNA repression without causing a change in the steady-state levels of the microRNA.86,87 Intriguingly, decreased uridylation levels are associated with an increase in nontemplated addition of adenosine residues, or adenylation, which rather stabilizes the level of

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Fig. 6.5 Proposed model for platelet inheritance of a functional microRNA pathway from megakaryocyte precursor cells. Harboring a complete microRNA pathway, megakaryocytes are believed to pass on the cytosolic components of the pathway to the platelets in formation, which inherit of a functional microRNA pathway devoid of its habitual nuclear initiation step. (From Landry et al.,76 with permission from the Nature Publishing Group).

functional microRNAs. In vitro enzymatic assays demonstrated the ability of human platelets to uridylate microRNAs, which correlated with the presence of the uridyltransferase enzyme TUT4,80 suggesting that platelets may themselves regulate the level and function of their microRNAs. The most important contributor to the complexity of microRNA regulation of gene expression in human cells, however, is the extremely high diversity of microRNA sequences unveiled

by RNA-Seq. This approach revealed that each pri-microRNA or pre-microRNA substrate can yield numerous microRNA sequences of various lengths and nucleotide composition.80 These microRNA isoforms are known as isomiRs and result from imprecise Drosha and/or Dicer processing. In some cases, isomiRs are even more abundant than the reference microRNA sequence annotated in miRBase, as demonstrated in human platelets for miR-140-3p (Fig. 6.6).80 Notably, these variants

Fig. 6.6 Detection of multiple microRNA isoforms in human platelets. As shown on the left, most of the miR-140-3p isoforms may result from a combination of imprecise processing by Drosha and/or Dicer, including the most abundant. The miR-140-3p microRNA sequence annotated in miRBase is highlighted in bold. Two major miR-140-3p isomiR populations coexist in platelets depending on 50 cleavage by Dicer, either at the
et al.80 (open-access article)). canonical position (blue bars) or harboring a 1 nt cleavage shift (red bars). (From Ple

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MicroRNAs as Possible Regulators of mRNA Translation in Platelets

Fig. 6.7 Redirection of platelet miR-140-3p targeting upon a 1 nt shift of the 50 cleavage site. (A) mRNA target prediction for the reference mature miR-140-3p sequence and the miR-140-3p isomiR harboring a 1-nt 50 shift using TargetScan. (B) In contrast to the reference miR-140-3p sequence, the 1-nt 50 shifted isomiR specifically downregulated mRNA translation controlled by adenylate cyclase
et al.80 (open-access associated protein 1 (CAP1) 3’UTR. (From Ple article)). 0

include 5 -shifted isomiRs with redirected mRNA targeting abilities (Fig. 6.7),80 due to the shift of the microRNA seed region. Together, these microRNA variants markedly expand the repertoire of mRNA targets placed under microRNA control and highlight the complexity of microRNA regulation of gene expression in human cells and platelets.

Function of MicroRNAs in Platelets The abundance and diversity of microRNAs in platelets80 raises the question of their functionality, biological role and significance. Known to be involved in hematopoietic cell differentiation and megakaryopoiesis,88,89 why would microRNAs not continue to function in platelets ultimately produced by that process? In order to be functional in gene regulation, a microRNA needs to be incorporated into a complex containing a member of the Ago family of proteins, such as Ago2.90 Human platelets do harbor minimal miRNP effector complexes, formed of Ago2 and its associated microRNA (e.g., miR-223), exhibiting miR-223-directed RNA cleavage activity.76 Therefore, one of the primary functions of platelet microRNAs may well be similar to that largely documented in nucleated cells, i.e., the control of mRNA translation.

The initial evidence supporting that possibility came from the detection of an endogenous platelet mRNA, namely that encoding the purinergic receptor P2Y12, in Ago2 immunoprecipitates.76 Complemented by the functional validation of the microRNA binding site(s) present in the platelet mRNA 3’UTR using reporter gene activity assays, such as that for miR-223 in the 3’UTR of P2Y12 mRNA,76 this biochemical approach is directly relevant to, and is regarded as a gold standard for, the identification of microRNA-targeted mRNAs.91–93 This strategy also addressed the major limitation associated with working with primary human platelets, i.e., their relative refractoriness to transfection. Additional platelet mRNAs previously shown to template de novo protein synthesis are associated to Ago2 complexes, and thus presumably controlled by microRNAs, such as SERPINE1 (encodes Plasminogen activator inhibitor-1; PAI-1), ITGB3 (GPIIIa), ITGA2B (GPIIb), SVCT2 (Sodium-dependent vitamin C transporter 2), TLN1 (Talin-1), PTGS1 (Prostaglandin G/H synthase 1, also known as Cyclooxygenase 1; COX-1) and CYTB (Cytochrome B).94 Under conditions favoring de novo synthesis of PAI-1 protein, a rapid dissociation of the encoding platelet SERPINE1 mRNA from Ago2 protein complexes as well as from the translational repressor protein T-cell-restricted intracellular antigen-1 (TIA-1) was documented.94 These findings are consistent with a scenario by which lifting of the repressive effects of Ago2 and TIA-1 protein complexes, involving a rearrangement of protein•mRNA complexes rather than disassembly of Ago2•microRNA complexes, would allow translation of SERPINE1 mRNA into PAI-1 in response to platelet activation. Cimmino et al.95 reported a significant reprograming of the platelet microRNA profile during activation, with consequent significant changes in almost half of >700 proteins quantified by quantitative proteomics. The platelet transcriptome remained largely unaffected, suggesting that the changes in mature microRNA expression appear to be the main driver of the observed discrepancy between transcriptome and proteome changes.95

A Role for MicroRNAs in Platelet Function? Some evidence suggests that platelet microRNAs are important for platelet biogenesis96 and function.97–99 Elgheznawy et al.100 reported that the levels of Dicer were altered in platelets from diabetic mice and patients, a change that could be attributed to the cleavage of the enzyme by calpain, resulting in loss of function and a decrease in the levels of platelet miR-142, miR-143, miR-155, and miR-223. Deletion of miR-223 in mice modestly enhanced platelet aggregation, the formation of large thrombi and delayed clot retraction compared with wild-type littermates.100 A similar deregulation was detected in platelets from diabetic patients. Proteomics analysis of platelets from miR-223 knockout mice revealed increased levels of several proteins, including the miR223 target coagulation factor XIII, which was also altered in diabetic platelets.100 Treatment of diabetic mice with a calpain inhibitor prevented loss of platelet Dicer as well as the diabetes mellitus–induced decrease in platelet microRNA levels and the upregulation of miR-223 target proteins.100 To determine the contribution of microRNAs to platelet function, Rowley et al.101 created a murine megakaryocytespecific knockdown of Dicer1, which reduced the level of the majority of microRNAs in platelets and altered platelet mRNA expression profile. The fibrinogen receptor integrin subunits alpha-IIb and beta3 mRNAs were among the differentially expressed transcripts that are increased in microRNA-depleted

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platelets and whose translation can be regulated by microRNAs miR-326, miR-128, miR-331, and miR-500.101 Consistent with these molecular changes, the deletion of Dicer1 resulted in increased surface expression of integrins αIIb and β3, and enhanced platelet binding to fibrinogen in vivo and in vitro.101 These findings indicate that Dicer1-dependent generation of mature microRNAs modulates the expression of target mRNAs important for the hemostatic and thrombotic function of platelets.101 Together, these results suggest that fine-tuning of resident mRNA translation by microRNAs is a key event in the platelet pathophysiological response to stimuli and disease conditions.

A Role for Platelet MicroRNAs in Other Cell Types The abundance of microRNAs may explain the very low rate of protein synthesis observed in platelets, which does not exclude a possible role of microRNAs outside of platelets, through their release in lipid vesicles. A type of lipid vesicle, called exosomes, has been shown previously to contain mRNAs as well as microRNAs,102,103 suggesting that microRNAs could be packaged and transferred between different cell types. Platelets are no exception, as the microRNA-containing microparticles they release upon activation may transfer signaling and gene regulatory molecules between platelets and the other cells of the cardiovascular system, such as endothelial cells104,105 and macrophages,106 and harbor functional106,107 and clinical108 significance. Platelet-derived exosomes are discussed in more detail in Chapter 22, whereas the interaction between platelets, leukocytes and the endothelium is presented in Chapter 16.

Platelet MicroRNA Data: Between Mice and Men A number of mouse models aimed at studying platelet function and/or thrombosis have been developed and are very useful in platelet research, since data emerging from such studies may be transposed to humans. However, although mRNA and microRNA sequences are generally well conserved, several sequences diverge between mice and men.60 Knowing that microRNA function is often modified upon changes of a single nucleotide, as it occurs in SNPs, caution must be taken before transposing platelet microRNA data from mice to humans, or vice-versa.

development of serious, platelet-related cardiovascular diseases, including atherothrombosis.109 It is tempting to envision and speculate about the eventual use of platelet microRNAs as biomarkers of specific platelet disorders, whereby platelet microRNA profiling would be indicative of underlying diseases and have predictive, diagnostics and/or therapeutic values to physicians (Fig. 6.8), as in the fields of cancer110–113 and cardiovascular disease,114 whether it be thrombotic episode115 or acute myocardial infarction.116 In that perspective, it is interesting to point out the relatively strong correlation observed between the microRNA profile of human platelets isolated from healthy subjects of different geographic locations (e.g., Quebec City, QC, Canada versus Philadelphia, PA),97 suggesting the applicability of platelet microRNA profiling for medical applications.

Platelet MicroRNAs and Reactivity The laboratory of Paul F. Bray was the first to investigate the relationship between microRNAs and platelet reactivity in order to identify potential biomarkers for thrombotic risk.97,117,118 One of their studies focused on the VAMP8 gene, which encodes an essential SNARE protein involved in platelet granule exocytosis and bears a single nucleotide polymorphism in its 3’UTR that has been associated with coronary artery disease. Kondkar et al.117 have reported that VAMP8 mRNA may be down-regulated by miR-96, both of which are differentially expressed between platelets of differing reactivity to epinephrine-induced aggregation in a manner that is consistent with an effect of miR-96 on VAMP8 expression. This group also demonstrated the use of differentially expressed mRNAmiRNA pairs for identifying functional microRNAs, as assessed by the ability of the microRNA of interest to target and downregulate target mRNA levels in cell culture system.118 These studies also suggest that some of the reported heterogeneity of platelet reactivity may be related to interindividual variations in specific microRNA-mRNA pairs of interest. Validation of this assertion would indicate a role for microRNAs in governing platelet reactivity through the regulation of specific mRNAs. Some studies suggest that the monitoring of platelet-derived microRNAs may also inform on the platelet response to pharmacological agents/inhibition, as decreased levels of platelet119 or plasma120 miR-223 may indicate a poor pharmacological response to clopidogrel (Chapter 36).

USE OF PLATELET MICRORNAs AS BIOMARKERS Likely involved in finely tuning expression of specific gene products that may govern platelet reactivity, a dysfunctional microRNA-based regulatory system may lead to deregulated platelet gene expression and function, leading to the

Fig. 6.8 Future perspectives for platelet microRNAs in clinical practice. Diagnostic and therapeutic applications of platelet microRNAs—personalizing diagnosis and medical treatment of patients. (From Perron et al.,44 with permission from Springer).

Platelet MicroRNAs and Cancer At the interface of exchanges with immune cells, cancer cells and endothelial cells, platelets are increasingly recognized as important contributors to the pathogenesis of cancer (Chapter 30).

Platelet MicroRNAs

In metastatic hematogenous lung cancer, for example, patients see their platelet count increase, although the underlying mechanism remains unknown.121 A possible role for platelet microRNAs has been studied in the context of BCR-ABL-negative myeloproliferative neoplasms (MPNs).122 This includes polycythemia vera,73 a clonal hematopoietic stem cell disorder in which the JAK2 V617F mutation is observed in >95% of patients. An initial study by Bruchova et al.73 reported differential expression of platelet microRNAs in polycythemia vera,73 including an upregulation of miR-26b, compared to controls. Specific microRNAs could also be correlated with JAK2 V617F frequency. Investigating the upstream components of the JAK2 signaling cascade, Girardot et al.122 found that the thrombopoietin receptor (TpoR, MPL) is down-regulated in certain MPN patients. Investigating negative regulators of MPL expression, they identified miR-28. miR-28 was found to be overexpressed in platelets of a fraction of MPN patients, while it was expressed at constant low levels in platelets from healthy subjects.122 These findings prompted the authors to conclude that aberrant microRNA expression may underlie the pathogenesis of MPNs.122 An oncogenic role of platelet microRNA miR-223 was proposed when it was found to (i) promote the invasiveness of lung cancer cells, by targeting the tumor suppressor EPB41L3,121 and (ii) promote cell growth and inhibit apoptosis, through regulation of FBXW7, in testicular germ cell tumor.123 Further clinically oriented studies are required in order to clarify the role and possible implication of microRNAs in specific platelet phenotypes, which would be expected to accelerate the transfer of knowledge from the bench to the bedside of patients.

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mRNA/protein levels also supports a miR-103-mediated regulation of SFRP4 expression in platelets of pre-DM2 subjects.126 One year later, the same group demonstrated that plasminogen activator inhibitor (PAI-1) could be regulated by platelet miR-30c.127

Platelet MicroRNAs and Chronic Kidney Disease A disease in which circulating platelets are exposed to extreme conditions is chronic kidney disease (CKD). Platelet exposure to uremic toxins and contact with artificial surfaces during dialysis had been reported to induce platelet abnormalities and to alter the platelet proteome. The platelet mRNA and microRNA profiles are altered in CKD patients, and appeared to be corrected by dialysis.128 Reduced in platelets of uremic patients, WD repeat-containing protein is regulated by miR-19b, a microRNA increased in platelets of uremic patients and involved in platelet reactivity.128 These results suggest that an alteration of microRNA-based mRNA regulatory mechanisms may underlie the platelet response to uremia and entail the development of platelet-related complications in CKD.128

Clinical Monitoring of Platelet MicroRNAs Using platelet microRNAs as biomarkers would imply collecting blood and isolating platelets prior to RNA extraction and qPCR detection/monitoring, a relatively tedious series of operations that may not be easily adapted to high-throughput screening in a clinical setting. A biomarker assay, such as the thrombomiR™ kit,129 based on the measurement of platelet microRNAs in plasma, which is a clinically available biofluid, would be more straightforward.

THERAPEUTIC APPLICATIONS Platelet MicroRNAs and Diabetes In type 2 diabetes mellitus (DM2), hyperglycemia reduced the platelet level of miR-223,124 miR-26b, miR-126, and miR-140, thereby causing upregulation of purinergic receptor P2Y12 (P2RY12) and P-selectin (SELP) mRNAs, which may contribute to adverse platelet function.125 An inverse correlation between miR-103 and secreted frizzled-related protein 4 (SFRP4)

The existence of functional microRNA machinery in human platelets opens up the possibility of modulating platelet gene expression using therapeutic RNAs. Indeed, the synthetic, microRNA duplex mimetic small interfering RNA (siRNA) species can be incorporated into Ago2 effector complexes and mediate potent gene regulatory effects through the recognition, cleavage and degradation of specific mRNAs (Fig. 6.9).

Fig. 6.9 Possible use of small therapeutic RNAs to modulate platelet gene expression through RNA interference. Small interfering RNAs (siRNAs) are assembled into a mature RNA-induced silencing complex (RISC) and guide specific mRNA target cleavage.

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Synthetic single-stranded microRNAs could also be developed and administered in order to restore the level (and function) of a defective platelet microRNA (Fig. 6.8). Alternatively, synthetic 2’OMe antisense oligonucleotides (AS) may be used to modulate, i.e., inhibits, the function of specific microRNAs (Fig. 6.8). These strategies based on therapeutic RNAs may represent promising approaches for the treatment of platelet-related diseases caused by gene deregulation and/or defective microRNAs and may prove to be effective in achieving desirable therapeutic benefits. In view of the relative refractoriness of platelets to transfection, significant delivery issues may be anticipated, which could limit the applicability of therapeutic RNAs to platelet biology. However, a report by Hong et al.130 suggests that this hurdle may not be unsurmountable, as the authors were able to introduce siRNAs into washed human platelets by using lipofectamine as a lipid carrier. Although the transfection efficiency was relatively low (
8%) and the degree of GAPDH mRNA knockdown rather modest (
26%), future technical improvements may help bring to reality the promises of manipulating gene expression in platelets. More recently, modulation of miR-326 levels in apheresis platelets, using transfection of synthetic single-stranded microRNAs (agomiR and antagomir), suggested that miR-326 may influence platelet apoptosis, by modulating Bcl-xL, without affecting platelet activation.131 The same year, Zhou et al.132 reported an interesting sequence of events, whereby antisense inhibition of miR-148a upregulated platelet T-cell ubiquitin ligand-2 mRNA expression, reduced platelet FcλRIIa signaling and decreased thrombosis in vivo in mice. These data suggest that modulating miR-148a expression is a potential therapeutic approach for thrombosis.108

CONCLUSION AND PERSPECTIVES Further investigations are required in order to improve our understanding of the platelet microRNA pathway and to define the biological role and importance of microRNAs in regulating the proteome and function of platelets in health and disease.62 As for neurological3 and infectious41 diseases, research advances in the field of platelet microRNAs may not only provide new perspectives to the etiology of platelet-related disorders (thrombotic or bleeding), but may be key to ensuring the development of efficient therapeutic tools and strategies aimed at modulating platelet function by preserving, restoring or neutralizing global and/or specific microRNA function in the platelets of patients at risk of developing, or suffering from, cardiovascular and platelet-related diseases.112 Acknowledgments The author thanks the past and current members of the Provost Lab for their contributions and fruitful discussions, including M. Jonathan Laugier, and the CHUQ Research Center Computer Graphics Department for the illustrations. This work was supported by the CBS/Canadian Institutes of Health Research Partnership—Blood Utilization and Conservation Initiative.

REFERENCES 1. Ouellet DL, Perron MP, Gobeil LA, Plante P, Provost P. MicroRNAs in gene regulation: when the smallest governs it all. J Biomed Biotechnol 2006;2006(4): 69616. 2. Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell 2009;136(2):215–33. 3. Provost P. MicroRNAs as a molecular basis for mental retardation, Alzheimer’s and prion diseases. Brain Res 2010;1338:58–66.

4. Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T. Identification of novel genes coding for small expressed RNAs. Science 2001;294(5543):853–8. 5. Lau NC, Lim LP, Weinstein EG, Bartel DP. An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 2001;294(5543):858–62. 6. Lee RC, Ambros V. An extensive class of small RNAs in Caenorhabditis elegans. Science 2001;294(5543):862–4. 7. Miranda KC, Huynh T, Tay Y, et al. A pattern-based method for the identification of MicroRNA binding sites and their corresponding heteroduplexes. Cell 2006;126(6):1203–17. 8. Friedman RC, Farh KK, Burge CB, Bartel DP. Most mammalian mRNAs are conserved targets of microRNAs. Genome Res 2009;19(1):92–105. 9. Lee Y, Kim M, Han J, et al. MicroRNA genes are transcribed by RNA polymerase II. EMBO J 2004;23(20):4051–60. 10. Lee Y, Ahn C, Han J, et al. The nuclear RNase III Drosha initiates microRNA processing. Nature 2003;425(6956):415–9. 11. Denli AM, Tops BB, Plasterk RH, Ketting RF, Hannon GJ. Processing of primary microRNAs by the microprocessor complex. Nature 2004;432(7014):231–5. 12. Gregory RI, Yan KP, Amuthan G, et al. The microprocessor complex mediates the genesis of microRNAs. Nature 2004;432 (7014):235–40. 13. Han J, Lee Y, Yeom KH, Kim YK, Jin H, Kim VN. The DroshaDGCR8 complex in primary microRNA processing. Genes Dev 2004;18(24):3016–27. 14. Partin AC, Ngo TD, Herrell E, Jeong BC, Hon G, Nam Y. Heme enables proper positioning of Drosha and DGCR8 on primary microRNAs. Nat Commun 2017;8(1):1737. 15. Lund E, Guttinger S, Calado A, Dahlberg JE, Kutay U. Nuclear export of microRNA precursors. Science 2004;303(5654):95–8. 16. Yi R, Qin Y, Macara IG, Cullen BR. Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev 2003;17(24):3011–6. 17. Bernstein E, Caudy AA, Hammond SM, Hannon GJ. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 2001;409(6818):363–6. 18. Provost P, Dishart D, Doucet J, Frendewey D, Samuelsson B, Radmark O. Ribonuclease activity and RNA binding of recombinant human dicer. EMBO J 2002;21(21):5864–74. 19. Zhang H, Kolb FA, Jaskiewicz L, Westhof E, Filipowicz W. Single processing center models for human dicer and bacterial RNase III. Cell 2004;118(1):57–68. 20. Chendrimada TP, Gregory RI, Kumaraswamy E, et al. TRBP recruits the dicer complex to Ago2 for microRNA processing and gene silencing. Nature 2005;436(7051):740–4. 21. Fareh M, Yeom KH, Haagsma AC, Chauhan S, Heo I, Joo C. TRBP ensures efficient dicer processing of precursor microRNA in RNAcrowded environments. Nat Commun 2016;7: 13694. 22. Lopez-Orozco J, Pare JM, Holme AL, Chaulk SG, Fahlman RP, Hobman TC. Functional analyses of phosphorylation events in human Argonaute 2. RNA 2015;21(12):2030–8. 23. Guo H, Ingolia NT, Weissman JS, Bartel DP. Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature 2010;466(7308):835–40. 24. Bhattacharyya SN, Habermacher R, Martine U, Closs EI, Filipowicz W. Relief of microRNA-mediated translational repression in human cells subjected to stress. Cell 2006;125(6):1111–24. 25. Liu J, Rivas FV, Wohlschlegel J, Yates 3rd JR, Parker R, Hannon GJ. A role for the P-body component GW182 in microRNA function. Nat Cell Biol 2005;7(12):1261–6. 26. Yang JS, Phillips MD, Betel D, et al. Widespread regulatory activity of vertebrate microRNA* species. RNA 2011;17(2):312–26. 27. Okamura K, Phillips MD, Tyler DM, Duan H, Chou YT, Lai EC. The regulatory activity of microRNA* species has substantial influence on microRNA and 3’ UTR evolution. Nat Struct Mol Biol 2008;15(4):354–63. 28. Griffiths-Jones S, Grocock RJ, van Dongen S, Bateman A, Enright AJ. miRBase: microRNA sequences, targets and gene nomenclature. Nucleic Acids Res 2006;34(Database issue):D140–4. 29. Griffiths-Jones S, Saini HK, van Dongen S, Enright AJ. miRBase: tools for microRNA genomics. Nucleic Acids Res 2008;36(Database issue):D154–8.

Platelet MicroRNAs 30. Kozomara A, Griffiths-Jones S. miRBase: annotating high confidence microRNAs using deep sequencing data. Nucleic Acids Res 2014;42(Database issue):D68–73. 31. Jo MH, Shin S, Jung SR, Kim E, Song JJ, Hohng S. Human Argonaute 2 has diverse reaction pathways on target RNAs. Mol Cell 2015;59(1):117–24. 32. Vasudevan S, Tong Y, Steitz JA. Switching from repression to activation: microRNAs can up-regulate translation. Science 2007;318 (5858):1931–4. 33. Bukhari SI, Truesdell SS, Lee S, et al. A specialized mechanism of translation mediated by FXR1a-associated MicroRNP in cellular quiescence. Mol Cell 2016;61(5):760–73. 34. Wong N, Wang X. miRDB: an online resource for microRNA target prediction and functional annotations. Nucleic Acids Res 2015;43 (Database issue):D146–52. 35. Agarwal V, Bell GW, Nam JW, Bartel DP. Predicting effective microRNA target sites in mammalian mRNAs. elife 2015;4. 36. Dweep H, Gretz N. miRWalk2.0: a comprehensive atlas of microRNA-target interactions. Nat Methods 2015;12(8):697. 37. Chou CH, Shrestha S, Yang CD, et al. miRTarBase update 2018: a resource for experimentally validated microRNA-target interactions. Nucleic Acids Res 2018;46(D1):D296–302. 38. Paraskevopoulou MD, Georgakilas G, Kostoulas N, et al. DIANAmicroT web server v5.0: service integration into miRNA functional analysis workflows. Nucleic Acids Res 2013;41(Web Server issue): W169–73. 39. Karagkouni D, Paraskevopoulou MD, Chatzopoulos S, et al. DIANA-TarBase v8: a decade-long collection of experimentally supported miRNA-gene interactions. Nucleic Acids Res 2018;46 (D1):D239–45. 40. Riffo-Campos AL, Riquelme I, Brebi-Mieville P. Tools for sequence-based miRNA target prediction: what to choose? Int J Mol Sci 2016;17(12). 41. Ouellet DL, Provost P. Current knowledge of MicroRNAs and noncoding RNAs in virus-infected cells. Methods Mol Biol 2010;623:35–65. 42. Ouellet DL, Plante I, Barat C, Tremblay MJ, Provost P. Emergence of a complex relationship between HIV-1 and the microRNA pathway. Methods Mol Biol 2009;487:415–33. 43. Perron MP, Provost P. Protein components of the microRNA pathway and human diseases. Methods Mol Biol 2009;487:369–85. 44. Perron MP, Boissonneault V, Gobeil LA, Ouellet DL, Provost P. Regulatory RNAs: future perspectives in diagnosis, prognosis, and individualized therapy. Methods Mol Biol 2007;361:311–26. 45. Isaacs SR, Wang J, Kim KW, et al. MicroRNAs in type 1 diabetes: complex interregulation of the immune system, beta cell function and viral infections. Curr Diab Rep 2016;16(12):133. 46. Paul P, Chakraborty A, Sarkar D, et al. Interplay between miRNAs and human diseases. J Cell Physiol 2018;233(3):2007–18. 47. Moszynska A, Gebert M, Collawn JF, Bartoszewski R. SNPs in microRNA target sites and their potential role in human disease. Open Biol 2017;7(4). 48. Elgheznawy A, Fleming I. Platelet-enriched microRNAs and cardiovascular homeostasis. Antioxid Redox Signal 2018;29(9):902–21. 49. Ts’ao CH. Rough endoplasmic reticulum and ribosomes in blood platelets. Scand J Haematol 1971;8(2):134–40. 50. Kieffer N, Guichard J, Farcet JP, Vainchenker W, Breton-Gorius J. Biosynthesis of major platelet proteins in human blood platelets. Eur J Biochem/FEBS 1987;164(1):189–95. 51. Booyse FM, Rafelson ME,J. Studies on human platelets. I. Synthesis of platelet protein in a cell-free system. Biochim Biophys Acta 1968;166(3):689–97. 52. Warshaw AL, Laster L, Shulman NR. Protein synthesis by human platelets. J Biol Chem 1967;242(9):2094–7. 53. Newman PJ, Gorski J, White GC, 2nd, Gidwitz S, Cretney CJ, Aster RH. Enzymatic amplification of platelet-specific messenger RNA using the polymerase chain reaction. J Clin Invest 1988; 82(2):739–43. 54. Roth GJ, Hickey MJ, Chung DW, Hickstein DD. Circulating human blood platelets retain appreciable amounts of poly (A)+ RNA. Biochem Biophys Res Commun 1989;160(2):705–10. 55. Weyrich AS, Dixon DA, Pabla R, et al. Signal-dependent translation of a regulatory protein, Bcl-3, in activated human platelets. Proc Natl Acad Sci U S A 1998;95(10):5556–61.

137

56. Evangelista V, Manarini S, Di Santo A, et al. De novo synthesis of cyclooxygenase-1 counteracts the suppression of platelet thromboxane biosynthesis by aspirin. Circ Res 2006;98(5):593–5. 57. Allam O, Samarani S, Jenabian MA, et al. Differential synthesis and release of IL-18 and IL-18 binding protein from human platelets and their implications for HIV infection. Cytokine 2017;90:144–54. 58. Hu Q, Cho MS, Thiagarajan P, Aung FM, Sood AK, AfsharKharghan V. A small amount of cyclooxygenase 2 (COX2) is constitutively expressed in platelets. Platelets 2017;28(1):99–102. 59. Mills EW, Green R, Ingolia NT. Slowed decay of mRNAs enhances platelet specific translation. Blood 2017;129(17):e38–48. 60. Rowley JW, Oler AJ, Tolley ND, et al. Genome-wide RNA-seq analysis of human and mouse platelet transcriptomes. Blood 2011; 118(14):e101–11. 61. Bray PF, McKenzie SE, Edelstein LC, et al. The complex transcriptional landscape of the anucleate human platelet. BMC Genomics 2013;14(1). 62. Macaulay IC, Carr P, Gusnanto A, Ouwehand WH, Fitzgerald D, Watkins NA. Platelet genomics and proteomics in human health and disease. J Clin Invest 2005;115(12):3370–7. 63. Bugert P, Kluter H. Profiling of gene transcripts in human platelets: an update of the platelet transcriptome. Platelets 2006; 17(7):503–4. 64. Dittrich M, Birschmann I, Stuhlfelder C, et al. Understanding platelets. Lessons from proteomics, genomics and promises from network analysis. Thromb Haemost 2005;94(5):916–25. 65. Bugert P, Dugrillon A, Gunaydin A, Eichler H, Kluter H. Messenger RNA profiling of human platelets by microarray hybridization. Thromb Haemost 2003;90(4):738–48. 66. Gnatenko DV, Dunn JJ, McCorkle SR, Weissmann D, Perrotta PL, Bahou WF. Transcript profiling of human platelets using microarray and serial analysis of gene expression. Blood 2003;101(6): 2285–93. 67. McRedmond JP, Park SD, Reilly DF, et al. Integration of proteomics and genomics in platelets: a profile of platelet proteins and platelet-specific genes. Mol Cell Proteomics 2004;3(2):133–44. 68. Rowley JW, Weyrich AS. Coordinate expression of transcripts and proteins in platelets. Blood 2013;121(26):5255–6. 69. Denis MM, Tolley ND, Bunting M, et al. Escaping the nuclear confines: signal-dependent pre-mRNA splicing in anucleate platelets. Cell 2005;122(3):379–91. 70. Schubert S, Weyrich AS, Rowley JW. A tour through the transcriptional landscape of platelets. Blood 2014;124(4):493–502. 71. Lai EC. Micro RNAs are complementary to 3’ UTR sequence motifs that mediate negative post-transcriptional regulation. Nat Genet 2002;30(4):363–4. 72. Dittrich M, Birschmann I, Pfrang J, et al. Analysis of SAGE data in human platelets: features of the transcriptome in an anucleate cell. Thromb Haemost 2006;95(4):643–51. 73. Bruchova H, Merkerova M, Prchal JT. Aberrant expression of microRNA in polycythemia vera. Haematologica 2008;93(7): 1009–16. 74. Merkerova M, Belickova M, Bruchova H. Differential expression of microRNAs in hematopoietic cell lineages. Eur J Haematol 2008; 81(4):304–10. 75. Kannan M, Mohan KV, Kulkarni S, Atreya C. Membrane arraybased differential profiling of platelets during storage for 52 miRNAs associated with apoptosis. Transfusion 2009;49(7):1443–50. 76. Landry P, Plante I, Ouellet DL, Perron MP, Rousseau G, Provost P. Existence of a microRNA pathway in anucleate platelets. Nat Struct Mol Biol 2009;16(9):961–6. 77. Nimmo RA, Slack FJ. An elegant miRror: microRNAs in stem cells, developmental timing and cancer. Chromosoma 2009;118(4): 405–18. 78. Roush S, Slack FJ. The let-7 family of microRNAs. Trends Cell Biol 2008;18(10):505–16. 79. Boyerinas B, Park SM, Hau A, Murmann AE, Peter ME. The role of let-7 in cell differentiation and cancer. Endocr Relat Cancer 2010;17(1):F19–36. 80. Ple H, Landry P, Benham A, Coarfa C, Gunaratne PH, Provost P. The repertoire and features of human platelet microRNAs. PLoS One 2012;7(12). e50746. 81. Ouellet DL, Plante I, Landry P, et al. Identification of functional microRNAs released through asymmetrical processing of HIV-1 TAR element. Nucleic Acids Res 2008;36(7):2353–65.

6

138

PART I Platelet Biology

82. Osman A, Falker K. Characterization of human platelet microRNA by quantitative PCR coupled with an annotation network for predicted target genes. Platelets 2011;22(6):433–41. 83. Kahr WH, Hinckley J, Li L, et al. Mutations in NBEAL2, encoding a BEACH protein, cause gray platelet syndrome. Nat Genet 2011; 43(8):738–40. 84. Lauziere V, Lessard M, Meunier AJ, McCoy M, Bergeron LJ, Corbin F. Unusual subcellular confinement of the fragile X mental retardation protein (FMRP) in circulating human platelets: complete polyribosome dissociation. Biochimie 2012;94(4): 1069–73. 85. Heo I, Joo C, Kim YK, et al. TUT4 in concert with Lin28 suppresses microRNA biogenesis through pre-microRNA uridylation. Cell 2009;138(4):696–708. 86. Jones MR, Quinton LJ, Blahna MT, et al. Zcchc11-dependent uridylation of microRNA directs cytokine expression. Nat Cell Biol 2009;11(9):1157–63. 87. Jones MR, Blahna MT, Kozlowski E, et al. Zcchc11 uridylates mature miRNAs to enhance neonatal IGF-1 expression, growth, and survival. PLoS Genet 2012;8(11). e1003105. 88. Garzon R, Pichiorri F, Palumbo T, et al. MicroRNA fingerprints during human megakaryocytopoiesis. Proc Natl Acad Sci U S A 2006;103(13):5078–83. 89. Opalinska JB, Bersenev A, Zhang Z, et al. MicroRNA expression in maturing murine megakaryocytes. Blood 2010;116(23):e128–38. 90. Hutvagner G, Simard MJ. Argonaute proteins: key players in RNA silencing. Nat Rev Mol Cell Biol 2008;9(1):22–32. 91. Beitzinger M, Peters L, Zhu JY, Kremmer E, Meister G. Identification of human microRNA targets from isolated argonaute protein complexes. RNA Biol 2007;4(2):76–84. 92. Easow G, Teleman AA, Cohen SM. Isolation of microRNA targets by miRNP immunopurification. RNA 2007;13(8):1198–204. 93. Karginov FV, Conaco C, Xuan Z, et al. A biochemical approach to identifying microRNA targets. Proc Natl Acad Sci U S A 2007; 104(49):19291–6. 94. Corduan A, Ple H, Laffont B, et al. Dissociation of SERPINE1 mRNA from the translational repressor proteins Ago2 and TIA-1 upon platelet activation. Thromb Haemost 2015;113(5): 1046–59. 95. Cimmino G, Tarallo R, Nassa G, et al. Activating stimuli induce platelet microRNA modulation and proteome reorganisation. Thromb Haemost 2015;114(1):96–108. 96. Dangwal S, Thum T. MicroRNAs in platelet biogenesis and function. Thromb Haemost 2012;108(4):599–604. 97. Edelstein LC, Bray PF. MicroRNAs in platelet production and activation. Blood 2011;117(20):5289–96. 98. Edelstein LC, McKenzie SE, Shaw C, Holinstat MA, Kunapuli SP, Bray PF. MicroRNAs in platelet production and activation. J Thromb Haemost 2013;11(Suppl 1):340–50. 99. Dangwal S, Thum T. MicroRNAs in platelet physiology and pathology. Hamostaseologie 2013;33(1):17–20. 100. Elgheznawy A, Shi L, Hu J, et al. Dicer cleavage by calpain determines platelet microRNA levels and function in diabetes. Circ Res 2015;117(2):157–65. 101. Rowley JW, Chappaz S, Corduan A, et al. Dicer1-mediated miRNA processing shapes the mRNA profile and function of murine platelets. Blood 2016;127(14):1743–51. 102. Skog J, Wurdinger T, van Rijn S, et al. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat Cell Biol 2008;10(12): 1470–6. 103. Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 2007;9(6):654–9. 104. Laffont B, Corduan A, Ple H, et al. Activated platelets can deliver mRNA regulatory Ago2•microRNA complexes to endothelial cells via microparticles. Blood 2013;122(2):253–61. 105. Li J, Tan M, Xiang Q, Zhou Z, Yan H. Thrombin-activated plateletderived exosomes regulate endothelial cell expression of ICAM-1 via microRNA-223 during the thrombosis-inflammation response. Thromb Res 2017;154:96–105. 106. Laffont B, Corduan A, Rousseau M, et al. Platelet microparticles reprogram macrophage gene expression and function. Thromb Haemost 2016;115(2):311–23. 107. Pan Y, Liang H, Liu H, et al. Platelet-secreted microRNA-223 promotes endothelial cell apoptosis induced by advanced glycation

108. 109. 110. 111. 112. 113. 114. 115. 116. 117. 118. 119. 120.

121. 122.

123.

124. 125.

126. 127. 128. 129. 130. 131. 132.

end products via targeting the insulin-like growth factor 1 receptor. J Immunol 2014;192(1):437–46. Provost P. The clinical significance of platelet microparticleassociated microRNAs. Clin Chem Lab Med: CCLM/FESCC 2017;55(5):657–66. Ouwehand WH. Platelet genomics and the risk of atherothrombosis. J Thromb Haemost 2007;5(Suppl 1):188–95. Thomson JM, Parker J, Perou CM, Hammond SM. A custom microarray platform for analysis of microRNA gene expression. Nat Methods 2004;1(1):47–53. He L, Thomson JM, Hemann MT, et al. A microRNA polycistron as a potential human oncogene. Nature 2005;435(7043):828–33. Landry P, Provost P. MicroRNAs as modulators of the platelet proteome. Curr Proteom 2011;8(3):193–9. Sol N, Wurdinger T. Platelet RNA signatures for the detection of cancer. Cancer Metastasis Rev 2017;36(2):263–72. McManus DD, Freedman JE. MicroRNAs in platelet function and cardiovascular disease. Nat Rev Cardiol 2015;12(12):711–7. Bijak M, Dzieciol M, Rywaniak J, Saluk J, Zielinska M. Platelets miRNA as a prediction marker of thrombotic episodes. Dis Markers 2016;2016: 2872507. Li S, Guo LZ, Kim MH, Han JY, Serebruany V. Platelet microRNA for predicting acute myocardial infarction. J Thromb Thrombolysis 2017;44(4):556–64. Kondkar AA, Bray MS, Leal SM, et al. VAMP8/endobrevin is overexpressed in hyperreactive human platelets: suggested role for platelet microRNA. J Thromb Haemost 2010;8(2):369–78. Nagalla S, Shaw C, Kong X, et al. Platelet microRNA-mRNA coexpression profiles correlate with platelet reactivity. Blood 2011; 117(19):5189–97. Shi R, Ge L, Zhou X, et al. Decreased platelet miR-223 expression is associated with high on-clopidogrel platelet reactivity. Thromb Res 2013;131(6):508–13. Zhang YY, Zhou X, Ji WJ, et al. Decreased circulating microRNA223 level predicts high on-treatment platelet reactivity in patients with troponin-negative non-ST elevation acute coronary syndrome. J Thromb Thrombolysis 2014;38(1):65–72. Liang H, Yan X, Pan Y, et al. MicroRNA-223 delivered by plateletderived microvesicles promotes lung cancer cell invasion via targeting tumor suppressor EPB41L3. Mol Cancer 2015;14:58. Girardot M, Pecquet C, Boukour S, et al. miR-28 is a thrombopoietin receptor targeting microRNA detected in a fraction of myeloproliferative neoplasm patient platelets. Blood 2010;116(3):437–45. Liu J, Shi H, Li X, Chen G, Larsson C, Lui W-O. miR-223-3p regulates cell growth and apoptosis via FBXW7 suggesting an oncogenic role in human testicular germ cell tumors. Int J Oncol 2017;50(2):356–64. Duan X, Zhan Q, Song B, et al. Detection of platelet microRNA expression in patients with diabetes mellitus with or without ischemic stroke. J Diabetes Complicat 2014;28(5):705–10. Fejes Z, Poliska S, Czimmerer Z, et al. Hyperglycaemia suppresses microRNA expression in platelets to increase P2RY12 and SELP levels in type 2 diabetes mellitus. Thromb Haemost 2017;117(3): 529–42. Luo M, Li R, Deng X, et al. Platelet-derived miR-103b as a novel biomarker for the early diagnosis of type 2 diabetes. Acta Diabetol 2015;52(5):943–9. Luo M, Li R, Ren M, et al. Hyperglycaemia-induced reciprocal changes in miR-30c and PAI-1 expression in platelets. Sci Rep 2016;6: 36687. Ple H, Maltais M, Corduan A, Rousseau G, Madore F, Provost P. Alteration of the platelet transcriptome in chronic kidney disease. Thromb Haemost 2012;108(4):605–15. Schoergenhofer C, Hobl EL, Schellongowski P, et al. Clopidogrel in critically ill patients. Clin Pharmacol Ther 2018;103(2):217–23. Hong W, Kondkar AA, Nagalla S, et al. Transfection of human platelets with short interfering RNA. Clin Transl Sci 2011;4 (3):180–2. Yu S, Huang H, Deng G, et al. miR-326 targets antiapoptotic BclxL and mediates apoptosis in human platelets. PLoS One 2015;10 (4): e0122784. Zhou Y, Abraham S, Andre P, et al. Anti-miR-148a regulates platelet FcgammaRIIA signaling and decreases thrombosis in vivo in mice. Blood 2015;126(26):2871–81.