Revealing the diversity of extracellular vesicles using high-dimensional flow cytometry analyses

Extracellular vesicles (EV) are small membrane vesicles produced by cells upon activation and apoptosis. EVs are heterogeneous according to their origin, mode of release, membrane composition, organelle and biochemical content, and other factors. Whereas it is apparent that EVs are implicated in intercellular communication, they can also be used as biomarkers. Continuous improvements in pre-analytical parameters and flow cytometry permit more efficient assessment of EVs; however, methods to more objectively distinguish EVs from cells and background, and to interpret multiple single-EV parameters are lacking. We used spanning-tree progression analysis of density-normalized events (SPADE) as a computational approach for the organization of EV subpopulations released by platelets and erythrocytes. SPADE distinguished EVs, and logically organized EVs detected by high-sensitivity flow cytofluorometry based on size estimation, granularity, mitochondrial content, and phosphatidylserine and protein receptor surface expression. Plasma EVs were organized by hierarchy, permitting appreciation of their heterogeneity. Furthermore, SPADE was used to analyze EVs present in the synovial fluid of patients with inflammatory arthritis. Its algorithm efficiently revealed subtypes of arthritic patients based on EV heterogeneity patterns. Our study reveals that computational algorithms are useful for the analysis of high-dimensional single EV data, thereby facilitating comprehension of EV functions and biomarker development

Detection and Quantification of Microparticles from Different Cellular Lineages Using Flow Cytometry. Evaluation of the Impact of Secreted Phospholipase A2 on Microparticle Assessment

Microparticles, also called microvesicles, are submicron extracellular vesicles produced by plasma membrane budding and shedding recognized as key actors in numerous physio(patho)logical processes. Since they can be released by virtually any cell lineages and are retrieved in biological fluids, microparticles appear as potent biomarkers. However, the small dimensions of microparticles and soluble factors present in body fluids can considerably impede their quantification. Here, flow cytometry with improved methodology for microparticle resolution was used to detect microparticles of human and mouse species generated from platelets, red blood cells, endothelial cells, apoptotic thymocytes and cells from the male reproductive tract. A family of soluble proteins, the secreted phospholipases A2 (sPLA2), comprises enzymes concomitantly expressed with microparticles in biological fluids and that catalyze the hydrolysis of membrane phospholipids. As sPLA2 can hydrolyze phosphatidylserine, a phospholipid frequently used to assess microparticles, and might even clear microparticles, we further considered the impact of relevant sPLA2 enzymes, sPLA2 group IIA, V and X, on microparticle quantification. We observed that if enriched in fluids, certain sPLA2 enzymes impair the quantification of microparticles depending on the species studied, the source of microparticles and the means of detection employed (surface phosphatidylserine or protein antigen detection). This study provides analytical considerations for appropriate interpretation of microparticle cytofluorometric measurements in biological samples containing sPLA2 enzymes

The efficacy of therapeutic plasma exchange in COVID-19 patients on endothelial tightness in vitro is hindered by platelet activation

Coronavirus disease (COVID)-19 is characterised in particular by vascular inflammation with platelet activation and endothelial dysfunction. During the pandemic, therapeutic plasma exchange (TPE) was used to reduce the cytokine storm in the circulation and delay or prevent ICU admissions. This procedure consists in replacing the inflammatory plasma by fresh frozen plasma from healthy donors and is often used to remove pathogenic molecules from plasma (autoantibodies, immune complexes, toxins, etc.). This study uses an in vitro model of platelet-endothelial cell interactions to assess changes in these interactions by plasma from COVID-19 patients and to determine the extent to which TPE reduces such changes. We noted that exposure of an endothelial monolayer to plasmas from COVID-19 patients post-TPE induced less endothelial permeability compared to COVID-19 control plasmas. Yet, when endothelial cells were co-cultured with healthy platelets and exposed to the plasma, the beneficial effect of TPE on endothelial permeability was somewhat reduced. This was linked to platelet and endothelial phenotypical activation but not with inflammatory molecule secretion. Our work shows that, in parallel to the beneficial removal of inflammatory factors from the circulation, TPE triggers cellular activation which may partly explain the reduction in efficacy in terms of endothelial dysfunction. These findings provide new insights for improving the efficacy of TPE using supporting treatments targeting platelet activation, for instance

Bioactive lipids as biomarkers of adverse reactions associated with apheresis platelet concentrate transfusion

Platelet concentrate (PC) transfusion seeks to provide haemostasis in patients presenting severe central thrombocytopenia or severe bleeding. PCs may induce adverse reactions (AR) that can occasionally be severe (SAR). PCs contain active biomolecules such as cytokines and lipid mediators. The processing and storage of PCs creates so-called structural and biochemical storage lesions that accumulate when blood products reach their shelf life. We sought to investigate lipid mediators as bioactive molecules of interest during storage and review associations with adverse reactions post-transfusion. To facilitate understanding, we focused on single donor apheresis (SDA) PCs with approximately 31.8% of PCs being delivered in our setting. Indeed, pooled PCs are the most widely transfused products, but the study of a single donor lipid mediator is easier to interpret. We are investigating key lipid mediators involved in AR. Adverse reactions were closely monitored in accordance with current national and regional haemovigilance protocols. Residual PCs were analysed post-transfusion in a series of observations, both with and without severe reactions in recipients. A decrease in the lysophosphatidylcholine species to produce the lysophosphatidic acid species has been observed during storage and in the case of AR. Lysophosphatidic acid increased with primarily platelet-inhibitor lipids. Anti-inflammatory platelet-induced inhibition lipids were weakly expressed in cases of severe adverse reactions. We therefore propose that a decrease in lysophosphatidylcholine and an increase in lysophosphatidic acid can prospectively predict serious adverse transfusion reactions

Portrait of blood-derived extracellular vesicles in patients with Parkinson's disease.

The production of extracellular vesicles (EV) is a ubiquitous feature of eukaryotic cells but pathological events can affect their formation and constituents. We sought to characterize the nature, profile and protein signature of EV in the plasma of Parkinson's disease (PD) patients and how they correlate to clinical measures of the disease. EV were initially collected from cohorts of PD (n = 60; Controls, n = 37) and Huntington's disease (HD) patients (Pre-manifest, n = 11; manifest, n = 52; Controls, n = 55) - for comparative purposes in individuals with another chronic neurodegenerative condition - and exhaustively analyzed using flow cytometry, electron microscopy and proteomics. We then collected 42 samples from an additional independent cohort of PD patients to confirm our initial results. Through a series of iterative steps, we optimized an approach for defining the EV signature in PD. We found that the number of EV derived specifically from erythrocytes segregated with UPDRS scores corresponding to different disease stages. Proteomic analysis further revealed that there is a specific signature of proteins that could reliably differentiate control subjects from mild and moderate PD patients. Taken together, we have developed/identified an EV blood-based assay that has the potential to be used as a biomarker for PD

The 20S proteasome core, active within apoptotic exosome-like vesicles, induces autoantibody production and accelerates rejection

Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rejection in organ transplant recipients. However, mechanisms of immunization to apoptotic components remain largely uncharacterized. We used large-scale proteomics, with validation by electron microscopy and biochemical methods, to compare the protein profiles of apoptotic bodies and apoptotic exosome-like vesicles, smaller extracellular vesicles released by endothelial cells downstream of caspase-3 activation. We identified apoptotic exosome-like vesicles as a central trigger for production of anti-perlecan antibodies and acceleration of rejection. Unlike apoptotic bodies, apoptotic exosome-like vesicles triggered the production of anti-perlecan antibodies in naïve mice and enhanced anti-perlecan antibody production and allograft inflammation in mice transplanted with an MHC (major histocompatibility complex)–incompatible aortic graft. The 20S proteasome core was active within apoptotic exosome-like vesicles and controlled their immunogenic activity. Finally, we showed that proteasome activity in circulating exosome-like vesicles increased after vascular injury in mice. These findings open new avenues for predicting and controlling maladaptive humoral responses to apoptotic cell components that enhance the risk of rejection after transplantation

Rôle de la phospholipase A₂ sécrétée de type IIA dans l'arthrite

L’arthrite rhumatoïde est une maladie auto-immune systémique affectant près de 1% de la population mondiale. L’inflammation articulaire est caractérisée par une infiltration leucocytaire majoritaire de neutrophiles, la formation d’un pannus et la destruction du cartilage et de l’os. De nombreux acteurs cellulaires et moléculaires dont les plaquettes et leurs microparticules (MPs) ainsi que la phospholipase A2 sécrétée de type IIA (sPLA2-IIA) contribuent à cette pathologie. Récemment, nous avons mis en évidence que les plaquettes activées libèrent certes des MPs mais aussi des mitochondries libres que nous avons appelées freeMitos. Les MPs et les freeMitos sont détectées dans les fluides synoviaux de patients arthritiques. Au cours de nos recherches, nos résultats indiquent que la sPLA2-IIA hydrolyse les phospholipides membranaires des MPs et des freeMitos. Elle libère des lysophospholipides et des acides gras, dont l’acide arachidonique (AA). L’ADN mitochondrial est aussi relâché à la suite de l’hydrolyse des freeMitos par la sPLA2-IIA. Ces différents produits induisent la libération de leucotriènes et de cytokines pro-inflammatoires ainsi que la formation de neutrophil extracellular trap (NET) par les neutrophiles. La sPLA2-IIA cible aussi les MPs qui sont riches en enzymes du métabolisme de l’acide arachidonique (AA). Cet AA est majoritairement métabolisé en 12-Hydroxyeicosatetraenoic acide (12-HETE) par la 12-lipoxygénase (12-LO) des MPs. Le 12(S)-HETE issue de l’action concertée de la sPLA2-IIA et la 12-LO, induit l’internalisation des MPs par les neutrophiles in vitro et in vivo. Les MPs transfèrent leur cargaison en facteurs de transcription, en acides nucléiques et en mitochondries aux neutrophiles. Les MPs modulent le transcriptome et les fonctions du neutrophile. Les MPs et les produits d’hydrolyse par la sPLA2-IIA induisent une augmentation de la génération de leucotriènes, la formation de NETs et une résistance à l’apoptose. Ces deux enzymes sont aussi impliqués dans la sévérité de l’arthrite inflammatoire murine. En somme, nos études apportent une meilleure connaissance sur le contenu des MPs de plaquette. Un mécanisme finement régulé, d’internalisation des MPs par les neutrophiles, a été mis en évidence.Rheumatoid arthritis is a systemic autoimmune disease affecting 1% of the world population. This pathology is characterized by a symmetric articular achievement where takes place a synovial hyperplasia accompanied with an infiltration of leukocytes, mainly neutrophils, and a destruction of cartilage and bone. Several cellular and molecular actors including platelets, platelet microparticles (MPs) and the secreted phospholipase A2 group IIA (sPLA2-IIA) contribute to this pathology. Recently, we highlighted that activated platelets produce also extracellular mitochondria (freeMitos) which we detected in the synovial fluids from arthritic patients. In our research, our results indicate that sPLA2-IIA hydrolyzes membrane phosopholipids of freeMitos and MPs, releasing lysophospholipids and arachidonic acid (AA). Mitochondrial DNA is also liberated after sPLA2-IIA hydrolysis. These products induce leukotrienes production, proinflammatory cytokine release and neutrophil extracellular trap (NET) formation by neutrophils. sPLA2-IIA also targets MPs that contain enzyme involved in AA metabolism. AA is mainly metabolized in 12-Hydroxyeicosatetraenoic acid (12-HETE) by 12-lipoxygenase (12-LO) from MPs. It induces MP internalization in the human and murin neutrophils. MPs transfer their elaborated cargo, rich in transcription factors, nucleic acids and mitochondria, to neutrophils. MPs modulate transcriptome and functions of neutrophils. MPs and the products of hydrolysis by sPLA2-IIA, induce increase of leukotrienes production, NET release and apoptosis resistance. 12-LO and sPLA2-IIA are involved in inflammatory murine arthritis severity. Our work brings a better knowledge on the content of the platelet MPs. It highlights a tightly regulated mechanism implicated in MP internalization in neutrophils

The diversity of platelet microparticles

PURPOSE OF REVIEW: Platelet microparticles are small extracellular vesicles abundant in blood. The present review will introduce the mechanisms underlying the generation of microparticles, and will describe the diverse microparticle subtypes identified to date. The most appropriate methodologies used to distinguish microparticle subtypes will be also presented. RECENT FINDINGS: Both the megakaryocytes and platelets can generate microparticles. Circulating microparticles originating from megakaryocytes are distinguished from those derived from activated platelets by the presence of CD62P, LAMP-1, and immunoreceptor-based activation motif receptors. Close examination of platelet activation has shed light on a novel mechanism leading to microparticle production. Under physiologic flow, microparticles bud off from long membrane strands formed by activated platelets. Furthermore, mounting evidence supports the notion of microparticle heterogeneity. Platelet microparticles are commonly characterized by the expression of surface platelet antigens and phosphatidylserine. In fact, only a fraction of platelet microparticles harbor phosphatidylserine, and a distinct subset contains respiratory-competent mitochondria. During disease, the microparticle surface may undergo posttranslational modifications such as citrullination, further supporting the concept of microparticle diversity. SUMMARY: An appreciation of the microparticle heterogeneity will support their development as potential biomarkers and may reveal functions unique to each microparticle subtype in health and disease

Immunothrombosis and the Role of Platelets in Venous Thromboembolic Diseases

Venous thromboembolism (VTE) is the third leading cardiovascular cause of death and is conventionally treated with anticoagulants that directly antagonize coagulation. However, recent data have demonstrated that also platelets play a crucial role in VTE pathophysiology. In the current review, we outline how platelets are involved during all stages of experimental venous thrombosis. Platelets mediate initiation of the disease by attaching to the vessel wall upon which they mediate leukocyte recruitment. This process is referred to as immunothrombosis, and within this novel concept inflammatory cells such as leukocytes and platelets directly drive the progression of VTE. In addition to their involvement in immunothrombosis, activated platelets can directly drive venous thrombosis by supporting coagulation and secreting procoagulant factors. Furthermore, fibrinolysis and vessel resolution are (partly) mediated by platelets. Finally, we summarize how conventional antiplatelet therapy can prevent experimental venous thrombosis and impacts (recurrent) VTE in humans

Red blood cell-derived phosphatidylserine positive extracellular vesicles are associated with past thrombotic events in patients with systemic erythematous lupus

Background Extracellular vesicles (EVs) released by blood cells have proinflammation and procoagulant action. Patients with systemic lupus erythematosus (SLE) present high vascular inflammation and are prone to develop cardiovascular diseases. Therefore, we postulated that the EV populations found in blood, including platelet EVs (PEVs) and red blood cell EVs (REVs), are associated with SLE disease activity and SLE-associated cardiovascular accidents.Method We assessed autotaxin (ATX) plasma levels by ELISA, the platelet activation markers PAC1 and CD62P, ATX bound to platelets and the amounts of plasma PEVs and REVs by flow cytometry in a cohort of 102 patients with SLE, including 29 incident cases of SLE and 30 controls. Correlation analyses explored the associations with the clinical parameters.Result Platelet activation markers were increased in patients with SLE compared with healthy control, with the marker CD62P associated with the SLE disease activity index (SLEDAI). The incident cases show additional associations between platelet markers (CD62P/ATX and PAC1/CD62P) and the SLEDAI. Compared with controls, patients with SLE presented higher levels of PEVs, phosphatidylserine positive (PS+) PEVs, REVs and PS+ REVs, but there is no association with disease activity. When stratified according to the plasma level of PS+ REVs, the group of patients with SLE with a high level of PS+ REVs presented a higher number of past thrombosis events and higher ATX levels.Conclusion Incident and prevalent forms of SLE cases present similar levels of platelet activation markers, with CD62P correlating with disease activity. Though EVs are not associated with disease activity, the incidence of past thrombotic events is higher in patients with a high level of PS+ REVs