Activity Of Microparticles Research Articles

Ethnic Minority Microparticles have Distinct Pro-Thrombotic and Pro-Oxidative Phenotypes and Interact Differentially with Endothelial Cells in vitro: Implications for Risk to Cardiovascular Disease

Ethnic minority individuals are disproportionately susceptible to endothelial dysfunction and cardiovascular disease (CVD). Microparticles (MP) are biologically active membrane-bound nanovesicles released from cells that act as biomolecular shuttles. Plasma MP was isolated from healthy White, Black African, and South Asian individuals and analysed using flow cytometry. Their effects and interactions were assessed using fluorescence, confocal, and scanning electron microscopy. Total MP and a sub-population of smaller MP associated with dysfunction and disease progression were significantly increased in Black African individuals. Pro-thrombotic and pro-oxidant MP were substantially more numerous in Black African individuals. The tissue factor activity of ethnic minority MP was significantly greater than White MP. Ethnic minority MP induced significantly greater functional changes and morphology to an endothelial cell line in vitro and integrated into endothelial cells noticeably more than White MP. These data imply distinct differences in ethnic minority MP, suggesting a role in CVD susceptibility.

MPs-ACT, an Assay to Evaluate the Procoagulant Activity of Microparticles.

The procoagulant effect of microparticles (MPs) contributes to hypercoagulability-induced thrombosis. We provide preliminary findings of the MPs-Activated Clotting Time (MPs-ACT) assay to determine the procoagulant activity of MPs. MPs-rich plasma was obtained and recalcified. Changes in plasma viscoelasticity were evaluated and the time to the peak viscoelastic changes was defined as the MPs-ACT. MPs concentration was measured by flow cytometry. Coagulation products produced during plasma clotting were identified by fibrin and fibrinopeptide A. MPs were prepared in vitro and added to standard plasma to simulate pathological samples. In addition, reproducibility and sensitivity were evaluated. We confirmed the linear relationship between MPs-ACT and MP concentrations. Dynamic changes in fibrin production were depicted. We simulated the correlation between MPs-ACT and standard plasma containing MPs prepared in vitro. The reproducibility of high-value and low-value samples was 6.0% and 10.8%, respectively. MPs-ACT sensitively detected hypercoagulable samples from patients with pre-eclampsia, hip fractures, and lung tumors. MPs-ACT largely reflects the procoagulant effect of MPs. MPs-ACT sensitively and rapidly detects hypercoagulability with MPs-rich plasma. It may be promising for the diagnosis of hypercoagulable states induced by MPs.

Enhancement in Site-Specific Delivery of Chloramphenicol Using Bacterially Sensitive Microparticle Loaded Into Dissolving Microneedle: Potential For Enhanced Effectiveness Treatment of Cellulitis.

One of the biggest challenges in infectious disease treatment is the existence of bacterial infections in underskin wound tissue, such as cellulitis. Compared to other treatments, it is harder for antibacterial drugs to penetrate the physical barrier on the affected skin with a nonspecific target, making conventional therapy for cellulitis infection more difficult and considered. In this novel research, we pioneer a combined strategy of dissolving microneedles (MNs) and bacteria-sensitive microparticles (MPs) for enhanced penetration and targeted delivery of chloramphenicol (CHL) to the infection site specifically. The polycaprolactone polymer was used to make MPs because of its sensitivity to bacterial enzyme stimuli. The best microparticle formulation was discovered and optimized using the Design-Expert application. Furthermore, this study evaluated the antibacterial activity of MPs in vitro and in vivo on the mutant Drosophila larval infection model. This strategy shows improvement in the antibacterial activity of MPs and higher retention duration compared to conventional cream formulation, and the inclusion of these MPs into dissolving MNs was able to greatly improve the dermatokinetic characteristics of CHL in ex vivo evaluation. Importantly, the antimicrobial efficacy in an ex vivo infection model demonstrated that, following the use of this strategy, bacterial bioburdens decreased by up to 99.99% after 24 h. The findings offered a proof of concept for the enhancement of CHL dermatokinetic profiles and antimicrobial activities after its preparation into bacteria-sensitive MPs and distribution by MNs. Future research should investigate in vivo effectiveness in an appropriate animal model.

Preliminary study of microparticle coagulation properties in septic patients with disseminated intravascular coagulation.

BackgroundSepsis typically results in enhanced coagulation system activation and microthrombus formation. Microparticle (MP) production promotes coagulation and enhances pro-coagulation. This study investigated how circulating MP levels and tissue factor-bearing MP (TF+-MP) activity caused coagulation in patients with septic disseminated intravascular coagulation (DIC).MethodsThirty patients with septic DIC and 30 healthy controls were studied from December 2017 to March 2019. Patient blood samples were collected at enrolment (day 1) and on days 3 and 5; DIC scores and Sequential Organ Failure Assessment (SOFA) scores were recorded. TF+-MP activity was measured using TF-dependent factor Xa generation experiments. Circulating MP concentrations were determined by MP capture assay. Clotting factor activity, antithrombin level, soluble thrombomodulin, and serum tissue factor pathway inhibitor (TFPI) concentrations were measured.ResultsPatients with septic DIC had lower circulating MP levels than healthy control patients. Circulating MP levels in patients with septic DIC were positively correlated with DIC scores and negatively correlated with coagulation factors, but TF+-MP activity did not correlate with clotting factor levels and TFPI.ConclusionsIn patients with septic DIC, circulating MP levels are important in promoting coagulation activation and increasing clotting factor consumption. TF+-MP activity may not be the main form of active TF.

Improvement in the cardiovascular profile of patients with morbid obesity following bariatric surgery: Effect on hypercoagulability.

Obesity is an inflammatory state related to vascular endothelium dysfunction. It generates a biological situation of hypercoagulability increasing the risk of thrombosis. This prothrombotic condition could be improved by bariatric surgery.The main objective was to analyze the impact of bariatric surgery on cardiovascular risk factors (CVRF) associated with changes in thrombin generation and procoagulant activity of microparticles (MP).We present a prospective longitudinal study including consecutive patients candidate for bariatric surgery. We performed 3 sequential clinical visits: at inclusion, before surgery after completing the modified fasting phase, and 6 months after surgery. We analyzed CVRF, thrombin generation, and MP activity. The data analysis was performed using a logistic regression model to determine changes over time of hemostatic parameters and body mass index (BMI). McNemar test for binary variables was used to analyze the CVRF.We included 94 patients (66 women), with an average age of 45.7 ± 10.1 years. The mean BMI reduction at the end of the follow-up was 15.5 ± 4.2 kg/m2. We detected a statistically significant improvement in CVRF: hypertension, diabetes mellitus, dyslipidemia, and obstructive sleep apnea, as well as a significant reduction in thrombin generation capacity and procoagulant MP activity.Massive weight loss induced by bariatric surgery improves the cardiovascular profile, associated with a reduction in the hypercoagulable status.

Particulate β-glucan activates early and delayed phagosomal maturation and autophagy within macrophage in a NOX-2 dependent manner

AimsMacrophage is known to readily engulf any particulate material they encounter, including invading microbes and nano- or micro-particles. While recent studies show that some microparticles (MP) are immunogenic even without drug-cargo, the mechanism underlying this phenomenon is yet unclear. Phagocytosis induces NADPH oxidase-2 (NOX-2) mediated ROS generation that is reported to regulate antibacterial autophagy. We therefore, investigated the role of NOX-2 derived ROS in phagosomal maturation and autophagy induction in response to phagocytic uptake of two kinds of polymeric biodegradable and biocompatible microparticles: yeast-derived β-glucan particles (YDGP) and poly-(D, L-Lactic Acid) microparticles (PMP). Main methodsJ774A.1 macrophage wereas exposed to polymeric particles and the immune responses: ROS, phagosomal maturation and autophagy induction, were examined by assays including NBT, DCFH-DA, NADPH-Oxidase activity, Lysotracker and Acridine Orange. Further, the LC3 and NOX-2 expression were validated by RT-PCR, immunofluorescence assay and Western blotting. Antimicrobial activity of both MP was examined by CFU counting after administration to Mycobacterium tuberculosis and Salmonella typhimurium infected macrophage. Key findingsYDGP induces phagosomal maturation and acidic vesicle accumulation at 30 min and 24 h post-exposure, much more proficiently than that by PMP. YDGP exposure also induced NOX-2 dependent expression of light chain 3 (LC3-II), further confirmed as autophagy activation via autophagic flux assay with autophagolysosome inhibitor bafilomycin A1. Additionally, YDGP displayed superior anti-microbial activity than that by PMP. SignificanceThe induction of NOX-2-dependent autophagy and antimicrobial activity exhibited by particulate glucans has significant implications in harnessing these drug delivery vehicles as potential ‘value-added’ autophagy-mediated therapeutics in future.

Procoagulant microparticles are associated with arterial disease in patients with systemic lupus erythematosus.

Microparticles (MPs) have been associated with inflammatory and thrombotic disease. High levels of MPs have been identified in patients with systemic lupus erythematosus (SLE) and associated with cardiovascular disease. We analyzed the procoagulant activity of MPs and itscorrelation with arteriosclerosis and arterial thrombosis in SLE patients. Eighty-seven patients with SLE were included: 22 (25.3%) with associated antiphospholipid syndrome (APS), 32 (36.8%) without antiphospholipid antibodies (aPL) and 33 (37.9%) with aPL but without APS. Subclinical arteriosclerosis, defined as the presence and number of plaques, was evaluated by ultrasonography of carotid arteries. Thrombotic events were confirmed by objective methods. The procoagulant activity of MPs was determined by a functional assay with annexin V. Subclinical arteriosclerosis was found in 19 (21.8%) patients. Thirteen episodes of arterial thrombosis and eight of venous thrombosis were recorded. The procoagulant activity of MPs was greater in patients with arterial thrombosis (17.28 ± 8.29nM vs 12.96 ± 7.90nM, p < 0.05). In patients without arterial thrombosis, greater procoagulant activity of MPs was identified in patients with multiple (≥ 2) carotid plaques (17.26 ± 10.63nM vs 12.78 ± 7.15nM, p = 0.04). In the multivariate analysis, the procoagulant activity of MPs was independently associated with multiple (≥ 2) carotid plaques and arterial thrombosis [OR = 1.094 (95%CI 1.010-1.185), p = 0.027 and OR = 1.101 (95%CI 1.025-1.182), p = 0.008; respectively]. In conclusion, the procoagulant activity of MPs is associated with arteriosclerosis burden and arterial thrombosis in patients with SLE.

High Level of Circulating Microparticles in Patients with BCR/ABL Negative Myeloproliferative Neoplasm - a Pilot Study.

Microparticles (MPs) are small (0.1-1 &#956;m) cell-derived vesicles released during activation or apoptosis, with a surface-exposed phosphatidylserine along with antigens indicating the cellular origin. The level of MPs is known to be elevated in thromboembolic diseases and malignancies; it is believed that MPs are not only amplifying but can also initiate the thrombogenesis processes. BCR/ABL negative myeloproliferative neoplasms (MPNs) are clonal haematopoietic diseases, which include polycythemia vera, essential thrombocythemia and primary myelofibrosis. One of the main problems of MPN patients is high risk and incidence of thrombosis which affect the survival, quality of life and life expectancy. The clinical significance of circulating MPs was assessed in a group of 179 patients with BCR/ABL-negative MPNs. Analysis of MPs was done using flow cytometry on 417 samples, and MPs procoagulation activity was performed using a functional assay called Zymuphen MP-activity (Hyphen Biomed, Neuville-sur-oise, France) on 274 samples. Significantly higher absolute and relative count of platelet MPs was found in MPN patients when compared with healthy group, respectively (p = 0.001, p = 0.043). Erythrocyte MPs were also significantly higher in MPN patients than in the healthy group (p &lt; 0.001). Procoagulation activity of MPs was as well significantly higher in patients compared to the control group (p &lt; 0.001). Patients with primary myelofibrosis had decreased absolute and relative count of platelet MPs compared to polycythemia vera and essential thrombocythemia patients, respectively (p = 0.008, p = 0.014). Presence of JAK2V617F mutation was associated with higher absolute and relative count of platelet MPs, respectively (p = 0.045, p = 0.029). Although some literature data support the hypothesis of a direct relation between MPs and thrombotic events in MPN patients, further studies are needed to evaluate the clinical implication of MPs in the hypercoagulation state of MPN patients.

The Role of Exposed Phosphatidylserine on Microparticles and Blood Cells in Coagulant Activity in Acute Kidney Injury Patients on Continuous Renal Replacement Therapy

Introduction: Serving as the preferred treatment for patients with acute kidney injury (AKI), continuous renal replacement therapy (CRRT) is mostly interrupted by clotting caused by procoagulant state despite of persistent improvements in anticoagulant technology, reducing therapy time, enhancing cost and resulting in blood lost. Due to hemodynamics disorders and foreign material circuit, blood cells undergo definite activation. However, considered as markers of cell activation, the role of phosphatidylserine (PS) and microparticles (MPs) in the hypercoagulability remains largely unexplored. The aim of this study is to measure PS exposure on blood cells and MPs at baseline and after different therapy duration in AKI patients.Methods: Fifty AKI patients admitted to the First Affiliated Hospital of Harbin Medical University between March 2017 and May 2018 and forty healthy controls were enrolled in this study. Plasma samples were collected at baseline from patients were diagnosed with AKI and after receiving CRRT (Mode: CVVH; dose: 25mL/kg/h; Citrate which has relative little influence on PS exposure and MPs count was used as regional anticoagulant.) for 6hours, 12hours, 18hours, as well as from healthy controls. After being isolated, MPs were measured by flow cytometry (FCM) while procoagulant activity (PCA) of erythrocytes, platelets and MPs was assessed through coagulation time and purified coagulation complex. Lactadherin was applied as a probe and inhibitor of PS.Results: Between AKI baseline group and healthy controls, there was no obvious difference in lactadherin+ MPs, exposure on platelets and erythrocytes (Table 1,2), coagulation time and protein production (Fig. 1). While with therapy time prolonged to 6h, 12h and 18h, lactadherin+ MPs and blood cells showed a significant increase compared with baseline group (Table 1,2). Additionally, coagulation time shortened significantly (Fig. 1A) and purified coagulation complex increased visibly in parallel with total therapy time (Fig. 1B-D). Based on previous analysis, we used lactadherin to suppress PS exposure at time point 18h. Compared with the baseline, lactadherin+ MPs count declined by 75.5% (P < 0.001), coagulation time for MPs, platelets and erythrocytes was shortened by 74.6%, 77.2% and 71.9% (all p < 0.01) and procoagulant enzyme complexes were also reduced by 73.8, 78.7% and 72.9% (all p < 0.01). (Figure. 1)Conclusions: Our results first reveal that upon AKI patients on CRRT, a procoagulant role of PS-driven PCA and an immediate time-dependent elevation in PS+ MPs and blood cells is observed. What's more, suppression of PS obviously inhibits the PCA. Accordingly, PS shows potential to be a new predictor and new strategies focusing on blockade of PS may provide a novel way of anticoagulants in CRRT process. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Release of Monocyte-Derived Tissue Factor-Positive Microparticles Contributes to a Hypercoagulable State in Idiopathic Membranous Nephropathy.

Aim: Idiopathic membranous nephropathy (IMN) is an immune-mediated inflammatory disease characterized by a high risk of thromboembolic complications. Microparticles (MPs), a type of extracellular vesicles, have procoagulant properties, especially when they display tissue factor (TF). This study aimed to investigate whether circulating TF-positive MPs contributed to the hypercoagulable state in patients with IMN.Methods: Twenty adult IMN patients and fourteen healthy subjects were included in the study. The basic indexes of a routine biochemical examination and coagulative function were determined. The plasma levels of MPs were detected by flow cytometry, and TF activity of MPs was examined using an assay kit. The plasma levels of lipopolysaccharide (LPS) were measured by an enzyme-linked immunosorbent assay.Results: Total circulating MPs were not increased in patients with IMN compared with healthy controls. Circulating CD14+/TF+MPs were significantly increased in IMN patients, but this achieved significance was not observed in CD41+/TF+MPs between the two groups. Interestingly, the circulating TF-positive MPs were increased significantly. Plasma MPs TF assays revealed high procoagulant activity, which was positively associated with the D-dimer level in IMN. In addition, circulating LPS in IMN patients were significantly higher than those in the controls. Furthermore, after two hours' incubation with healthy whole blood, LPS enhanced the release of circulating TF-positive MPs and the TF activity of MPs.Conclusion: Increased circulating LPS may mediate the release of monocyte-derived TF-positive MPs, which further contributes to the hypercoagulable state in IMN patients. These findings provide an additional mechanism by which patients with IMN have a higher risk of thromboembolic complication.

Influence of annealing temperature on visible-light photocatalytic activities of Ag3PO4 particulates synthesized using CH3COOAg as a precursor

Ag3PO4 microparticles (MPs) were prepared through a facile chemical precipitation route and using silver acetate (AgAc) as metal salt. The effect of annealing temperature (Ta) and time (τa) on the actual photocatalytic (PC) activity of Ag3PO4 MPs is investigated. The optimal annealing parameters are Ta of 400 °C and τa of 90 min. The enhanced PC activity by annealing at 400 °C is ascribed to the increase of electron mobility. Besides, an Ag3PO4 photoelectrode was fabricated through a drop-coating deposition route, which demonstrates a photocurrent density of 80 μA/cm2 and acceptable stability. The n-type conduction behavior of Ag3PO4 is verified by a Mott-Schottky (M-S) plot.

Microparticle Release from Cell Lines and Its Anti-Influenza Activity.

Microparticles (MPs) are vesicles that are released by budding from plasma membrane of living cells. Recently, the role of MPs in antiviral activity has been proposed. We investigated quantity and anti-influenza activity of MPs from human alveolar epithelial cells A549, human bronchial epithelial cells BEAS-2B, human colon adenocarcinoma cells HT-29, and the human lung fibroblast cells MRC-5. MPs were found from all four cell lines. However, anti-influenza activity against an H1N1 influenza virus was found only from MPs of A549 and BEAS-2B. BEAS-2B cell differentiation did not increase MP release. Methyl-β-cyclodextrin (MβCD) increased MP release and anti-influenza activity in HT-29 and A549. MP release increased after calcium ionophore A23187 treatment in three cell lines but only in HT-29 after forskolin treatment. These findings provide in vitro data supporting the role of MPs as an innate defense against influenza virus and as an approach to enhance the defense.

Platelet function and microparticle levels in atrial fibrillation: Changes during the acute episode

BackgroundThrombotic risk constitutes a major complication of atrial fibrillation (AF). Platelets and microparticles (MPs) are important for hemostasis and thrombosis, however their participation during AF is not well known. The aim of this study was to characterize platelet function and MPs procoagulant and fibrinolytic activity in AF patients and to determine the effects of an acute-AF episode. MethodsBlood was collected from paroxysmal (21) and persistent (16) AF patients referred for AF catheter ablation. Ten patients in sinus rhythm for 10days were induced in AF allowing comparisons of left atrium samples before and after induction. Platelet aggregation with ADP, TRAP, collagen, and ristocetin was studied. Platelet surface expression of PAR-1, αIIbβ3, GPIb and P-selectin were evaluated by flow cytometry, and MPs-associated procoagulant and fibrinolytic activity levels were determined by functional assays. ResultsA specific reduction in platelet aggregation to TRAP, activating the thrombin receptor PAR-1, was found in all AF patients. No differences in platelet receptor expression were found. Yet, after acute-induced AF, the platelet response was improved. Furthermore, a significant decrease of left atrium tissue factor-dependent procoagulant activity of MPs was observed. ConclusionAcute episodes of AF results in a decrease in MPs-associated tissue factor activity, possibly corresponding to consumption, which in turn favors coagulation and the local production of thrombin. A decreased platelet basal aggregation to TRAP may result from PAR1 desensitization, whereas the improved response after an induced episode of AF suggests activation of coagulation and PAR1 re-sensitization.

Influence of Hydroxyurea on Neutrophil Microparticles: A SCA Model

Introduction: MPs are stable units that express on their surface specific proteins of the cells that were originated. In this sense, neutrophils MPs have a singular importance since they present in their surface myeloperoxidase (MPO), an enzyme that are involved with vascular inflammatory conditions. Studies have shown that the use of HU, currently the only pharmacological drug approved to treat SCA patients, leads to reduction in count neutrophils, monocytes and reticulocytes, and the slight increase in total hemoglobin concentration. However, some studies have shown that MPs levels are not affected by HU treatment. Recently, our group showed that nod-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome and interleukin (IL)-1β could contribute to the inflammatory condition in SCA patients. This study tested the hypothesis that neutrophils challenged with serum of sickle cell anemia (SCA) patients, treated with hydroxyurea (HU), decrease neutrophils microparticles (MPs) production. Therefore, the aim of this study was to evaluate the inflammatory state of neutrophils from healthy volunteers by NLRP3 and IL1B gene expression and of SCA patients treated or untreated with HU and to measure the production of MPs derived from neutrophils challenged with SCA patient's serum, treated or not with HU.Methods: We enrolled 10 SCA patients (all in steady state with age of 8.0 ± 1.9 years), from the Fundação de Hematologia e Hemoterapia do Estado da Bahia (HEMOBA). Neutrophils were isolated from one healthy donor as previously described (Pitanga et al, 2014). The protein concentration of neutrophils MPs was evaluated using the BCA method (Thermo Scientific,Waltham, MA, USA) according to the manufacturer’s protocol and described by Pitanga et al (2014). NLRP3 and IL1Bgene expressions genes were performed by real time PCR with SYBR Green assays from neutrophils RNA in trizol reagent. Relative expression was estimated through the 2ddct method, with GAPDH and ACTB genes. Serum biochemical analysis was evaluated using commercially available biochemical kits. This study was conducted in accordance and approved by the Research Ethics Committee of the Fundação Oswaldo Cruz - FIOCRUZ, Brazil; and also with the Helsinki Declaration of 1975, and its revisions. The informed consents were signed by patients or official responsible.Results: Our findings showed that NLRP3 (6.95 ± 1.77)and IL1B (6.46 ± 3.31) genes are highly expressed in neutrophils of SCA patients comparing to healthy volunteers (2.43 ± 0.73, p=0.0286; 2.65 ± 1.30, p=0.0081 respectively). Additionally, neutrophils from SCA patients treated with HU decrease NLRP3 gene expression (4.06 ± 1.12; p= 0.0357), but it was not observed for IL1B gene expression (6.03 ± 3.13; p=0.3472). We demonstrated that serum from SCA patients increased neutrophils MPs production (181.90 ± 53.00) comparing to healthy volunteer’s serum (37.63 ± 13.06; p=0.0011). However, HU treatment decreased this scenario (30.33 ± 13.70; p=0.0022).Conclusions: This study highlighted that HU acts decreasing NLRP3 gene expression and neutrophils MPs production suggesting that NLRP3 protein could participate of cellular activation and production of MPs, contributing to the maintenance of inflammation as well as vascular activation. DisclosuresNo relevant conflicts of interest to declare.

Elevated thrombopoietin and platelet indices confirm active thrombopoiesis but fail to predict clinical severity of puumala hantavirus infection.

We evaluated the mechanisms of thrombocytopenia and procoagulant changes in relation with clinical variables in a cohort of patients with acute hantavirus disease.Blood samples of 33 prospectively recruited, consecutive, hospitalized patients with acute Puumala virus–induced hemorrhagic fever with renal syndrome (HFRS) were collected acutely and at the recovery visit (control). Serum thrombopoietin (TPO) and activity of plasma microparticles (MPs) from various cell sources were measured with enzyme-linked immunosorbent assay-based methods. The results were related to data on platelet indices and functions, coagulation variables, and clinical disease.Serum TPO was nearly 4-fold higher acutely compared with the control (median 207 pg/mL, range 56–1258 pg/mL vs. median 58 pg/mL, range 11–241 pg/mL, P < 0.001) and coincided with high mean platelet volume (MPV) and immature platelet fraction (IPF%). Prothrombin fragments and D-dimer were high acutely compared with the control (F1 + 2 median 704 pmol/L, range 284–1875 pmol/L vs. median 249 pmol/L, range 118–556 pmol/L, P < 0.001; d-dimer median 2.8 mg/L, range 0.6–34.0 mg/L vs. median 0.4 mg/L, range 0.2–1.1 mg/L, P < 0.001), and associated with low platelet count and severe acute kidney injury (AKI). MPs’ procoagulant activity was high acutely only among patients with mild AKI (plasma creatinine below the median at the time of the measurement).Upregulated TPO together with high MPV and IPF% confirm active thrombopoiesis, but do not predict severity of HFRS. Simultaneously, elevated prothrombin fragments and d-dimer suggest increased consumption of platelets in patients with severe AKI. Activity of platelet-derived MPs in HFRS should be studied with flow cytometry in a larger cohort of patients.

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.

Contributions of phosphatidylserine-positive platelets and leukocytes and microparticles to hypercoagulable state in gastric cancer patients.

Hypercoagulability in gastric cancer is a common complication and a major contributor to poor prognosis. This study aimed to determine procoagulant activity of blood cells and microparticles (MPs) in gastric cancer patients. Phosphatidylserine-positive blood cells and MPs, and their procoagulant properties in particular, were assessed in 48 gastric cancer patients and 35 healthy controls. Phosphatidylserine-positive platelets, leukocytes, and MPs in patients with tumor-node-metastasis stage III/IV gastric cancer were significantly higher than those in stage I/II patients or healthy controls. Moreover, procoagulant activity of platelets, leukocytes, and MPs in stage III/IV patients was significantly increased compared to the controls, as indicated by shorter clotting time, higher intrinsic and extrinsic factor tenase, and prothrombinase complex activity. Interestingly, lactadherin, which competes with factors V and VIII to bind phosphatidylserine, dramatically prolonged clotting time of the cells and MPs by inhibiting factor tenase and prothrombinase complex activity. Although anti-tissue factor antibody significantly attenuated extrinsic tenase complex activity of leukocytes and MPs, it only slightly prolonged clotting times. Meanwhile, treatment with radical resection reduced phosphatidylserine-positive platelets, leukocytes, and MPs, and prolonged the clotting times of the remaining cells and MPs. Our results suggest that phosphatidylserine-positive platelets, leukocytes, and MPs contribute to hypercoagulability and represent a potential therapeutic target to prevent coagulation in patients with stage III/IV gastric cancer.

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.

Procoagulant microparticles are increased in patients with Behçet's disease but do not define a specific subset of clinical manifestations.

Microparticles (MP) are considered a key component in the haemostatic response. Beyond their in vitro procoagulant properties, a number of pieces of evidence points to procoagulant MP as efficient effectors in the haemostatic response and as pathogenic markers of thrombotic disorders and vascular damage. The aim of the present study was to analyze the procoagulant activity of MP and its correlation with clinical manifestations focusing on vascular involvement in patients with Behçet's disease (BD). We analyzed 55 BD patients in inactive phase of the disease (26 men; mean age, 35 ± 15 years) of which 19 had previously suffered from thrombosis (deep venous thrombosis in 17 and ischemic stroke in 2), and 73 healthy controls matched for age and sex. Procoagulant MP were assessed by a functional assay. BD patients showed higher procoagulant MP values than controls (22.89 ± 15.74 nM versus 14.47 ± 7.34 nM; p < 0.0001). Conversely, we did not find differences in the levels of procoagulant MP according to the gender of patients (22.22 ± 16.23 nM for men versus 21.46 ± 16.47 for women; p = 0.846) or to previous and current treatments. Moreover, the plasmatic concentration of MP does not define any clinical phenotype and it was not related to the time of evolution of the disease. Although inactive BD patients had high values of procoagulant MP, they did not differentiate between BD patients with or without thrombosis.