A Platelet Function Modulator of Thrombin Activation Is Causally Linked to Cardiovascular Disease and Affects PAR4 Receptor Signaling.

Dual antiplatelet therapy reduces ischemic events in cardiovascular disease, but it increases bleeding risk. Thrombin receptors PAR1 and PAR4 are drug targets, but the role of thrombin in platelet aggregation remains largely unexplored in large populations. We performed a genome-wide association study (GWAS) of platelet aggregation in response to full-length thrombin, followed by clinical association analyses, Mendelian randomization, and functional characterization including iPSC-derived megakaryocyte and platelet experiments. We identified a single sentinel variant in the GRK5 locus (rs10886430-G, p = 3.0 × 10-42) associated with increased thrombin-induced platelet aggregation (ß = 0.70, SE = 0.05). We show that disruption of platelet GRK5 expression by rs10886430-G is associated with enhanced platelet reactivity. The proposed mechanism of a GATA1-driven megakaryocyte enhancer is confirmed in allele-specific experiments. Utilizing further data, we demonstrate that the allelic effect is highly platelet- and thrombin-specific and not likely due to effects on thrombin levels. The variant is associated with increased risk of cardiovascular disease outcomes in UK BioBank, most strongly with pulmonary embolism. The variant associates with increased risk of stroke in the MEGASTROKE, UK BioBank, and FinnGen studies. Mendelian randomization analyses in independent samples support a causal role for rs10886430-G in increasing risk for stroke, pulmonary embolism, and venous thromboembolism through its effect on thrombin-induced platelet reactivity. We demonstrate that G protein-coupled receptor kinase 5 (GRK5) promotes platelet activation specifically via PAR4 receptor signaling. GRK5 inhibitors in development for the treatment of heart failure and cancer could have platelet off-target deleterious effects. Common variants in GRK5 may modify clinical outcomes with PAR4 inhibitors, and upregulation of GRK5 activity or signaling in platelets may have therapeutic benefits.

Integrative analysis of HASMCs gene expression profile revealed the role of thrombin in the pathogenesis of atherosclerosis.

We explored the effect of thrombin on human aortic smooth muscle cells (HASMCs) and further analyzed its role in the pathogenesis of atherosclerosis (AS). Thrombin-induced differentially expressed genes (DEGs) in HASMCs were identified by analyzing expression profiles from the GEO. Subsequently, enrichment analysis, GSEA, PPI network, and gene-microRNAs networks were interrogated to identify hub genes and associated pathways. Enrichment analysis results indicated that thrombin causes HASMCs to secrete various pro-inflammatory cytokines and chemokines, exacerbating local inflammatory response in AS. Moreover, we identified 9 HUB genes in the PPI network, which are closely related to the inflammatory response and the promotion of the cell cycle. Additionally, we found that thrombin inhibits lipid metabolism and autophagy of HASMCs, potentially contributing to smooth muscle-derived foam cell formation. Our study deepens a mechanistic understanding of the effect of thrombin on HASMCs and provides new insight into treating AS.

Using CIVT-SELEX to Select Aptamers as Genetic Parts to Regulate Gene Circuits in a Cell-Free System.

The complexity of genetic circuits has not seen a significant increase over the last decades, even with the rapid development of synthetic biology tools. One of the bottlenecks is the limited number of orthogonal transcription factor-operator pairs. Researchers have tried to use aptamer-ligand pairs as genetic parts to regulate transcription. However, most aptamers selected using traditional methods cannot be directly applied in gene circuits for transcriptional regulation. To that end, we report a new method called CIVT-SELEX to select DNA aptamers that can not only bind to macromolecule ligands but also undergo significant conformational changes, thus affecting transcription. The single-stranded DNA library with affinity to our example ligand human thrombin protein is first selected and enriched. Then, these ssDNAs are inserted into a genetic circuit and tested in the in vitro transcription screening to obtain the ones with significant inhibitory effects on downstream gene transcription when thrombins are present. These aptamer-thrombin pairs can inhibit the transcription of downstream genes, demonstrating the feasibility and robustness of their use as genetic parts in both linear DNAs and plasmids. We believe that this method can be applied to select aptamers of any target ligands and vastly expand the genetic part library for transcriptional regulation.

How Thrombomodulin Enables W215A/E217A Thrombin to Cleave Protein C but Not Fibrinogen.

The W215A/E217A mutant thrombin is called "anticoagulant thrombin" because its activity toward its procoagulant substrate, fibrinogen, is reduced more than 500-fold whereas in the presence of thrombomodulin (TM) its activity toward its anticoagulant substrate, protein C, is reduced less than 10-fold. To understand how these mutations so dramatically alter one activity over the other, we compared the backbone dynamics of wild type thrombin to those of the W215A/E217A mutant thrombin by hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS). Our results show that the mutations cause the 170s, 180s, and 220s C-terminal ß-barrel loops near the sites of mutation to exchange more, suggesting that the structure of this region is disrupted. Far from the mutation sites, residues at the N-terminus of the heavy chain, which need to be buried in the Ile pocket for correct structuring of the catalytic triad, also exchange much more than in wild type thrombin. TM binding causes reduced H/D exchange in these regions and also alters the dynamics of the ß-strand that links the TM binding site to the catalytic Asp 102 in both wild type thrombin and in the W215A/E217A mutant thrombin. In contrast, whereas TM binding reduces the dynamics the 170, 180 and 220 s C-terminal ß-barrel loops in WT thrombin, this region remains disordered in the W215A/E217A mutant thrombin. Thus, TM partially restores the catalytic activity of W215A/E217A mutant thrombin by allosterically altering its dynamics in a manner similar to that of wild type thrombin.

Regulation of factor V and factor V-short by TFPIα: Relationship between B-domain proteolysis and binding.

Coagulation factor V (FV) plays an anticoagulant role but serves as a procoagulant cofactor in the prothrombinase complex once activated to FVa. At the heart of these opposing effects is the proteolytic removal of its central B-domain, including conserved functional landmarks (basic region, BR; 963-1008 and acidic region 2, AR2; 1493-1537) that enforce the inactive FV procofactor state. Tissue factor pathway inhibitor α (TFPIα) has been associated with FV as well as FV-short, a physiologically relevant isoform with a shortened B-domain missing the BR. However, it is unclear which forms of FV are physiologic ligands for TFPIα. Here, we characterize the binding and regulation of FV and FV-short by TFPIα via its positively charged C-terminus (TFPIα-BR) and examine how bond cleavage in the B-domain influences these interactions. We show that FV-short is constitutively active and functions in prothrombinase like FVa. Unlike FVa, FV-short binds with high affinity (Kd ∼1 nM) to TFPIα-BR, which blocks procoagulant function unless FV-short is cleaved at Arg1545, removing AR2. Importantly, we do not observe FV binding (µM detection limit) to TFPIα. However, cleavage at Arg709 and Arg1018 displaces the FV BR, exposing AR2 and allowing TFPIα to bind via its BR. We conclude that for full-length FV, the detachment of FV BR from AR2 is necessary and sufficient for TFPIα binding and regulation. Our findings pinpoint key forms of FV, including FV-short, that act as physiologic ligands for TFPIα and establish a mechanistic framework for assessing the functional connection between these proteins.

Molecular basis of anticoagulant and anticomplement activity of the tick salivary protein Salp14 and its homologs.

During feeding, a tick's mouthpart penetrates the host's skin and damages tissues and small blood vessels, triggering the extrinsic coagulation and lectin complement pathways. To elude these defense mechanisms, ticks secrete multiple anticoagulant proteins and complement system inhibitors in their saliva. Here, we characterized the inhibitory activities of the homologous tick salivary proteins tick salivary lectin pathway inhibitor, Salp14, and Salp9Pac from Ixodesscapularis in the coagulation cascade and the lectin complement pathway. All three proteins inhibited binding of mannan-binding lectin to the polysaccharide mannan, preventing the activation of the lectin complement pathway. In contrast, only Salp14 showed an appreciable effect on coagulation by prolonging the lag time of thrombin generation. We found that the anticoagulant properties of Salp14 are governed by its basic tail region, which resembles the C terminus of tissue factor pathway inhibitor alpha and blocks the assembly and/or activity of the prothrombinase complex in the same way. Moreover, the Salp14 protein tail contributes to the inhibition of the lectin complement pathway via interaction with mannan binding lectin-associated serine proteases. Furthermore, we identified BaSO4-adsorbing protein 1 isolated from the tick Ornithodoros savignyi as a distant homolog of tick salivary lectin pathway inhibitor/Salp14 proteins and showed that it inhibits the lectin complement pathway but not coagulation. The structure of BaSO4-adsorbing protein 1, solved here using NMR spectroscopy, indicated that this protein adopts a noncanonical epidermal growth factor domain-like structural fold, the first such report for tick salivary proteins. These data support a mechanism by which tick saliva proteins simultaneously inhibit both the host coagulation cascade and the lectin complement pathway.

Laboratory Testing for the Evaluation of Phenotypic Activated Protein C Resistance.

Activated protein C (APC) resistance (APCR) is considered a risk factor of venous thromboembolism (VTE). The most common genetic disorder conferring APCR is a factor (F) V Leiden mutation, but many other factors are also implicated, such as other F5 mutations (e.g., FV Hong-Kong and FV Cambridge), protein S deficiency, elevated factor VIII, exogenous hormone use, pregnancy and postpartum, depending on how APCR is defined. Considering the large population affected, the detection of this phenotype is crucial. Two types of tests are currently available: clotting time-based assays (with several versions) and thrombin generation-based assays with the endogenous thrombin potential (ETP)-based assay. The purpose of this review is therefore to discuss the performances of these tests and the cases in which it would be appropriate to use one over the other. Initially, as APCR was thought to be solely related to the FV Leiden mutation, the objective was to obtain a 100% specific assay. Clotting-time based assays were thus specifically designed to detect this inherited condition. Later on, an APCR condition without a FV Leiden mutation was identified and highlighted as an independent risk factor of VTE. Therefore, the development of a less specific assay was needed and a global coagulation test was proposed, known as the ETP-based APCR assay. In light of the above, these tests should not be used for the same purpose. Clotting time-based assays should only be recommended as a screening test for the detection of FV mutations prior to confirmation by genetic testing. On the other hand, the ETP-based APC resistance assay, in addition to being able to detect any type of APCR, could be proposed as a global screening test as it assesses the entire coagulation process.

Nafamostat protects against early brain injury after subarachnoid hemorrhage in mice.

This study aimed to evaluate the effects of nafamostat, a serin protease inhibitor, in the management of subarachnoid hemorrhage (SAH). SAH was induced by endovascular perforation in male mice. Nafamostat was administered intraperitoneally four times immediately after SAH induction. Cerebral blood flow, neurological behavior tests, SAH grade and protein expression were evaluated at 24 h after SAH induction. In the in vitro model, human brain microvascular endothelial cells (HBMVECs), HBVECs were exposed to thrombin and hypoxia for 24 h; nafamostat was administered and the protein expression was evaluated. Eighty-eight mice were included in the in vivo study. Fifteen mice (17%) were excluded because of death or procedure failure. Nafamostat exerted no significant effect on the SAH grade or cerebral blood flow; however, it improved the neurological behavior and suppressed the thrombin and MMP-9 expression. In addition, nafamostat suppressed the ICAM-1 expression and p38 phosphorylation in the in vitro study. Nafamostat has a protective effect against HBMVEC after exposure to thrombin and hypoxia, suggesting its role in improving the neurological outcomes after SAH. These findings indicate that nafamostat has the potential to be a novel therapeutic drug in the management of SAH.

Structural properties and anticoagulant/cytotoxic activities of heterochiral enantiomeric thrombin binding aptamer (TBA) derivatives.

The thrombin binding aptamer (TBA) possesses promising antiproliferative properties. However, its development as an anticancer agent is drastically impaired by its concomitant anticoagulant activity. Therefore, suitable chemical modifications in the TBA sequence would be required in order to preserve its antiproliferative over anticoagulant activity. In this paper, we report structural investigations, based on circular dichroism (CD) and nuclear magnetic resonance spectroscopy (NMR), and biological evaluation of four pairs of enantiomeric heterochiral TBA analogues. The four TBA derivatives of the d-series are composed by d-residues except for one l-thymidine in the small TT loops, while their four enantiomers are composed by l-residues except for one d-thymidine in the same TT loop region. Apart from the left-handedness for the l-series TBA derivatives, CD and NMR measurements have shown that all TBA analogues are able to adopt the antiparallel, monomolecular, 'chair-like' G-quadruplex structure characteristic of the natural D-TBA. However, although all eight TBA derivatives are endowed with remarkable cytotoxic activities against colon and lung cancer cell lines, only TBA derivatives of the l-series show no anticoagulant activity and are considerably resistant in biological environments.

Rapid assembly and profiling of an anticoagulant sulfoprotein library.

Hematophagous organisms produce a suite of salivary proteins which interact with the host's coagulation machinery to facilitate the acquisition and digestion of a bloodmeal. Many of these biomolecules inhibit the central blood-clotting serine proteinase thrombin that is also the target of several clinically approved anticoagulants. Here a bioinformatics approach is used to identify seven tick proteins with putative thrombin inhibitory activity that we predict to be posttranslationally sulfated at two conserved tyrosine residues. To corroborate the biological role of these molecules and investigate the effects of amino acid sequence and sulfation modifications on thrombin inhibition and anticoagulant activity, a library of 34 homogeneously sulfated protein variants were rapidly assembled using one-pot diselenide-selenoester ligation (DSL)-deselenization chemistry. Downstream functional characterization validated the thrombin-directed activity of all target molecules and revealed that posttranslational sulfation of specific tyrosine residues crucially modulates potency. Importantly, access to this homogeneously modified protein library not only enabled the determination of key structure-activity relationships and the identification of potent anticoagulant leads, but also revealed subtleties in the mechanism of thrombin inhibition, between and within the families, that would be impossible to predict from the amino acid sequence alone. The synthetic platform described here therefore serves as a highly valuable tool for the generation and thorough characterization of libraries of related peptide and/or protein molecules (with or without modifications) for the identification of lead candidates for medicinal chemistry programs.

Glial cells maintain synapses by inhibiting an activity-dependent retrograde protease signal.

Glial cells regulate multiple aspects of synaptogenesis. In the absence of Schwann cells, a peripheral glial cell, motor neurons initially innervate muscle but then degenerate. Here, using a genetic approach, we show that neural activity-regulated negative factors produced by muscle drive neurodegeneration in Schwann cell-deficient mice. We find that thrombin, the hepatic serine protease central to the hemostatic coagulation cascade, is one such negative factor. Trancriptomic analysis shows that expression of the antithrombins serpin C1 and D1 is significantly reduced in Schwann cell-deficient mice. In the absence of peripheral neuromuscular activity, neurodegeneration is completely blocked, and expression of prothrombin in muscle is markedly reduced. In the absence of muscle-derived prothrombin, neurodegeneration is also markedly reduced. Together, these results suggest that Schwann cells regulate NMJs by opposing the effects of activity-regulated, muscle-derived negative factors and provide the first genetic evidence that thrombin plays a central role outside of the coagulation system.

Complement component factor B has thrombin-like activity.

Serine proteases are fundamental components of biology, including innate immunity, which is systematically orchestrated in an orderly, balanced fashion in the healthy host. Such serine proteases are found in two well-recognized pathways of an innate immune network, coagulation and complement. Both pathways, if uncontrolled due to a variety of causes, are pathogenic in numerous diseases, including coagulation disorders and infectious diseases. Previous studies have reported sequence homologies, functional similarities and interplay between these two pathways with some implications in health and disease. The current study newly reveals that complement component factor B (Bf), the second component of the alternative complement pathway, has thrombin-like activity, which is supported by a characteristic homology of the trypsin-like domain of Bf to that of thrombin. Moreover, we newly report that the trypsin-like domain of Bf is closely related to Limulus clotting factor C, the LPS sensitive clotting factor of the innate immune system. We will also discuss potential implications of our findings in diseases.

A mechanism for hereditary angioedema with normal C1 inhibitor: an inhibitory regulatory role for the factor XII heavy chain.

The plasma proteins factor XII (FXII) and prekallikrein (PK) undergo reciprocal activation to the proteases FXIIa and kallikrein by a process that is enhanced by surfaces (contact activation) and regulated by the serpin C1 inhibitor. Kallikrein cleaves high-molecular-weight kininogen (HK), releasing the vasoactive peptide bradykinin. Patients with hereditary angioedema (HAE) experience episodes of soft tissue swelling as a consequence of unregulated kallikrein activity or increased prekallikrein activation. Although most HAE cases are caused by reduced plasma C1-inhibitor activity, HAE has been linked to lysine/arginine substitutions for Thr309 in FXII (FXII-Lys/Arg309). Here, we show that FXII-Lys/Arg309 is susceptible to cleavage after residue 309 by coagulation proteases (thrombin and FXIa), resulting in generation of a truncated form of FXII (δFXII). The catalytic efficiency of δFXII activation by kallikrein is 15-fold greater than for full-length FXII. The enhanced rate of reciprocal activation of PK and δFXII in human plasma and in mice appears to overwhelm the normal inhibitory function of C1 inhibitor, leading to increased HK cleavage. In mice given human FXII-Lys/Arg309, induction of thrombin generation by infusion of tissue factor results in enhanced HK cleavage as a consequence of δFXII formation. The effects of δFXII in vitro and in vivo are reproduced when wild-type FXII is bound by an antibody to the FXII heavy chain (HC; 15H8). The results contribute to our understanding of the predisposition of patients carrying FXII-Lys/Arg309 to angioedema after trauma, and reveal a regulatory function for the FXII HC that normally limits PK activation in plasma.

A SERS-LFA biosensor combined with aptamer recognition for simultaneous detection of thrombin and PDGF-BB in prostate cancer plasma.

An innovative surface-enhanced Raman spectroscopy and lateral flow assay (SERS-LFA) biosensor combined with aptamer recognition had been developed for the convenient, rapid, sensitive and accurate detection of thrombin and platelet-derived growth factor-BB (PDGF-BB) associated with prostate cancer simultaneously. During the biosensor operation, thrombin and PDGF-BB in the sample were recognized and combined by thiol-modified aptamers immobilized on Au-Ag hollow nanoparticles (Au-Ag HNPs) surface and biotinylated aptamers immobilized on the test lines of the biosensor. Thus, thrombin and PDGF-BB were simultaneously captured between detection aptamers and capture aptamers in a sandwich structure. Finite difference time domain simulation confirmed that 'hot spots' appeared at the gaps of Au-Ag HNPs dimer in the enhanced electromagnetic field compared to that of a single Au-Ag HNP, indicating that the aggregated Au-Ag HNPs owned a good SERS signal amplification effect. The detection limits of thrombin and PDGF-BB in human plasma were as low as 4.837 pg ml-1and 3.802 pg ml-1, respectively. Moreover, the accuracy of the biosensor which was applied to detect thrombin and PDGF-BB in prostate cancer plasma had been verified. This designed biosensor had broad application prospects in the clinical diagnosis of prostate cancer.

Thrombin-deficient mutant of medaka, a model fish, displays serious retardation in blood coagulation.

At the last stage of the blood coagulation cascade, thrombin plays a central role in the processing of fibrinogen for the polymerization and in the additional activation of Factor XIII for the stable cross-linking of fibrin. In addition, thrombin carries out possible multiple roles via processing or interaction with various functional proteins. Several studies conducted in order to elucidate additional physiological significance are ongoing. To clarify further significance of thrombin and to establish an associated disease model, we characterized the orthologue gene for medaka (Oryzias latipes), a research model fish. Tissue distribution of medaka prothrombin has been immunotechnically analyzed. Furthermore, thrombin-deficient medaka mutants were viably established by utilizing a genome-editing method. The established gene-deficient mutants exhibited retarded blood coagulation even in the heterozygous fish. Taking advantage of their ease of handling, this specific model is useful for further investigation in medical research areas on human coagulation diseases.

G protein-coupled receptors activate p38 MAPK via a non-canonical TAB1-TAB2- and TAB1-TAB3-dependent pathway in endothelial cells.

Endothelial dysfunction is induced by inflammatory mediators including multiple G protein-coupled receptor (GPCR) agonists. However, the GPCR signaling pathways that promote endothelial dysfunction are incompletely understood. We previously showed that thrombin promotes endothelial barrier disruption through autophosphorylation and activation of p38 mitogen-activated protein kinase (MAPK) via a non-canonical transforming growth factor-ß-activated protein kinase-1-binding protein-1 (TAB1) and TAB2-dependent pathway rather than the canonical three-tiered kinase cascade. Here, we sought to determine whether other GPCR agonists stimulate p38 MAPK activation via this non-canonical pathway in human endothelial cells derived from different vascular beds. Using primary human umbilical vein endothelial cells (HUVECs), HUVEC-derived EA.hy926 cells, and human dermal microvascular endothelial cells (HDMECs), we found that both non-canonical and canonical p38 activation pathways components are expressed in these various endothelial cell types, including TAB3, a structurally-related TAB2 homolog. Moreover, multiple GPCRs agonists, including thrombin, histamine, prostaglandin E2, and ADP, stimulated robust p38 autophosphorylation, whereas phosphorylation of the upstream MAPKs MAP kinase kinase 3 (MKK3) and MKK6, was virtually undetectable, indicating that non-canonical p38 activation may exist for other GPCRs. Indeed, in EA.hy926 cells, thrombin- and histamine-stimulated p38 activation depended on TAB1-TAB2, whereas in primary HUVECs, both TAB1-TAB2 and TAB1-TAB3 were required for p38 activation. In HDMECs, thrombin-induced p38 activation depended on TAB1-TAB3, but histamine-induced p38 activation required TAB1-TAB2. Moreover, thrombin- and histamine-stimulated interleukin-6 production required both TAB1-TAB2 and TAB1-TAB3 in HUVEC. We conclude that multiple GPCR agonists utilize non-canonical TAB1-TAB2 and TAB1-TAB3-dependent p38 activation to promote endothelial inflammatory responses.

Protein Z-dependent protease inhibitor (ZPI) is a physiologically significant inhibitor of prothrombinase function.

Current thought holds that factor Xa (FXa) bound in the prothrombinase complex is resistant to regulation by protein protease inhibitors during prothrombin activation. Here we provide evidence that, contrary to this view, the FXa-specific serpin inhibitor, protein Z-dependent protease inhibitor (ZPI), complexed with its cofactor, protein Z (PZ), functions as a physiologically significant inhibitor of prothrombinase-bound FXa during prothrombin activation. Kinetics studies showed that the rapid rate of inhibition of FXa by the ZPI-PZ complex on procoagulant membrane vesicles (ka(app) ∼107 m-1 s-1) was decreased ∼10-fold when FXa was bound to FVa in prothrombinase and a further ∼3-4-fold when plasma levels of S195A prothrombin were present (ka(app) 2 × 105 m-1 s-1). Nevertheless, the ZPI-PZ complex produced a major inhibition of thrombin generation during prothrombinase-catalyzed activation of prothrombin under physiologically relevant conditions. The importance of ZPI-PZ complex anticoagulant regulation of FXa both before and after incorporation into prothrombinase was supported by thrombin generation assays in plasma. These showed enhanced thrombin generation when the inhibitor was neutralized with a PZ-specific antibody and decreased thrombin generation when exogenous ZPI-PZ complex was added whether prothrombin was activated directly by FXa or through extrinsic or intrinsic pathway activators. Moreover, the PZ antibody enhanced thrombin generation both in the absence and presence of activated protein C (APC) anticoagulant activity. Taken together, these results suggest an important anticoagulant role for the ZPI-PZ complex in regulating both free FXa generated in the initiation phase of coagulation as well as prothrombinase-bound FXa in the propagation phase that complement prothrombinase regulation by APC.

Targeting anticoagulant protein S to improve hemostasis in hemophilia.

Improved treatments are needed for hemophilia A and B, bleeding disorders affecting 400 000 people worldwide. We investigated whether targeting protein S could promote hemostasis in hemophilia by rebalancing coagulation. Protein S (PS) is an anticoagulant acting as cofactor for activated protein C and tissue factor pathway inhibitor (TFPI). This dual role makes PS a key regulator of thrombin generation. Here, we report that targeting PS rebalances coagulation in hemophilia. PS gene targeting in hemophilic mice protected them against bleeding, especially when intra-articular. Mechanistically, these mice displayed increased thrombin generation, resistance to activated protein C and TFPI, and improved fibrin network. Blocking PS in plasma of hemophilia patients normalized in vitro thrombin generation. Both PS and TFPIα were detected in hemophilic mice joints. PS and TFPI expression was stronger in the joints of hemophilia A patients than in those of hemophilia B patients when receiving on-demand therapy, for example, during a bleeding episode. In contrast, PS and TFPI expression was decreased in hemophilia A patients receiving prophylaxis with coagulation factor concentrates, comparable to osteoarthritis patients. These results establish PS inhibition as both controller of coagulation and potential therapeutic target in hemophilia. The murine PS silencing RNA approach that we successfully used in hemophilic mice might constitute a new therapeutic concept for hemophilic patients.

MicroRNA-126 is a regulator of platelet-supported thrombin generation.

Circulating microRNA (miRNA) expression profiles correlate with platelet reactivity. MiR-126 is a promising candidates in this regard. We generated a transgenic zebrafish line with thrombocyte-specific overexpression of miR-126. Laser injury of the posterior cardinal vein of 5 day-old larvae was performed with or without antithrombotic pre-treatment. Platelet-like structures (PLS) derived from human megakaryocytes transfected with miR-126 were also evaluated for procoagulant activity. Finally, we studied the correlation between miR-126 level and thrombin generation markers in a cohort of stable cardiovascular patients. Control zebrafish developed small thrombocyte-rich thrombi at the site of vessel injury, without vessel occlusion. The miR-126 transgenic line developed an occluding thrombus in 75% (95% CI: 51-91%) of larvae. Pre-treatment with the direct thrombin inhibitor argatroban, but not aspirin, prevented vessel occlusion in the transgenic line (0% occlusion, 95%CI: 0-18%). Upon activation, human PLS showed an increased procoagulant profile after miR-126 transfection compared to control. Finally, the plasma levels of miR-126, but not a control platelet-derived miRNA, correlated with markers of in vivo thrombin generation in a cohort of 185 cardiovascular patients. Our results from three complementary approaches support a key role for miR-126 in platelet-supported thrombin generation and open new avenues in the tailoring of antithrombotic treatment.

In Silico Evaluation of the Effectivity of Approved Protease Inhibitors against the Main Protease of the Novel SARS-CoV-2 Virus.

The coronavirus disease, COVID-19, caused by the novel coronavirus SARS-CoV-2, which first emerged in Wuhan, China and was made known to the World in December 2019 turned into a pandemic causing more than 126,124 deaths worldwide up to April 16th, 2020. It has 79.5% sequence identity with SARS-CoV-1 and the same strategy for host cell invasion through the ACE-2 surface protein. Since the development of novel drugs is a long-lasting process, researchers look for effective substances among drugs already approved or developed for other purposes. The 3D structure of the SARS-CoV-2 main protease was compared with the 3D structures of seven proteases, which are drug targets, and docking analysis to the SARS-CoV-2 protease structure of thirty four approved and on-trial protease inhibitors was performed. Increased 3D structural similarity between the SARS-CoV-2 main protease, the HCV protease and α-thrombin was found. According to docking analysis the most promising results were found for HCV protease, DPP-4, α-thrombin and coagulation Factor Xa known inhibitors, with several of them exhibiting estimated free binding energy lower than -8.00 kcal/mol and better prediction results than reference compounds. Since some of the compounds are well-tolerated drugs, the promising in silico results may warrant further evaluation for viral anticipation. DPP-4 inhibitors with anti-viral action may be more useful for infected patients with diabetes, while anti-coagulant treatment is proposed in severe SARS-CoV-2 induced pneumonia.