Overall haemostatic potential assay for prediction of outcomes in venous and arterial thrombosis and thrombo-inflammatory diseases.

Thromboembolic diseases including arterial and venous thrombosis are common causes of morbidity and mortality globally. Thrombosis frequently recurs and can also complicate many inflammatory conditions through the process of 'thrombo-inflammation,' as evidenced during the COVID-19 pandemic. Current candidate biomarkers for thrombosis prediction, such as D-dimer, have poor predictive efficacy. This limits our capacity to tailor anticoagulation duration individually and may expose lower risk individuals to undue bleeding risk. Global coagulation assays, such as the Overall Haemostatic Potential (OHP) assay, that investigate fibrin generation and fibrinolysis, may provide a more accurate and functional assessment of hypercoagulability. We present a review of fibrin's critical role as a central modulator of thrombotic risk. The results of our studies demonstrating the OHP assay as a predictive biomarker in venous thromboembolism, chronic renal disease, diabetes mellitus, post-thrombotic syndrome, and COVID-19 are discussed. As a comprehensive and global measurement of fibrin generation and fibrinolytic capacity, the OHP assay may be a valuable addition to future multi-modal predictive tools in thrombosis.

Persistent complement dysregulation with signs of thromboinflammation in active Long Covid.

Long Covid is a debilitating condition of unknown etiology. We performed multimodal proteomics analyses of blood serum from COVID-19 patients followed up to 12 months after confirmed severe acute respiratory syndrome coronavirus 2 infection. Analysis of >6500 proteins in 268 longitudinal samples revealed dysregulated activation of the complement system, an innate immune protection and homeostasis mechanism, in individuals experiencing Long Covid. Thus, active Long Covid was characterized by terminal complement system dysregulation and ongoing activation of the alternative and classical complement pathways, the latter associated with increased antibody titers against several herpesviruses possibly stimulating this pathway. Moreover, markers of hemolysis, tissue injury, platelet activation, and monocyte-platelet aggregates were increased in Long Covid. Machine learning confirmed complement and thromboinflammatory proteins as top biomarkers, warranting diagnostic and therapeutic interrogation of these systems.

Extracellular Vesicles and Citrullinated Histone H3 in Coronavirus Disease 2019 Patients.

OBJECTIVES: Pulmonary thrombus formation is a hallmark of coronavirus disease 2019 (COVID-19). A dysregulated immune response culminating in thromboinflammation has been described, but the pathomechanisms remain unclear. METHODS: We studied 41 adult COVID-19 patients with positive results on reverse-transcriptase polymerase-chain-reaction assays and 37 sex- and age-matched healthy controls. Number and surface characteristics of extracellular vesicles (EVs) and citrullinated histone H3 levels were determined in plasma upon inclusion by flow cytometry and immunoassay. RESULTS: In total, 20 patients had severe and 21 nonsevere disease. The number of EV (median [25th, 75th percentile]) was significantly higher in patients compared with controls (658.8 [353.2, 876.6] vs. 435.5 [332.5, 585.3], geometric mean ratio [95% confidence intervals]: 2.6 [1.9, 3.6]; p < 0.001). Patients exhibited significantly higher numbers of EVs derived from platelets, endothelial cells, leukocytes, or neutrophils than controls. EVs from alveolar-macrophages and alveolar-epithelial cells were detectable in plasma and were significantly higher in patients. Intercellular adhesion molecule-1-positive EV levels were higher in patients, while no difference between tissue factor-positive and angiotensin-converting enzyme-positive EV was seen between both groups. Levels of EV did not differ between patients with severe and nonsevere COVID-19. Citrullinated histone H3 levels (ng/mL, median [25th, 75th percentile]) were higher in patients than in controls (1.42 [0.6, 3.4] vs. 0.31 [0.1, 0.6], geometric mean ratio: 4.44 [2.6, 7.7]; p < 0.001), and were significantly lower in patients with nonsevere disease compared with those with severe disease. CONCLUSION: EV and citrullinated histone H3 are associated with COVID-19 and could provide information regarding pathophysiology of the disease.

A unique circulating miRNA profile highlights thrombo-inflammation in Behçet's syndrome.

OBJECTIVES: Behçet's syndrome (BS) is a rare systemic vasculitis often complicated by thrombotic events. Given the lack of validated biomarkers, BS diagnosis relies on clinical criteria.In search of novel biomarkers for BS diagnosis, we determined the profile of plasmatic circulating microRNAs (ci-miRNAs) in patients with BS compared with healthy controls (HCs). METHODS: ci-miRNA profile was evaluated by microarray in a screening cohort (16 patients with BS and 18 HCs) and then validated by poly(T) adaptor PCR (PTA-PCR) in a validation cohort (30 patients with BS and 30 HCs). Two disease control groups (30 patients with systemic lupus erythematosus (SLE) and 30 patients with giant cell arteritis (GCA) were also analysed. RESULTS: From the microarray screening, 29 deregulated (differentially expressed (DE)) human ci-miRNAs emerged. A hierarchical cluster analysis indicated that DE ci-miRNAs clearly segregated patients from controls, independently of clinical features. PTA-PCR analysis on the validation cohort confirmed the deregulation of miR-224-5p, miR-206 and miR-653-5p. The combined receiver operating characteristic (ROC) curve analyses showed that such ci-miRNAs discriminate BS from HCs (and BS with active vs inactive disease), as well as BS from patients with SLE and GCA.The functional annotation analyses (FAAs) showed that the most enriched pathways affected by DE ci-miRNAs (ie, cell-matrix interaction, oxidative stress and blood coagulation) are related to thrombo-inflammatory mechanisms. Accordingly, the expression of the three ci-miRNAs from the validation cohort significantly correlated with leucocyte reactive oxygen species production and plasma lipid peroxidation. CONCLUSIONS: The ci-miRNA profile identified in this study may represent a novel, poorly invasive BS biomarker, while suggesting an epigenetic control of BS-related thrombo-inflammation.

Angiotensin II triggers release of neutrophil extracellular traps, linking thromboinflammation with essential hypertension.

Innate immunity and chronic inflammation are involved in atherosclerosis and atherothrombosis, leading to target organ damage in essential hypertension (EH). However, the role of neutrophils in EH is still elusive. We investigated the association between angiotensin II (Ang II) and neutrophil extracellular traps (NETs) in pathogenesis of EH. Plasma samples, kidney biopsies, and surgical specimens of abdominal aortic aneurysms (AAAs) from patients with EH were used. Cell-based assays, NETs/human aortic endothelial cell cocultures, and in situ studies were performed. Increased plasma levels of NETs and tissue factor (TF) activity were detected in untreated, newly diagnosed patients with EH. Stimulation of control neutrophils with plasma from patients with untreated EH generated TF-enriched NETs promoting endothelial collagen production. Ang II induced NETosis in vitro via an ROS/peptidylarginine deiminase type 4 and autophagy-dependent pathway. Circulating NETs and thrombin generation levels were reduced substantially in patients with EH starting treatment with Ang II receptor blockers, whereas their plasma was unable to trigger procoagulant NETs. Moreover, TF-bearing NETotic neutrophils/remnants accumulated in sites of interstitial renal fibrosis and in the subendothelial layer of AAAs. These data reveal the important pathogenic role of an Ang II/ROS/NET/TF axis in EH, linking thromboinflammation with endothelial dysfunction and fibrosis.

P2X1: a unique platelet receptor with a key role in thromboinflammation.

Thromboinflammation involves complex interactions between actors of inflammation and immunity and components of the hemostatic system, which are elicited upon infection or tissue injury. In this context, the interplay between platelets and innate immune cells has been intensively investigated. The ATP-gated P2X1 ion channel, expressed on both platelets and neutrophils is of particular interest. On platelets, this ion channel contributes to platelet activation and thrombosis, especially under high shear stress conditions of small arteries, whereas on neutrophils, it is involved in chemotaxis and in mitigating the activation of circulating cells. In vitro studies indicate that it may also be implicated in platelet-dependent immune responses during bacterial infection. More recently, in a mouse model of intestinal epithelial barrier disruption causing systemic inflammation, it has been reported that neutrophil P2X1 ion channel could play a protective role against exaggerated inflammation-associated thrombosis. This review will focus on this unique role of the ATP-gated P2X1 ion channel in thromboinflammation, highlighting possible implications and pointing to the need for further investigation of the role of P2X1 ion channels in the interplay between platelets and neutrophils during thrombus formation under various sterile or infectious inflammatory settings and in distinct vascular beds.

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.