Erythrocyte-derived Microvesicles Research Articles

Association of Circulating Procoagulant Microvesicles with Painful Vaso-Occlusive Crisis in Sickle Cell Disease

Introduction: Thrombotic complication is one of the features of sickle cell disease (SCD), characterized by appearance of phosphatidylserine on the outer membrane of sickle-shaped red blood cells and most abundantly on membrane protrusions called microvesicles (MVs). However, the exact mechanism by which MVs may enhance coagulant activity in SCD patients has not been fully addressed. The aim of this study was to further investigate the procoagulant activity of circulating MVs in sickle cell crises. Materials and Methods: Subjects included in this cross-sectional study were 47 patients with SCD and 25 normal subjects with written informed consent obtained from all the participants. MV analysis was conducted by using CD61, CD235α, and Annexin-V monoclonal antibodies. The coagulant activity of MVs was determined by an ELISA-based procoagulant activity assay. Results: The majority of MVs were originated from platelets (CD61+) and erythrocytes (CD235+). These MVs demonstrated significantly enhanced levels during the painful crisis when compared with the steady-state period (p < 0.001) and controls (p < 0.001). Also, the procoagulant activity of MVs was significantly higher in crisis compared to those of steady state (p < 0.001) and positively correlated with the number of Annexin-V+ MVs (p < 0.001). Significant correlations were found between erythrocyte-derived MVs with hemolysis marker (r = 0.51, p < 0.001) and the hemoglobin level (r = −0.63, p < 0.001). Conclusion: The numbers of platelet- and erythrocyte-derived MVs are related to painful crisis, and their quantification in SCD may be helpful for identifying cases at increased risk of thrombotic complications.

The potential effect of leukocyte filtration methods on erythrocyte-derived microvesicles: One step forward.

By harmonizing the pre-preparation conditions and also removing some donors’ variations, the current study took one step forward to investigate whether different leukocyte filtration sets influence the quality of RBCs throughout the storage time. Twelve whole blood units were collected, and each unit was split into three equal parts. Thirty-six divided bags were filtered using three different leukocyte-filtration sets including Red Cell and Whole Blood Filters (12 units per filter). The prepared RBCs were refrigerated for up to 42 days and assessed for microvesicle count and size, clotting- and prothrombin time, hemolysis index, and biochemical parameters. A significant increment in erythrocytes microvesicle count (EMVs/μL) was observed during the time in the three filtration sets. The number of EMVs in WBF-RBCs was higher (~1.6 fold) than in F-RCF on day 42 (p=0.035). Interestingly the median fluorescence intensity of EMVs decreased during the storage. The size of MVs rose during the time without any significant differences among the filters. Coagulation time decreased in RBCs over the storage, with no significant differences among the filters. Hemolysis index and lactate concentration increased while glucose level decreased significantly throughout the time. The changes in WBF-RBCs were more drastic rather than RCF-RBCs. The only significant difference in the count of EMVs was between WBF and F-RCF components on day 42. Though the changes in WBF products were more drastic, all the values fell within the standard limits. Accordingly, all three filtration sets can be considered.

Erythrocyte-derived microvesicles induce arterial spasms in JAK2V617F myeloproliferative neoplasm.

Arterial cardiovascular events are the leading cause of death in patients with JAK2V617F myeloproliferative neoplasms (MPNs). However, their mechanisms are poorly understood. The high prevalence of myocardial infarction without significant coronary stenosis or atherosclerosis in patients with MPNs suggests that vascular function is altered. The consequences of JAK2V617F mutation on vascular reactivity are unknown. We observe here increased responses to vasoconstrictors in arteries from Jak2V617F mice resulting from a disturbed endothelial NO pathway and increased endothelial oxidative stress. This response was reproduced in WT mice by circulating microvesicles isolated from patients carrying JAK2V617F and by erythrocyte-derived microvesicles from transgenic mice. Microvesicles of other cellular origins had no effect. This effect was observed ex vivo on isolated aortas, but also in vivo on femoral arteries. Proteomic analysis of microvesicles derived from JAK2V617F erythrocytes identified increased expression of myeloperoxidase as the likely mechanism accounting for their effect. Myeloperoxidase inhibition in microvesicles derived from JAK2V617F erythrocytes suppressed their effect on oxidative stress. Antioxidants such as simvastatin and N-acetyl cysteine improved arterial dysfunction in Jak2V617F mice. In conclusion, JAK2V617F MPNs are characterized by exacerbated vasoconstrictor responses resulting from increased endothelial oxidative stress caused by circulating erythrocyte-derived microvesicles. Simvastatin appears to be a promising therapeutic strategy in this setting.

A STUDY OF MICROVESICLES IN THE ASSESSMENT OF THE THROMBOTIC MICROENVIRONMENT IN NEWLY DIAGNOSED DIABETES MELLITUS TYPE 2

Objective:We investigated the thrombotic microenvironment at the very early stages of Type 2 Diabetes Mellitus (T2DM) by measuring Platelet-derived Microvesicles (PMVs) and Erythrocyte-derived Microvesicles (ErMVs) in newly-diagnosed patients. Microvesicles (MVs) are small, membrane vesicles (0.1 to

Increased erythrocyte- and platelet-derived microvesicles in newly diagnosed type 2 diabetes mellitus.

To investigate the thrombotic microenvironment in early stages of type 2 diabetes mellitus measuring platelet-derived, endothelial-derived and erythrocyte-derived microvesicles. We recruited 50 newly diagnosed type 2 diabetes mellitus patients who did not receive glucose-lowering treatment except for metformin and 25 matched non-type 2 diabetes mellitus volunteers. Microvesicles were measured with flow cytometry, glycated haemoglobin with high-performance liquid chromatography and advanced glycation end products with enzyme-linked immunosorbent assay. Type 2 diabetes mellitus patients showed significantly higher levels of platelet-derived microvesicles [195/μL (115-409) vs 110/μL (73-150), p = 0.001] and erythrocyte-derived microvesicles [26/μL (9-100) vs 9/μL (4-25), p = 0.007] compared to non-type 2 diabetes mellitus individuals. Platelet-derived microvesicles were positively associated with fasting blood glucose (p = 0.026) and glycated haemoglobin (p = 0.002). Erythrocyte-derived microvesicles were also positively associated with fasting blood glucose (p = 0.018) but not with glycated haemoglobin (p = 0.193). No significant association was observed between platelet-derived microvesicles (p = 0.126) or erythrocyte-derived microvesicles (p = 0.857) and advanced glycation end products. Erythrocyte-derived microvesicles predicted the presence of type 2 diabetes mellitus, independently of platelet-derived microvesicles. In newly diagnosed type 2 diabetes mellitus, ongoing atherothrombosis is evident during the early stages as evidenced by increased microvesicles levels. Furthermore, the association with glycemic profile suggests that microvesicles represent not only a novel mechanism by which hyperglycemia amplifies thrombotic tendency in type 2 diabetes mellitus but also early markers of thrombosis highlighting the need for earlier management of hyperglycemia.

Protective effects of circulating microvesicles derived from ischemic preconditioning on myocardial ischemia/reperfusion injury in rats by inhibiting endoplasmic reticulum stress

Microvesicles (MVs) have been shown to be involved in pathophysiology of ischemic heart diseases. However, the underlying mechanisms are still unclear. Here we investigated the effects of MVs derived from ischemic preconditioning (IPC-MVs) on myocardial ischemic/reperfusion (I/R) injury in rats. Myocardial IPC model was elicited by three cycles of ischemia and reperfusion of the left anterior descending (LAD) coronary artery. IPC-MVs from the peripheral blood of the above animal model were isolated by ultracentrifugation and characterized by flow cytometry and transmission electron microscopy. IPC-MVs were administered intravenously (7mg/kg) at 5min before reperfusion procedure in I/R injury model which was induced by 30-min ischemia and 120-min reperfusion of LAD in rats. We found that total IPC-MVs and different phenotypes, including platelet-derived MVs (PMVs), endothelial cell-derived MVs (EMVs), leucocyte-derived MVs and erythrocyte-derived MVs (RMVs) were all isolated which were identified membrane vesicles (< 1µm) with corresponding antibody positive. The numbers of PMVs, EMVs and RMVs were significantly increased in circulation of IPC treated rats respectively. Additionally, treatment with IPC-MVs significantly alleviated damage of myocardium, and restored cardiac function of I/R injury rats, as evidenced by increased heart rate, and decreased the elevation of ST-segment. The size of myocardial infarction, lactate dehydrogenase activity, and the number of apoptotic cardiomyocytes were also reduced significantly with IPC-MVs treatment, coincident with the above function amelioration. Moreover, IPC-MVs decreased the activity of caspase 3, and the expression of endoplasmic reticulum stress (ERS) markers, GRP78, CHOP and caspase 12 indicating the involvement of ERS-specific apoptosis in I/R injury, and cardioprotective effects of IPC-MVs. In summary, our study demonstrated a novel mechanism of IPC in which circulating IPC-MVs could protect hearts from I/R injury in rats through attenuation of ERS-induced apoptosis. These findings provide new insight into therapeutic potential of IPC-induced MVs in cardioprotection against I/R injury.

Cellular Microvesicles in the Blood of Patients with Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disease with a complex and largely unclear pathogenesis. Cellular phospholipid microvesicles released upon activation and/or death of a cell have been proposed to play a role in inflammatory autoimmune pathologies, including SLE. Here, circulating microvesicles of various cellular origins were marked with fluorescently labeled cell-specific antibodies and enumerated by flow cytometry in platelet-free plasma obtained from the heparinized blood of 29 SLE patients and 19 normal subjects. Significantly higher concentrations of endothelial-, monocyte-, and erythrocyte-derived microvesicles were found in the SLE patients compared to normal subjects with prevalence of microvesicles originating from endothelial cells. No significant difference was found for platelet-derived microvesicles. A correlation analysis of microvesicle counts with laboratory parameters and clinical features of SLE suggest differential implications of various cell-derived microvesicles in the pathogenesis of SLE. These data suggest that SLE is associated with functional alterations of endotheliocytes, monocytes, and erythrocytes followed by enhanced release of microvesicles that may contribute to inflammation and hypercoagulability.

Antithrombin Activity of Erythrocyte Microvesicles.

Coagulation and optical (based on chromogenic substrate) methods were employed to examine antithrombin activity of erythrocytes and erythrocyte-derived microvesicles isolated days 7, 14, 21, and 28 on erythrocyte storage. The erythrocyte-derived microvesicles decelerated fibrin clot formation from fibrinogen in the presence of exogenous thrombin both with and without heparin. Microvesicles reduced optical density of chromogenic substrate. These data suggest that erythrocyte-derived microvesicles display a prominent antithrombin activity, which significantly increases during erythrocyte storage.

Peripheral blood microvesicles secretion is influenced by storage time, temperature, and anticoagulants.

Microvesicles (MVs) are small membrane bound vesicles released from various cell types after activation or apoptosis. In the last decades, MVs received an increased interest as biomarkers in inflammation, coagulation and cancer. However, standardized pre-analytical steps are crucial for the minimization of artifacts in the MV analysis. Thus, this study evaluated the MV release in whole blood samples under the influence of different anticoagulants, storage time and various temperature conditions. Samples were collected from healthy probands and processed immediately, after 4, 8, 24 and 48 hours at room temperature (RT) or 4°C. To identify MV subpopulations, platelet free plasma (PFP) was stained with Annexin V, calcein AM, CD15, CD41 and CD235a. Analysis was performend on a CytoFLEX flow cytometer. Procoagulatory function of MVs was measured using a phospholipid dependent activity and a tissue factor MVactivity assay. Without prior storage, sodium citrate showed the lowest MV count compared to heparin and EDTA. Interestingly, EDTA showed a significant release of myeloid-derived MVs (MMVs) compared to sodium citrate. Sodium citrate showed a stable MV count at RT in the first 8 hours after blood collection. Total MV counts increased after 24 hours in sodium citrated or heparinzed blood which was related to all subpopulations. Interestingly, EDTA showed stable platelet-derived MV (PMV) and erythrocyte-derived MV (EryMV) count at RT over a 48 h period. In addition, the procoagulatory potential increased significantly after 8-hour storage. Based on both, this work and literature data, the used anticoagulant, storage time and storage temperature differently influence the analysis of MVs within 8 hours. To date, sodium citrated tubes are recommended for MV enumeration and functional analysis. EDTA tubes might be an option for the clinical routine due to stable PMV and EryMV counts. These new approaches need to be validated in a clinical laboratory setting before being applied to patient studies. © 2016 International Society for Advancement of Cytometry.

The influence of thermal trauma on pro- and anticoagulant activity of erythrocyte-derived microvesicles

The goal of this research was to study the influence of erythrocyte-derived microvesicles on hemostasis parameters during burn. It was found that the number of microvesicles derived from washed erythrocytes of burn patients after 1 day of storage at 37°C was 4.2 times bigger than the number of microvesicles derived from erythrocytes of healthy donors. Hemocoagulation properties of erythrocyte-derived microvesicles of burn patients also change: according to the results of thromboelastography their procoagulant activity increases significantly, at the same time their antithrombin and fibrinilytic activity decrease. Thus, we can conclude that hepercoagulation during burn is to a certain extent caused by the disruption of the balance between procoagulant activity of erythrocyte-derived microvesicles and their antithrombin and fibrinolytic activity. Hypercoagulation effect of erythrocyte-derived microvesicles increases during burn not just because of their changed properties but also due to their increased number after thermal trauma.

Differences between calcium-stimulated and storage-induced erythrocyte-derived microvesicles

Microvesicles (MVs), or microparticles, are a complex, dynamic and functional part of cells. Red blood cell (RBC)-derived MVs are naturally produced in vivo (during normal aging processes or in several diseases) as well as ex vivo during cold storage of RBCs, or in vitro by ATP depletion or treatment with Ca2+ and calcium ionophore. All these MVs are equivalently classified according to their size and/or surface markers. Nevertheless, their content in proteins can differ and a few differences in terms of lipid raft proteins, notably stomatin and flotillin-2, have been reported. Based on two-dimensional gel electrophoreses, the present study highlights the differences between MVs induced during storage of RBCs (storage-MVs) and MVs stimulated by Ca2+ entry (Ca-MVs). Upon treatment, Ca-MVs are formed following a clear recruitment of Ca2+-binding proteins (sorcin, grancalcin, PDCD6) and particularly annexins (4 and 5). Therefore, it emerges that different molecular pathways are available to produce similar MVs by disturbing the membrane/cytoskeleton interactions. Interestingly, these differences provide non-negligible pieces of information on the parent cells, and the mechanisms and modes of actions involved in the formation of MVs. In addition to biophysical characterization, protein analysis is important to classify these cellular corpuscles and evaluate their potential impacts in diseases or transfusion medicine.

Erythrocyte-Derived Microvesicles Amplify Systemic Inflammation by Thrombin-Dependent Activation of Complement

Transfusion of aged blood has been associated with increased morbidity and mortality in critically ill patients. During storage, erythrocytes release increasing numbers of microvesicles (red blood cell-derived microvesicles [RBC-MV]). We hypothesized that RBC-MV mediate some of the deleterious effects of aged blood transfusions. We established a murine transfusion model using RBC-MV purified from aged mouse erythrocytes. Injection of RBC-MV into healthy mice had no effect. However, they aggravated pulmonary leukocyte sequestration and peripheral blood leukopenia induced by lipopolysaccharides. Lipopolysaccharide-induced proinflammatory cytokines were significantly increased in plasma after RBC-MV injection. These effects were not seen in C5aR-deficient mice. In vitro, RBC-MV bound C3 fragments after incubation with plasma but failed to bind immunoglobulins, C1q, or mannose-binding lectin. Preventing thrombin generation inhibited complement activation in vitro and in vivo and reversed the proinflammatory effects of RBC-MV in lipopolysaccharide-primed mice. Finally, the RBC-MV-induced phenotype was recapitulated using phosphatidylserine-expressing liposomes, suggesting that surface expression of phosphatidylserine by RBC-MV was mechanistically involved. These results point toward a thrombin-dependent mechanism of complement activation by RBC-MV independent of the classical, lectin, or alternative pathway. Besides identifying RBC-MV as potential mediators of transfusion-related morbidity, our findings may be relevant for other inflammatory disorders involving intravascular microvesicle release, for example, sickle cell disease or thrombotic microangiopathy.

Magnetic Nanosensor for Detection and Profiling of Erythrocyte-Derived Microvesicles

During the course of their lifespan, erythrocytes actively shed phospholipid-bound microvesicles (MVs). In stored blood, the number of these erythrocyte-derived MVs has been observed to increase over time, suggesting their potential value as a quality metric for blood products. The lack of sensitive, standardized MV assays, however, poses a significant barrier to implementing MV analyses into clinical settings. Here, we report on a new nanotechnology platform capable of rapid and sensitive MV detection in packed red blood cell (pRBC) units. A filter-assisted microfluidic device was designed to enrich MVs directly from pRBC units, and label them with target-specific magnetic nanoparticles. Subsequent detection using a miniaturized nuclear magnetic resonance system enabled accurate MV quantification as well as the detection of key molecular markers (CD44, CD47, CD55). When the developed platform was applied, MVs in stored blood units could also be monitored longitudinally. Our results showed that MV counts increase over time and, thus, could serve as an effective metric of blood aging. Furthermore, our studies found that MVs have the capacity to generate oxidative stress and consume nitric oxide. By advancing our understanding of MV biology, we expect that the developed platform will lead to improved blood product quality and transfusion safety.

Malaria-Infected Erythrocyte-Derived Microvesicles Mediate Cellular Communication within the Parasite Population and with the Host Immune System

Humans and mice infected with different Plasmodium strains are known to produce microvesicles derived from the infected red blood cells (RBCs), denoted RMVs. Studies in mice have shown that RMVs are elevated during infection and have proinflammatory activity. Here we present a detailed characterization of RMV composition and function in the human malaria parasite Plasmodium falciparum. Proteomics profiling revealed the enrichment of multiple host and parasite proteins, in particular of parasite antigens associated with host cell membranes and proteins involved in parasite invasion into RBCs. RMVs are quantitatively released during the asexual parasite cycle prior to parasite egress. RMVs demonstrate potent immunomodulatory properties on human primary macrophages and neutrophils. Additionally, RMVs are internalized by infected red blood cells and stimulate production of transmission stage parasites in a dose-dependent manner. Thus, RMVs mediate cellular communication within the parasite population and with the host innate immune system.