Co-isolation of extracellular vesicles and high-density lipoproteins using density gradient ultracentrifugation

Extracellular vesicles (EVs) facilitate intercellular communication by carrying bioactive molecules such as proteins, messenger RNA, and micro (mi)RNAs. Recently, high-density lipoproteins (HDL) isolated from human plasma were also reported to transport miRNA to other cells. HDL, when isolated from human plasma, ranges in density between 1.063 and 1.21 g/mL, which grossly overlap with the reported density of EVs. Consequently, HDL and EV will be co-isolated when using density gradient ultracentrifugation. Thus, more stringent isolation/separation procedures of EV and HDL are essential to know their relative contribution to the pool of circulating bioactive molecules

Single-step isolation of extracellular vesicles by size-exclusion chromatography

Background: Isolation of extracellular vesicles from plasma is a challenge due to the presence of proteins and lipoproteins. Isolation of vesicles using differential centrifugation or density-gradient ultracentrifugation results in co-isolation of contaminants such as protein aggregates and incomplete separation of vesicles from lipoproteins, respectively. Aim: To develop a single-step protocol to isolate vesicles from human body fluids. Methods: Platelet-free supernatant, derived from platelet concentrates, was loaded on a sepharose CL-2B column to perform size-exclusion chromatography (SEC; n=3). Fractions were collected and analysed by nanoparticle tracking analysis, resistive pulse sensing, flow cytometry and transmission electron microscopy. The concentrations of high-density lipoprotein cholesterol (HDL) and protein were measured in each fraction. Results: Fractions 9–12 contained the highest concentrations of particles larger than 70 nm and platelet-derived vesicles (46%±6 and 61%±2 of totals present in all collected fractions, respectively), but less than 5% of HDL and less than 1% of protein (4.8%±1 and 0.65%±0.3, respectively). HDL was present mainly in fractions 18–20 (32%±2 of total), and protein in fractions 19–21 (36%±2 of total). Compared to the starting material, recovery of platelet-derived vesicles was 43%±23 in fractions 9–12, with an 8-fold and 70-fold enrichment compared to HDL and protein. Conclusions: SEC efficiently isolates extracellular vesicles with a diameter larger than 70 nm from platelet-free supernatant of platelet concentrates. Application SEC will improve studies on the dimensional, structural and functional properties of extracellular vesicles

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.Peer reviewe

Extracellular vesicles in physiological and pathological conditions

Body fluids contain surprising numbers of cell-derived vesicles which are now thought to contribute to both physiology and pathology. Tools to improve the detection of vesicles are being developed and clinical applications using vesicles for diagnosis, prognosis, and therapy are under investigation. The increased understanding why cells release vesicles, how vesicles play a role in intercellular communication, and how vesicles may concurrently contribute to cellular homeostasis and host defense, reveals a very complex and sophisticated contribution of vesicles to health and diseas

Clinical Significance of Tissue Factor-Exposing Microparticles in Arterial and Venous Thrombosis

Microparticles (MP) are small extracellular vesicles (30-1,000 nm) that are released from activated cells or platelets. Exposure of negatively charged phospholipids and tissue factor (TF) renders MP procoagulant. Normal plasma levels of intravascular TF-exposing MP (TFMP) are low, but their number may rise in pathological conditions, including cancer and infectious disease. Emerging evidence indicates an important role for these circulating TFMP in the pathogenesis of thrombotic complications such as venous thromboembolism and disseminated intravascular coagulation, whereas their contribution to arterial thrombosis is less studied. Despite serious limitations of the currently available assays for measuring TFMP levels or the procoagulant activity associated with TFMP with respect to sensitivity and specificity, the scientific interest in TFMP is rapidly growing because their application as prognostic biomarkers for thrombotic complications is promising. Future advances in detection methods will likely provide more insight into TFMP and eventually improve their clinical utilit

Microparticles in vascular disorders: How tissue factor-exposing vesicles contribute to pathology and physiology

Coagulation is initiated by tissue factor (TF). Coagulant TF is constitutively expressed by extravascular cells, but there is increasing evidence that TF can also be present within the blood, in particular during pathological conditions. Such TF is exposed on circulating cell-derived vesicles, and its presence has been associated with development of disseminated intravascular coagulation and venous thrombosis. For example, the presence of TF-exposing vesicles in the blood of cancer patients may be associated with their high risk of developing venous thromboembolism. Remarkably, high levels of coagulant TF-exposing vesicles are present in other body fluids such as saliva and urine of healthy persons, suggesting that these vesicles play a physiological role. We postulate that the presence of TF-exposing vesicles in body fluids as saliva and urine provides an additional source of coagulant TF that promotes coagulation, thereby reducing blood loss and contributing to host defence by reducing the risk of microorganisms entering the "milieu interieur". (C) 2012 Elsevier Ltd. All rights reserve

Platelet microparticles contain active caspase 3

During storage, platelets undergo processes resembling apoptosis, including microparticle release, aminophospholipid exposure, and procaspase 3 processing. Recently, we showed that microparticles from endothelial cells contain caspase 3, one of the executioner enzymes of apoptosis. In this study we determined whether platelet-derived microparticles (PMP) contain caspase 3 in vitro (stored platelet concentrate) and ex vivo (plasma from healthy humans). In addition, we studied the underlying mechanism of caspase 3 formation in PMP, and the ability of such PMP to induce apoptosis in human macrophages (THP-1 cells). The presence of caspase 3 (antigen) was studied by Western blot and flowcytometry, and activity was determined by Ac-DEVD-pNA and ROCK I cleavage. In vitro, PMP numbers increased during storage. From day one onwards, PMP contained procaspase 3, whereas caspase 3 (antigen and activity) was detectable after 5-7 days of storage. PMP contained caspase 9 but not caspase 8, and the time course of caspase 9 formation paralleled procaspase 3 disappearance and caspase 3 appearance. In addition, PMP in human plasma also contained detectable quantities of caspase 3. Incubation of THP-1 cells with PMP induced apoptosis. Taken together, PMP contain caspase 3 in vitro and ex vivo. Our data implicate that procaspase 3 is likely to be processed by caspase 9 in PMP during storage. PMP induce apoptosis of human macrophages, but whether this induction is due to the transfer of caspase 3 remains to be determine

Co-isolation of extracellular vesicles and high-density lipoproteins using density gradient ultracentrifugation

Extracellular vesicles (EVs) facilitate intercellular communication by carrying bioactive molecules such as proteins, messenger RNA, and micro (mi)RNAs. Recently, high-density lipoproteins (HDL) isolated from human plasma were also reported to transport miRNA to other cells. HDL, when isolated from human plasma, ranges in density between 1.063 and 1.21 g/mL, which grossly overlap with the reported density of EVs. Consequently, HDL and EV will be co-isolated when using density gradient ultracentrifugation. Thus, more stringent isolation/separation procedures of EV and HDL are essential to know their relative contribution to the pool of circulating bioactive molecules