Isolation Of Microparticles Research Articles

Correlations between the Circulating Level of Cell-Derived Microparticles and Surgical Variables in Heart Valve Surgery with Cardiopulmonary Bypass.

Cell-derived microparticles (MPs) as membrane vesicles are procoagulant. They play a role in surgical hemostasis. In this study, the correlations between the circulating level of cell-derived MPs and surgical variables in heart valve surgery were investigated. The present prospective case-series study was conducted in Rajaie Cardiovascular Medical and Research Center from January through March 2021. Forty patients undergoing heart valve surgery with cardiopulmonary bypass (CPB) were enrolled. Before the induction of anesthesia and 30 minutes after the administration of protamine sulfate, venous blood samples were collected. After MP isolation, the concentration of MPs was determined via the Bradford method. Flow cytometry analysis was performed to determine the MP count and phenotype. Intraoperative variables and postoperative routine coagulation tests were defined as surgical variables. Postoperative coagulopathy was defined as an activated partial thromboplastin time (aPTT) ≥48 seconds or an international normalized ratio (INR) >1.5. The total concentration of MPs and the MP count increased significantly after surgery compared with before surgery. The postoperative concentration of MPs was positively correlated with the CPB time (P=0.030, ρ=0.40). The preoperative concentration of MPs was significantly lower in patients with higher postoperative aPTT and INR (P=0.003, P= -0.50 and P=0.020, P= -0.40, respectively). In multivariate logistic regression analysis, the preoperative MP concentration (OR, 1.00; 95% CI, 1.00 to 1.01; P=0.017) was considered a risk factor for postoperative coagulopathy. The levels of MPs, especially platelet-derived MPs, rose after surgery, in correlation with the CPB time. Given the role of MPs in the induction of coagulation and inflammation, they can be considered therapeutic goals for preventing postoperative complications. In addition, the preoperative levels of MPs are a risk factor for predicting the occurrence of postoperative coagulopathy in heart valve surgery.

Microparticles in Autoimmunity: Cause or Consequence of Disease?

Microparticles (MPs) are small (100 nm – 1 um) extracellular vesicles derived from the plasma membrane of dying or activated cells. MPs are important mediators of intercellular communication, transporting proteins, nucleic acids and lipids from the parent cell to other cells. MPs resemble the state of their parent cells and are easily accessible when released into the blood or urine. MPs also play a role in the pathogenesis of different diseases and are considered as potential biomarkers. MP isolation and characterization is technically challenging and results in different studies are contradictory. Therefore, uniform guidelines to isolate and characterize MPs should be developed. Our understanding of MP biology and how MPs play a role in different pathological mechanisms has greatly advanced in recent years. MPs, especially if derived from apoptotic cells, possess strong immunogenic properties due to the presence of modified proteins and nucleic acids. MPs are often found in patients with autoimmune diseases where MPs for example play a role in the break of immunological tolerance and/or induction of inflammatory conditions. In this review, we describe the main techniques to isolate and characterize MPs, define the characteristics of MPs generated during cell death, illustrate different mechanism of intercellular communication via MPs and summarize the role of MPs in pathological mechanisms with a particular focus on autoimmune diseases.

Microparticles profiling in trauma patients: high level of microparticles induce activation of platelets in vitro.

Trauma-induced coagulopathy (TIC) is recognised as an own clinical entity which includes all components of haemostasis following rapidly tissue injury, hypoperfusion and shock. Microparticles (MP) are known to be released in large quantities from different cell types after trauma. The present study aimed to perform a phenotypic MP profiling after major trauma and to elucidate potential procoagulative function of MP under simulated conditions of lethal triad. For MP isolation, 20 trauma patients (median ISS 24) were included. To produce a Standard MP Phenotype Profile after trauma, samples were pooled, extracted and concentrated by using an ultracentrifuge protocol. Specific cell surface markers were measured by flow cytometry. Our Standard MP Phenotype Profile was subsequently added in high and low concentration to an in vitro lethal triad assay, simulating coagulopathy via induced hypothermia, dilution and acidosis. A comprehensive analysis of coagulation function was performed. Within our Standard MP Phenotype Profile, PDMP (56%) were found as predominant phenotype followed by EDMP (33%) and MDMP (11%). EDMP characterized by CD144, CD62E and Annexin were determined most frequently but also EDMP expressing CD62P. In addition, tissue factor (TF) was expressed on all MP entities (EDMP 63%, PDMP 30%, MDMP 7%). Within our lethal triad simulation assay, the addition of low and high concentrated MP did not cause any significant alteration in standard coagulation assays, coagulation initiation, clot kinetics or stability. Addition of high concentrated MP increased platelet function and P-selectin expression significantly. Our data confirm the assumption that there is a characteristic MP phenotype pattern in trauma, which may alter haemostatic capacity at least in part mediated via augmenting in primary haemostasis resulting in an improved contribution of platelets to clot formation. There are indications that expression of selectins on MP surface is involved in this activation process, but this pathway needs to be investigated in more detail.

Impact of sample processing on the measurement of circulating microparticles: storage and centrifugation parameters.

Microparticles (MPs) have been shown to be markers of cellular activation and interactions. Pre-analytical conditions such as the centrifugation protocol and sample storage conditions represent an important source of variability in determining MPs values. The objectives of this study were to evaluate the influence of sample storage conditions and centrifugation speed and temperature on the determination of MPs in plasma. Citrate-anticoagulated blood samples obtained from 21 healthy subjects were centrifuged under four different protocols involving different speeds (2500 g or 1500 g) and temperatures (4 °C or 20 °C) to isolate platelet-poor plasma (PPP). The number of MPs in fresh and frozen-thawed PPP were analyzed by flow cytometry, and MPs-mediated procoagulant activity was determined by a thrombin generation test and phospholipid-dependent procoagulant tests. The number of MPs and their procoagulant activity were affected by freeze-thaw cycling and centrifugation speed but not by centrifugation temperature. Sample freezing increased MPs number (six-fold) and thrombin generation (four-fold), and decreased clotting time (two-fold). Low centrifugation speed caused an increase in MPs number and a parallel increase in MP-mediated procoagulant activity. Sample storage conditions and centrifugation speed are important processing conditions affecting MPs number and activity. Before any study, the protocol for MPs isolation should be optimized to ensure a reliable characterization of MPs, which could provide important information for diagnostic purposes and for understanding the pathogenesis of diseases.

Microparticles from Patients with the Acute Coronary Syndrome Impair Vasodilatation by Inhibiting the Akt/eNOS-Hsp90 Signaling Pathway

Objectives: Endothelial dysfunction is involved in the development of the acute coronary syndrome (ACS). Plasma microparticles (MPs) from other diseases have been demonstrated to initiate coagulation and endothelial dysfunction. However, whether MPs from ACS patients impair vasodilatation and endothelial function remains unclear. Methods: Patients (n = 62) with ACS and healthy controls (n = 30) were recruited for MP isolation. Rat thoracic aortas were incubated with MPs from ACS patients or healthy controls to determine the effects of MPs on endothelial-dependent vasodilatation, the phosphorylation of Akt and endothelial nitric oxide synthase (eNOS), the interaction of eNOS with heat shock protein 90 (Hsp90), and nitric oxide (NO) and superoxide anion (O₂^-) production. The origin of MPs was assessed by flow cytometry. Results: MP concentrations were increased in patients with ACS compared with healthy controls. They were positively correlated with the degree of coronary artery stenosis. MPs from ACS patients impair endothelial-dependent vasodilatation, decrease both Akt and eNOS phosphorylation, decrease the interaction between eNOS and Hsp90, and decrease NO production but increase O₂^- generation in rat thoracic aortas. Endothelial-derived MPs and platelet-derived MPs made up nearly 75% of MPs. Conclusions: Our data indicate that MPs from ACS patients negatively affect endothelial-dependent vasodilatation via Akt/eNOS-Hsp90 pathways.

Comparison Of Autologous Microparticles and Fucosyltransferase-7 For Adhesion Improvement Of Hematopoietic Stem Cells To Bone Marrow Endothelial Cells In a Microfluidic Flow Chamber

The improvement of graft function and time to engraftment might help to reduce infection-related mortality in stem cell transplantation (SCT). While the concept of stem cells fucosylation for accelerated engraftment has already reached clinical study phase (for cord blood transplantation; NCT01471067), own previous work has shown an association between engraftment time and circulating microparticles bearing P-Selectin and P-Selectin glycoprotein ligand 1 (PSGL-1). PSGL-1 contains the sialyl Lewis x (CD15s) antigen that requires fucosylation for optimal binding of P- and E-Selectin on endothelial cells. We therefore hypothesized that addition of microparticles (MP) might enhance adhesion of human stem cells (HSC) to bone marrow endothelial cells and that MP might have synergistic effects in combination with stem cell fucosylation. HSC were obtained from apheresis products of allogeneic donors, purified by Ficoll and magnetic bead separation for CD34, stained with calcein AM and perfused through an automated microfluidic flow chamber (Bioflux 200, Fluxionbio, USA) covered with a confluent layer of an immortalized human bone marrow endothelial cell line (HBMEC). Photos (and videos) were taken using a fluorescence microscope at start, 5 min and 10 min and analyzed for adherent HSC across the whole chamber (about 1.5 sqmm) using ImageJ software. Autologous MP were generated by addition of calcimycin to apheresis and isolation of MP by centrifugation. For control experiments, one part of the MP solution was passed through a 0,2µm-filter to remove MP. MP concentration (mean: 1362/µl) was assessed by detection of Annexin V binding in flow cytometry, using TrucountBeads® for quantification. Fucosylation was performed by 1h incubation of isolated CD34+ stem cells with GDP-fucose and fucosyltransferase 7 (FUT7). Successful fucosylation was controlled by CD15s staining of HSC in flow cytometry. Results of seven experiments (in duplicate) demonstrated the highest number of adherent HSC in the MPpositiv/FUT7negativ preparation (median: 32 HSC/sqmm; range: 15-78), followed by MPpositiv/FUT7positiv (30 HSC/sqmm; range: 16-38), MPnegativ/FUT7positiv (median: 25/sqmm; range: 11-27) and MPnegativ/FUT7negativ (20 HSC/sqmm; range: 0-22). Comparison of the MPpositiv/FUT7negativ and MPnegativ/FUT7negativ as well as the MPpositiv/FUT7positiv and MPpositiv/FUT7negativ preparations showed statistically significant differences in Wilcoxon rank test (p<.05) while comparison of MPpositiv/FUT7positiv vs. MPnegativ/FUT7positiv and MPnegativ/FUT7positiv vs. MPnegativ/FUT7negativ preparations did not. In summary, these results demonstrate that MP can improve HSC adhesion to bone marrow endothelial cells similar to fucosylation. The effect of fucosylation on HSC adhesion appears to be mediated by MP. However, there is not a synergistic effect between MP and fucosylation. Disclosures:No relevant conflicts of interest to declare.

Gateway to Understanding Microparticles: Standardized Isolation and Identification of Plasma Membrane-Derived Vesicles

Microparticles (MPs) are small plasma membrane-derived vesicles that can expose molecules originating from their parental cells. As vectors of biological information they are likely to play an active role in both homeostasis and pathogenesis, making them promising biomarkers and nanomedicine tools. Therefore, there is an urgent need for standardization of MP isolation and analysis protocols to propel our understanding of MP biology to the next level. Based on current methodology and recent insights, this review proposes an optimized protocol for the isolation and biochemical characterization of MPs.

Microparticles: Modulators and biomarkers of liver disease

Division of Molecular and Translational Medicine,Dept. of Medicine I, Johannes Gutenberg University, Mainz, GermanyEmerging role of microparticlesMicroparticles (MP) have gained increasing attention as biomark-ers for various diseases. First described as platelet dust, MP wereregarded as unspecific debris [1]. However, it has become appar-ent that cell derived MP or ectosomes represent a novel route ofhorizontal communication between cells. MP are between 100–1000 nm in size and generated through cell membrane shedding(ectocytosis), a process that can be triggered by the activation ofthe complement C5b-9 complex, as shown for platelet derivedMP, or by inhibition of flippase activitythroughCa

Microparticles as a Predictor of Acute Deep Venous Thrombosis in a Patient with Antiphospholipid Antibody Syndrome.

Microparticles (MP) are cell membrane derived fragments released as a result of apoptosis or cellular activation that may be prothrombotic. Elevated numbers of microparticles may circulate in inflammatory and thrombophilic disorders. Here, we report a patient with Antiphospholipid Antibody Syndrome (APS) in whom increased levels of circulating microparticles preceded the onset of clinically-detectable deep venous thrombosis (DVT). The patient is a 36 year old Hispanic male with previously diagnosed APS (DRVVT ratio 2.6, ACA IgG > 120, and β2GPI IgG >100, IgA 74) complicated by several DVTs and transient ischemic attacks (TIA). Due to thrombosis on warfarin and enoxaparin, he was maintained on chronic therapy with fondaparinux. Microparticle analysis on three separate occasions was performed as part of an ongoing study of MPs in patients with antiphospholipid antibodies (aPL). The first analysis was performed during routine follow-up when the patient was asymptomatic. The second analysis was performed when the patient presented with complaints of left lower extremity tightness. Physical exam was normal, and Doppler ultrasound of the left lower extremity revealed chronic venous changes but no evidence of acute thrombosis. The patient was followed with plans for serial ultrasound examination. However, two days after this visit, the patient presented with acute DVT, confirmed by venous ultrasound. He was treated with five days of intravenous unfractionated heparin followed by reinstitution of fondaparinux. The third MP analysis was performed two weeks after the diagnosis of DVT. Isolation of MP was performed using a modification of previously described methods [Dignat-George et al, Thromb Haemost 91:668, 2004]. Platelet free plasma (PFP) was labeled with monoclonal antibodies for CD 144 and CD 105 (against endothelial cell VE cadherin and endoglin, respectively), and CD 41 (against platelet integrin αIIb) and analyzed by flow cytometry the same day as collection. Results of these analyses, expressed as number of MP/ml of PFP, are listed below.Antibody2 Months Prior to DVT2 Days Prior to DVT2 Weeks After DVT DiagnosisNormal RangeCD 1443650977041612814815 ± 17714 n=13CD 105n/a352656246245890 ± 8754 n=5CD 41255042624188641607 ± 2627 n=20These results demonstrate normal levels of circulating MPs in this APS patient while asymptomatic. However, endothelial cell and platelet MPs increased 20–35 fold prior to the development of venous-ultrasound detectable DVT, and decreased following therapy. While previous reports have documented elevated levels of MP in the setting of established thrombosis, we believe that this is the first description of a serial analysis of microparticles in an APS patient during an asymptomatic period, during incipient thrombosis not otherwise clinically diagnosable, and after treatment of acute DVT. MPs may prove to be a sensitive marker of incipient DVT in patients with negative vascular ultrasound studies.