microRNAs and thrombo-inflammation: relationship in sight.

PURPOSE OF REVIEW: Thrombo-inflammation is a multifaceted pathologic process involving various cells such as platelets, neutrophils, and monocytes. In recent years, microRNAs have been consistently implicated as regulators of these cells. RECENT FINDINGS: MicroRNAs play a regulatory role in several platelet receptors that have recently been identified as contributing to thrombo-inflammation and neutrophil extracellular trap (NET) formation. In addition, a growing body of evidence has shown that several intracellular and extracellular microRNAs directly promote NET formation. SUMMARY: Targeting microRNAs is a promising therapeutic approach to control thrombosis in patients with both infectious and noninfectious inflammatory diseases. Future research efforts should focus on elucidating the specific roles of microRNAs in thrombo-inflammation and translating these findings into tangible benefits for patients.

Increased galactose expression and enhanced clearance in patients with low von Willebrand factor.

Glycan determinants on von Willebrand factor (VWF) play critical roles in regulating its susceptibility to proteolysis and clearance. Abnormal glycosylation has been shown to cause von Willebrand disease (VWD) in a number of different mouse models. However, because of the significant technical challenges associated with accurate assessment of VWF glycan composition, the importance of carbohydrates in human VWD pathogenesis remains largely unexplored. To address this, we developed a novel lectin-binding panel to enable human VWF glycan characterization. This methodology was then used to study glycan expression in a cohort of 110 patients with low VWF compared with O blood group-matched healthy controls. Interestingly, significant interindividual heterogeneity in VWF glycan expression was seen in the healthy control population. This variation included terminal sialylation and ABO(H) blood group expression on VWF. Importantly, we also observed evidence of aberrant glycosylation in a subgroup of patients with low VWF. In particular, terminal α(2-6)-linked sialylation was reduced in patients with low VWF, with a secondary increase in galactose (Gal) exposure. Furthermore, an inverse correlation between Gal exposure and estimated VWF half-life was observed in those patients with enhanced VWF clearance. Together, these findings support the hypothesis that loss of terminal sialylation contributes to the pathophysiology underpinning low VWF in at least a subgroup of patients by promoting enhanced clearance. In addition, alterations in VWF carbohydrate expression are likely to contribute to quantitative and qualitative variations in VWF levels in the normal population. This trial was registered at www.clinicaltrials.gov as #NCT03167320.

TNF-α-driven inflammation and mitochondrial dysfunction define the platelet hyperreactivity of aging.

Aging and chronic inflammation are independent risk factors for the development of atherothrombosis and cardiovascular disease. We hypothesized that aging-associated inflammation promotes the development of platelet hyperreactivity and increases thrombotic risk during aging. Functional platelet studies in aged-frail adults and old mice demonstrated that their platelets are hyperreactive and form larger thrombi. We identified tumor necrosis factor α (TNF-α) as the key aging-associated proinflammatory cytokine responsible for platelet hyperreactivity. We further showed that platelet hyperreactivity is neutralized by abrogating signaling through TNF-α receptors in vivo in a mouse model of aging. Analysis of the bone marrow compartments showed significant platelet-biased hematopoiesis in old mice reflected by increased megakaryocyte-committed progenitor cells, megakaryocyte ploidy status, and thrombocytosis. Single-cell RNA-sequencing analysis of native mouse megakaryocytes showed significant reprogramming of inflammatory, metabolic, and mitochondrial gene pathways in old mice that appeared to play a significant role in determining platelet hyperreactivity. Platelets from old mice (where TNF-α was endogenously increased) and from young mice exposed to exogenous TNF-α exhibited significant mitochondrial changes characterized by elevated mitochondrial mass and increased oxygen consumption during activation. These mitochondrial changes were mitigated upon TNF-α blockade. Similar increases in platelet mitochondrial mass were seen in platelets from patients with myeloproliferative neoplasms, where TNF-α levels are also increased. Furthermore, metabolomics studies of platelets from young and old mice demonstrated age-dependent metabolic profiles that may differentially poise platelets for activation. Altogether, we present previously unrecognized evidence that TNF-α critically regulates megakaryocytes resident in the bone marrow niche and aging-associated platelet hyperreactivity and thrombosis.

miR-146a is a pivotal regulator of neutrophil extracellular trap formation promoting thrombosis.

Neutrophil extracellular traps (NETs) induce a procoagulant response linking inflammation and thrombosis. Low levels of miR-146a, a brake of inflammatory response, are involved in higher risk for cardiovascular events, but the mechanisms explaining how miR-146a exerts its function remain largely undefined. The aim of this study was to explore the impact of miR-146a deficiency in NETosis both, in sterile and non-sterile models in vivo, and to inquire into the underlying mechanism. Two models of inflammation were performed: 1) Ldlr-/- mice transplanted with bone marrow from miR-146a-/- or wild type (WT) were fed high-fat diet, generating an atherosclerosis model; and 2) an acute inflammation model was generated by injecting lipopolysaccharide (LPS) (1 mg/Kg) into miR-146a-/- and WT mice. miR-146a deficiency increased NETosis in both models. Accordingly, miR-146a-/- mice showed significant reduced carotid occlusion time and elevated levels of NETs in thrombi following FeCl3-induced thrombosis. Infusion of DNAse I abolished arterial thrombosis in WT and miR-146a-/- mice. Interestingly, miR-146a deficient mice have aged, hyperreactive and pro-inflammatory neutrophils in circulation that are more prone to form NETs independently of the stimulus. Furthermore, we demonstrated that community acquired pneumonia (CAP) patients with reduced miR-146a levels associated with the T variant of the functional rs2431697, presented an increased risk for cardiovascular events due in part to an increased generation of NETs.

N-Glycosylation as a Tool to Study Antithrombin Secretion, Conformation, and Function.

N-linked glycosylation is a crucial post-translational modification involved in protein folding, function, and clearance. N-linked glycosylation is also used therapeutically to enhance the half-lives of many proteins. Antithrombin, a serpin with four potential N-glycosylation sites, plays a pivotal role in hemostasis, wherein its deficiency significantly increases thrombotic risk. In this study, we used the introduction of N-glycosylation sites as a tool to explore what effect this glycosylation has on the protein folding, secretion, and function of this key anticoagulant. To accomplish this task, we introduced an additional N-glycosylation sequence in each strand. Interestingly, all regions that likely fold rapidly or were surrounded by lysines were not glycosylated even though an N-glycosylation sequon was present. The new sequon in the strands of the A- and B-sheets reduced secretion, and the B-sheet was more sensitive to these changes. However, the mutations in the strands of the C-sheet allowed correct folding and secretion, which resulted in functional variants. Therefore, our study revealed crucial regions for antithrombin secretion and could potentially apply to all serpins. These results could also help us understand the functional effects of natural variants causing type-I deficiencies.

MicroRNAs as New Regulators of Neutrophil Extracellular Trap Formation.

Neutrophil extracellular traps (NETs) are formed after neutrophils expelled their chromatin content in order to primarily capture and eliminate pathogens. However, given their characteristics due in part to DNA and different granular proteins, NETs may induce a procoagulant response linking inflammation and thrombosis. Unraveling NET formation molecular mechanisms as well as the intracellular elements that regulate them is relevant not only for basic knowledge but also to design diagnostic and therapeutic tools that may prevent their deleterious effects observed in several inflammatory pathologies (e.g., cardiovascular and autoimmune diseases, cancer). Among the potential elements involved in NET formation, several studies have investigated the role of microRNAs (miRNAs) as important regulators of this process. miRNAs are small non-coding RNAs that have been involved in the control of almost all physiological processes in animals and plants and that are associated with the development of several pathologies. In this review, we give an overview of the actual knowledge on NETs and their implication in pathology with a special focus in cardiovascular diseases. We also give a brief overview on miRNA biology to later focus on the different miRNAs implicated in NET formation and the perspectives opened by the presented data.

A novel role for the macrophage galactose-type lectin receptor in mediating von Willebrand factor clearance.

Previous studies have shown that loss of terminal sialic acid causes enhanced von Willebrand factor (VWF) clearance through the Ashwell-Morrell receptor (AMR). In this study, we investigated (1) the specific importance of N- vs O-linked sialic acid in protecting against VWF clearance and (2) whether additional receptors contribute to the reduced half-life of hyposialylated VWF. α2-3-linked sialic acid accounts for <20% of total sialic acid and is predominantly expressed on VWF O-glycans. Nevertheless, specific digestion with α2-3 neuraminidase (α2-3Neu-VWF) was sufficient to cause markedly enhanced VWF clearance. Interestingly, in vivo clearance experiments in dual VWF-/-/Asgr1-/- mice demonstrated enhanced clearance of α2-3Neu-VWF even in the absence of the AMR. The macrophage galactose-type lectin (MGL) is a C-type lectin that binds to glycoproteins expressing terminal N-acetylgalactosamine or galactose residues. Importantly, the markedly enhanced clearance of hyposialylated VWF in VWF-/-/Asgr1-/- mice was significantly attenuated in the presence of an anti-MGL inhibitory antibody. Furthermore, dose-dependent binding of human VWF to purified recombinant human MGL was confirmed using surface plasmon resonance. Additionally, plasma VWF:Ag levels were significantly elevated in MGL1-/- mice compared with controls. Collectively, these findings identify MGL as a novel macrophage receptor for VWF that significantly contributes to the clearance of both wild-type and hyposialylated VWF.

Platelet activation and neutrophil extracellular trap (NET) formation in immune thrombocytopenia: is there an association?

No biological predictors for the increased risk of thrombosis in patients with immune thrombocytopenia (ITP) have been identified. The aim of the study was to investigate platelet and neutrophil activation as well neutrophil extracellular trap (NET) formation in 63 ITP patients and 30 healthy volunteers. Platelet and neutrophil activation was assessed during steady state using flow cytometry analysis, and NETs were evaluated by quantitation of cell free DNA (cfDNA), and citrullinated histone-3-DNA (CitH3-DNA). Patient platelets and neutrophils showed increased CD62 and CD11b expression compared to controls (p = .038, and p = .022, respectively). In patients, platelet activation inversely correlated with platelet count and platelet size (p < .001), and positively correlated with neutrophil degranulation (p = .024). More NET formation, both CitH3-DNA (p = .025) and cfDNA(p < .001), were present in ITP patients compared to controls. CitH3-DNA inversely correlated with CD62 expression on platelets (p = .042), but higher levels of cfDNA were observed in individuals with classical cardiovascular risk factors for thrombosis, and in those with a previous history of thrombotic events. In this disease, the increased platelet activation and plasma NET levels seem to be separable processes that associate (either positively or inversely in the case of CitH3-DNA or platelet degranulation, respectively) to platelet mass.

Novel insights into the clinical phenotype and pathophysiology underlying low VWF levels.

Critical clinical questions remain unanswered regarding diagnosis and management of patients with low von Willebrand factor (VWF) levels (30-50 IU/dL). To address these questions, the Low VWF Ireland Cohort (LoVIC) study investigated 126 patients registered with low VWF levels. Despite marginally reduced plasma VWF levels, International Society of Thrombosis and Haemostasis Bleeding Assessment Tool (ISTH BAT) confirmed significant bleeding phenotypes in the majority of LoVIC patients. Importantly, bleeding tendency did not correlate with plasma VWF levels within the 30 to 50 IU/dL range. Furthermore, bleeding phenotypes could not be explained by concurrent hemostatic defects. Plasma factor VIII to VWF antigen (VWF:Ag) ratios were significantly increased in LoVIC patients compared with controls (P < .0001). In contrast, VWF propeptide to VWF:Ag ratios >3 were observed in only 6% of the LoVIC cohort. Furthermore, platelet-VWF collagen binding activity levels were both significantly reduced compared with controls (P < .05). In response to 1-desamino-8-D-arginine vasopressin (DDAVP), peak VWF:Ag levels exceeded 100 IU/dL in 88% of patients and was sustained >100 IU/dL after 4 hours in 72% of subjects. In conclusion, our novel data suggest that low VWF levels can be associated with significant bleeding and are predominantly due to reductions in VWF synthesis and/or constitutive secretion. Although enhanced VWF clearance may contribute to the pathophysiology in some individuals, the absolute reduction in VWF plasma half-life is usually mild and not sufficient to significantly impact upon the duration of DDAVP-induced VWF response. This trial was registered at www.clinicaltrials.gov as #NCT03167320.

Disease-causing mutations in the serpin antithrombin reveal a key domain critical for inhibiting protease activities.

Antithrombin mainly inhibits factor Xa and thrombin. The reactive center loop (RCL) is crucial for its interactions with its protease targets and is fully inserted into the A-sheet after its cleavage, causing translocation of the covalently linked protease to the opposite end of the A-sheet. Antithrombin variants with altered RCL hinge residues behave as substrates rather than inhibitors, resulting in stoichiometries of inhibition greater than one. Other antithrombin residues have been suggested to interfere with RCL insertion or the stability of the antithrombin-protease complex, but available crystal structures or mutagenesis studies have failed to identify such residues. Here, we characterized two mutations, S365L and I207T, present in individuals with type II antithrombin deficiency and identified a new antithrombin functional domain. S365L did not form stable complexes with thrombin or factor Xa, and the I207T/I207A variants inhibited both proteases with elevated stoichiometries of inhibition. Close proximity of Ile-207 and Ser-365 to the inserted RCL suggested that the preferred reaction of these mutants as protease substrates reflects an effect on the rate of the RCL insertion and protease translocation. However, both residues lie within the final docking site for the protease in the antithrombin-protease complex, supporting the idea that the enhanced substrate reactions may result from an increased dissociation of the final complexes. Our findings demonstrate that the distal end of the antithrombin A-sheet is crucial for the last steps of protease inhibition either by affecting the rate of RCL insertion or through critical interactions with proteases at the end of the A-sheet.

N-linked glycans within the A2 domain of von Willebrand factor modulate macrophage-mediated clearance.

Enhanced von Willebrand factor (VWF) clearance is important in the etiology of von Willebrand disease. However, the molecular mechanisms underlying VWF clearance remain poorly understood. In this study, we investigated the role of VWF domains and specific glycan moieties in regulating in vivo clearance. Our findings demonstrate that the A1 domain of VWF contains a receptor-recognition site that plays a key role in regulating the interaction of VWF with macrophages. In A1-A2-A3 and full-length VWF, this macrophage-binding site is cryptic but becomes exposed following exposure to shear or ristocetin. Previous studies have demonstrated that the N-linked glycans within the A2 domain play an important role in modulating susceptibility to ADAMTS13 proteolysis. We further demonstrate that these glycans presented at N1515 and N1574 also play a critical role in protecting VWF against macrophage binding and clearance. Indeed, loss of the N-glycan at N1515 resulted in markedly enhanced VWF clearance that was significantly faster than that observed with any previously described VWF mutations. In addition, A1-A2-A3 fragments containing the N1515Q or N1574Q substitutions also demonstrated significantly enhanced clearance. Importantly, clodronate-induced macrophage depletion significantly attenuated the increased clearance observed with N1515Q and N1574Q in both full-length VWF and A1-A2-A3. Finally, we further demonstrate that loss of these N-linked glycans does not enhance clearance in VWF in the presence of a structurally constrained A2 domain. Collectively, these novel findings support the hypothesis that conformation of the VWF A domains plays a critical role in modulating macrophage-mediated clearance of VWF in vivo.

Conformational activation of antithrombin by heparin involves an altered exosite interaction with protease.

Heparin allosterically activates antithrombin as an inhibitor of factors Xa and IXa by enhancing the initial Michaelis complex interaction of inhibitor with protease through exosites. Here, we investigate the mechanism of this enhancement by analyzing the effects of alanine mutations of six putative antithrombin exosite residues and three complementary protease exosite residues on antithrombin reactivity with these proteases in unactivated and heparin-activated states. Mutations of antithrombin Tyr(253) and His(319) exosite residues produced massive 10-200-fold losses in reactivity with factors Xa and IXa in both unactivated and heparin-activated states, indicating that these residues made critical attractive interactions with protease independent of heparin activation. By contrast, mutations of Asn(233), Arg(235), Glu(237), and Glu(255) exosite residues showed that these residues made both repulsive and attractive interactions with protease that depended on the activation state and whether the critical Tyr(253)/His(319) residues were mutated. Mutation of factor Xa Arg(143), Lys(148), and Arg(150) residues that interact with the exosite in the x-ray structure of the Michaelis complex confirmed the importance of all residues for heparin-activated antithrombin reactivity and Arg(150) for native serpin reactivity. These results demonstrate that the exosite is a key determinant of antithrombin reactivity with factors Xa and IXa in the native as well as the heparin-activated state and support a new model of allosteric activation we recently proposed in which a balance between attractive and repulsive exosite interactions in the native state is shifted to favor the attractive interactions in the activated state through core conformational changes induced by heparin binding.

Amelioration of the severity of heparin-binding antithrombin mutations by posttranslational mosaicism.

The balance between actions of procoagulant and anticoagulant factors protects organisms from bleeding and thrombosis. Thus, antithrombin deficiency increases the risk of thrombosis, and complete quantitative deficiency results in intrauterine lethality. However, patients homozygous for L99F or R47C antithrombin mutations are viable. These mutations do not modify the folding or secretion of the protein, but abolish the glycosaminoglycan-induced activation of antithrombin by affecting the heparin-binding domain. We speculated that the natural ß-glycoform of antithrombin might compensate for the effect of heparin-binding mutations. We purified α- and ß-antithrombin glycoforms from plasma of 2 homozygous L99F patients. Heparin affinity chromatography and intrinsic fluorescence kinetic analyses demonstrated that the reduced heparin affinity of the α-L99F glycoform (K(D), 107.9 ± 3nM) was restored in the ß-L99F glycoform (K(D), 53.9 ± 5nM) to values close to the activity of α-wild type (K(D), 43.9 ± 0.4nM). Accordingly, the ß-L99F glycoform was fully activated by heparin. Similar results were observed for recombinant R47C and P41L, other heparin-binding antithrombin mutants. In conclusion, we identified a new type of mosaicism associated with mutations causing heparin-binding defects in antithrombin. The presence of a fully functional ß-glycoform together with the activity retained by these variants helps to explain the viability of homozygous and the milder thrombotic risk of heterozygous patients with these specific antithrombin mutations.

Control of post-translational modifications in antithrombin during murine post-natal development by miR-200a.

BACKGROUND: Developmental haemostatic studies may help identifying new elements involved in the control of key haemostatic proteins like antithrombin, the most relevant endogenous anticoagulant. RESULTS: In this study, we showed a significant reduction of sialic acid content in neonatal antithrombin compared with adult antithrombin in mice. mRNA levels of St3gal3 and St3gal4, two sialyltransferases potentially involved in antithrombin sialylation, were 85% lower in neonates in comparison with adults. In silico analysis of miRNAs overexpressed in neonates revealed that mir-200a might target these sialyltransferases. Moreover, in vitro studies in murine primary hepatocytes sustain this potential control. CONCLUSIONS: These data suggest that in addition to the direct protein regulation, microRNAs may also modulate qualitative traits of selected proteins by an indirect control of post-translational processes.

Inflammatory microRNAs in cardiovascular pathology: another brick in the wall.

The regulatory role of microRNAs (miRNAs) is mainly mediated by their effect on protein expression and is recognized in a multitude of pathophysiological processes. In recent decades, accumulating evidence has interest in these factors as modulatory elements of cardiovascular pathophysiology. Furthermore, additional biological processes have been identified as new components of cardiovascular disease etiology. In particular, inflammation is now considered an important cardiovascular risk factor. Thus, in the present review, we will focus on the role of a subset of miRNAs called inflamma-miRs that may regulate inflammatory status in the development of cardiovascular pathology. According to published data, the most representative candidates that play functional roles in thromboinflammation are miR-21, miR-33, miR-34a, miR-146a, miR-155, and miR-223. We will describe the functions of these miRNAs in several cardiovascular pathologies in depth, with specific emphasis on the molecular mechanisms related to atherogenesis. We will also discuss the latest findings on the role of miRNAs as regulators of neutrophil extracellular traps and their impact on cardiovascular diseases. Overall, the data suggest that the use of miRNAs as therapeutic tools or biomarkers may improve the diagnosis or prognosis of adverse cardiovascular events in inflammatory diseases. Thus, targeting or increasing the levels of adequate inflamma-miRs at different stages of disease could help mitigate or avoid the development of cardiovascular morbidities.

microRNAs as biomarkers of risk of major adverse cardiovascular events in atrial fibrillation.

Atrial fibrillation is a complex and multifactorial disease. Although prophylactic anticoagulation has great benefits in avoiding comorbidities, adverse cardiovascular events still occur and thus in recent decades, many resources have been invested in the identification of useful markers in the prevention of the risk of MACE in these patients. As such, microRNAs, that are small non-coding RNAs whose function is to regulate gene expression post-transcriptionally, have a relevant role in the development of MACE. miRNAs, have been investigated for many years as potential non-invasive biomarkers of several diseases. Different studies have shown their utility in the diagnosis and prognosis of cardiovascular diseases. In particular, some studies have associated the presence of certain miRNAs in plasma with the development of MACE in AF. Despite these results, there are still many efforts to be done to allow the clinical use of miRNAs. The lack of standardization concerning the methodology in purifying and detecting miRNAs, still provides contradictory results. miRNAs also have a functional impact in MACE in AF through the dysregulation of immunothrombosis. Indeed, miRNAs may be a link between MACE and inflammation, through the regulation of neutrophil extracellular traps that are a key element in the establishment and evolution of thrombotic events. The use of miRNAs as therapy against thromboinflammatory processes should also be a future approach to avoid the occurrence of MACE in atrial fibrillation.

The infective polymerization of conformationally unstable antithrombin mutants may play a role in the clinical severity of antithrombin deficiency.

Mutations affecting mobile domains of antithrombin induce conformational instability resulting in protein polymerization that associates with a severe clinical phenotype, probably by an unknown gain of function. By homology with other conformational diseases, we speculated that these variants might infect wild-type (WT) monomers reducing the anticoagulant capacity. Infective polymerization of WT polymers and different P1 mutants (p.R425del, p.R425C and p.R425H) were evaluated by using native gels and radiolabeled WT monomers and functional assays. Human embryonic kidney cells expressing the Epstein-Barr nuclear antigen 1 (HEK-EBNA) cells expressing inducible (p.R425del) or two novel constitutive (p.F271S and p.M370T) conformational variants were used to evaluate intracellular and secreted antithrombin under mild stress (pH 6.5 and 39°C for 5 h). We demonstrated the conformational sensitivity of antithrombin London (p.R425del) to form polymers under mild heating. Under these conditions purified antithrombin London recruited WT monomers into growing polymers, reducing the anticoagulant activity. This process was also observed in the plasma of patients with p.R425del, p.R425C and p.R425H mutations. Under moderate stress, coexpression of WT and conformational variants in HEK-EBNA cells increased the intracellular retention of antithrombin and the formation of disulfide-linked polymers, which correlated with impaired secretion and reduction of anticoagulant activity in the medium. Therefore, mutations inducing conformational instability in antithrombin allow its polymerization with the subsequent loss of function, which under stress could sequestrate WT monomers, resulting in a new prothrombotic gain of function, particularly relevant for intracellular antithrombin. The in vitro results suggest a temporal and severe plasma antithrombin deficiency that may contribute to the development of the thrombotic event and to the clinical severity of these mutations.

MiR-146a Contributes to Thromboinflammation and Recurrence in Young Patients with Acute Myocardial Infarction.

Studies on older patients have established notable conceptual changes in the etiopathogenesis of acute coronary syndrome (ACS), but little is known about this disease in young patients (<45 years). Of special interest is thromboinflammation, key at onset, evolution and therapy of cardiovascular pathology. Therefore, we explored whether ACS at an early age is a thromboinflammatory disease by analyzing NETs and rs2431697 of miR-146a (a miRNA considered as a brake of TLR/NF-kB pathway), elements previously related to higher rates of recurrence in atrial fibrillation and sepsis. We included 359 ACS patients (<45 years) and classified them for specific analysis into G1 (collected during the hospitalization of the first event), G2 and G3 (retrospectively collected from patients with or without ACS recurrence, respectively). cfDNA and citH3−DNA were quantified, and rs2431697 was genotyped. Analysis in the overall cohort showed a moderate but significant correlation between cfDNA and citH3−DNA and Killip−Kimball score. In addition, patients with citH3−DNA > Q4 more frequently had a history of previous stroke (6.1% vs. 1.6%). In turn, rs2431697 did not confer increased risk for the onset of ACS, but T carriers had significantly higher levels of NET markers. By groups, we found that cfDNA levels were similarly higher in all patients, but citH3−DNA was especially higher in G1, suggesting that in plasma, this marker may be attenuated over time. Finally, patients from G2 with the worst markers (cfDNA and citH3−DNA > Q2 and T allele) had a two-fold increased risk of a new ischemic event at 2-year follow-up. In conclusion, our data confirm that ACS is younger onset with thromboinflammatory disease. In addition, these data consolidate rs2431697 as a silent proinflammatory factor predisposing to NETosis, and to a higher rate of adverse events in different cardiovascular diseases.

von Willebrand factor links primary hemostasis to innate immunity.

The plasma multimeric glycoprotein von Willebrand factor (VWF) plays a critical role in primary hemostasis by tethering platelets to exposed collagen at sites of vascular injury. Recent studies have identified additional biological roles for VWF, and in particular suggest that VWF may play an important role in regulating inflammatory responses. However, the molecular mechanisms through which VWF exerts its immuno-modulatory effects remain poorly understood. In this study, we report that VWF binding to macrophages triggers downstream MAP kinase signaling, NF-κB activation and production of pro-inflammatory cytokines and chemokines. In addition, VWF binding also drives macrophage M1 polarization and shifts macrophage metabolism towards glycolysis in a p38-dependent manner. Cumulatively, our findings define an important biological role for VWF in modulating macrophage function, and thereby establish a novel link between primary hemostasis and innate immunity.

miR-146a in Cardiovascular Diseases and Sepsis: An Additional Burden in the Inflammatory Balance?

The new concept of thrombosis associated with an inflammatory process is called thromboinflammation. Indeed, both thrombosis and inflammation interplay one with the other in a feed forward manner amplifying the whole process. This pathological reaction in response to a wide variety of sterile or non-sterile stimuli eventually causes acute organ damage. In this context, neutrophils, mainly involved in eliminating pathogens as an early barrier to infection, form neutrophil extracellular traps (NETs) that are antimicrobial structures responsible of deleterious side effects such as thrombotic complications. Although NETosis mechanisms are being unraveled, there are still many regulatory elements that have to be discovered. Micro-ribonucleic acids (miRNAs) are important modulators of gene expression implicated in human pathophysiology almost two decades ago. Among the different miRNAs implicated in inflammation, miR-146a is of special interest because: (1) it regulates among others, Toll-like receptors/nuclear factor-κB axis which is of paramount importance in inflammatory processes, (2) it regulates the formation of NETs by modifying their aging phenotype, and (3) it has expression levels that may decrease among individuals up to 50%, controlled in part by the presence of several polymorphisms. In this article, we will review the main characteristics of miR-146a biology. In addition, we will detail how miR-146a is implicated in the development of two paradigmatic diseases in which thrombosis and inflammation interact, cardiovascular diseases and sepsis, and their association with the presence of miR-146a polymorphisms and the use of miR-146a as a marker of cardiovascular diseases and sepsis.