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.

Granulocyte microvesicles with a high plasmin generation capacity promote clot lysis and improve outcome in septic shock.

Microvesicles (MVs) have previously been shown to exert profibrinolytic capacity, which is increased in patients with septic shock (SS) with a favorable outcome. We, therefore, hypothesized that the plasmin generation capacity (PGC) could confer to MVs a protective effect supported by their capacity to lyse a thrombus, and we investigated the mechanisms involved. Using an MV-PGC kinetic assay, ELISA, and flow cytometry, we found that granulocyte MVs (Gran-MVs) from SS patients display a heterogeneous PGC profile driven by the uPA (urokinase)/uPAR system. In vitro, these MVs lyse a thrombus according to their MV-PGC levels in a uPA/uPAR-dependent manner, as shown in a fluorescent clot lysis test and a lysis front retraction assay. Fibrinolytic activators conveyed by MVs contribute to approximately 30% of the plasma plasminogenolytic capacity of SS patients. In a murine model of SS, the injection of high PGC Gran-MVs significantly improved mouse survival and reduced the number of thrombi in vital organs. This was associated with a modification of the mouse coagulation and fibrinolysis properties toward a more fibrinolytic profile. Interestingly, mouse survival was not improved when soluble uPA was injected. Finally, using a multiplex array on plasma from SS patients, we found that neutrophil elastase correlates with the effect of high-PGC-capacity plasma and modulates the Gran-MV plasmin generation capacity by cleaving uPA-PAI-1 complexes. In conclusion, we show that the high PGC level displayed by Gran-MVs reduces thrombus formation and improves survival, conferring to Gran-MVs a protective role in a murine model of sepsis.

[Extracellular vesicles-associated biomarkers: Opportunities and challenges in cardiovascular diseases and cancer]. / Biomarqueurs vésiculaires - Opportunités et défis dans les maladies cardiovasculaires et les cancers.

Cardiovascular diseases and cancer are the leading causes of mortality and morbidity in the world. The search for pertinent biomarkers for risk stratification and treatment monitoring is a challenge. Rapid advances in the identification of the molecular and functional content of extracellular vesicles (EV) and ongoing progress in developing highly sensitive methodologies, identify EV as promising biomarkers easily accessible in liquid biopsies. Thanks to robust and sensitive methodologies, the measurability of biological targets on EV allows to define vesicular biomarkers pertinent for disease management. Adaptation of the pre-analytical and analytical steps to each EV-associated biomarker, technological improvement and standardization efforts driven by scientific societies are essential prerequisites to accelerate the transfer of these EV-associated biomarkers to the clinics and to support the development of personalized medicine.

TITLE: Biomarqueurs vésiculaires - Opportunités et défis dans les maladies cardiovasculaires et les cancers. ABSTRACT: Les maladies cardiovasculaires et les cancers sont les premières causes de mortalité et de morbidité dans le monde. La recherche de biomarqueurs pertinents est un réel enjeu pour améliorer leur prise en charge clinique et thérapeutique. Les progrès rapides faits sur l'identification du contenu moléculaire et fonctionnel des vésicules extracellulaires et le développement de méthodologies sensibles et robustes identifient ces dernières comme des biomarqueurs prometteurs, accessibles dans les biopsies liquides. L'adaptation des étapes pré-analytiques, analytiques et une standardisation adaptée à chaque cible vésiculaire sont des prérequis indispensables pour accélérer le transfert de ces biomarqueurs vésiculaires vers la clinique et accompagner le développement d'une médecine personnalisée.

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.

Increasing the sensitivity of the human microvesicle tissue factor activity assay.

INTRODUCTION: The TF-FVIIa complex is the primary activator of coagulation. Elevated levels of microvesicle (MV) bearing tissue factor (TF)-dependent procoagulant activity are detectable in patients with an increased risk of thrombosis. Several methods have been described to measure MV TF activity but they are hampered by limited sensitivity and specificity. The aim of this work was to increase the sensitivity of the MV TF activity assay (called Chapel Hill assay). MATERIAL AND METHODS: Improvements of the MV TF activity assay included i/ speed and time of centrifugation, ii/ use of a more potent inhibitory anti-TF antibody iii/ use of FVII and a fluorogenic substrate to increase specificity. RESULTS: The specificity of the MV TF activity assay was demonstrated by the absence of activity on MV derived from a knock-out-TF cell line using an anti-human TF monoclonal antibody called SBTF-1, which shows a higher TF inhibitory effect than the anti-human TF monoclonal antibody called HTF-1. Experiments using blood from healthy individuals, stimulated or not by LPS, or plasma spiked with 3 different levels of MV, demonstrated that the new assay was more sensitive and this allowed detection of MV TF activity in platelet free plasma (PFP) samples from healthy individuals. However, the assay was limited by an inter-assay variability, mainly due to the centrifugation step. CONCLUSIONS: We have improved the sensitivity of the MV TF activity assay without losing specificity. This new assay could be used to evaluate levels of TF-positive MV as a potential biomarker of thrombotic risk in patients.

Microvesicles and Cancer Associated Thrombosis.

Microvesicles (MVs) are small membrane enclosed structures released into the extracellular space by virtually all cell types. Their composition varies according to the cell origin and the stimulus which caused their formation. They harbor functional molecules and participate in intercellular communication. Endothelium, inflammatory cells, and cancer cells produce procoagulant MVs which contribute to cancer-associated thrombosis (CAT) in animal models. The tissue factor (TF) conveyed by these MVs was shown to play a key role in different animal models of experimental CAT. Alternatively, other molecular mechanisms involving polyphosphates or phosphatidylethanolamine could also be involved. In clinical practice, an association between an increase in the number of TF-positive or the procoagulant activity of these MVs and the occurrence of CAT has indeed been demonstrated in pancreatic-biliary cancers, suggesting that they could behave as a biomarker predictive for CAT. However, to date, this association was not confirmed in other types of cancer. Potential causes explaining this limited associated between MVs and CAT are (1) the diversity of mechanisms associating MVs and different types of cancer; (2) a more complex role of MVs in hemostasis integrating their anticoagulant and fibrinolytic activity; and (3) the lack of sensitivity, reproducibility, and standardization of current methodologies permitting measurement of MVs. Each of these hypotheses constitutes an interesting exploration path for a future reassessment of the clinical interest of the MVs in CAT.

A new assay to evaluate microvesicle plasmin generation capacity: validation in disease with fibrinolysis imbalance.

Among extracellular vesicles, leukocyte-derived microvesicles (LMVs) have emerged as complex vesicular structures. Primarily identified as procoagulant entities, they were more recently ascribed to plasmin generation capacity (MV-PGC). The objectives of this work were (1) to develop a new hybrid bio-assay combining the specific isolation of LMVs and measurement of their PGC, and compare its performance to the original method based on centrifugation, (2) to validate MV-PGC in septic shock, combining increased levels of LMVs and fibrinolytic imbalance. Using plasma sample spiked with LMVs featuring different levels of PGC, we demonstrated that CD15-beads specifically extracted LMVs. The MV dependency of the test was demonstrated using electron microscopy, high speed centrifugation, nanofiltration and detergent-mediated solubilization and the MV-PGC specificity using plasmin-specific inhibitors, or antibodies blocking elastase or uPA. Thanks to a reaction booster (ε-ACA), we showed that the assay was more sensitive and reproducible than the original method. Moreover, it exhibited a good repeatability, inter-operator and inter-experiment reproducibility. The new immunomagnetic bio-assay was further validated in patients with septic shock. As a result, we showed that MV-PGC values were significantly lower in septic shock patients who died compared to patients who survived, both at inclusion and 24 h later (1.4 [0.8-3.0] vs 3.1 [1.7-18] A405 × 10-3/min, p = 0.02; 1.4 [1-1.6] vs 5.2 [2.2-16] A405 × 10-3/min, p = 0.004). Interestingly, combining both MV-PGC and PAI-1 in a ratio significantly improved the predictive value of PAI-1. This strategy, a hybrid capture bioassay to specifically measure LMV-PGC using for the first time, opens new perspectives for measuring subcellular fibrinolytic potential in clinical settings with fibrinolytic imbalance.

Microparticles and Fibrinolysis.

Microparticles (MPs) are submicronic vesicles which are formed by budding of the cellular membrane of virtually any cell type in response to cell activation or apoptosis. Both circulating MPs and MPs generated within tissues harbor molecules with a large repertoire of biological activities and transfer material to target cells. Depending on their cellular origin, the stimuli triggering their formation, or their localization, they may participate in the maintenance of organ or vascular homeostasis as well as inducing dysfunction. MPs have mostly been described as having procoagulant properties. However, the fact that some MP subsets are able to efficiently generate plasmin suggests that the role of MPs in hemostasis is more complex than initially thought. In this review, we summarize key findings showing that MPs provide a heterogeneous catalytic surface for plasmin generation, according to their cellular origin. We further address the specific features of the MP-dependent fibrinolytic system. Potential consequences of this MP-associated fibrinolytic activity in pathology are illustrated in cancer.