VE-Cadherin is involved in endothelial cell detachment during Thrombotic Thrombocytopenic Purpura.

BACKGROUND AND OBJECTIVES: Immune Thrombotic thrombocytopenic purpura (i-TTP) is a life-threatening thrombotic microangiopathie linked to ADAMTS13 deficiency. It has long been assumed that the activation of endothelial cells is the triggering factor for the TTP crisis. Circulating endothelial cells (CEC) have been shown to be a biomarker of vascular damage and are associated with the clinical severity of i-TTP. However, the mechanisms leading to endothelial cell detachment remain unclear. OBJECTIVES: We have investigated junctional destabilization and the mechanisms underlying cell detachment in TTP. METHODS AND RESULTS: In plasma from i-TTP patients, we show that CEC count is associated with severity and correlated to intracellular calcium influx (p<0.01). In vitro, serum from i-TTP patients induced stronger detachment of human umbilical vein endothelial cells (HUVECs) than serum from control patients (p<0.001). Plasma from i-TTP patients induced a higher calcium-dependent phosphorylation (p<0.05) and internalization (p<0.05) of VE-cadherin compared to plasma from control patients. This effect could be reproduced by IgG fraction isolated from patient plasma and in particular by the F(ab)'2 fragments of the corresponding IgG. In addition, subcutaneous injection of i-TTP plasma into mice resulted in higher vascular permeability than plasma from control patients. An inhibitor of endothelial calcium influx, ITF1697, normalized this increase in permeability. CONCLUSIONS: Our results suggest that plasma-induced endothelial activation also leads to an increase in vascular permeability. They contribute to the understanding of the mechanisms behind the presence of elevated CECs in patients' blood by linking endothelial activation to endothelial injury.

Macrophage IL-1ß-positive microvesicles exhibit thrombo-inflammatory properties and are detectable in patients with active juvenile idiopathic arthritis.

Objective: IL-1ß is a leaderless cytokine with poorly known secretory mechanisms that is barely detectable in serum of patients, including those with an IL-1ß-mediated disease such as systemic juvenile idiopathic arthritis (sJIA). Leukocyte microvesicles (MVs) may be a mechanism of IL-1ß secretion. The first objective of our study was to characterize IL-1ß-positive MVs obtained from macrophage cell culture supernatants and to investigate their biological functions in vitro and in vivo. The second objective was to detect circulating IL-1ß-positive MVs in JIA patients. Methods: MVs were purified by serial centrifugations from PBMCs, or THP-1 differentiated into macrophages, then stimulated with LPS ± ATP. MV content was analyzed for the presence of IL-1ß, NLRP3 inflammasome, caspase-1, P2X7 receptor, and tissue factor (TF) using ELISA, Western blot, or flow cytometry. MV biological properties were studied in vitro by measuring VCAM-1, ICAM-1, and E-selectin expression after HUVEC co-culture and factor-Xa generation test was realized. In vivo, MVs' ability to recruit leukocytes in a murine model of peritonitis was evaluated. Plasmatic IL-1ß-positive MVs were studied ex vivo in 10 active JIA patients using flow cytometry. Results: THP-1-derived macrophages stimulated with LPS and ATP released MVs, which contained NLRP3, caspase-1, and the 33-kDa precursor and 17-kDa mature forms of IL-1ß and bioactive TF. IL-1ß-positive MVs expressed P2X7 receptor and released soluble IL-1ß in response to ATP stimulation in vitro. In mice, MVs induced a leukocyte peritoneal infiltrate, which was reduced by treatment with the IL-1 receptor antagonist. Finally, IL-1ß-positive MVs were detectable in plasma from 10 active JIA patients. Conclusion: MVs shed from activated macrophages contain IL-1ß, NLRP3 inflammasome components, and TF, and constitute thrombo-inflammatory vectors that can be detected in the plasma from active JIA patients.

Circulating microvesicles correlate with radiation proctitis complication after radiotherapy.

In a large retrospective study, we assessed the putative use of circulating microvesicles (MVs), as innovative biomarkers of radiation toxicity in a cohort of 208 patients with prostate adenocarcinoma overexposed to radiation. The level of platelet (P)-, monocyte (M)- and endothelial (E)-derived MVs were assessed by flow cytometry. Rectal bleeding toxicity scores were collected at the time of blood sampling and during the routine follow-up and were tested for association with MVs using a multivariate logistic regression. MVs dosimetric correlation was investigated using dose volume histograms information available for a subset of 36 patients. The number of PMVs was significantly increased in patients with highest toxicity grades compared to lower grades. Risk prediction analysis revealed that increased numbers of PMVs, and an increased amount of MMVs relative to EMVs, were associated with worst rectal bleeding grade compared to the time of blood sampling. Moreover, a significant correlation was found between PMV and MMV numbers, with the range of doses up to the median exposure (40 Gy) of bladder/rectum and anterior rectal wall, respectively. MVs could be considered as new biomarkers to improve the identification of patients with high toxicity grade and may be instrumental for the prognosis of radiation therapy complications.

Signaling Pathways and Potential Therapeutic Strategies in Cardiac Fibrosis.

Cardiac fibrosis constitutes irreversible necrosis of the heart muscle as a consequence of different acute (myocardial infarction) or chronic (diabetes, hypertension, …) diseases but also due to genetic alterations or aging. Currently, there is no curative treatment that is able to prevent or attenuate this phenomenon that leads to progressive cardiac dysfunction and life-threatening outcomes. This review summarizes the different targets identified and the new strategies proposed to fight cardiac fibrosis. Future directions, including the use of exosomes or nanoparticles, will also be discussed.

Soluble CD146, a biomarker and a target for preventing resistance to anti-angiogenic therapy in glioblastoma.

RATIONALE: Glioblastoma multiforme (GBM) is a primary brain tumor with poor prognosis. The U.S. food and drug administration approved the use of the anti-VEGF antibody bevacizumab in recurrent GBM. However, resistance to this treatment is frequent and fails to enhance the overall survival of patients. In this study, we aimed to identify novel mechanism(s) responsible for bevacizumab-resistance in CD146-positive glioblastoma. METHODS: The study was performed using sera from GBM patients and human GBM cell lines in culture or xenografted in nude mice. RESULTS: We found that an increase in sCD146 concentration in sera of GBM patients after the first cycle of bevacizumab treatment was significantly associated with poor progression free survival and shorter overall survival. Accordingly, in vitro treatment of CD146-positive glioblastoma cells with bevacizumab led to a high sCD146 secretion, inducing cell invasion. These effects were mediated through integrin αvß3 and were blocked by mucizumab, a novel humanized anti-sCD146 antibody. In vivo, the combination of bevacizumab with mucizumab impeded CD146 + glioblastoma growth and reduced tumor cell dissemination to an extent significantly higher than that observed with bevacizumab alone. CONCLUSION: We propose sCD146 to be 1/ an early biomarker to predict and 2/ a potential target to prevent bevacizumab resistance in patients with glioblastoma.

CD146 at the Interface between Oxidative Stress and the Wnt Signaling Pathway in Systemic Sclerosis.

CD146 involvement was recently described in skin fibrosis of systemic sclerosis through its regulation of the Wnt pathway. Because the interaction between Wnt and ROS signaling plays a major role in fibrosis, we hypothesized that in systemic sclerosis, CD146 may regulate Wnt/ROS crosstalk. Using a transcriptomic and western blot analysis performed on CD146 wild-type or knockout mouse embryonic fibroblasts, we showed a procanonical Wnt hallmark in the absence of CD146 that is reversed when CD146 expression is restored. We found an elevated ROS content in knockout cells and an increase in DNA oxidative damage in the skin sections of knockout mice compared with those of wild-type mice. We also showed that ROS increased CD146 and its noncanonical Wnt ligand, WNT5A, only in wild-type cells. In humans, fibroblasts from patients with systemic sclerosis presented higher ROS content and expressed CD146, whereas control fibroblasts did not. Moreover, CD146 and its ligand were upregulated by ROS in both human fibroblasts. The increase in bleomycin-induced WNT5A expression was abrogated when CD146 was silenced. We showed an interplay between Wnt and ROS signaling in systemic sclerosis, regulated by CD146, which promotes the noncanonical Wnt pathway and prevents ROS signaling, opening the way for innovative therapeutic strategies.

Soluble CD146 is increased in preeclampsia and interacts with galectin-1 to regulate trophoblast migration through VEGFR2 receptor.

OBJECTIVE: To explore the regulatory role of soluble CD146 (sCD146) and its interaction with galectin-1 (Gal1) in placenta-mediated complications of pregnancy. DESIGN: Prospective pilot and experimental studies. SETTING: University-affiliated hospital and academic research laboratory. PATIENT(S): One hundred fifteen women divided into three groups: 30 healthy, nonpregnant women, 50 women with normal pregnancies, and 35 with placenta-mediated pregnancy complications. INTERVENTION(S): Wound-healing experiments were conducted to study trophoblast migration. MAIN OUTCOME MEASURE(S): Quantification of sCD146 and Gal1 by enzyme-linked immunosorbent assay. Analysis of trophoblast migration by wound closure. RESULT(S): Concomitant detection of sCD146 and Gal1 showed lower sCD146 and higher Gal1 concentrations in women with normal pregnancies compared with nonpregnant women. In addition, follow-up of these women revealed a decrease in sCD146 associated with an increase in Gal1 throughout pregnancy. In contrast, in women with preeclampsia, we found significantly higher sCD146 concentrations compared with women with normal pregnancies and no modification of Gal1. We emphasize the opposing effects of sCD146 and Gal, since, unlike Gal1, sCD146 inhibits trophoblast migration. Moreover, the migratory effect of Gal1 was abrogated with the use of an anti-CD146 blocking antibody or the use of small interfering RNA to silence VEGFR2 expression. This suggests that trophoblast migration is mediated though the interaction of Gal1 with CD146, further activating the VEGFR2 signaling pathway. Significantly, sCD146 blocked the migratory effects of Gal1 on trophoblasts and inhibited its secretion, suggesting that sCD146 acts as a ligand trap. CONCLUSION(S): Soluble CD146 could be proposed as a biomarker in preeclampsia and a potential therapeutic target. CLINICAL TRIAL REGISTRATION NUMBER: NCT 01736826.

Soluble CD146 as a Potential Target for Preventing Triple Negative Breast Cancer MDA-MB-231 Cell Growth and Dissemination.

Background: Triple Negative Breast Cancers (TNBC) are the most aggressive breast cancers and lead to poor prognoses. This is due to a high resistance to therapies, mainly because of the presence of Cancer Stem Cells (CSCs). Plasticity, a feature of CSCs, is acquired through the Epithelial to Mesenchymal Transition (EMT), a process that has been recently shown to be regulated by a key molecule, CD146. Of interest, CD146 is over-expressed in TNBC. Methods: The MDA-MB-231 TNBC cell line was used as a model to study the role of CD146 and its secreted soluble form (sCD146) in the development and dissemination of TNBC using in vitro and in vivo studies. Results: High expression of CD146 in a majority of MDA-MB-231 cells leads to an increased secretion of sCD146 that up-regulates the expression of EMT and CSC markers on the cells. These effects can be blocked with a specific anti-sCD146 antibody, M2J-1 mAb. M2J-1 mAb was able to reduce tumour development and dissemination in a model of cells xenografted in nude mice and an experimental model of metastasis, respectively, in part through its effects on CSC. Conclusion: We propose that M2J-1 mAb could be used as an additional therapeutic approach to fight TNBC.

Involvement of Multiple Variants of Soluble CD146 in Systemic Sclerosis: Identification of a Novel Profibrotic Factor.

OBJECTIVE: Systemic sclerosis (SSc) is an autoimmune disorder characterized by excessive fibrosis, immune dysfunction, and vascular damage, in which the expression of many growth factors is deregulated. CD146 was recently described as a major actor in SSc. Since CD146 also exists as a circulating soluble form (sCD146) that acts as a growth factor in numerous angiogenic- and inflammation-related pathologies, we sought to identify the mechanisms underlying the generation of sCD146 and to characterize the regulation and functions of the different variants identified in SSc. METHODS: We performed in vitro experiments, including RNA-Seq and antibody arrays, and in vivo experiments using animal models of bleomycin-induced SSc and hind limb ischemia. RESULTS: Multiple forms of sCD146, generated by both shedding and alternative splicing of the primary transcript, were discovered. The shed form of sCD146 was generated from the cleavage of both long and short membrane isoforms of CD146 through ADAM-10 and TACE metalloproteinases, respectively. In addition, 2 novel sCD146 splice variants, I5-13-sCD146 and I10-sCD146, were identified. Of interest, I5-13-sCD146 was significantly increased in the sera of SSc patients (P < 0.001; n = 117), in particular in patients with pulmonary fibrosis (P < 0.01; n = 112), whereas I10-sCD146 was decreased (P < 0.05; n = 117). Further experiments revealed that shed sCD146 and I10-sCD146 displayed proangiogenic activity through the focal adhesion kinase and protein kinase Cε signaling pathways, respectively, whereas I5-13-sCD146 displayed profibrotic effects through the Wnt-1/ß-catenin/WISP-1 pathway. CONCLUSION: Variants of sCD146, and in particular the novel I5-13-sCD146 splice variant, could constitute novel biomarkers and/or molecular targets for the diagnosis and treatment of SSc and other angiogenesis- or fibrosis-related disorders.

Mast cells drive pathologic vascular lesions in Takayasu arteritis.

BACKGROUND: Takayasu arteritis (TAK) is a large vessel vasculitis resulting in artery wall remodeling with segmental stenosis and/or aneurysm formation. Mast cells (MCs) are instrumental in bridging cell injury and inflammatory response. OBJECTIVES: This study sought to investigate the contribution of MCs on vessel permeability, angiogenesis, and fibrosis in patients with TAK. METHODS: MC activation and their tissue expression were assessed in sera and in aorta from patients with TAK and from healthy donors (HDs). In vivo permeability was assessed using a modified Miles assay. Subconfluent cultured human umbilic vein endothelial cells and fibroblasts were used in vitro to investigate the effects of MC mediators on angiogenesis and fibrogenesis. RESULTS: This study found increased levels of MC activation markers (histamine and indoleamine 2,3-dioxygenase) in sera of patients with TAK compared with in sera of HDs. Marked expression of MCs was shown in aortic lesions of patients with TAK compared with in those of noninflammatory aorta controls. Using Miles assay, this study showed that sera of patients with TAK significantly increased vascular permeability in vivo as compared with that of HDs. Vessel permeability was abrogated in MC-deficient mice. MCs stimulated by sera of patients with TAK supported neoangiogenesis (increased human umbilic vein endothelial cell proliferation and branches) and fibrosis by inducing increased production of fibronectin, type 1 collagen, and α-smooth muscle actin by fibroblasts as compared to MCs stimulated by sera of HD. CONCLUSIONS: MCs are a key regulator of vascular lesions in patients with TAK and may represent a new therapeutic target in large vessel vasculitis.

The Role of the Adhesion Receptor CD146 and Its Soluble Form in Human Embryo Implantation and Pregnancy.

CD146 is an adhesion molecule essentially located in the vascular system, which has been described to play an important role in angiogenesis. A soluble form of CD146, called sCD146, is detected in the bloodstream and is known as an angiogenic factor. During placental development, CD146 is selectively expressed in extravillous trophoblasts. A growing body of evidence shows that CD146 and, in particular, sCD146, regulate extravillous trophoblasts migration and invasion both in vitro and in vivo. Hereby, we review expression and functions of CD146/sCD146 in the obstetrical field, mainly in pregnancy and in embryo implantation. We emphasized the relevance of quantifying sCD146 in the plasma of pregnant women or in embryo supernatant in the case of in vitro fertilization (IVF) to predict pathological pregnancy such as preeclampsia or implantation defect. This review will also shed light on some major results that led us to define CD146/sCD146 as a biomarker of placental development and paves the way toward identification of new therapeutic targets during implantation and pregnancy.

Endothelial-Specific Deletion of CD146 Protects Against Experimental Glomerulonephritis in Mice.

[Figure: see text].

Multifaceted role of extracellular vesicles in atherosclerosis.

Extracellular vesicles (EVs) are small vesicles released by the majority of cells in response to cell activation or death stimuli. They are grouped as small EVs or exosomes, large EVs such as microvesicles (MVs) and apoptotic bodies, resulting from distinct mechanisms of generation. EVs are released into the extracellular space, in most human biological fluids and tissues, including atherosclerotic plaques. They transport complex cargo of bioactive molecules, including proteins, lipids and genetic material and are therefore involved in pathophysiological pathways of cell-cell communication. Indeed, EVs are involved in several processes such as inflammation, coagulation, vascular dysfunction, angiogenesis and senescence, contributing to the initiation and progression of atherothrombotic diseases. Consequently, they behave as a determinant of atherosclerotic plaque vulnerability leading to major cardiovascular disorders. Over the last decade, the field of EVs research has grown, highlighting their involvement in atherosclerosis. However, limitations in both detection methodologies and standardisation have hindered implementation of EVs in the clinical settings. This review summarizes the effect of EVs in atherosclerosis development, progression and severity, with specific attention devoted to their ambivalent roles in senescence and hemostasis. This review will also highlight the role of MVs as multifaceted messengers, able to promote or to attenuate atherosclerosis progression. Finally, we will discuss the main technical challenges and prerequisites of standardization for driving EVs to the clinics and delineate their relevance as emergent biomarkers and innovative therapeutic approaches in atherosclerosis.

CD146/sCD146 in the Pathogenesis and Monitoring of Angiogenic and Inflammatory Diseases.

CD146 is a cell adhesion molecule expressed on endothelial cells, as well as on other cells such as mesenchymal stem cells and Th17 lymphocytes. This protein also exists in a soluble form, whereby it can be detected in biological fluids, including the serum or the cerebrospinal fluid (CSF). Some studies have highlighted the significance of CD146 and its soluble form in angiogenesis and inflammation, having been shown to contribute to the pathogenesis of many inflammatory autoimmune diseases, such as systemic sclerosis, mellitus diabetes, rheumatoid arthritis, inflammatory bowel diseases, and multiple sclerosis. In this review, we will focus on how CD146 and sCD146 contribute to the pathogenesis of the aforementioned autoimmune diseases and discuss the relevance of considering it as a biomarker in these pathologies.

Role of CD146 (MCAM) in Physiological and Pathological Angiogenesis-Contribution of New Antibodies for Therapy.

The fundamental role of cell adhesion molecules in mediating various biological processes as angiogenesis has been well-documented. CD146, an adhesion molecule of the immunoglobulin superfamily, and its soluble form, constitute major players in both physiological and pathological angiogenesis. A growing body of evidence shows soluble CD146 to be significantly elevated in the serum or interstitial fluid of patients with pathologies related to deregulated angiogenesis, as autoimmune diseases, obstetric and ocular pathologies, and cancers. To block the undesirable effects of this molecule, therapeutic antibodies have been developed. Herein, we review the multifaceted functions of CD146 in physiological and pathological angiogenesis and summarize the interest of using monoclonal antibodies for therapeutic purposes.

Immunomodulatory role of Interleukin-33 in large vessel vasculitis.

The mechanisms regulating inflammation in large vessels vasculitis (LVV) are poorly understood. Interleukin 33 (IL-33) has been shown to license innate and adaptive immunity by enhancing Th2 cytokines production. We aimed to examine the role of IL-33 in the immunomodulation of T cell activation in LVV. T cell homeostasis and cytokines production were determined in peripheral blood from 52 patients with giant cell arteritis (GCA) and 50 healthy donors (HD), using Luminex assay, flow cytometry, quantitative RT-PCR and by immunofluorescence analysis in inflammatory aorta lesions. We found increased level of IL-33 and its receptor ST2/IL-1R4 in the serum of patient with LVV. Endothelial cells were the main source of IL-33, whereas Th2 cells, Tregs and mast cells (MC) express ST2 in LVV vessels. IL-33 had a direct immunomodulatory impact by increasing Th2 and Tregs. IL-33 and MC further enhanced Th2 and regulatory responses by inducing a 6.1 fold increased proportion of Tregs (p = 0.008). Stimulation of MC by IL-33 increased indoleamine 2 3-dioxygenase (IDO) activity and IL-2 secretion. IL-33 mRNA expression was significantly correlated with the expression of IL-10 and TGF-ß within aorta inflammatory lesions. To conclude, our findings suggest that IL-33 may exert a critical immunoregulatory role in promoting Tregs and Th2 cells in LVV.

Therapeutic targeting of soluble CD146/MCAM with the M2J-1 monoclonal antibody prevents metastasis development and procoagulant activity in CD146-positive invasive tumors.

Initially discovered in human melanoma, CD146/MCAM is expressed on many tumors and is correlated with cancer progression and metastasis. However, targeting CD146 remains challenging since it is also expressed on other cell types, as vessel cells, where it displays important physiological functions. We previously demonstrated that CD146 is shed as a soluble form (sCD146) that vectorizes the effects of membrane CD146 on tumor angiogenesis, growth and survival. We thus generated a novel monoclonal antibody, the M2J-1 mAb, which specifically targets sCD146, but not membrane CD146, and counteracts these effects. In our study, we analyzed the effects of sCD146 on the dissemination and the associated procoagulant phenotype in two highly invasive human CD146-positive cancer cell lines (ovarian and melanoma). Results show that sCD146 induced epithelial to mesenchymal transition, favored the generation of cancer stem cells and increased the membrane expression of tissue factor. Treatment of cancer cells with sCD146 in two experimental models (subcutaneous xenografting and intracardiac injection of cancer cells in nude mice) led to increased tumor dissemination and procoagulant activity. The M2J-1 mAb drastically reduced metastasis but also procoagulant activity, in particular by decreasing the number of circulating tumor microparticles, and blocked the relevant signaling pathways as demonstrated by RNA expression profiling experiments. Thus, our findings demonstrate that sCD146 mediates important pro-metastatic and procoagulant effects in two CD146-positive tumors. Targeting sCD146 with the newly generated M2J-1 mAb could constitute an innovative strategy for preventing dissemination and thromboembolism in many CD146-positive tumors.

Randomized controlled trial protocol to investigate the antiplatelet therapy effect on extracellular vesicles (AFFECT EV) in acute myocardial infarction.

Activated platelets contribute to thrombosis and inflammation by the release of extracellular vesicles (EVs) exposing P-selectin, phosphatidylserine (PS) and fibrinogen. P2Y12 receptor antagonists are routinely administered to inhibit platelet activation in patients after acute myocardial infarction (AMI), being a combined antithrombotic and anti-inflammatory therapy. The more potent P2Y12 antagonist ticagrelor improves cardiovascular outcome in patients after AMI compared to the less potent clopidogrel, suggesting that greater inhibition of platelet aggregation is associated with better prognosis. The effect of ticagrelor and clopidogrel on the release of EVs from platelets and other P2Y12-exposing cells is unknown. This study compares the effects of ticagrelor and clopidogrel on (1) the concentrations of EVs from activated platelets (primary end point), (2) the concentrations of EVs exposing fibrinogen, exposing PS, from leukocytes and from endothelial cells (secondary end points) and (3) the procoagulant activity of plasma EVs (tertiary end points) in 60 consecutive AMI patients. After the percutaneous coronary intervention, patients will be randomized to antiplatelet therapy with ticagrelor (study group) or clopidogrel (control group). Blood will be collected from patients at randomization, 48 hours after randomization and 6 months following the index hospitalization. In addition, 30 age- and gender-matched healthy volunteers will be enrolled in the study to investigate the physiological concentrations and procoagulant activity of EVs using recently standardized protocols and EV-dedicated flow cytometry. Concentrations of EVs will be determined by flow cytometry. Procoagulant activity of EVs will be determined by fibrin generation test. The compliance and response to antiplatelet therapy will be assessed by impedance aggregometry. We expect that plasma from patients treated with ticagrelor (1) contains lower concentrations of EVs from activated platelets, exposing fibrinogen, exposing PS, from leukocytes and from endothelial cells and (2) has lower procoagulant activity, when compared to patients treated with clopidogrel. Antiplatelet therapy effect on EVs may identify a new mechanism of action of ticagrelor, as well as create a basis for future studies to investigate whether lower EV concentrations are associated with improved clinical outcomes in patients treated with P2Y12 antagonists.

Ticagrelor attenuates the increase of extracellular vesicle concentrations in plasma after acute myocardial infarction compared to clopidogrel.

BACKGROUND: Platelet P2Y12 antagonist ticagrelor reduces mortality after acute myocardial infarction (AMI) compared to clopidogrel, but the underlying mechanism is unknown. Because activated platelets, leukocytes, and endothelial cells release proinflammatory and prothrombotic extracellular vesicles (EVs), we hypothesized that the release of EVs is more efficiently inhibited by ticagrelor compared to clopidogrel. OBJECTIVES: We compared EV concentrations and EV procoagulant activity in plasma of patients after AMI treated with ticagrelor or clopidogrel. METHODS: After percutaneous coronary intervention, 60 patients with first AMI were randomized to ticagrelor or clopidogrel. Flow cytometry was used to determine concentrations of EVs from activated platelets (CD61+ , CD62p+ ), fibrinogen+ , phosphatidylserine (PS+ ), leukocytes (CD45+ ), endothelial cells (CD31+ , 146+ ), and erythrocytes (CD235a+ ) in plasma at randomization, after 72 hours and 6 months of treatment. A fibrin generation test was used to determine EV procoagulant activity. RESULTS: Concentrations of platelet, fibrinogen+ , PS+ , leukocyte, and erythrocyte EVs increased 6 months after AMI compared to the acute phase of AMI (P ≤ .03). Concentrations of platelet EVs were lower on ticagrelor compared to clopidogrel after 6 months (P = .03). Concentrations of fibrinogen+ , PS+ , and leukocyte EVs were lower on ticagrelor compared to clopidogrel both after 72 hours and 6 months (P ≤ .03). Concentrations of endothelial EVs and EV procoagulant activity did not differ between patient groups and over time (P ≥ .17). CONCLUSIONS: Ticagrelor attenuates the increase of EV concentrations in plasma after acute myocardial infarction compared to clopidogrel. The ongoing release of EVs despite antiplatelet therapy might explain recurrent thrombotic events after AMI and worse clinical outcomes on clopidogrel compared to ticagrelor.