Total Microparticles Research Articles

37 1,25(OH)2D3 suppresses oxidative stress – Induced microparticle release by placental trophoblasts

Introduction Emerging evidence showed that microparticles (MPs) derived from placental trophoblasts (TC) contribute to maternal vascular dysfunction and promote endothelial inflammatory response in preeclampsia (PE). Vitamin D insufficiency has been linked to placental dysfunction in pregnancy disorders such as PE and IUGR. Objective To determine: (1) if TC from PE placentas release more MPs than TC from normal placentas; (2) if vitamin D could suppress oxidative stress-induced MP release by placental TC; and (3) potential mechanism of vitamin D protective effects on placental TCs. Methods TC were isolated from normal and PE placentas by trypsin digestion and purified with Percoll gradient centrifugation. Freshly isolated TC (5×06cells/well) were seeded into 6 well plates and cultured with DMEM containing 5% FBS and antibiotics for 72h. For the oxidative stress experiment, cobalt chloride (CoCl 2 , a hypoxic mimicking agent) was used to induce TC oxidative stress and 1,25(OH)2D3 was used as bioactive vitamin D. MPs were isolated from culture supernatants by a two-step centrifugation procedure (420g for 20min and then 20,000g for 60min) and quantified by annexin V positive staining measured by flow cytometry with TruCount beads to determine absolute counts of MP for each sample. Data are expressed as annexin V positive MP/μg cell protein/well. Total cellular protein and MP protein were also collected and protein expression for caveolin-1, eNOS, pro-caspase-3, cleaved caspase-3, and ROCK1 were determined by Western blot. Data were analyzed by paired or unpaired t-test or ANOVA with Newman-Keuls test as a post hoc test. A p level Results TC isolated from PE placentas ( n =6) released significantly more MP than cells isolated from normal placentas ( n =6), 295.20±47.67 vs. 117.20±18.02/μg protein/well, p 2 compared to control TC, p 2 was significantly reduced in TC treated with 1,25(OH)2D3+CoCl 2 , p 2 . This phenomenon could be reversed when cells were treated with 1,25(OH)2D3. Moreover, reduced pro-caspase-3 and ROCK1 expression in TC induced by CoCl 2 could also be attenuated in cells treated with1,25(OH)2D3. Conclusions MP release was significantly increased in TC from PE placenta. 1,25(OH)2D3 suppresses oxidative stress-induced MP release by placental TC. Caveolin-1 is an essential structural component of caveolae and eNOS is associated with plasma membrane caveolae. Therefore, reduced caveolin-1 and increased eNOS expression together with reduced MP release in TC treated with 1,25(OH)2D3 demonstrate that vitamin D could protect placental TC from oxidative stress-induced injury during pregnancy. Since caspase-3 cleavage and ROCK1 activation are key steps of MP formation and release, suppression of caspase-3 cleavage and ROCK1 activation by vitamin D could be a plausible mechanism of vitamin D inhibition of increased MP release induced by oxidative stress.

Enrichment of extracellular vesicles from tissues of the central nervous system by PROSPR.

BackgroundExtracellular vesicles (EVs) act as key mediators of intercellular communication and are secreted and taken up by all cell types in the central nervous system (CNS). While detailed study of EV-based signaling is likely to significantly advance our understanding of human neurobiology, the technical challenges of isolating EVs from CNS tissues have limited their characterization using ‘omics’ technologies. We therefore developed a new Protein Organic Solvent Precipitation (PROSPR) method that can efficiently isolate the EV repertoire from human biological samples.ResultsIn the current report, we present a novel experimental workflow that outlines the process of sample extraction and enrichment of CNS-derived EVs using PROSPR. Subsequent LC-MS/MS-based proteomic profiling of EVs enriched from brain homogenates successfully identified 86 of the top 100 exosomal markers. Proteomic profiling of PROSPR-enriched CNS EVs indicated that > 75 % of the proteins identified matched previously reported exosomal and microvesicle cargoes, while also expanded the known human EV-associated proteome with 685 novel identifications. Similarly, lipidomic characterization of enriched CNS vesicles not only identified previously reported EV-specific lipid families (PS, SM, lysoPC, lysoPE) but also uncovered novel lipid isoforms not previously detected in human EVs. Finally, dedicated flow cytometry of PROSPR-CNS-EVs revealed that ~80 % of total microparticles observed were exosomes ranging in diameter from ≤100 nm to 300 nm.ConclusionsThese data demonstrate that the optimized use of PROSPR represents an easy-to-perform and inexpensive method of enriching EVs from human CNS tissues for detailed characterization by ‘omics’ technologies. We predict that widespread use of the methodology described herein will greatly accelerate the study of EVs biology in neuroscience.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-016-0108-1) contains supplementary material, which is available to authorized users.

Procoagulant microparticles promote coagulation in a factor XI‐dependent manner in human endotoxemia

Essentials The procoagulant effects of microparticles (MPs) on coagulation in endotoxemia are not known. MPs from endotoxemia volunteers were evaluated for procoagulant activity in a plasma milieu. MPs from endotoxemia volunteers shortened clotting times and enhanced thrombin generation. MP procoagulant effects were mediated in a factor XI-dependent manner. Background Human endotoxemia is characterized by acute inflammation and activation of coagulation, as well as increased numbers of circulating microparticles (MPs). Whether these MPs directly promote coagulation and through which pathway their actions are mediated, however, has not been fully explored. Objectives In this study, we aimed to further characterize endotoxin-induced MPs and their procoagulant properties using several approaches. Methods Enumeration and characterization of MPs were performed using a new-generation flow cytometer. Relative contributions of the extrinsic and intrinsic pathways in MP-mediated procoagulant activity were assessed using plasmas deficient in factor (F) VII or FXI or with blocking antibodies to tissue factor (TF) or FXIa. Results Total MPs and platelet MPs were significantly elevated in plasma at 6 h after infusion of endotoxin in healthy human subjects. MPs isolated from plasma following endotoxin infusion also demonstrated increased TF activity in a reconstituted buffer system. When added to recalcified platelet-poor plasma, these MPs also promoted coagulation, as judged by a decreased clotting time with shortening of the lag time and time to peak thrombin using calibrated automated thrombography (CAT). However, the use of FVII-deficient plasma or blocking antibody to TF did not inhibit these procoagulant effects. In contrast, plasma clotting time was prolonged in FXI-deficient plasma and a blocking antibody to FXIa inhibited all MP-mediated parameters in the CAT assay. Conclusions The initiation of coagulation by cellular TF in endotoxemia is in contrast to (and presumably complemented by) the intrinsic pathway-mediated procoagulant effects of circulating MPs.

Effect of Irradiation on Microparticles in Red Blood Cell Concentrates.

Changes in microparticles (MP) from red blood cell (RBC) concentrates in the context of irradiation have not been investigated. The aim of this study was to evaluate how irradiation affects the number of MPs within transfusion components. Twenty RBC concentrates, within 14 days after donation, were exposed to gamma rays (dose rate: 25 cGy) from a cesium-137 irradiator. Flow cytometry was used to determine the numbers of MPs derived from RBC concentrates before and 24 hr after irradiation. The mean number of MPs (±standard deviation) in RBC concentrates was 21.9×109/L (±22.7×109/L), and the total number of MPs ranged from 2.6×109/L to 96.9×109/L. The mean number of MPs increased to 22.6×109/L (±31.6×109/L) after irradiation. Before irradiation, the CD41-positive and CD235a-positive MPs constituted 9.5% (1.0×109/L) and 2.2% (263×106/L) of total MPs, respectively. After irradiation, CD41-positive MPs increased to 12.1% (1.5×109/L) (P=0.014), but the CD235a-positive MPs decreased to 2.0% (214×106/L) of the total MPs (P=0.369). Irradiation increases the number of CD41-positive MPs within RBC concentrates, suggesting the irradiation of RBC concentrates could be associated with thrombotic risk of circulating blood through the numerical change.

Circulating microparticles in pancreatic tumor patients and their roles in mediating leukocyte adhesion in intact microvessels

Pancreatic cancer is one of the nation's deadliest cancers with a dismal 6 percent five‐year survival rate. Pancreatic cancer characteristically creates an abnormal inflammatory or desmoplastic response, which may play a role in its resistance to systemic therapy and aggressive metastasis. Recent studies in circulating microparticles (MPs), the small plasma membrane‐derived vesicles (0.1–1um) released during cell activation, indicated that elevated MPs occur in many cardiovascular diseases, as well as in cancer patients. Since the released MPs carry a range of antigens depending on the type and activation state of their parental cells, they have been considered as biomarker to indicate the disease status and predict the prognosis. The objectives of our study are to investigate the relationship between pancreatic cancer and vascular inflammation and evaluate whether circulating MPs can be a potential biomarker for pancreatic cancer prognosis and treatment outcomes. In this study we characterized the profile of circulating MPs in patients with malignant and non‐malignant pancreatic diseases, investigated the interaction of cultured pancreatic cancer cell‐derived MPs (PACMPs) with blood and endothelial cell‐derived MPs, and explored the roles of PACMPs in mediating vascular inflammation. Flow cytometry was used to quantify plasma MPs, and identify MP cell origins using antibodies directly against cell specific antigens. Compared with healthy controls (n=8), patients with pancreatic cancer (n=11) and pancreatic cyst (nonmalignant, n=7) all had elevated levels of MPs. About 90% of total plasma MPs were from platelets, leukocytes, and endothelial cells. Over 50% of the MPs existed as aggregated units expressing multiple parental cell surface antigens. Electron microscopy and atomic force microscopy delineated the aggregation profiles of plasma MPs. When PACMPs were introduced into cancer patients’ plasma, more than 50% of the plasma MPs bound with PACMPs, and 32% of the plasma MPs that bound with PACMPs express platelet specific antigen. The interaction between PACMPs and vascular endothelial cells were investigated in individually perfused rat venules. After perfusing vessels with PACMPs followed by annexin V staining, confocal images show a significant number of adhered PATMPs in the microvessel walls. After resuming blood flow in PACMP perfused vessels, leukocyte adhesion was significantly increased from 3 ± 1 (control) to 13 ± 3 per 100 um of vessel length. Our results showed that both patients with malignant and non‐malignant pancreatic diseases had elevated circulating MPs, indicating an association between pancreatic diseases and inflammation. The increased plasma MPs, mainly from blood cells and ECs origins, and PACMPS are highly adhesive. They not only interact with each other, but also interact with normal ECs and mediate leukocyte adhesion, which may play important roles in propagation of the inflammation and contribute to pancreatic cancer progression and metastasis.Support or Funding InformationSupported by HL056237, HL084338 and DK097391.

Microparticles variability in fresh frozen plasma: preparation protocol and storage time effects.

Extracellular vesicles or microparticles exhibiting procoagulant and thrombogenic activity may contribute to the haemostatic potential of fresh frozen plasma. Fresh frozen plasma was prepared from platelet-rich plasma at 20 °C (Group-1 donors) or directly from whole blood at 4 °C (Group-2 donors). Each unit was aseptically divided into three parts, stored frozen for specific periods of time, and analysed by flow cytometry for procoagulant activity immediately after thaw or following post-thaw storage for 24 h at 4 °C. Donors' haematologic, biochemical and life-style profiles as well as circulating microparticles were analysed in parallel. Circulating microparticles exhibited a considerable interdonor but not intergroup variation. Fresh frozen plasma units were enriched in microparticles compared to plasma in vivo. Duration of storage significantly affected platelet- and red cell-derived microparticles. Fresh frozen plasma prepared directly from whole blood contained more residual platelets and more platelet-derived microparticles compared to fresh frozen plasma prepared from platelet-rich plasma. Consequently, there was a statistically significant difference in total, platelet- and red cell-derived microparticles between the two preparation protocols over storage time in the freezer. Preservation of the thawed units for 24 h at 4 °C did not significantly alter microparticle accumulation. Microparticle accumulation and anti-oxidant capacity of fresh frozen plasma was positively or negatively correlated, respectively, with the level of circulating microparticles in individual donors. The preparation protocol and the duration of storage in the freezer, independently and in combination, influenced the accumulation of microparticles in fresh frozen plasma units. In contrast, storage of thawed units for 24 h at 4 °C had no significant effect on the concentration of microparticles.

Placental Microparticles and MicroRNAs in Pregnant Women with Plasmodium falciparum or HIV Infection.

BackgroundDuring pregnancy, syncytiotrophoblast vesicles contribute to maternal tolerance towards the fetus, but also to pathologies such as pre-eclampsia. The aim of the study was to address whether Plasmodium falciparum and HIV infections in pregnancy affect the secretion, microRNA content and function of trophoblast microparticles.MethodsMicroparticles were isolated and characterized from 122 peripheral plasmas of Mozambican pregnant women, malaria- and/or HIV-infected and non-infected. Expression of placenta-related microRNAs in microparticles was analysed by qPCR and the effect of circulating microparticles on dendritic cells assessed by phenotype analysis and cytokine/chemokine measurement.ResultsConcentrations of total and trophoblast microparticles detected by flow cytometry were higher in HIV-positive (P = 0.005 and P = 0.030, respectively) compared to non-infected mothers, as well as in women delivering low birthweight newborns (P = 0.032 and P = 0.021, respectively). miR-517c was overexpressed in mothers with placental malaria (P = 0.034), compared to non-infected. Microparticles from HIV-positive induced a higher expression of MHCII (P = 0.021) and lower production of MCP1 (P = 0.008) than microparticles from non-infected women.ConclusionsIn summary, alterations in total and trophoblast microparticles associated with malaria and HIV in pregnant women may have an immunopathogenic role. The potential for placental-derived vesicles and microRNAs as biomarkers of adverse outcomes during pregnancy and malaria infection should be confirmed in future studies.

Distinct features of circulating microparticles and their relationship with disease activity in inflammatory bowel disease.

BackgroundThere is evidence that circulating microparticles (MPs) and annexin (+) platelet-derived MPs (PDMPs) are increased in inflammatory bowel disease (IBD). The aim of our study was to characterize the abundance, origin, and annexin V binding of MPs in patients with IBD and correlate them with the disease characteristics.MethodsCase-control study of 46 IBD patients (23 Crohn’s disease, 23 ulcerative colitis) and 40 matched healthy controls (HC). MPs were divided according to annexin V binding, their origin was estimated based on specific cell membrane markers in plasma samples and their number was calculated via flow cytometry. Clinical and laboratory activity indices were also analyzed.ResultsAnnexin (-) PDMPs (P=0.0004), total (P=0.04) and annexin (+) monocyte-derived MPs (P=0.02) were increased and annexin (-) total MPs (P=0.0007) were decreased in IBD patients compared to HC. The annexin (+)/(-) ratio of all MP types were significantly elevated in IBD patients compared to HC (P<0.003). IBD patients with active disease displayed elevated total and annexin (+) total MPs, total, annexin (+) and (-) PDMPs compared with those in remission (P<0.05). Annexin (-) PDMPs were considerably increased in IBD patients with active compared to those with inactive disease (P=0.0013). Total and annexin (-) PDMPs were significantly correlated with most of the disease activity indices (P<0.05).ConclusionThe majority of circulating MPs, their counterparts and particularly annexin (-) PDMPs are increased in active IBD patients. Annexin (+)/(-) ratio proved to be the most reliable distinctive MP index between HC and IBD patients.

Cell-Derived Microparticles in Patients with Type 2 Diabetes Mellitus: a Systematic Review and Meta-Analysis

Background/Aims: The aim of this study was to assess the association between circulating cell-derived microparticles (MPs) and type 2 diabetes mellitus (T2DM). Methods: A literature search was performed systematically in PubMed and Embase to identify available case-control or cross-sectional studies that compared different types of cell-derived MPs in patients with T2DM and non-diabetic controls. Pooled standardized mean differences (SMDs) of each MP type were pooled using meta-analysis. Results: Forty-eight studies involving 2,460 patients with T2DM and 1,880 non-diabetic controls were included for systematic review and 34 of which were included for quantitative study by meta-analysis. In the overall analysis, the levels of circulating total MPs (TMPs), platelet-derived MPs (PMPs), monocyte-derived MPs (MMPs) and endothelium-derived MPs (EMPs) were significantly higher in T2DM patients than those in controls (TMPs: SMD, 0.64; 95%CI, 0.12∼1.15; P=0.02; PMPs: SMD, 1.19; 95%CI, 0.88∼1.50; P <0.00001; MMPs: SMD, 0.92; 95%CI, 0.66∼1.17; P <0.00001; EMPs: SMD, 0.73; 95%CI, 0.50∼0.96; P <0.00001). Meanwhile, no significant difference was shown in leukocyte-derived MPs (LMPs) level between diabetic and non-diabetic groups (SMD, 0.37; 95%CI, -0.15∼0.89; P=0.17). Conclusions: The counts of TMPs, PMPs, MMPs and EMPs elevated in patients with T2DM. And cell-derived MPs may play a role in the pathogenesis of T2DM.

P67: Use of extracellular RNA from urine for the diagnosis of prostate cancer

Differently sized microparticles, including exosomes (30–100 nm), prostasomes (50–500 nm), oncosomes (50–500 nm) and other microparticles (100–1000 nm) were found in blood and urine. Exosomes from prostate cancer (PCa) patients can potentially contain cancer-specific nucleic acids, and thus can represent a valuable source of diagnostic material. In this study, we have investigated microvesicles and miRNA from urine of healthy donors and PCa patients. To isolate miRNAs from urine and microparticles, novel methods for miRNA isolation were elaborated (Rus. patent application No 2014137763, priority date 17.09.2014). The study population included 14 patients with PCa (63–82 years, T2-3NxMx1) and control group of 20 healthy volunteers with no previous history of prostate disease (48–73 years). Urine was clarified by two serial centrifugations at 400 g , 20 °C, 20 min and at 17000 g , 20 °C, 20 min. Microparticles were precipitated from the resulting supernatant by high-speed centrifugation at 100000 g , 18 °C, 90 min, the pellet was resuspended and pelleted by centrifugation under the same conditions. To isolate exosomes, total microparticles were filtered through 0.1 μm pore filters and reprecipitated. The resulting pellets were resuspended and exosome samples were investigated by transmission electron microscopy (TEM). MiRNAs were isolated by one-step single-phase protocol and purified using “BioSilica” spin- columns (Zaporozhchenko et al., Anal. Biochem, upcoming, doi: 10.1016/j.ab.2015.03.028) and by recently developed method based on precipitation of excess biopolymers, allowing to isolate miRNAs with better efficiency than commercially available kits. The size and quantity assessment of extracellular RNA were performed using capillary electrophoresis system on Agilent 2100 Bioanalyzer. Concentrations of miRNAs (miR19b, miR25, miR205, miR125b, miR126) were measured by qRT-PCR and normalized to miR-16 using dCq method. TEM demonstrated the presence of 20–300 nm microparticles in urine of healthy donors and PCa patients. Approximately 50–70% of all urine microparticles are represented by 30–100 nm exosomes and residual 30–50% by particles larger than 100 nm. (The pool of urine microvesicles consists of 50–70% exosomes and 30–50% particles larger than 100 nm.). The major part of extracellular RNA found both in exosomes and total microparticles fraction of healthy donors and patients with PCa, is 25–200 nt long and can include tRNA (73–93 n.), 5.8 rRNA (∼150 n.), snoRNK (10–20 n.), snRNA (60–300 n.) piRNAs (29–30 n.), miRNAs (20–25 n.), siRNA (21–25 n.). Concentration of extracellular urine RNA in exosomes and total microparticles of healthy donors and patients with PCa amounts to100 pg/ml of urine on average.Receiver Operating Characteristic (ROC) curve analysis of all miRNAs in samples isolated from total urine did not demonstrate any diagnostic significance. In contrast, in training cohort concentration of miR-19b in exosomes and total microparticles fraction provide 95%/75% and 93%/100% sensitivity and specificity, respectively. Thus, microparticles, isolated from urine of PCa patients represent a valuable source of diagnostically significant miRNAs. To investigate miR-19b, a large testing cohort is required. Search for other potential miRNA-markers by microarray profiling of cell-free miRNA isolated from urine of PCa patients is also required to reveal a sold set of markers for confident PCa diagnostics.

Polylactide-based microspheres prepared using solid-state copolymerized chitosan and d,l-lactide

Amphiphilic chitosan-g-poly(d,l-lactide) copolymers have been manufactured via solid-state mechanochemical copolymerization and tailored to design polyester-based microspheres for tissue engineering. A single-step solid-state reactive blending (SSRB) using low-temperature co-extrusion has been used to prepare these copolymers. These materials have been valorized to stabilize microspheres processed by an oil/water emulsion evaporation technique. Introduction of the copolymers either in water or in the oil phase of the emulsion allowed to replace a non-degradable emulsifier typically used for microparticle preparation. To enhance cell adhesion, these copolymers were also tailored to bring amino-saccharide positively charged segments to the microbead surface. Size distribution, surface morphology, and total microparticle yield have been studied and optimized as a function of the copolymer composition.

Effects of lipid-lowering treatment on circulating microparticles in patients with diabetes mellitus and chronic kidney disease.

Elevated levels of circulating microparticles (MPs) may contribute to the high cardiovascular risk in diabetes mellitus (DM) and chronic kidney disease (CKD). Therefore, we investigated the effects of lipid-lowering treatment (LLT) with simvastatin alone (S) or with ezetimibe (S+E) on MPs in DM patients with or without CKD. After a placebo run-in period, 18 DM patients with an estimated glomerular filtration rate (eGFR) of 15-59 mL/min (CKD stages 3-4) (DM-CKD) and 21 DM patients with eGFR >75 mL/min (DM-only) were treated with S and S+E in a randomized, double-blind, crossover study. MPs from platelets, monocytes and endothelial cells (PMPs, MMPs and EMPs), and their expression of phosphatidylserine (PS), P-selectin, CD40 ligand (CD40L) and tissue factor (TF) were measured by flow cytometry. At baseline, all types of MPs, except TF-positive MMPs, were elevated in DM-CKD compared with DM-only patients. All MPs, regardless of origin and phenotype, were inversely correlated with eGFR. S reduced the expression of P-selectin, TF and CD40L on PMPs and of TF on MMPs in both patient groups. S+E had no further effect. S also reduced total PS-positive procoagulant MPs, PMPs and MMPs in DM-CKD but not in DM-only patients. DM patients with CKD stages 3-4 had elevated PMPs, EMPs and MMPs compared with DM patients with normal GFR. Simvastatin reduced procoagulant MPs, MMPs and PMPs in DM-CKD patients, suggesting a beneficial reduction of hypercoagulability in this high-risk patient group. Differences between DM-CKD and DM-only patients were counteracted by LLT.

Once versus twice daily injection of enoxaparin for thromboprophylaxis in bariatric surgery: effects on antifactor Xa activity and procoagulant microparticles. A randomized controlled study.

The optimal scheme of thromboprophylaxis in bariatric surgery remains uncertain, because clinical practice is different between countries and randomized trials are lacking. The primary objective of this randomized multicenter study was to determine the optimal regimen of enoxaparin providing an antifactor Xa peak activity between .3 and .5 IU/mL at equilibrium and to evaluate the course of procoagulant microparticles (MPs). University hospital. A total of 164 patients scheduled for gastric bypass were allocated to 3 groups (A, B, and C) of enoxaparin treatment (4000, 6000, or 2×4000 IU, respectively). Antifactor Xa activity was measured before and 4 hours after each injection from D0 to D2. Doppler screening of the lower limbs was performed at D1, D9, and D30. Bleeding (BE) and thrombotic events (TE) were recorded during the first postoperative month. Total MPs were measured at D0, D9, and D30. MPs of leucocyte, platelet, and granulocyte origin were assessed in one third of the patients from each group. The 3 groups were compared by ANOVA. A total of 135 patients were analyzed. The equilibrium of antifactor Xa peak levels was obtained 52 hours after the presurgery injection and 12.8%, 56.4%, and 27.3% of the patients reached the target in groups A, B, and C, respectively (P<.001). No TE was detected. BE occurred in 1, 2, and 6 patients in groups A, B, and C, respectively). Total MPs remained unchanged over time. While no significant variation was observed in the other groups, platelet GP1 b(+)-MPs increased (P = .01) at D9 in group C, suggesting an incomplete control of anticoagulation leading to cell activation and procoagulant MP release that was confirmed by the higher MP levels measured at D30 (P = .04). CD66(+)-MPs were also highly elevated at J9 and D30 in group C indicating a granulocyte contribution. This study shows that a single dose of enoxaparin 6000 IU/d allowed most of the patients to reach the target range of antifactor Xa activity without increasing the bleeding risk, with the most likely efficient reduction of procoagulant MPs. (Surg Obes Relat Dis 2015;0:000-000.) © 2015 American Society for Metabolic and Bariatric Surgery. All rights reserved.

High correlation between 2 flow cytometry platforms in the microparticles analysis using a new calibrated beads strategy

Microparticles (MPs) are potential noninvasive biomarkers for diagnosis or prognosis in pathologic conditions. However, the lack of standardization of the preanalytical and analytical methods leads to a wide variability in MPs results. The recently developed Megamix-Plus beads, a new bead-based standardization tool optimized to specific types of flow cytometers, could help circumvent this problem. The aim of the present study was to determine whether the number of total MPs and platelet-derived MPs (PMPs) is similar using 2 different cytometer platforms calibrated with the Megamix-Plus beads. Blood samples from 65 patients with deep venous thrombosis were collected and processed to obtain platelet poor plasma (PPP). The number of total MPs and PMPs in each PPP sample was measured using 2 flow cytometers. Megamix-Plus side scatter channel beads were used to calibrate the LSRFortessa flow cytometer from Becton Dickinson, whereas Megamix-Plus forward scatter channel beads were applied to the Navios flow cytometer from Beckman Coulter. High correlation of total MPs and PMPs values between the flow cytometers was found (r = 0.908, P < 0.01 and r = 0.910, P < 0.001, respectively). However, the absolute numbers of total MPs and PMPs were significantly higher measured with the Navios flow cytometer compared with the LSRFortessa cytometer. Therefore, both platforms are valid for MPs determination in general, although a similar platform with the same calibration tool could be a better choice for multicenter studies.

Cell-derived microparticles after exercise inindividuals with G6PD Viangchan.

Glucose-6-phospate dehydrogenase (G6PD) deficient cells are sensitive to oxidative damage leading to the formation of microparticles (MPs). Therefore, we examined the concentration of MPs and changes in the antioxidant balance after an acute strenuous exercise (SEx) and moderate-intensity exercise (MEx). Eighteen healthy females (18-24 years) with G6PD normal and eighteen age-matched females with G6PD Viangchan (871G>A) were tested by running on a treadmill at their maximal oxygen uptake for SEx and at 75% of their maximal heart rate for MEx. It was found that SEx triggered the release of total microparticles (TTMPs) above baseline levels and remained significantly higher 45 minutes after the exercise in G6PD normal individuals. However, SEx-induced increase in TTMPs was significantly higher in G6PD Viangchan as compared to G6PD normal. In contrast, MEx did not to alter the release of TTMPs in both G6PD normal and Viangchan. Moreover, TTMPs concentrations were inversely correlated with G6PD activity (r =-0.82, P < 0.05) but positively correlated with MDA concentrations (r = 0.74, P < 0.05). Using cell specific antibodies, we determined that MPs were mainly derived from platelets and erythrocytes. Altogether, the present study indicates that G6PD Viangchan may participate in MEx without higher MPs concentration and oxidative stress compared with G6PD normal.

Origin and levels of circulating microparticles in normal pregnancy: A longitudinal observation in healthy women

Objective. Microparticles (MP) are actively involved in the hypercoagulable state reported both in normal pregnancies and in pregnancy diagnosed with placenta-mediated complications. In this study the origin and the levels of plasma MP as well as MP activity were evaluated in a group of healthy women during the three trimesters of a normal pregnancy. Materials and methods. Seventy-five healthy normotensive pregnant women were enrolled and blood samples were prospectively collected at three different time points corresponding to 1st trimester, 2nd trimester, 3rd trimester of pregnancy. A group of age- matched healthy non-pregnant women acted as controls. Both standard clotting parameters and MP of different origin were measured. MP were identified by size and annexin V- FITC labelling using flow-cytometer. MP subtypes were identified using specific monoclonal antibodies. Procoagulant activity of MP was assessed using the STA® Procoag PPL assay. Results. The levels of total, platelet-, endothelial-, leukocyte-derived and tissue factor-bearing MP, as well as the MP procoagulant activity, in non-complicated pregnancy were higher in the 1st trimester as compared to non-pregnant age-matched women. Regardless of the origin, MP levels gradually increase during pregnancy, with the highest values reached in the 3rd trimester. Conclusions. MP levels gradually increase during normotensive pregnancy. All types of MP including TF+ present with the highest levels in the 3rd trimester. MP convey prothrombotic and proinflammatory antigens already from the first trimester of normal pregnancy. This may contribute to the global hypercoagulable state observed, particularly in the last months of pregnancy, also in healthy women.

Circulating microparticles in umbilical cord blood in normal pregnancy and pregnancy with preeclampsia

Placenta microthrombi being one of the prevalent recurrent histological findings in women with preeclampsia (PE), it is reasonable to think that the study of coagulation alterations in cord blood could be more informative than that observed in maternal blood. The aim of the present study was to measure different subtypes of microparticles (MP) plasma levels in the maternal peripheral blood at labour and in the venous cord blood of pregnant women with PE compared to those in a group of women without PE. Thirty-two pregnant women in labour, 16 with and 16 without PE, were enrolled. Blood samples were collected immediately after delivery from cord blood and from maternal peripheral blood. Total, cellular-derived and tissue factor- bearing MP were analyzed using flow-cytometry. Procoagulant activity of MP was assessed using the STA® Procoag PPL assay. Total MP, platelet activated-derived (P-Selectin+), leukocyte-derived and TF+MP were higher in pregnancies complicated by PE as compared with normotensive women (p<0.05). Platelet-derived MP (CD61+) levels were lower in PE than in healthy women and no difference was found in endothelial-derived MP levels between the two groups. The PPL clotting time was significantly shorter in PE compared with controls. When only venous cord blood was analysed, all MP detected were significantly higher in PE than in healthy normotensive women (p<0.05). MP are very likely involved in the hypercoagulable and pro-inflammatory intravascular reactions during PE. Prospective studies in a larger population are needed to define the clinical meaning of MP measurement in the PE setting.

Microparticles impact coagulation after traumatic brain injury

BackgroundThe pathophysiology that drives the subacute hypercoagulable state commonly seen after traumatic brain injury (TBI) is not well understood. Alterations caused by TBI in platelet and microparticle (MP) numbers and function have been suggested as possible causes; however, the contributions of platelets and MPs are currently unknown. Materials and methodsA weight-drop technique of TBI using a murine model of moderate head injury was used. Blood was collected at intervals after injury. MP enumeration and characterization were performed using Nanoparticle Tracking Analysis, and platelet counts and coagulation parameters were determined using thromboelastometry. A MP procoagulant assay was used to compare activity between injured and sham mice. ResultsAt 24 h after injury, there were no changes in circulating platelet numbers. However, there was a decrease in platelet contribution to clot formation. In contrast, there was a decline in circulating total MP numbers. When MPs from sham mice were added to the blood from head-injured animals, there was a normalization of platelet contribution to clot formation. Conversely, when MPs from TBI mice were added to sham blood, there was a significant decrease in platelet contribution to clot formation. Notably, there was an increase in MP procoagulant activity in head-injured mice. ConclusionsMPs generated after TBI likely contribute to altered coagulation after head injury and may play a key role in the development of a posttraumatic hypercoagulable state in TBI patients.

Bone Marrow Contains High Levels of Microparticles

Background: Microparticles (MP) are shed in humans from a broad variety of cells and play an important role in activation of coagulation, cell to cell interaction and transport of membrane components. Alternations in plasma levels have been found in a variety of diseases, mostly associated with thrombosis and inflammation. However, little is known about tissue levels of MP. The aim of this pilot study was to investigate the bone marrow compartment as a possible source of circulating MP.Material and Methods: MP were analysed in bone marrow blood and peripheral blood in 12 healthy stem cell donors. MPs were isolated and measured by flow-cytometric double-staining (FACScalibur, BD) with Anexin V and cell-specific antibodies for hematopoietic cells, platelets, white blood cells, red blood cells and endothelial cells. Statistical analysis was performed with SPSS for Windows 20.0.Results: Total MP levels differed between bone marrow and peripheral blood: 14.8 x109/l [8.5–19.3] vs. 9.2 x109/l [3.8–14.8] (median value [25-75 percentile]), p=0.060. Pattern of MP´s origin varied likewise: in bone marrow blood (bmb), main cell source of MP were CD235a positive red blood cells/erythropoetic cells (6.4x109/l [3.7–13.7], 43.2% of total bmb MP), CD61 positive platelets/megacaryocytes (4.1x109/l [1.7–10.1], 27.7% of total bmb MP), CD45 positive leucocytes/myeloic and lymphatic progenitor cells (3.8x109/l [2.7-4.9], 25.7% of total bmb MP) and CD62e positive endothelial cells (0.3x109/l [0.1-1.0], 2.0% of total bmb MP). In contrast, MP in peripheral blood (pb) mainly derived from CD61 positive platelets/megacaryocytes (8.5x109/l [2.4–14.4], 92.0% of total pb MP), CD45 positive leucocytes/myeloic and lymphatic progenitor cells (0.5x109/l [0.2–1.1], 4.5% of total pb MP), CD235a positive red blood cells/erythropoetic cells (0.2x109/l [0.1–0.6], 1.8% of total pb MP), and CD62e positive endothelial cells (0.1x109/l [0.0–0.1], 0.9% of total pb MP). Mean levels of tissue factor bearing MP were low in bone marrow blood as well as in peripheral blood (0.19 x109/l [0.16 – 0.29] and 0.08 x109/l [0.05 – 0.12], respectively, p=0.004).Conclusion: Within our study we were able to detect MP in bone marrow blood. When comparing MP from bone marrow and peripheral blood, major differences with regard to cell source and concentration was present. Our data suggest that the blood – bone marrow barrier is not only existentant for cells, but also for circulating MP. DisclosuresNo relevant conflicts of interest to declare.

Microparticles Modulate Formation, Structure, and Properties of Fibrin Clots

Microparticles (MPs) are phospholipid vesicles about 0.1-1 μm in size released into blood from vascular and blood cells upon activation and apoptosis. The mechanism of MP formation by the exocytic budding of the outer cell membrane provides them with procoagulant activity, mainly due to phosphatidylserine exposure and tissue factor expression. MPs are present in the peripheral blood of healthy subjects but their level is elevated in a variety of vascular, infectious, and immune-mediated pathologies. While there are extensive studies on multiple roles of MPs in various diseases, including thrombotic disorders, the question remains open as to whether MPs are involved in the formation and properties of fibrin, the scaffold of hemostatic clots and obstructive thrombi. To investigate this problem, we studied in vitro the effects of MPs on the kinetics of fibrin polymerization, fibrin clot structure and susceptibility to fibrinolysis.Blood was drawn from healthy donors according to an IRB protocol. MPs were removed from citrated platelet-free plasma (PFP) by filtration through 0.1-μm pores, yielding microparticle-depleted plasma (MDP). To restore the phospholipids eliminated, MDP was replenished with cephalin (MDP-C). Fresh samples of PFP, MDP, and MDP-C from the same donor were analyzed in parallel. Concentrations of MPs and expression of platelet-specific CD61 were studied by flow cytometry and confocal microscopy. A thrombin generation test in recalcified defibrinated plasma was performed using a chromogenic substrate. Fibrin polymerization induced by recalcification of PFP, MDP, and MDP-C was studied by dynamic turbidimetry. tPA-induced fibrinolysis was also followed optically as a decrease of a plasma clot turbidity. Fibrin ultrastructure was studied with scanning electron microscopy (SEM) in a FEI Quanta 250 microscope. Each test was performed with plasma samples from at least 3 donors and the results were averaged.Enumeration of MPs in PFP vs. MDP showed that about 90% of total MPs were removed by filtration, including 99.6% of CD61-positive platelet-derived MPs. The rate of thrombin generation was significantly reduced in MDP vs. PFP and was fully restored in MDP-C, confirming an essential role of MPs in the intrinsic coagulation pathway. Based on the lag times and slopes of the turbidimetric curves, fibrin formation in recalcified plasma samples was significantly delayed in the absence of MPs (MDP) and was much faster in their presence (PFP and MDP-C). The maximum optical density was consistently and significantly higher in MDP than in PFP and MDP-C, suggesting that fibrin clots formed in the absence and presence of MPs have fibers of different thickness. This presumption was confirmed by SEM of fibrin clots formed upon recalcification of the plasma samples. Clots formed in PFP were less porous and had thinner fibers (170 ± 38 nm), while clots from MDP had larger pores and were built of thicker fibers (214 ± 53 nm, p<0.01). Addition of phospholipids to MDP (MDP-C) resulted in the formation of densely packed thin fibrin fibers (141 ± 34 nm), similar to the initial clots from PFP. Consistent with the dissimilarity in ultrastructure, the clots displayed a distinct susceptibility to fibrinolysis: the rate of clot lysis was significantly higher in MDP than in PFP and MDP-PL. A novel and important finding from the SEM of fibrin clots is that fibers formed in MDP were quite smooth, while fibers formed in the presence of MPs (PFP and MDP-C) had relatively rough surfaces with multiple sub-micron size particles attached, suggesting that MPs might directly bind fibrin. Consistent with the SEM of the appearance of MPs on the fibers, confocal microscopy of PFP-clots stained for CD61 with FITC-labeled antibodies, unlike MDP-clots, revealed fluorescent spots associated with fibrin, indicating that MPs were adsorbed on fibrin fibers.In conclusion, MPs have profound effects on fibrin formation and on the final structure and characteristics of fibrin clots via at least two mechanisms. One is an indirect kinetic effect based on the MP-dependent rate of thrombin generation. The other is direct binding of MPs to fibrin fibers during and after fibrin assembly. Both mechanisms underlie a previously underappreciated potential role of MPs in hemostasis and thrombosis as modulators of fibrin formation and properties.(Research supported by NIH grant HL090774 and the Program of Competitive Growth of Kazan Federal University) DisclosuresNo relevant conflicts of interest to declare.