Blood Coagulation and Beyond: Position Paper from the Fourth Maastricht Consensus Conference on Thrombosis.

The Fourth Maastricht Consensus Conference on Thrombosis included the following themes. Theme 1: The "coagulome" as a critical driver of cardiovascular disease. Blood coagulation proteins also play divergent roles in biology and pathophysiology, related to specific organs, including brain, heart, bone marrow, and kidney. Four investigators shared their views on these organ-specific topics. Theme 2: Novel mechanisms of thrombosis. Mechanisms linking factor XII to fibrin, including their structural and physical properties, contribute to thrombosis, which is also affected by variation in microbiome status. Virus infection-associated coagulopathies perturb the hemostatic balance resulting in thrombosis and/or bleeding. Theme 3: How to limit bleeding risks: insights from translational studies. This theme included state-of-the-art methodology for exploring the contribution of genetic determinants of a bleeding diathesis; determination of polymorphisms in genes that control the rate of metabolism by the liver of P2Y12 inhibitors, to improve safety of antithrombotic therapy. Novel reversal agents for direct oral anticoagulants are discussed. Theme 4: Hemostasis in extracorporeal systems: the value and limitations of ex vivo models. Perfusion flow chamber and nanotechnology developments are developed for studying bleeding and thrombosis tendencies. Vascularized organoids are utilized for disease modeling and drug development studies. Strategies for tackling extracorporeal membrane oxygenation-associated coagulopathy are discussed. Theme 5: Clinical dilemmas in thrombosis and antithrombotic management. Plenary presentations addressed controversial areas, i.e., thrombophilia testing, thrombosis risk assessment in hemophilia, novel antiplatelet strategies, and clinically tested factor XI(a) inhibitors, both possibly with reduced bleeding risk. Finally, COVID-19-associated coagulopathy is revisited.

A new strategy to count and sort neutrophil-derived extracellular vesicles: Validation in infectious disorders.

Newly recognized polymorphonuclear neutrophil (PMNs) functions include the ability to release subcellular mediators such as neutrophil-derived extracellular vesicles (NDEVs) involved in immune and thrombo-inflammatory responses. Elevation of their plasmatic level has been reported in a variety of infectious and cardiovascular disorders, but the clinical use of this potential biomarker is hampered by methodological issues. Although flow cytometry (FCM) is currently used to detect NDEVs in the plasma of patients, an extensive characterization of NDEVs has never been done. Moreover, their detection remains challenging because of their small size and low antigen density. Therefore, the objective of the present study was first to establish a surface antigenic signature of NDEVs detectable by FCM and therefore to improve their detection in biological fluids by developing a strategy allowing to overcome their low fluorescent signal and reduce the background noise. By testing a large panel of 54 antibody specificities already reported to be positive on PMNs, we identified a profile of 15 membrane protein markers, including 4 (CD157, CD24, CD65 and CD66c) never described on NDEVs. Among them, CD15, CD66b and CD66c were identified as the most sensitive and specific markers to detect NDEVs by FCM. Using this antigenic signature, we developed a new strategy combining the three best antibodies in a cocktail and reducing the background noise by size exclusion chromatography (SEC). This strategy allowed a significant improvement in NDEVs enumeration in plasma from sepsis patients and made it feasible to efficiently sort NDEVs from COVID-19 patients. Altogether, this work opens the door to a more valuable measurement of NDEVs as a potential biomarker in clinical practice. A similar strategy could also be applied to improve detection by FCM of other rare subpopulations of EVs generated by tissues with limited access, such as vascular endothelium, cancer cells or placenta.

Dissemination of extreme levels of extracellular vesicles: tissue factor activity in patients with severe COVID-19.

Coronavirus disease 2019 (COVID-19) has become one of the biggest public health challenges of this century. Severe forms of the disease are associated with a thrombo-inflammatory state that can turn into thrombosis. Because tissue factor (TF) conveyed by extracellular vesicles (EVs) has been implicated in thrombosis, we quantified the EV-TF activity in a cohort of hospitalized patients with COVID-19 (n = 111) and evaluated its link with inflammation, disease severity, and thrombotic events. Patients with severe disease were compared with those who had moderate disease and with patients who had septic shock not related to COVID-19 (n = 218). The EV-TF activity was notably increased in patients with severe COVID-19 compared with that observed in patients with moderate COVID-19 (median, 231 [25th to 75th percentile, 39-761] vs median, 25 [25th to 75th percentile, 12-59] fM; P < .0001); EV-TF was correlated with leukocytes, D-dimer, and inflammation parameters. High EV-TF values were associated with an increased thrombotic risk in multivariable models. Compared with patients who had septic shock, those with COVID-19 were characterized by a distinct coagulopathy profile with significantly higher EV-TF and EV-fibrinolytic activities that were not counterbalanced by an increase in plasminogen activator inhibitor-1 (PAI-1). Thus, this article is the first to describe the dissemination of extreme levels of EV-TF in patients with severe COVID-19, which supports the international recommendations of systematic preventive anticoagulation in hospitalized patients and potential intensification of anticoagulation in patients with severe disease.

Les microvésicules cellulaires: biomarqueurs émergents en pathologie cardiovasculaire : intérêt dans le risque thrombotique de la COVID 19*

Résumé Les maladies cardiovasculaires sont une des premières causes de mortalité et de morbidité dans le monde et la recherche de biomarqueurs permettant de stratifier le risque est un enjeu majeur. Les microvesicules sont des fragments de membranes générés par toutes les cellules de l’organisme. Elles sont reconnues aujourd’hui comme des vecteurs d'information biologiques participant à la communication intercellulaire et des biomarqueurs dont la pertinence clinique a été démontrée. En effet, l’élévation des taux plasmatiques des microvésicules a été rapportée dans différents contextes cardiovasculaires incluant l’athérosclérose, les syndromes coronariens aigus, les accidents vasculaires cérébraux, les thromboses associées au cancer ou aux infections comme la COVID- 19. Dans ce contexte infectieux émergeant, les microvésicules ont récemment été identifiées comme des biomarqueurs prometteurs dans l’identification précoce des patients à risque et dans la prédiction du risque thrombotique. Les progrès accomplis ces dernières années pour développer des techniques ultrasensibles permettant d’énumérer ou de mesurer l’activité fonctionnelle des microvésicules, associés aux efforts de standardisation impulsés par les sociétés savantes, permettent aujourd’hui d’accélérer leur transfert vers la clinique, afin de les positionner comme des candidats prometteurs pour une médecine personnalisée à visée cardiovasculaire. Cardiovascular diseases are one of the leading causes of mortality and morbidity, and the search for biomarkers for risk stratification is a challenge. Microvesicles are cell fragments generated by all cells in the body. They are considered not only as vectors of biological information involved in cell communication but also as emerging biomarkers with clinical relevance. Indeed, elevated plasmatic levels of microvesicles have been reported in various cardiovascular settings including atherosclerosis, acute coronary syndromes, ischemic strokes, as well as in thrombosis associated with cancer or infection as COVID 19, where microvesicles were identified as promising biomarkers for early detection of patient with severe forms of the disease and for prediction of thrombotic complications.Despite the fact that the field has long been hampered by technical difficulties, the ongoing progress in developing highly sensitive techniques and standardization efforts impulsed by Scientific societies working in partnership with research teams are crucial for implementing microvesicles analyses in the clinic. Large prospective studies are required to confirm which microvesicle subpopulations are of prognostic relevance and can be used as a promising biomarker to develop personalized cardiovascular medicine.

Circulating Endothelial Cells as a Marker of Endothelial Injury in Severe COVID -19.

Beside the commonly described pulmonary expression of the coronavirus disease 2019 (COVID-19), major vascular events have been reported. The objective of this study was to investigate whether increased levels of circulating endothelial cells (CECs) might be associated with severe forms of COVID-19. Ninety-nine patients with COVID-19 were enrolled in this retrospective study. Patients in the intensive care units (ICU) had significantly higher CEC counts than non-ICU patients and the extent of endothelial injury was correlated with putative markers of disease severity and inflammatory cytokines. Together, these data provide in vivo evidence that endothelial injury is a key feature of COVID-19.