Sven Danckwardt
email: sven.danckwardt@unimedizin-mainz.de

David-Alexandre Tregouet

Elisabetta Castoldi
email: e.castoldi@maastrichtuniversity.nl

David-Alexandre Trégouët

hemostatic genes: mechanisms and emerging therapeutic concepts in thrombo-inflammatory disorders

Keywords: noncoding RNAs (miRNA, lncRNA, circRNA, ceRNA), RNA-binding proteins, RNA (m 6 A) and posttranslational protein modification, splicing, polyadenylation, biomarker

Introduction

In light of the current SARS-CoV2 pandemic, the mechanisms underlying the crosstalk between the hemostatic system and the immune system have received unprecedented attention. This interplay plays a central role in many pathological processes, ranging from sepsis to cardiovascular disease.

Perturbations of the hemostatic system are common in sepsis, the leading cause of death in critically ill patients worldwide [START_REF] Angus | Severe sepsis and septic shock[END_REF] . As a systemic inflammatory response to severe infections, sepsis involves excessive activation of the coagulation system [START_REF] Hotchkiss | The pathophysiology and treatment of sepsis[END_REF] . This can result in severe complications such as disseminated intravascular coagulation (DIC), which eventually leads to tissue necrosis, multiple organ failure and death, illustrating that inappropriate amplification of protective host-defense mechanisms can become a devastating alliance of harm [START_REF] Rittirsch | Harmful molecular mechanisms in sepsis[END_REF] .

Cardiovascular disorders including myocardial infarction, ischemic stroke and venous thromboembolism are the leading global cause of mortality with over 17 million deaths annually [START_REF] Lozano | Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: A systematic analysis for the global burden of disease study 2010[END_REF] . The incidence of cardiovascular disorders increases markedly with age, starting in the late 40s, with a dramatic increase occurring at 60 years of age [START_REF] Berry | Lifetime risks of cardiovascular disease[END_REF] . They account for approximately 32% of all deaths worldwide, underscoring the need of illuminating underlying mechanisms and devising therapeutic interventions to treat and prevent cardiovascular disorders [START_REF] Nabel | Cardiovascular disease[END_REF] .

The immune system and the hemostatic system are closely linked [START_REF] Levi | Bidirectional relation between inflammation and coagulation[END_REF] and their responses tend to reinforce each other [START_REF] Borissoff | The hemostatic system as a modulator of atherosclerosis[END_REF][START_REF] Antoniak | The coagulation system in host defense[END_REF] . Activation of coagulation and fibrin deposition in response to inflammation is well known. This led to the emergence of the concept of immunothrombosis, a defense mechanism in which inflammatory cells participate in thrombotic processes, and thrombosis in turn acts as an intravascular effector of innate immunity by limiting the spread of invading pathogens [START_REF] Engelmann | Thrombosis as an intravascular effector of innate immunity[END_REF] . However, a derailed hemostatic response can lead to a situation where coagulation, fibrin deposition and thrombosis contribute to disease, as evidenced by the propagation and exacerbation of atherosclerotic plaques 11 . Another example is the systemic activation of coagulation combined with microvascular failure resulting from the systemic inflammatory response to severe infection or sepsis, which eventually contributes to multiple organ dysfunction, such as in septicemia [START_REF] Rittirsch | Harmful molecular mechanisms in sepsis[END_REF] or COVID-19 [START_REF] Jose | Covid-19 cytokine storm: The interplay between inflammation and coagulation[END_REF] .

The multifaceted and intricate link between hemostasis and inflammation involves crosstalk between both systems at multiple levels [START_REF] Rittirsch | Harmful molecular mechanisms in sepsis[END_REF][START_REF] Levi | Bidirectional relation between inflammation and coagulation[END_REF][START_REF] Borissoff | The hemostatic system as a modulator of atherosclerosis[END_REF][START_REF] Antoniak | The coagulation system in host defense[END_REF][START_REF] Engelmann | Thrombosis as an intravascular effector of innate immunity[END_REF][11] , including coordinated changes in gene expression in megakaryocytes, immune cells, the vessel wall and/or the liver. A notable example is the acute phase response, in which central hemostatic components such as fibrinogen [START_REF] Gruys | Acute phase reaction and acute phase proteins[END_REF][START_REF] Fish | Fibrinogen gene regulation[END_REF] , Von Willebrand factor [START_REF] Pottinger | Von willebrand factor is an acute phase reactant in man[END_REF][START_REF] Chen | Inflammation, von willebrand factor, and adamts13[END_REF] and factor VIII [START_REF] Rennie | Changes in coagulation factors following acute myocardial infarction in man[END_REF][START_REF] Vaziri | Changes of coagulation factors IX, VIII, VII, X and V in nephrotic syndrome[END_REF][START_REF] Wyshock | Cofactors V and VIII after endotoxin administration to human volunteers[END_REF][START_REF] Stirling | Transcriptional activation of the factor VIII gene in liver cell lines by interleukin-6[END_REF][START_REF] Begbie | The factor VIII acute phase response requires the participation of NFkappab and c/ebp[END_REF] are induced in response to inflammatory signals. Such changes in gene expression are primarily regulated at the level of transcription, and the transcriptional regulation of hemostasis-related genes in physiological and pathological conditions has been well studied [START_REF] Fish | Fibrinogen gene regulation[END_REF][START_REF] Bancroft | The human prothrombin gene: Transcriptional regulation in HEPG2 cells[END_REF][START_REF] Parry | Transcriptional regulation of tissue factor expression in human endothelial cells[END_REF][START_REF] Zhang | Genetic mechanisms of age regulation of protein c and blood coagulation[END_REF][START_REF] Hung | Regulation of human coagulation factor X gene expression by GATA-4 and the SP family of transcription factors[END_REF][START_REF] Peghaire | The transcription factor ERG regulates a low shear stress-induced anti-thrombotic pathway in the microvasculature[END_REF] . In the present review we focus on emerging concepts of posttranscriptional mechanisms underlying the control of hemostasis and its crosstalk to other systems. In doing so, we discuss examples of the complexity of the transcriptome architecture arising from the use of alternative transcription start sites, exons and polyadenylation sites, as well as gene regulation by noncoding RNAs (miRNAs, lncRNAs, circRNAs), RNA-binding proteins and mechanisms of RNA modification. Remarkably, many of these regulatory principles also play an important functional role in tuning the immune system [START_REF] Chen | Gene regulation in the immune system by long noncoding RNAs[END_REF][START_REF] Shulman | The RNA modification n6-methyladenosine as a novel regulator of the immune system[END_REF][START_REF] Chen | AU-rich elements: Characterization and importance in mRNA degradation[END_REF][START_REF] Anderson | Post-transcriptional control of cytokine production[END_REF][START_REF] Carpenter | Post-transcriptional regulation of gene expression in innate immunity[END_REF] , suggesting conserved regulatory links between both systems. Finally, we also illustrate the emerging therapeutic opportunities on the cusp of a new era of targeted therapeutic approaches [START_REF] Neil | Reprogramming RNA processing: An emerging therapeutic landscape[END_REF] , exemplified by the recent introduction of novel RNA therapeutics in the hemostatic system [START_REF] Büller | Factor xi antisense oligonucleotide for prevention of venous thrombosis[END_REF] .

Role of splicing regulation in the hemostatic system

With the completion of the human genome project in 2003, it became apparent that the human genome comprises around 22.000 protein-coding genes, far less than actually required for the functional complexity in higher eukaryotes [START_REF] Esea | Initial sequencing and analysis of the human genome[END_REF] . On the other hand, next generation RNA sequencing and particularly the recently introduced long-read sequencing technologies [START_REF] Paoli-Iseppi | Isoform age -splice isoform profiling using long-read technologies[END_REF][START_REF] Glinos | Transcriptome variation in human tissues revealed by long-read sequencing[END_REF] are uncovering a perplexingly complex transcriptome architecture that arises from the use of alternative transcription start sites, exons and polyadenylation sites [START_REF] Carninci | The transcriptional landscape of the mammalian genome[END_REF][START_REF] Wang | Alternative isoform regulation in human tissue transcriptomes[END_REF] . The combinatorial use of such elements considerably expands genomic information and is subject to dynamic spatial and temporal modulation during development and adaptation (Figure 1). Pre-mRNA splicing, i.e. the accurate removal of introns and ligation of exons, is a pivotal step in the co-and posttranscriptional regulation of gene expression [START_REF] Lee | Mechanisms and regulation of alternative pre-mRNA splicing[END_REF] . Depending on how the exon/intron structure of the pre-mRNA is decoded by the spliceosome, the same primary transcript may be processed into different mature mRNAs (alternative splicing), encoding different isoforms of the same protein. In fact, the recognition of exon/intron boundaries in the pre-mRNA is critically dependent on the engagement of nearby splicing enhancer and silencer sequences by trans-acting proteins (splicing factors) whose availability varies in different cell types and disease states. As a consequence, splicing patterns are typically regulated in a tissue-specific manner and may change according to the developmental stage or in response to pathological processes. Moreover, they can be disrupted by genetic variants that weaken (or strengthen) the consensus sequences recognized by the spliceosome on the pre-mRNA. This is a well-known mechanism of disease in mendelian disorders [START_REF] Danckwardt | Abnormally spliced beta-globin mRNAs: A single point mutation generates transcripts sensitive and insensitive to nonsense-mediated mRNA decay[END_REF] , but it is increasingly appreciated that much of the genetic variation associated with complex traits also acts by altering splicing patterns [START_REF] Li | RNA splicing is a primary link between genetic variation and disease[END_REF][START_REF] Castaldi | Bridging the splicing gap in human genetics with long-read RNA sequencing: Finding the protein isoform drivers of disease[END_REF] Like most human genes [START_REF] Reyes | Alternative start and termination sites of transcription drive most transcript isoform differences across human tissues[END_REF] , many genes encoding proteins of the hemostatic system are alternatively spliced [START_REF] Yuan | Role of RNA splicing in mediating lineage-specific expression of the von Willebrand factor gene in the endothelium[END_REF][START_REF] Hsu | Molecular cloning of platelet factor XI, an alternative splicing product of the plasma factor XI gene[END_REF][START_REF] Asselta | Molecular characterization of in-frame and out-of-frame alternative splicings in coagulation factor XI pre-mRNA[END_REF][START_REF] Podmore | Real-time quantitative pcr analysis of factor XI mRNA variants in human platelets[END_REF][START_REF] Fu | Carboxy-terminal-extended variant of the human fibrinogen alpha subunit: A novel exon conferring marked homology to beta and gamma subunits[END_REF][START_REF] Chung | Gamma and gamma' chains of human fibrinogen are produced by alternative mRNA processing[END_REF][START_REF] Vincent | Coagulation factor v(a2440g) causes east texas bleeding disorder via TFPIiα[END_REF][START_REF] Hagen | Characterization of a cdna coding for human factor VII[END_REF][START_REF] O'hara | Nucleotide sequence of the gene coding for human factor VII, a vitamin k-dependent protein participating in blood coagulation[END_REF][START_REF] Shovlin | Endothelial cell processing and alternatively spliced transcripts of factor VIII: Potential implications for coagulation cascades and pulmonary hypertension[END_REF][START_REF] Zamolodchikov | A short isoform of coagulation factor XII mRNA is expressed by neurons in the human brain[END_REF][START_REF] Rehman | Identification of a novel alternatively spliced isoform of antithrombin containing an additional RCL-like loop[END_REF][START_REF] Bano | Identification and characterization of a novel isoform of heparin cofactor II in human liver[END_REF] . This often results in isoforms with distinct structural and functional

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characteristics, as exemplified by two major components of the extrinsic coagulation pathway (Figure 2). Tissue factor (TF), the main trigger of blood coagulation, acts as cofactor of the circulating serine protease factor VIIa (FVIIa) and comes in two isoforms: as membrane-bound (fulllength) protein and as a shorter, alternatively spliced variant that is secreted in soluble form (Figure 2) [START_REF] Bogdanov | Alternatively spliced human tissue factor: A circulating, soluble, thrombogenic protein[END_REF] . The two isoforms are identical at the N-terminal end, but the soluble form, which arises from exon 5 skipping, lacks the transmembrane and cytoplasmic domains, and has a completely different C-terminal sequence [START_REF] Bogdanov | Alternatively spliced human tissue factor: A circulating, soluble, thrombogenic protein[END_REF] . Just as full-length TF, alternatively spliced TF is produced by a variety of cell types [START_REF] Bogdanov | Alternatively spliced human tissue factor: A circulating, soluble, thrombogenic protein[END_REF][START_REF] Witkowski | Tissue factor as a link between inflammation and coagulation[END_REF] , is induced by pro-inflammatory stimuli [START_REF] Witkowski | Tissue factor as a link between inflammation and coagulation[END_REF][START_REF] Szotowski | Procoagulant soluble tissue factor is released from endothelial cells in response to inflammatory cytokines[END_REF] and enhances factor X (FX) activation by FVIIa, albeit less potently than full-length TF [START_REF] Bogdanov | Alternatively spliced human tissue factor: A circulating, soluble, thrombogenic protein[END_REF] . However, while membrane-bound TF is essential for normal hemostasis, elevated intravascular levels of TF have been proposed to contribute to venous as well as arterial thrombosis [START_REF] Owens | Tissue factor and thrombosis: The clot starts here[END_REF] . Despite conflicting data, it has been suggested that soluble TF, which is most likely dispensable for normal hemostasis, may represent a preferential target for antithrombotic therapy than fulllength TF, due to a lower risk of bleeding [START_REF] Bogdanov | Soluble tissue factor" in the 21st century: Definitions, biochemistry, and pathophysiological role in thrombus formation[END_REF] .

Tissue factor pathway inhibitor (TFPI) is a glycoprotein that functions as an inhibitor of coagulation and of TF-dependent signaling [START_REF] Mast | Regulation of coagulation by tissue factor pathway inhibitor: Implications for hemophilia therapy[END_REF] . The TFPI gene encodes two main splicing isoforms that are generated by the alternative inclusion of exon 8 (TFPIβ) or exons 9-10 (TFPIα) in the mature mRNA (Figure 2). Both isoforms are expressed in endothelial cells, but TFPIα is also found in plasma, platelets and the extracellular matrix [START_REF] Peterson | Major reservoir for heparin-releasable TFPIα (tissue factor pathway inhibitor α) is extracellular matrix[END_REF] . Structurally, TFPIα comprises an acidic N-terminus, three Kunitz domains and a basic C-terminus, whereas TFPIβ lacks the third Kunitz domain and the basic C-terminus, which are replaced by a glycosylphosphatidylinositol-anchor that tethers the protein to the cell membrane [START_REF] Wood | Biology of tissue factor pathway inhibitor[END_REF] . Both TFPI isoforms inhibit TF/FVIIa and FXa with their Kunitz-1 and Kunitz-2 domains, respectively, but TFPIα has additional properties by virtue of its Kunitz-3 domain (which binds protein S) and basic C-terminus (which binds FV/FV-short). Binding to protein S and FV/FV-short prevents the clearance of plasma TFPIα from the circulation [START_REF] Vincent | Coagulation factor v(a2440g) causes east texas bleeding disorder via TFPIiα[END_REF][START_REF] Castoldi | Hereditary and acquired protein s deficiencies are associated with low TFPI levels in plasma[END_REF][START_REF] Duckers | Low plasma levels of tissue factor pathway inhibitor in patients with congenital factor V deficiency[END_REF] and promotes its association with biological membranes, enhancing its anti-FXa activity [START_REF] Hackeng | Protein s stimulates inhibition of the tissue factor pathway by tissue factor pathway inhibitor[END_REF][START_REF] Santamaria | Factor V has an anticoagulant cofactor activity that targets the early phase of coagulation[END_REF][START_REF] Dahlbäck | Factor v-short and protein s as synergistic tissue factor pathway inhibitor (TFPIα) cofactors[END_REF] . Moreover, the interaction with FV/FV-short allows TFPIα to inhibit FV activation [START_REF] Van Doorn | The c-terminus of tissue factor pathway inhibitor-α inhibits factor V activation by protecting the Arg(1545) cleavage site[END_REF] and early prothrombinase activity [START_REF] Wood | Tissue factor pathway inhibitor-alpha inhibits prothrombinase during the initiation of blood coagulation[END_REF][START_REF] Wood | Tfpiα interacts with FVa and FXa to inhibit prothrombinase during the initiation of coagulation[END_REF] , while TFPIβ lacks these anticoagulant functions.

These and other [START_REF] Vincent | Coagulation factor v(a2440g) causes east texas bleeding disorder via TFPIiα[END_REF][START_REF] De Willige | The pleiotropic role of the fibrinogen gamma' chain in hemostasis[END_REF] examples illustrate how alternative splicing can change the structural and hence functional properties of central components in the hemostatic system [START_REF] Girard | Re-evaluation of mouse tissue factor pathway inhibitor and comparison of mouse and human tissue factor pathway inhibitor physiology[END_REF] . Extracellular signals, such as pro-inflammatory cytokines, can modify global patterns of alternative splicing [START_REF] Louis | TNF-alpha regulates alternative splicing of genes participating in pathways of crucial metabolic syndromes; a transcriptome wide study[END_REF] and it will be interesting to explore how this plays out in different (disease) contexts, including COVID-19 [START_REF] Wang | Abnormal global alternative RNA splicing in covid-19 patients[END_REF] . Moreover, since alternative splicing is pervasive and there are increasingly new therapeutic means to (re)direct splicing [START_REF] Lee | Therapeutic targeting of splicing in cancer[END_REF]79 , modulation of alternative splicing may become relevant for the therapeutic manipulation of the hemostatic system. In particular, many studies support the utility of antisense oligonucleotides (ASOs) to mask specific splicing signals on the pre-mRNA and thus prevent the recognition of these sequences by spliceosomal components, thereby re-directing splicing [START_REF] Havens | Splice-switching antisense oligonucleotides as therapeutic drugs[END_REF] . Alternatively, ad hoc engineered U1snRNA can be employed to promote the usage of donor splice sites that are naturally weak or have been disrupted by mutation [START_REF] Alanis | An exonspecific U1 small nuclear RNA (snRNA) strategy to correct splicing defects[END_REF] .

Apart from diversifying the transcriptome and proteome, alternative splicing has been proposed to contribute to the overall regulation of gene expression through its coupling with nonsense mediated decay (NMD), a surveillance pathway that degrades mRNAs containing premature stop codons. In fact, it has been observed that up to one third of all human transcripts are normally spliced into non-viable mRNAs that are substrates for NMD. This phenomenon, known as "regulated unproductive splicing and translation" (RUST), has been interpreted as a mechanism for the post-transcriptional temporal and spatial fine-tuning of gene expression [START_REF] Lewis | Evidence for the widespread coupling of alternative splicing and nonsense-mediated mRNA decay in humans[END_REF] . Evidence that this control mechanism may apply within the realm of hemostasis has been provided for the F11 gene, encoding coagulation factor XI [START_REF] Asselta | Molecular characterization of in-frame and out-of-frame alternative splicings in coagulation factor XI pre-mRNA[END_REF] . Interestingly, targeting non-productive splicing by antisense oligonucleotides can be exploited for the upregulation of gene expression from wild-type or hypomorphic alleles in disease states [START_REF] Lim | Antisense oligonucleotide modulation of non-productive alternative splicing upregulates gene expression[END_REF] .

Role of polyadenylation in the hemostatic system

In addition to capping and splicing, almost all eukaryotic transcripts undergo further processing at the RNA 3'-end (Figure 1). For most genes, this involves endonucleolytic cleavage and nontemplated polyadenylation (CPA) before the mature RNA can be exported to the cytoplasm [START_REF] Danckwardt | 3' end mRNA processing: Molecular mechanisms and implications for health and disease[END_REF] .

As CPA controls almost all genes, regulation of CPA has evolved as an important layer of gene expression regulation. CPA is carried out by a multi-subunit complex involving over 80 transacting proteins organized in four core protein subcomplexes [START_REF] Shi | The end of the message: Multiple protein-RNA interactions define the mRNA polyadenylation site[END_REF] . The recruitment of these multimeric complexes to dedicated, but largely poorly conserved, RNA sequence elements [START_REF] Gruber | A comprehensive analysis of 3′ end sequencing data sets reveals novel polyadenylation signals and the repressive role of heterogeneous ribonucleoprotein C on cleavage and polyadenylation[END_REF] ensures that 3'-end processing of the nascent transcript occurs timely and at the right position [START_REF] Proudfoot | Transcriptional termination in mammals: Stopping the RNA polymerase II juggernaut[END_REF][START_REF] Reimer | Co-transcriptional splicing regulates 3' end cleavage during mammalian erythropoiesis[END_REF] . Perturbations of this process -due to mutations in RNA sequence elements or defects in the RNA processing machinery -have drastic consequences, as exemplified by numerous diseases [START_REF] Nourse | Emerging roles of RNA 3′-end cleavage and polyadenylation in pathogenesis, diagnosis and therapy of human disorders[END_REF][START_REF] Hollerer | mRNA 3'end processing: A tale of the tail reaches the clinic[END_REF] .

The common thrombophilia mutation in the prothrombin (F2) gene (F2 G20210A) is a prime example of how mutations in noncoding regions can become pathogenic [START_REF] Danckwardt | 3' end mRNA processing: Molecular mechanisms and implications for health and disease[END_REF] . This mutation affects the last nucleotide of the 3'-untranslated region (UTR), where the pre-mRNA is cleaved and polyadenylated. As a result of the mutation, the efficiency of endonucleolytic cleavage is increased, leading to more prothrombin mRNA and protein expression. Although this mutation merely increases the amount of the precursor of a central hemostatic component (i.e., thrombin), it already shifts the balance of the hemostatic system toward a procoagulant condition [START_REF] Poort | A common genetic variation in the 3'untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis[END_REF][START_REF] Ceelie | Polymorphisms in the prothrombin gene and their association with plasma prothrombin levels[END_REF][START_REF] Gehring | Increased efficiency of mRNA 3' end formation: A new genetic mechanism contributing to hereditary thrombophilia[END_REF] . Consequently, the expression of F2 must be tightly controlled: even small

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changes (1.5-to 1.7-fold) in gene expression due to mutations at this and other nearby positions (F2 C20209T and F2 G20221T) [START_REF] Gehring | Increased efficiency of mRNA 3' end formation: A new genetic mechanism contributing to hereditary thrombophilia[END_REF][START_REF] Danckwardt | The prothrombin 3'end formation signal reveals a unique architecture that is sensitive to thrombophilic gain-of-function mutations[END_REF] can result in clinically relevant thrombophilia 94- 97 .

Compared to other genes, the architecture of sequence determinants directing 3'-end processing in F2 is unconventional [START_REF] Danckwardt | 3' end processing of the prothrombin mRNA in thrombophilia[END_REF] . It consists of weak signals, which explains the unusual susceptibility to thrombophilic gain-of-function mutations [START_REF] Danckwardt | The prothrombin 3'end formation signal reveals a unique architecture that is sensitive to thrombophilic gain-of-function mutations[END_REF][START_REF] Danckwardt | The prothrombin 20209 c>t mutation in jewish-moroccan caucasians: Molecular analysis of gain-of-function of 3'end processing[END_REF] . At the same time, this configuration allows for mechanisms that enhance processing and thereby upregulate F2 expression when needed [START_REF] Danckwardt | Splicing factors stimulate polyadenylation via uses at non-canonical 3' end formation signals[END_REF] . This is achieved through complex, mutually exclusive binding of suppressive and stimulatory RNA binding proteins (RBPs), and is regulated by activation of p38 MAPK (Figure 3) [START_REF] Danckwardt | P38 mapk controls prothrombin expression by regulated RNA 3' end processing[END_REF] . After phosphorylation by p38 MAPK, inhibitory RBPs (FBP2, FBP3) can no longer bind to the processing sites in the F2 pre-mRNA, allowing 3'-end processing to proceed. Thus, virtually all types of 'environmental' conditions that lead to activation of p38 MAPK 100, 101 can induce F2 expression.

Inflammatory conditions are known to trigger F2 expression [102][103][104][105][106][107] . Consistently, the mechanism described here was found to account for the induction of F2 expression under inflammatory conditions, including septicemia [START_REF] Danckwardt | P38 mapk controls prothrombin expression by regulated RNA 3' end processing[END_REF]108 . While this may contribute to the initial onset and undesirable propagation of hemostatic perturbances during septicemia, such mechanisms may also play a compensatory role [START_REF] Rittirsch | Harmful molecular mechanisms in sepsis[END_REF] . After an initial hypercoagulable state, septicemia is often followed by a hemorrhagic phase, in part due to consumption of procoagulant components 109 . Such conditions of increased turnover and demand require mechanisms to restore the hemostatic balance and stockpile hemostatic components 110 .

In addition to the critical function in hemostasis, the role of thrombin in angiogenesis 111 suggests that regulatory mechanisms have evolved a sensor for low oxygen pressure. This could explain why F2 is overexpressed due to ischemic events 112 or in the tumor micromilieu [START_REF] Danckwardt | P38 mapk controls prothrombin expression by regulated RNA 3' end processing[END_REF] .

Consistent with its role in oxygen pressure sensing 100,101 , activation of p38 MAPK also drives F2 overexpression in the tumor microenvironment. This activates protease-activated receptors (PARs) that induce genes with a role in angiogenesis and tumor dissemination [START_REF] Danckwardt | P38 mapk controls prothrombin expression by regulated RNA 3' end processing[END_REF] .

Thus, regulated 3′-end processing emerged as an important mechanism of gene regulation in the control of the hemostatic system. While such mechanisms are desirable under physiological conditions (to replenish the amount of blood coagulation factors under high turnover, see above), they can be 'hijacked' under pathological conditions (such as inflammation or cancer), thereby leading to a thrombophilic state 108,113 . Since prothrombin is expressed in a wide variety of organs and cells 108 , this type of regulation may become relevant to numerous other thrombin-mediated diseases 113 . However, it also appears that tissuespecific mechanisms can be used to selectively target deleterious prothrombin expression without altering essential prothrombin expression in the liver 108 .

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Targeted interference with cleavage and polyadenylation is increasingly perceived as an important therapeutic means. This involves either redirection of aberrant RNA processing (through ASOs, U1snRNP interference or trans-splicing) or the elimination of faulty transcripts [START_REF] Nourse | Emerging roles of RNA 3′-end cleavage and polyadenylation in pathogenesis, diagnosis and therapy of human disorders[END_REF] to prevent the fatal consequences of aberrant 3'-end processing 114,115 .

Perturbations of 3'-end processing can, for example, act as nongenomic oncogenic drivers of tumorigenesis 115 , but they also play important roles in inflammatory conditions 116 . Deciphering the underlying mechanisms is of paramount importance for establishing targets with therapeutic selectivity and specificity.

RNA-protein interactome studies 117 and transcriptome-wide profiling of polyadenylation 118 are thus central to defining new therapeutic targets, their specificity and downstream consequences 119 . Since most miRNA binding sites are localized in the 3'-UTR, when and where a pre-mRNA is polyadenylated has a critical impact on the regulatory properties of the resulting mRNA molecule (see below). A significant proportion of genetic variants in 3'-UTRs, often dismissed as 'non-functional' polymorphisms, are therefore likely to disrupt important regulatory mechanisms, ultimately leading to pathologies including a dysbalanced hemostatic system [START_REF] Nourse | Emerging roles of RNA 3′-end cleavage and polyadenylation in pathogenesis, diagnosis and therapy of human disorders[END_REF] . This is supported by the thrombophilia variants discovered in the F2 gene. However, this also extends to other coagulation factor 3'-UTR variants that affect, for example, miRNA regulation 120,121 .

Role of microRNAs in the hemostatic system

MicroRNAs (miRNAs) are small single-stranded non-coding RNAs (17-25 nucleotides in length) that post-transcriptionally down-regulate target gene expression by RNA silencing 122 .

After transcription, miRNAs are processed in the nucleus by the microprocessor complex consisting of Drosha and DGCR8 to produce a pre-miRNA 123 . After export to the cytoplasm and further processing by Dicer 124 , the mature miRNA duplex is incorporated into the RNAinduced silencing complex (RISC) 125 . This complex is guided by miRNA base pairing to a target gene mRNA resulting in translational inhibition and/or transcript degradation 126 . Generally, miRNAs target mRNAs via the 3'-UTR. In a few cases, miRNAs can also carry out their inhibitory function by binding to the coding region or the 5'-UTR of target mRNAs 127 .

Over 2600 human miRNAs have been identified 128 , regulating the majority of human genes 129 .

Thus almost every biological process is modulated through miRNAs 130 . Although miRNAs generally fine-tune gene expression 131 , they can also function as master regulators 132 . For example, multiple miRNAs can cooperatively silence a single gene to gain regulatory specificity, with the targeting of particular network hub genes enabling the regulation of entire pathways 133 . In addition, a single miRNA can target multiple genes, allowing broad regulation of molecular networks 127 . Perturbations of miRNA expression are observed in most disorders,

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with some of them even causally contributing to the development and progression of disease 130 .

A growing number of studies document a contribution of miRNAs to the regulation of hemostatic [134][135][136][137][138] and thrombotic 121,135,[137][138][139][140] functions. miRNAs directly regulate multiple hemostatic factors through interactions with the 3'-UTR (Table 1). Additionally, miRNAs can tune hemostatic factors indirectly, for example fibrinogen via interleukin-6-mediated signaling 141 , factor IX by repressing NMD 142 , plasminogen activator inhibitor 1 (PAI-1) via SMAD2 signaling 143 and CXCL12 to reduce inflammatory response and thrombosis, altering the expression of multiple factors including TF, PAI-1 and VWF 144 .

Further evidence implicating miRNAs in the hemostatic system comes from the important roles that miRNAs play in the development of bleeding disorders and thrombosis. Blood miRNA levels are associated with hemostatic perturbations, suggesting their potential use as prognostic or diagnostic tools in VTE 145 and beyond 146 . These include aberrant coagulation in sepsis 147 , venous thromboembolism 140,[148][149][150][151][152][153][154][155][156][157][158] , trauma-induced coagulopathy 159 , atherosclerosis [160][161][162][163][164] , coronary artery disease [165][166][167] , ischemic stroke 168,169 and autoimmune inflammatory conditions such as systemic lupus erythematosus (SLE) [170][171][172] .

Recently, using an unbiased systematic search based on a biophysical miRNA interaction study coupled to high-throughput sequencing, the Atlas of the Hemostatic miRNA Targetome was released 135 . This screening identified more than 1500 miRNA/3'-UTR interactions with potential function in the hemostatic system from nearly 4500 miRNA/3'-UTR biophysical interactions 135 . A proof-of-concept, rigorous filtering combined with loss-of-function studies (limited to 96 of the 1500 miRNA/3'-UTR interactions with a potential function) identified dozens of miRNAs targeting 27 hemostasis-associated gene 3′-UTRs globally or in a gene-specific manner (Figure 4). This highlights the global importance of miRNAs in controlling the hemostatic system and suggests that many more functional miRNAs will be discovered in this system.

The unbiased view on miRNAs regulating the hemostatic system also sheds light on hitherto functionally poorly characterized connections between different physiological systems and diseases. These include the link between tumor formation and hemostatic perturbations 135 , or the intricate relationship between the hemostatic system and inflammatory processes (Table 1). For example, miR-181 family members that target the 3'-UTR of F11 mRNA 135 are involved in several aspects of hemostasis, including vascular inflammation On the other hand, polymorphisms affecting miRNA binding sites in hemostatic genes can be associated with disease. For example, deletion of the miR-759 binding site of FGA is associated with susceptibility to chronic thromboembolic pulmonary hypertension 178 , and SNPs in the 3′-UTR of the F2, F8 and F11 genes are associated with increased activity levels of these hemostatic components 120,[179][180][181][182] .

The importance of miRNAs in hemostasis is further corroborated by their role in platelet biology 136 . Here miRNAs modulate the expression of target mRNAs important for hemostatic and thrombotic function [183][184][185][186][187] . For example, miRNA levels are altered in platelets from patients with essential thrombocythemia and this in turn is associated with elevated platelet counts and an increased risk of thromboembolic events 188 . Additionally altered miRNA expression is often observed in atherosclerotic plaques 189 (and refs therein).

In light of the functional importance of miRNAs in the hemostatic system 135 and the increasingly recognized role of miRNA therapeutics 190 currently conquering the cardiovascular system 191 , it is tempting to turn this knowledge into new therapeutics (see targeting section below). In support of this, miRNA treatment has been demonstrated to result in therapeutic response in thrombosis and hemostasis. In murine models of venous thrombosis, overexpression of miRNAs contributes to thrombus resolution 193 , reduces thrombogenesis 194 , enhances endothelial progenitor cell migration and tubulogenic activity 195 , angiogenesis and thrombosis recanalization 196 . Furthermore, the use of antagomirs (i.e., molecules that silence miRNAs) has been shown to block miR-19b-3p-mediated silencing of SERPINC1 (antithrombin), resulting in increased antithrombin expression and activity in vivo 135 . This documents the inprinciple druggability of the hemostatic system in a miRNA-directed manner and opens opportunities to target other hemostatic components such as coagulation FXI 138 .

Other means of posttranscriptional regulation of the hemostatic system

RNA binding proteins beyond their function in the biogenesis of mRNAs

In addition to co-and posttranscriptional processing, much of the fate of RNAs from synthesis to decay depends on RNA-binding proteins 197 . RBPs regulate RNA localization, transport, translation, stabilization and degradation of bound RNA molecules. In fact, much of the rapid adjustment of gene expression in inflammation and the immune system [START_REF] Chen | AU-rich elements: Characterization and importance in mRNA degradation[END_REF][START_REF] Anderson | Post-transcriptional control of cytokine production[END_REF][START_REF] Carpenter | Post-transcriptional regulation of gene expression in innate immunity[END_REF] is executed via modulation of RNA stability and decay. The same is likely to apply to the hemostatic system as well, and the adaptation of prothrombin expression (Figure 3) may be a prototype for analogous occurrences 198 . It is interesting to note that even in apparently non-polar cells such as hepatocytes, the major source of most hemostatic components, localization of transcripts and thus protein output critically depends on UTR-RBP interactions 199 . This suggests that dynamic changes of 5' and 3'-UTR structures of mRNAs, due to the use of alternative transcription start sites and alternative splicing/polyadenylation, may have a critical impact on

RNA modification and networks of competitive RNA-RBP binding

As soon as the nascent RNA molecules emerge from the RNA polymerase during transcription, they are instantly decorated with RBPs. While this ensures that co-transcriptional processing takes place effectively and at the right position, RBP loading also prevents the hybridization of the nascent RNA molecule with the DNA strand. This helps to avoid the formation of reactive RNA:DNA hybrids (so called R-loops) 201,202 , which can lead to genomic instability 203,204 . Most importantly, binding of RBPs and non-coding RNAs to (pre-)mRNAs can occur in a complex, sometimes mutually exclusive manner, thereby determining the posttranscriptional fate of mRNAs selectively [START_REF] Danckwardt | 3' end mRNA processing: Molecular mechanisms and implications for health and disease[END_REF] or in a global manner 205,206 . This is supported by the observation that the density of RBP and miRNA binding to the UTRs of coagulation factor mRNAs is very high 138 , and that numerous RBP and miRNA binding sites are in close proximity (Figure 5).

Hence, there must be mechanisms that coordinate the binding of such molecules.

Although not yet studied in great detail, it is likely that modifications of both RNAs 207 and RBPs 208 can result in remodeling of the 3'-UTR-RBP architecture and thereby change the fate of RNAs encoding coagulation factors under inflammatory conditions. In support of this notion, posttranslational modifications of RBPs have been shown to change the fate of mRNAs encoding central hemostatic components (Figure 3) [START_REF] Danckwardt | P38 mapk controls prothrombin expression by regulated RNA 3' end processing[END_REF] . But also variations in N [START_REF] Nabel | Cardiovascular disease[END_REF] methyladenosine (m 6 A), the most prevalent RNA modification with a wide biological impact 209,210 , have been documented in various RNA transcripts in vascular tissues of septic rats 211 .

Additionally, there is growing evidence that m 6 A modification is closely related to the development and progression of CVD, including cardiac hypertrophy, heart failure, ischemic heart disease and pulmonary hypertension 212,213 . It is tempting to explore if therapeutic modulation of the cellular m 6 A machinery (for example in COVID-19 214 ) might be useful in preserving vascular integrity and function in sepsis and/or CVD. Interestingly, the fat mass and obesity-associated protein (FTO), one of the few m [START_REF] Nabel | Cardiovascular disease[END_REF] A erasers, has emerged as an important pharmaceutical target in many pathophysiological conditions 209 . As many more RNA modifications are currently being discovered 215 , this holds great potential for systematically uncovering their importance in human diseases and defining novel therapeutic avenues.

Long non-coding RNAs and circRNAs

Despite the unexpectedly small number of protein-coding genes identified by the human genome project, RNA sequencing has shown that up to 85% of the human genome is transcribed 216 . This led to the identification of a large number of non-coding RNA molecules with regulatory functions 217 . In contrast to small non-coding RNAs (such a miRNAs, snoRNAs Unlike miRNAs or proteins, lncRNA function cannot currently be simply inferred from sequence or structure, and the diversity of lncRNAs described to date precludes simple generalizations 219 . In the context of the hemostatic system, this hitherto poorly explored area deserves attention. This is also supported by the role lncRNAs have in platelets 226,227 , although Since much of the posttranscriptional regulation of gene expression takes place at the level of the 3'-UTR, to which RBPs and ncRNAs are abundantly recruited, the 3'-UTR architecture has an important regulatory function (Figure 6) [START_REF] Danckwardt | 3' end mRNA processing: Molecular mechanisms and implications for health and disease[END_REF] . Diversification of the transcriptome at the 3'-end by alternative polyadenylation (APA) has recently emerged as a pervasive and evolutionarily conserved layer of gene expression control 236 (Figure 1), which affects more than 70% of all genes. APA considerably expands the diversity of the transcriptome 3'-end, affecting protein output, isoform composition and protein localization 237 .

APA is globally regulated in various conditions, including developmental and adaptive programs [START_REF] Nourse | Emerging roles of RNA 3′-end cleavage and polyadenylation in pathogenesis, diagnosis and therapy of human disorders[END_REF] . It is thus likely that APA also tunes the hemostatic system, as exemplified by alternative processing of TF and TFPI, where alternative splicing also generates different 3'-UTRs (Figure 2). In addition, a recent large scale RNAi screen based on the depletion of more than 170 putative APA regulators revealed how individual regulators affect the APA landscape 115 , including the resulting impact on gene ontologies 119 . Several significantly enriched GO terms suggest a critical function of UTR structures in inflammatory processes and innate and adaptive immunity 119 . APA affects key components broadly involved in inflammation and blood coagulation (Table 2). This is consistent with findings that APA is a critical component in the control of inflammatory processes 116,238, ), that typically result in shorter mRNA isoforms (Figure 6).

Strikingly, several hemostatic components have alternative transcripts that differ not only in their exon composition but also in their 3'-UTR structure (see NCBI Ref seq). These include essential components of the protein C pathway (i.e., protein C and protein S) with established functions at the interface of coagulation and inflammation 241 . For the protein C cofactor protein S, 3'-UTR dynamics are already documented 119 , which appear to be regulated by specific RBPs (RNPS1) or other components (CDKN2D). This points to a regulatory function of APA at the interface of the hemostatic and the immune system. Due to the pervasive regulatory function of APA in various processes 119 (with perturbations leading to numerous diseases 89 ), it is plausible that much of this diversity in the hemostatic system is regulated in response to inflammatory signals. This is illustrated by inflammation-triggered alternative processing of the FGG mRNA 242 , resulting in gamma prime (γ') fibrinogen [START_REF] De Willige | The pleiotropic role of the fibrinogen gamma' chain in hemostasis[END_REF] . γ' fibrinogen is the fibrinogen fraction that contains the γ' chain, which arises when the FGG mRNA is polyadenylated at an alternative polyadenylation signal, resulting in a polypeptide with a unique 20-amino acid extension encoded by intron 9 [START_REF] De Willige | The pleiotropic role of the fibrinogen gamma' chain in hemostasis[END_REF] . Thanks to the strongly negatively charged C-terminus of the γ' chain, fibrinogen γ' can bind with high affinity to thrombin exosite II, decreasing thrombin activity on several substrates (antithrombin I activity) 243 . As a consequence, low γ' fibrinogen levels have been associated with an increased risk of venous thrombosis [START_REF] De Willige | The pleiotropic role of the fibrinogen gamma' chain in hemostasis[END_REF]244 , while a potential role in CVD 245 and ischemic stroke 246 is under debate 247 . This highlights how seemingly subtle changes through alterations of APA and 3'-UTR diversity can have most significant functional effects in the hemostatic system. It also serves as an example illustrating the complex interdependency of posttranscriptional processing of RNA molecules and hence functional output.

A C C E P T E D M A N U S C
Interrogating system-wide posttranscriptional gene regulation [START_REF] Carninci | The transcriptional landscape of the mammalian genome[END_REF][START_REF] Wang | Alternative isoform regulation in human tissue transcriptomes[END_REF] and transcriptome 3'-end diversity 118,119 , combined with unbiased RNA interactome studies 117,135 and strategies to disentangle the functional significance of genomic perturbations in non-coding elements 248 , therefore holds great potential to unravel novel layers of coupling of the hemostatic system with inflammatory processes. This could also open entirely new therapeutic perspectives [START_REF] Nourse | Emerging roles of RNA 3′-end cleavage and polyadenylation in pathogenesis, diagnosis and therapy of human disorders[END_REF] to combat medical threats centering around thromboinflammation such as sepsis, which is still the leading cause of death in the Western world and in critically ill patients worldwide 1 .

Targeting post-transcriptional regulation of the hemostatic system

The multiple layers of posttranscriptional control of gene expression offer various opportunities and targets for therapeutic intervention. For example, RNA-based therapeutics can be used not only to re-direct splicing [START_REF] Havens | Splice-switching antisense oligonucleotides as therapeutic drugs[END_REF] and polyadenylation 249 , but also to silence an mRNA or to prevent its interaction with other RNAs or RBPs 250,251 .

Compared to 'conventional' small therapeutic molecules, RNA-based therapeutics such as ASOs, siRNAs and miRNAs offer the advantage of being able to act on 'non-druggable' targets (i.e., proteins that lack enzymatic function or whose conformation is inaccessible to traditional drug molecules), as they can be designed to affect virtually any gene of interest 192 .

ASOs are relatively short, chemically modified single-stranded nucleic acids that selectively pair to specific regions of mRNA resulting in endonucleolytic cleavage and degradation 250 .

Currently, more than 60 ASO therapies are in or have completed phase I/II trials, with a substantial number of antithrombotic ASO therapeutics currently under development 138 .

The recent introduction of ASOs down-regulating FXI expression exemplifies the potential of such therapeutics to modulate the hemostatic system via post-transcriptional mechanisms [START_REF] Büller | Factor xi antisense oligonucleotide for prevention of venous thrombosis[END_REF] .

This phase II study in patients undergoing knee surgery revealed that the FXI-targeting ASO effectively protects patients against venous thrombosis with a relatively limited risk of bleeding.

However, this proof-of-concept trial was too small to assess the effect on other thrombotic end

A C C E P T E D M A N U S C R I P T
points. Other genes that are being explored as potential targets for antithrombotic therapy using silencing ASOs are FII, FVII, FXII, prekallikrein, plasmin activator inhibitor, thrombopoetin and FMO3 138 . A possible concern is that changes in platelet counts were observed in non-human primates treated with ASOs 252 , which has been attributed to peripheral clearance 253 and could potentially impact hemostasis.

miRNA therapeutics represent another highly versatile therapeutic means in the context of the hemostatic system 138 . MiRNA mimics may be employed to silence pro-coagulant genes to treat thrombosis (or alternatively, anticoagulant genes to treat bleeding). Conversely, antagomirs or target site blockers can be used to relieve silencing of anticoagulant genes to treat thrombosis.

Moreover, some miRNAs target several hemostatic components at the same time (Figure 4), and silencing of such miRNAs can be intentionally used to control several hemostatic components. On the other hand, undesired pleiotropy is one of the conceptual downsides of therapeutic miRNA targeting. Finally, there are increasingly strategies to modulate other facets of the RNA biogenesis. This concerns the targeted interference with splicing [START_REF] Havens | Splice-switching antisense oligonucleotides as therapeutic drugs[END_REF] or with cleavage and polyadenylation 249 , involving either redirection of aberrant RNA processing (through ASOs, U1snRNP interference or trans-splicing) or the elimination of aberrant transcripts 79,[START_REF] Nourse | Emerging roles of RNA 3′-end cleavage and polyadenylation in pathogenesis, diagnosis and therapy of human disorders[END_REF] . The characterization of the transcriptome dynamics thus becomes the next milestone to exploit the untapped therapeutic opportunities arising from the increasingly available RNA therapeutics.

Summary

Besides transcriptional control, posttranscriptional regulation of gene expression is taking center stage in the modulation of the hemostatic system. The highly regulated use of alternative transcription start sites, exons and polyadenylation sites makes the transcriptome highly dynamic in time, space and in response to pathological processes. Additional posttranscriptional regulation by non-coding RNAs, RNA-binding proteins and RNA modification mechanisms further modulate the functional output of numerous biological processes, including the hemostatic system. Many of these regulatory principles also play an important functional role in tuning the immune system [START_REF] Chen | Gene regulation in the immune system by long noncoding RNAs[END_REF][START_REF] Shulman | The RNA modification n6-methyladenosine as a novel regulator of the immune system[END_REF][START_REF] Chen | AU-rich elements: Characterization and importance in mRNA degradation[END_REF][START_REF] Anderson | Post-transcriptional control of cytokine production[END_REF][START_REF] Carpenter | Post-transcriptional regulation of gene expression in innate immunity[END_REF] , suggesting conserved regulatory links between both systems. It will be critical to characterize these links to identify rational targets for the emerging repertoire of RNA therapeutics to effectively combat the dangerous alliance of the hemostatic and the immune system. S4). Heatmap of miRNA/3′UTR interactions (only highly stringent interactions are depicted). Results of miTRAP assays were divided into three functional categories of procoagulant, anticoagulant and fibrinolytic components, and for miRNAs targeting multiple 3′UTRs each category subjected to unsupervised hierarchical clustering as indicated by tree on the left (modified from 135 ). For further information of miRNA-mediated regulation of hemostatic components see Table 1. Alternative polyadenylation is a pervasive gene regulatory mechanism that results in mRNA isoforms with different 3'-ends. This can result in mRNA isoforms encoding truncated proteins or in mRNA isoforms with distinct 3'-UTR properties altering RNA transport, localization, translation, and/or stability (through binding to non-coding RNAs (such as miRNAs, lncRNAs, ceRNA), through binding to RNA binding proteins (RBPs) and/or through complex, sometimes mutually exclusive, interactions of RNA motifs with RBPs and/or ncRNAs. Of note, modifications of RNAs (such as "m 6 A") or posttranslational modifications (PTMs) of RBPs introduce further layers of modulation). Inflammatory conditions tend to result in the generation of shorter mRNA isoforms (either lacking elements of 3'-UTR regulation or resulting in truncated proteins; 119 116 ). Alternative polyadenylation affects numerous genes involved in blood coagulation and inflammation (Table 2).

A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C

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  Downloaded from https://academic.oup.com/cardiovascres/advance-article/doi/10.1093/cvr/cvad046/7082876 by Universite de Bordeaux user on 17 April 2023 protein output and ultimately function. This is corroborated, for example, by the role of 5'-UTR variants that alter upstream open reading frames in cardiovascular disorders (CVD) 200 .

  long-noncoding (lnc)RNAs are around 200 nucleotides or more218 and often undergo alternative splicing, which further expands their repertoire. LncRNAs can bind to DNA, mRNAs, miRNAs and proteins depending on sequence and secondary structure, thereby modulating gene expression under physiological and pathological conditions 219 . Their modes of action include epigenetic, transcriptional and post-transcriptional mechanisms. Accordingly, this new class of ncRNAs is increasingly taking center stage in the modulation of the cardiovascular system. As an example, lncRNA H19 is involved in the pathogenesis of atherosclerosis 220 . The expression of lncRNA H19 is significantly increased in patients with ischemic stroke compared to healthy controls 221 . Genome-wide association studies have identified SNPs in the lncRNA ANRIL associated with CVD, such as coronary atherosclerosis and cardiac infarction222, 223 , while variants in lncRNA ZFAS1 are associated with susceptibility to ischemic stroke 224 . Recently, a transcriptome wide association study on VTE also revealed further lncRNA hits (RP11-747H7.3, RP4-737E23.2) 225 , corroborating their function in CVD.

  their role is still under active investigation. In analogy to the central regulatory function of noncoding RNAs in the immune system and because of the resulting therapeutic implications 228 , it will be important to better understand the pathophysiological dimension of this class of regulators in thrombosis and its connection to inflammation.Circular RNAs (circRNAs) are another class of endogenous non-coding regulatory biomolecules. They are prevalent and arise from a non-canonical splicing event called 'backsplicing' 229 . They exert important biological functions by acting as miRNA or protein sponges, by regulating protein function or by being translated 230 . As such, circRNAs regulate a plethora of biological functions including ROS formation and cardiovascular metabolic inflammation231 . Accordingly, perturbations of these process(es) can become pathogenic and result in CVD. For example, a haplotype on 9p21 that protects against coronary artery disease has been shown to be associated with the abundance of circRNA ANRIL, which in turn regulates ribosomal RNA maturation, conferring atheroprotection 232 . Accordingly, circANRIL has been proposed as a potential therapeutic target for the treatment of atherosclerosis. The in-principle therapeutic utility of circRNA is also supported by recent preclinical observations demonstrating their use, for example, to attenuate cell apoptosis in cerebral ischemiareperfusion233 . Finally, circulating circRNA may have diagnostic potential and serve as biomarkers for acute ischemic stroke 234 and even help distinguish different etiologies (i.e., atherothrombotic, cardiothrombotic vs undetermined stroke)235 . Downloaded from https://academic.oup.com/cardiovascres/advance-article/doi/10.1093/cvr/cvad046/7082876 by Universite de Bordeaux user on 17 April 2023 What comes next? Alternative polyadenylation and 3'-UTR diversity as central regulatory hubs Much of the posttranscriptional regulation of the hemostatic system depends on players that determine the fate of RNAs encoding the respective hemostatic components. The different layers of regulation are largely inter-dependent, as alternative splicing and polyadenylation are coupled to each other 84 and thereby determine not only the final open reading frame, but also the 3'-UTR sequence and hence the susceptibility of the mature mRNA to posttranscriptional control by RBPs and ncRNAs.

  MiRNA therapeutics are currently at an early stage of development and not yet applicable in the clinical setting254 . In preclinical studies, several miRNA mimics and antagomirs have been shown to reduce thrombus formation138 or increase the antithrombin activity in vivo135 . One of the biggest challenges in the clinical development of miRNA-based therapeutics is the identification of key miRNA candidates and targets, their specificity and effect size. There is currently a relatively small number of experimentally validated miRNA:mRNA interactions, making knowledge of the miRNA targetome in the hemostatic system a major trove for future targeted therapeutics135 .ASOs and most siRNAs exhibit perfect complementary to their targets, which usually results in degradation of the target mRNA255 . In contrast, partial base-pairing of miRNAs prevents the cleavage activity of RISC, predominately causing translational repression, and only in some cases deadenylation, decapping and finally mRNA degradation 256 . Although the proportion of mRNA target degradation varies widely 257 , a number of targets are almost exclusively repressed at the level of translation 258 . How much each mechanism contributes to downregulation depends on characteristics, such as seed-flanking nucleotides, of the individual miRNA-mRNA pair 259 .In the context of the hemostatic system, it is interesting to note that miRNA regulation of transcripts encoding secretory proteins results almost exclusively in translational repression, because miRNA translational repression is stronger for mRNAs translated at the endoplasmicreticulum compared to free cytosolic ribosomes 258 . Thus, miRNA-mediated therapeutic targeting without degradation of the target mRNAs preserves physiological cell intrinsic regulatory mechanisms carried out by 3'-UTRs and their binding partners (such as RBPs, miRNAs, lncRNAs, circRNA or miRNA sponges). This allows for 'compensatory' on-demand Downloaded from https://academic.oup.com/cardiovascres/advance-article/doi/10.1093/cvr/cvad046/7082876 by Universite de Bordeaux user on 17 April 2023 adjustments of protein output even in the presence of the miRNA therapeutic, and thus may represent a conceptual advantage of miRNA therapeutics over ASO-based approaches 138 .While RNA therapeutic approaches have been used in the development of new drugs and clinical trials are underway 260 , there are still concerns and challenges to be overcome. These include, but are not limited to, off-target effects 261 , triggering innate immune responses 262 , stability of the therapeutic RNA molecule and design of optimal delivery systems for diseasespecific release with minimal toxicity 190 .

Figure 1 .

 1 Figure 1. The functional complexity encoded by approximately 22.000 genes is substantially diversified by co-and posttranscriptional mechanisms involving alternative transcription initiation, alternative splicing and alternative polyadenylation (APA). Regulation by non-coding RNAs such as micro (mi)RNAs, long-non-coding (lnc)RNAs, circular (circ)RNAs, as well as RNA-binding proteins (RBPs) and RNA modifications, further tunes the functional output of the transcriptome. Modulation of the biogenesis and the posttranscriptional fate of RNAs (RNA localization, transport, translation, stability or decay, RNA modifications) are emerging therapeutic principles (further details see text; * 263 , ** 264 ).A C C E P T E D M A N U S C R I P T

Figure 2 Figure 2 .

 22 Figure 2

Figure 3 Figure 3 .

 33 Figure 3

Figure 4 Figure 4 .

 44 Figure 4

Figure 5

 5 Figure 5

Figure 5 .

 5 Figure 5. FXI 3'-UTR interactome. The graph depicts the density of sites for miRNA and RNAbinding proteins (RBPs) across the FXI 3'-UTR (based on 125 FXI 3'-UTR/miRNA interactions identified by miTRAP/RNA-seq 135 with 41 mapped to the FXI 3'-UTR using miRWalk target site prediction, and 392 FXI 3'-UTR/RBP interactions identified by miTRAP/MS and of which 66 are mapped to the FXI 3'-UTR using RBPDB target site prediction. Site density calculated by number of sites present in 50 nt windows over length of the FXI 3'-UTR). A C C E P T E D M A N U S C R I P T

Figure 6 Figure 6 .

 66 Figure 6

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	S C R I P T
	A N U
	M
	A C C E P T E D
	152, 173-175 and platelet
	activation 176 . Another example is miR-24 which controls the expression of VWF 177 . Here,
	hyperglycemia-induced repression of miR-24 increases VWF expression and secretion in
	diabetes mellitus, linking metabolic dysfunction to a miRNA-mediated mechanism of
	hemostatic deregulation.

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  R I P T

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Table 1 . Hemostatic components under miRNA control and relation to 1 thromboinflammation.

 1 For full Hemostatic miRNA Targetome Atlas see 135 . 2

	Procoagulant		Main miRNAs (functionally validated)	Ref.
	fibrinogen alpha	FGA	miR-193b-3p 135	135, 267
			miR-194-5p 135
			miR-759 267
	fibrinogen beta	FGB	miR-409-3p (miR-29 family)	268
	fibrinogen gamma	FGG	miR-99b-3p	135
			miR-193a-5p
	coagulation factor III,	F3	miR-19b	194, 269-
	tissue factor		• Anti-thrombotic protector in patients	272, 274
			with unstable angina 269
			miR-19b, miR-20a
			• Down-regulation contributes to a
			hypercoagulable state in SLE and
			APS 270
			miR-126
			• Reduces thrombogenicity in
			diabetes mellitus 271
			miR-145
			• Impedes thrombus formation in
			venous thrombosis 194
			miR-223
			• Partially blocks TNF-α-induced
			increase of TF activity in
			endothelial cells 272
			miR-365a-3p
			• Interacts with TF 3'-UTR to
			modulate TF-initiated thrombin
			generation 273
	coagulation factor VII	F7	miR-19a-3p	135
			miR-19b-3p
	coagulation factor VIII	F8	miR-7-5p 135	135, 275
			miR 454-3p 135
			miR-532-5p 135
			miR-1246 275
	coagulation factor XI	F11	miR-15b-5p 135
			• Biomarker for PAD 276
			• Influences platelet reactivity and
			clopidogrel response 277
			miR-24-3p 135
			• Biomarker for acute cerebral
			infarction, arteriosclerosis
			obliterans, atherosclerosis and
			severe trauma 278-281
			miR-30a-3p 135
			• Biomarker for AMI and ischemic
			stroke 282, 283
			miR-30d-3p 135
			miR-96-5p 135
			• Biomarker for DVT and DIC 284, 285
			miR-103a-3p 135
			• Involved in atherosclerosis and
			vascular inflammation by
			suppression of KLF4 286
			• Biomarker for VTE 287
			miR-145-5p 288

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Acknowledgments:

The authors would like to express their gratitude to current and former members of the Danckwardt and the Castoldi lab. Work in the Danckwardt lab is kindly supported by the German Research Foundation Priority Program SPP 1935, German Research Foundation grants DA 1189/2-1 and DA 1189/5-1, Germany Research Foundation Major Research Instrumentation Program INST 371/33-1, the German Research Foundation graduate school GRK 1591, Federal Ministry of Education and Research (BMBF01EO1003), German Society of Clinical and Laboratory Medicine and the Hella Bühler Award for Cancer Research. Work in the labs of S.D. and E.C. is supported by the EU Horizon 2020 Innovative training network

'Thromboinflammation in Cardiovascular disorders' (TICARDIO, Marie Skłodowska-Curie grant agreement No 813409). D.-A. T. is supported by the EPIDEMIOM-VT Senior Chair from the University of Bordeaux initiative of excellence IdEX.

• Biomarker for CAD, AMI, stroke, long-term outcome [289][290][291][292][293][294][295][296] • Impedes thrombus formation in atherosclerosis by targeting tissue factor and influencing platelet reactivity 277, 297 miR-148b-3p 135 miR-151a-3p 135 miR-181a-5p 298,299 • Biomarker for AMI and PAD 276, 300 miR-181b-5p 135 miR-191b-5p 298 miR-544a 302 miR-1255a Table 2. Alternative polyadenylation regulates components involved in blood coagulation and inflammation. Each column depicts genes belonging to the GO term "blood coagulation", "regulation of inflammation" and "complement" that are affected by alternative polyadenylation (APA) upon depletion of central APA regulators (CPSF6, NUDT21, PCF11).

Data obtained from TREND-DB 119 ; for further APA affected genes and -effectors: http://shiny.imbei.uni-mainz.de:3838/trend-db/.

affedcted GO term blood coagulation regulation of inflammation complement blood coagulation regulation of inflammation complement blood coagulation regulation of inflammation complement


Celia Sańchez-Girón
email: celia.sanchez-giron@uva.es

Luc Coca

Mathis Perera

Antoine Boiteau

Jérôme Costanza

France Emmanuel Grison

Michèle Sarnette

Frederic Kanhonou

Nga Fauberteau

Viet Thi

Pierre Jouvelot 2023 Nguyen

Jouvelot

Celia Sańchez-Girón Coca

Luc Perera
email: luc.perera@ensad.fr

Mathis Boiteau
email: mathis.boiteau@gmail.com

Antoine Costanza
email: antoine.costanza@ac-versailles.fr

Jérôme Grison

Emmanuel Sarnette

Michèle Kanhonou

Frédéric Fauberteau

Emmanuel Grison

Frédéric Kanhonou

Tala Box: an Interactive Embedded System to Accompany Patients with Cognitive Disorders

Keywords: Computer systems organization → Embedded systems, • Human-centered computing → Human-computer interaction (HCI), • Applied computing → Health informatics

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

International Conference on Connected Health: Applications, Systems and Engineering Technologies (CHASE '23), June 21-23, 2023, Orlando, FL, USA. ACM, New York, NY, USA, 2 pages. https://doi.org/10.1145/3580252.3589413

THE TALA SOUND PROJECT

With the aim of reducing the spread of Alzheimer's disease, impacting more than 30 million people worldwide [START_REF]Dementia[END_REF], or of at least mitigating its effects, many medical research studies have been carried out in recent years to find an effective cure. Although a few of these experiments using pharmaceutics turned out to show some limited success, their effectiveness was not as high as expected in many of the illness aspects [START_REF] Moreno-Morales | Music therapy in the treatment of dementia: A systematic review and meta-analysis[END_REF]. Other approaches [START_REF] Kenigsberg | Assistive Technologies to Address Capabilities of People with Dementia: From Research to Practice[END_REF] are thus emerging to attempt to address Alzheimer's disease and other cognitive disorders.

One of these non-drug-based approaches is music therapy, which seems particularly promising since the sense of music is one of the last human abilities to be affected in cognitive disorders [START_REF] Devere | Music and dementia: An overview[END_REF]. Several music-therapy studies have focused on the impact of the repetitive listening of rhythmic groups [START_REF] Dowling | Rhythmic groups and subjective chunks in memory for melodies[END_REF] or the effect of music already wellknown by patients [START_REF] Platel | Semantic and episodic memory of music are subserved by distinct neural networks[END_REF]. However, we have not found proper studies that analyze the effects of non-familiar music built upon complex rhythmic patterns on patients.

The Tala Sound multidisciplinary project intends to fill that gap by bringing together Indian music, embedded systems and health [START_REF] Perera | Tala Sound : un projet interdisciplinaire innovant en design social pour la santé[END_REF]. At the fundamental level, we want to test the scientific research hypothesis that, when interacting with Indian rhythms, or "talas", originating from the southern regions of India (Carnatic music), the rhythmic structure of the talas interferes in a positive way with cognitive processes and thus, at least partially, improves the state of mind of cognitively-impaired patients, even from Western countries. For this specific goal, we designed a dedicated interaction device, the Tala Box, a new multi-sensory device that mixes audio, visual and tactile stimuli to provide an immersive experience even to often-apathic older patients (see a short demo1 of the Tala Box being used by French seniors).

TALA BOX

The Tala Box is a wooden enclosure totally covered by a piece of fabric (see Figure 1) designed to be used in medical environments where the user is therefore a person considered at risk, which calls for specific requirements. In particular, COVID-19 new standards have been taken into account when building the prototype. We chose a removable flexible top based on a semi-synthetic fabric coated with a natural elastomere so that it can be easily disinfected after each session. Wood is a material that is easy to manipulate and repair; in addition, its rigidity helps avoiding spurious resonances, which is important given that the Tala Box sound module is enclosed within. The choice of a uniform blue color is motivated by its soothing appeal and lack of distinctly visible features, thus possibly helping patients to project their own memories during sessions.

Figure 1: Tala Box final design

The Tala Box core is composed of two mobile structures (the two balls and mountings at the left of Figure 2) designed via CAD and manufactured by 3D printing. Each structure can be moved via two stepper motors that allow vertical and horizontal displacements, while four limit switches have been installed on each movable arm in order to detect the vertical and horizontal bounds. The coordination of the different elements in the device is managed by two controllers. An Arduino UNO microcontroller is responsible for issuing the stepper-motor-movement commands, while a Raspberry PI 3B manages the rest of the modules in the device, including music storage, sound generation and user interfacing.

To play music, two HP928 speakers have been installed, while a Module LCD 2,15" touch screen and a double rotary potentiometer are used as user interface for music and volume selection.

As a sound-mediation device, the Tala Box movements are synced to the music being simultaneously played. This requires signal processing software to both analyze the sound files and generate those movements. The Tala Box software environment thus provides two core modules that are aimed at (1) extracting musical dynamic characteristics from .wav-formatted files and (2) designing and scheduling, from the musical characteristics previously obtained, the movements to be performed by the Tala Box . We used the librosa2 library to extract and study various parameters such as amplitude, tempo or frequency, which can be used as inputs to determine the path of locations that the balls in the Tala Box will ultimately follow.

EXPERIMENTS

Using surveys and questionnaires, we already confirmed, first, the positive impact of passive listening to Carnatic music on patients suffering from cognitive disorders in a hospital and, subsequently, the affordance of the Tala Box for healthy seniors in a residence. These two preliminary tests already hint at the potential of the Tala Box for this type of population.

Further experiments with more patients will assess whether using the Tala Box helps the most critically affected ones to reduce their anxiety and increase their overall awareness, thus improving their well-being. Also, we intend to add a force-sensor-based mechanism to the Tala Box for measuring the patients' strength and response time (pushing down on the balls, swiping the cover, etc.). This mechanism would provide a non-intrusive manner to gather quantitative evidence, e.g., via machine-learning techniques based on sensor data, regarding the patient's response to the practice.

Figure 2 :

 2 Figure 2: Tala Box mechanism

https://www.youtube.com/watch?v=zRQf40-VfI4

https://librosa.org