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Introduction: Pulmonary arterial hypertension (PAH) is a serious complication of systemic lupus erythematosus (SLE) with increased mortality. A prothrombotic state may contribute to pathogenesis of SLE-PAH. Extracellular vesicles (EVs) are known to be associated with thrombosis. Here, we investigated circulating EVs and their associations with SLE-PAH., Methods: Eighteen SLE-PAH patients, 36 SLE-non-PAH patients, and 36 healthy controls (HCs) were enrolled. Flow cytometry was used to analyze circulating EVs from leukocytes (LEVs), red blood cells (REVs), platelets (PEVs), endothelial cells (EEVs), and Annexin V + EVs with membrane phosphatidylserine (PS) exposure., Results: Plasma levels of all EV subgroups were elevated in SLE patients with or without PAH compared to HCs. Furthermore, plasma Annexin V + EVs, LEVs, PEVs, REVs, EEVs, and Annexin V + REVs were significantly elevated in SLE-PAH patients compared to SLE-non-PAH patients. Additionally, PAH patients with moderate/high SLE showed a significant increase in LEVs, PEVs, REVs, Annexin V + EVs, and Annexin V + REVs compared to SLE-non-PAH patients. However, PAH patients with inactive/mild SLE only exhibited elevations in Annexin V + EVs, REVs, and Annexin V + REVs. In the SLE-PAH patients, EEVs were positively correlated with pulmonary arterial systolic pressure, while PEVs and EEVs were positively correlated with right ventricular diameter. Moreover, the receiver operating characteristic curve indicated that Annexin V + EVs, LEVs, PEVs, REVs, EEVs and Annexin V + REVs could predict the presence of PAH in SLE patients. Importantly, multivariate logistic regression analysis showed that circulating levels of LEVs or REVs, anti-nRNP antibody, and serositis were independent risk factors for PAH in SLE patients., Discussion: Findings reveal that specific subgroups of circulating EVs contribute to the hypercoagulation state and the severity of SLE-PAH. Higher plasma levels of LEVs or REVs may serve as biomarkers for SLE-PAH., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ding, Qi, Liu, Wang, Zhang, Lyu, Sun, Du, Song, Hou, Guo, Wang, Liu and Wei.)

Background/aims: Defects in the Glucose-6-Phosphate Dehydrogenase (G6PD) enzyme enhance cellular oxidative damage, thus impairing erythrocytes and radically shortening their lifespan. We aimed to study programmed erythrocyte cell death in G6PD-deficient patients, describe the molecular genetics basis of G6PD and investigate phenotype-genotype correlations., Methods: We explored eryptosis using the annexin V-binding assay, taken as an indicator of PS exposure at the erythrocyte surface. We assessed reactive oxygen species (ROS) production, intracellular calcium concentrations and ceramide formation at the cell surface. Prior to and following treatments, cells were analyzed by flow cytometry. Finally, we explored G6PD gene mutations through PCR-Sanger sequencing., Results: Before stimulation, PS-exposing erythrocytes were significantly higher in G6PD-deficient patients than in healthy volunteers. This was paralleled by a significant increase in reactive oxygen species production, suggesting that oxidative stress is the main trigger of PS exposure in G6PD-deficient erythrocytes. Five previously described mutations were detected in our patients. Two genotypes correlated with a significantly higher percentage of PS-exposing cells., Conclusion: Our study uncovers a novel effect detected in G6PD-deficient erythrocytes which is cell membrane scrambling with PS translocation to the erythrocyte surface. Our findings shed a light on the mechanisms of premature erythrocyte clearance in G6PD deficiency., Competing Interests: The authors declare that no conflicts of interest exist., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Introduction: The antiphospholipid syndrome (APS) is characterized by thrombosis or pregnancy morbidity, and the detection in the blood of at least one of three antiphospholipid antibodies (lupus anticoagulant, or anticardiolipin or anti-β 2 -glycoprotein I antibodies). Diagnosing APS is important so that secondary prophylaxis may be administered to reduce risk of recurrent thrombosis and/or pregnancy morbidity. In addition to APS-defining antibodies, there may be additional autoantibodies that have a role in thrombosis and/or pregnancy morbidity. Furthermore, some patients have clinical manifestations highly suggestive of APS but are persistently negative for the APS-defining antibodies ("seronegative APS") and instead, have other autoantibodies. Antiannexin A5 (aANXA5) autoantibodies have been associated with increased risk of thrombosis and pregnancy morbidity; levels are also reportedly higher in patients with venous thrombosis compared with healthy controls. The prevalence of aANXA5 among patients with unprovoked venous thrombosis is not well-documented and determination of the frequency of aANXA5 is the objective of this study., Methods: We analysed sera from 148 patients with unprovoked venous thrombosis who had undergone routine laboratory testing for the present APS-defining antibodies., Results: aANXA5 IgG and IgM were present in 6% and 1%, respectively., Conclusion: Prevalence of these antibodies in unprovoked venous thrombosis is comparable with frequencies reported in healthy individuals and is far lower than the prevalence in women with pregnancy morbidity. This may indicate lack of association with venous thrombosis, however, adequately powered case-control studies will be required to resolve this and prevalence data from this study will assist in the design of such studies., (© 2021 John Wiley & Sons Ltd.)

Background/aims: Chronic kidney disease is frequently accompanied by anemia, hypoxemia, and hypoxia. It has become clear that the impaired erythropoietin production and altered iron homeostasis are not the sole causes of renal anemia. Eryptosis is a process of red blood cells (RBC) death, like apoptosis of nucleated cells, characterized by Ca 2+ influx and phosphatidylserine (PS) exposure to the outer RBC membrane leaflet. Eryptosis can be induced by uremic toxins and occurs before senescence, thus shortening RBC lifespan and aggravating renal anemia. We aimed to assess eryptosis and intracellular oxygen levels of RBC from hemodialysis patients (HD-RBC) and their response to hypoxia, uremia, and uremic toxins uptake inhibition., Methods: Using flow cytometry, RBC from healthy individuals (CON-RBC) and HD-RBC were subjected to PS (Annexin-V), intracellular Ca 2+ (Fluo-3/AM) and intracellular oxygen (Hypoxia Green) measurements, at baseline and after incubation with uremic serum and/or hypoxia (5% O 2 ), with or without ketoprofen. Baseline levels of uremic toxins were quantified in serum and cytosol by high performance liquid chromatography., Results: Here, we show that HD-RBC have less intracellular oxygen and that it is further decreased post-HD. Also, incubation in 5% O 2 and uremia triggered eryptosis in vitro by exposing PS. Hypoxia itself increased the PS exposure in HD-RBC and CON-RBC, and the addition of uremic serum aggravated it. Furthermore, inhibition of the organic anion transporter 2 with ketoprofen reverted eryptosis and restored the levels of intracellular oxygen. Cytosolic levels of the uremic toxins pCS and IAA were decreased after dialysis., Conclusion: These findings suggest the participation of uremic toxins and hypoxia in the process of eryptosis and intracellular oxygenation., Competing Interests: Dias, G.F. has been a consultant for Renal Research Institute, NY, USA. Kotanko, P. is an employee of the Renal Research Institute, a wholly owned subsidiary of Fresenius Medical Care; he holds stock in Fresenius Medical Care. The other authors have no conflicts of interest to declare., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Acute cellular rejection (ACR) after liver transplantation (LT) goes along with allograft dysfunction, which is diagnosed by liver biopsy and concomitant histological analysis, representing the gold standard in clinical practice. Yet, liver biopsies are invasive, costly, time-intensive and require expert knowledge. Herein we present substantial evidence that blood plasma residing peripheral liver-derived extracellular particles (EP) could be employed to diagnose ACR non-invasively. In vitro experiments showed organ-specific EP release from primary human hepatocytes under immunological stress. Secondly, analysis of consecutive LT patients (n=11) revealed significant heightened EP concentrations days before ACR. By conducting a diagnostic accuracy study (n = 69, DRKS00011631), we explored the viability of using EP as a liquid biopsy for diagnosing ACR following LT. Consequently, novel EP populations in samples were identified using visualization of t-distributed stochastic neighbor embedding (viSNE) and self-organizing maps (FlowSOM) algorithms. As a result, the ASGR1 + CD130 + Annexin V + EP subpopulation exhibited the highest accuracy for predicting ACR (area under the curve: 0.80, 95% confidence interval [CI], 0.70-0.90), with diagnostic sensitivity and specificity of 100% (95% CI, 81.67-100.0%) and 68.5% (95% CI, 55.3-79.3%), respectively. In summary, this new EP subpopulation presented the highest diagnostic accuracy for detecting ACR in LT patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kamali, Schmelzle, Kamali, Brunnbauer, Splith, Leder, Berndt, Hillebrandt, Raschzok, Feldbrügge, Felsenstein, Gaßner, Ritschl, Lurje, Schöning, Benzing, Pratschke and Krenzien.)

Introduction: Microvesicles (MVs) with procoagulant properties may favor liver parenchymal extinction, then cirrhosis-related complications and mortality. In a longitudinal cohort of cirrhotic patients, we measured plasma levels of platelet-derived MVs (PMVs), endothelial-derived MVs, and red blood cell-derived MVs, expressing phosphatidylserine (annexin V-positive [AV+]) or not, and evaluated their impact on Model for End-Stage Liver Disease (MELD) score and transplant-free survival., Methods: MVs were quantified using flow cytometry in plasma from 90 noninfected cirrhotic patients and 10 healthy volunteers matched for age and sex. Impact of plasma microvesicle levels on 6-month transplant-free survival was assessed using log-rank tests and logistic regression., Results: Microvesicle levels, mostly platelet-derived, were 2.5-fold higher in healthy volunteers compared with cirrhotic patients. Circulating small AV+ PMV levels were lower in cirrhotic patients (P = 0.014) and inversely correlated with MELD scores (R = -0.28; P = 0.0065). During 1-year follow-up, 8 patients died and 7 underwent liver transplantation. In the remaining patients, circulating microvesicle levels did not change significantly. Six-month transplant-free survival was lower in patients with low baseline small AV+ PMV levels (72.6% vs 96.2%; P = 0.0007). In multivariate analyses adjusted for age, ascites, esophageal varices, encephalopathy, clinical decompensation, total platelet counts, MELD score, and/or Child-Pugh C stage, patients with lower small AV+ PMV levels had a significant 5- to 8-fold higher risk of 6-month death or liver transplant. Other PMV levels did not impact on survival., Discussion: Decreased circulating small AV+ PMV levels are associated with significantly lower transplant-free survival in cirrhotic patients independently of MELD score and platelet counts., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The American College of Gastroenterology.)

In a global context of advanced aging, geriatric diseases such as frailty syndrome face challenges in the search for biomarkers and preventive strategies. Frailty has been associated with atherothrombotic pathologies. Circulating microvesicles (cMVs), phospholipid-rich vesicles with a size of 0.1-1.0 μm, have been shown to participate in atherothrombosis onset and progression. We have hypothesized that cMVs from platelets, and vascular and immune cells, are increased in frail older adults. To verify this, a prevalent-case control study was designed with 28 frail older and 27 nonfrail older adults older than 64 years. Frailty was defined by Fried's phenotype. Total cMVs, annexin V positive (AV+)-cMVs, and annexin V negative (AV - )-cMVs derived from blood and vascular cells were measured by flow cytometry. In the analysis of total cMVs, the frail group presented higher levels of CD14 + /CD142 + (p = .042), CD41a + /CD142 + (p = .041), and CD56 + (p = .025), CD14 + cMVs (p = .043), and CD16 + /CD14 + (p = .019) cMVs levels. Within the phosphatidylserine-exposing cMVs (AV + ), the frail group showed higher CD14 + /AV + (p = .044), CD9 + /AV + (p = .031), P2RY12 + /AV + (p = .028), and CD235a + /AV + (p = .043) cMVs concentrations. Finally, within AV - cMVs, the frail group showed higher CD142 + /CD41a + /AV - cMVs concentrations originated from platelets (p = .027), CD56 + /AV - originated from natural killer cells (p = .022), and CD34 + /AV - cMVs from hematopoietic stem cells (p = .037). In summary, frail older adults present higher concentrations of platelet-, leukocyte-, and hematopoietic cell-derived cMVs compared to robust age-matched older adults. These cMVs may be involved in the deregulation of the immune system, endothelial damage, and increased risk of thrombosis associated with frailty., (© 2020 Wiley Periodicals LLC.)

Objectives: Veno-arterial extracorporeal membrane oxygenation (VA-ECMO) is used in critically ill patients requiring haemodynamic support. Microvesicles (MV) are released by activated blood cells acting as mediators of intercellular communication. We aimed to determine MV count and composition over time in patients with VA-ECMO and explore what drives MV formation., Methods: VA-ECMO patients and healthy controls were recruited prospectively, and blood was taken at different time points (day 0, 1, 3 after ECMO placement and after explantation) for MV analysis., Results: Annexin V positive MV were increased in patients (n = 14, mean age = 61.4 ± 9.0 years, 11 males, 3 females) compared to healthy controls (n = 6, Annexin V positive MV count per millilitre day 1 versus healthy controls: 2.3 × 106 vs 1.3 × 105, P < 0.001). Furthermore, patients had higher proportions of endothelial and leukocyte MV [leukocyte MV day 1 versus healthy controls (%): 32.8 vs 17.5, P = 0.001; endothelial MV day 1 versus healthy controls (%): 10.5 vs 5.5, P = 0.01]. Annexin V positive and leucocyte MV correlated with the flow rate (r = 0.46, P = 0.01)., Conclusions: Patients on VA-ECMO have increased levels of circulating MV and a changed MV composition. Our data support the hypothesis that MV release may be driven by higher flow rate and cellular activation in the extracorporeal circuit leading to poor outcomes in these patients., Clinical Trial Registration Number: German Clinical Trials Register-ID: DRKS00011106., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

Background: Treatment with cyclosporine A (CsA), a calcineurin inhibitor, is effective in children with difficult idiopathic nephrotic syndrome (INS). Prolonged CsA treatment can result in several adverse effects, the most significant being nephrotoxicity (CsAN). The plasma and urine levels of the proteins annexin V (AnV) and uromodulin (UM) were investigated in order to assess their usefulness as indicators of early-stage CsAN. Uromodulin is considered a distal tubular damage marker. Annnexin V is present in the distal tubules., Objectives: To measure AnV in children with INS receiving CsA treatment and to assess the usefulness of this biomarker for monitoring CsAN and as an indicator of changes in the distal tubules of the nephron., Material and Methods: The prospective study included 30 patients with INS and 22 controls. Plasma and urinary AnV levels were measured 3 times: before CsA treatment, and after 6 and 12 months of therapy. The AnV levels were compared to those of UM., Results: The urinary AnV and UM levels were significantly higher in the INS patients before CsA therapy in comparison to the reference group. A progressive increase of urinary AnV was observed after 6 and 12 months of therapy. Urinary UM only increased after 6 months. No significant correlations were found between plasma and urinary concentrations of the proteins studied., Conclusions: The increased urinary excretion of AnV in children with INS receiving CsA treatment may suggest its usefulness as an early marker of subclinical CsAN. Annexin V seems to be a more sensitive indicator of tubular damage in the course of CsA therapy than UM, though large, multicenter studies are needed.

Background: We investigated the serum annexin V and anti-annexin V levels and their relationship with metabolic parameters in patients recently diagnosed type 2 diabetic., Methods: A total of 143 patients recently diagnosed type 2 diabetes and 133 control subjects were included in the study. Body mass index (BMI), hs-CRP, HOMA-IR, carotid intima-media thickness, and serum levels of annexin V and anti-annexin V were investigated., Results: HOMA-IR, serum hs-CRP, and carotid intima-media thickness were found to be statistically significant. The Pearson correlation analysis revealed a statistically significant positive relationship between the carotid intima-media thickness and the annexin V level (r=0.29, p=0.006*). A statistically significant positive relationship was also detected between the Annexin V level and level of serum hs-CRP (r=0.29 p=0.006*)., Conclusion: A positive relationship was observed between the carotid intima-media thickness and annexin V at the end of our investigation. In this regard, we also believe that serum levels of annexin V may be increased for cardiovascular protection in the elevation of carotid intima-media thickness.

Background: Although hemodialysis is a highly effective treatment for diffusive clearance of low molecular weight uremic toxins, its effect on circulating extracellular vesicles and submicron particles is less clear. The purpose of this study was to examine the impact of hemodialysis on circulating levels of submicron particles., Methods: Plasma samples from patients were collected immediately before and after the mid-week hemodialysis session. Total submicron particles were assessed by nanoparticle tracking analysis and levels of endothelial (CD144 + ), platelet (CD41 + ), leukocyte (CD45 + ), and total (Annexin V + ) membrane microparticles (MPs) were assessed by flow cytometry., Results: Total submicron particle number was significantly lower post-dialysis with reductions in particles < 40 nm, 40-100 nm, and 100-1000 nm in size. Circulating annexin V + MPs, platelet MPs, leukocyte MPs, and endothelial MPs were all reduced following dialysis. Assessment of protein markers suggested that extracellular vesicles were not present in the dialysate, but rather adsorbed to the dialysis membrane., Conclusions: In summary, hemodialysis is associated with reductions in circulating submicron particles including membrane MPs. Accordingly, there may be significant interdialytic variation in circulating submicron particles. Investigators interested in measuring extracellular vesicles in patients undergoing hemodialysis should therefore carefully consider the timing of biosampling.

Background: Hypobaric hypoxia has been reported to cause endothelial cell and platelet dysfunction implicated in the formation of microvascular lesions, and in its extremes may contribute to vascular leakage in high altitude pulmonary edema or blood brain barrier disruption leading to cerebral micro-hemorrhage (MH). Platelet function in the development of microvascular lesions remained ill defined, and is still incompletely understood. In this study platelet- and endothelial cell-derived extracellular vesicles (PEV and EEV, respectively) and cell adhesion molecules were characterized in plasma samples of members of a high altitude expedition to delineate the contribution of platelets and endothelial cells to hypobaric hypoxia-induced vascular dysfunction., Methods and Findings: In this observational study, platelet and endothelial cell-derived extracellular vesicles were analysed by flow-cytometry in plasma samples from 39 mountaineers participating in a medical research climbing expedition to Himlung Himal, Nepal, 7,050m asl. Megakaryocyte/platelet-derived AnnexinVpos, PECAM-1 (CD31) and glycoprotein-1b (GP1b, CD42b) positive extracellular vesicles (PEV) constituted the predominant fraction of EV in plasma samples up to 6,050m asl. Exposure to an altitude of 7,050m led to a marked decline of CD31pos CD42neg EEV as well as of CD31pos CD42bpos PEV at the same time giving rise to a quantitatively prevailing CD31neg CD42blow/neg subpopulation of AnnexinVpos EV. An almost hundredfold increase in the numbers of this previously unrecognized population of CD31neg CD42blow/neg EV was observed in all participants reaching 7,050m asl., Conclusions: The emergence of CD31neg CD42blow/neg EV was observed in all participants and thus represents an early hypoxic marker at extreme altitude. Since CD31 and CD42b are required for platelet-endothelial cell interactions, these hypobaric hypoxia-dependent quantitative and phenotypic changes of AnnexinVpos EV subpopulations may serve as early and sensitive indicators of compromised vascular homeostasis., Competing Interests: The authors have declared that no competing interests exist.

There is a great need for a noninvasive diagnosis for endometriosis. Several biomarkers and biomarker panels have been proposed. Biomarker models consisting of CA-125, VEGF, Annexin V, and glycodelin/sICAM-1 were previously developed by our group. The objective of our current study was to assess the impact of technical and biological variability on the performance of those previously developed prediction models in a technical verification and a validation setting. The technical verification cohort consisted of peripheral blood plasma samples from a subset of the patients included in the original study of Vodolazkaia et al. (99 women with and 37 women without endometriosis). The validation study was done in plasma samples of an independent patient cohort (170 women with and 86 women without endometriosis). Single immunoassays were used for CA-125, VEGF-A, sICAM-1, Annexin V, and glycodelin. Statistical analyses were done using univariate and multivariate (logistic regression) approaches. The previously reported prediction models for endometriosis had a low performance in both the technical verification and validation setting. New prediction models were developed, which included CA-125, Annexin V, and sICAM-1, but CA-125 was the only marker that was retained in the models across the technical verification and validation study. Overall, successful validation of a biomarker model depends on several factors such as patient selection, collection methods, assay selection/handling, stability of the marker, and statistical analysis and interpretation. There is a need for standardized studies in large, well-defined patient cohorts with robust assay methodologies., Competing Interests: The authors report financial and nonfinancial support from Roche Diagnostics GmbH, Penzberg, that provided and performed the immunological assays of the technical verification study. The author Thomas M. D'Hooghe reports only conflicts of interest outside the scope of the paper. He has served as advisor for Bayer Pharma, Proteomika, Pharmaplex, Astellas, Roche Diagnostics, Actavis, has received grants from Ferring, Merck Serono, MSD, Besins, Pharmaplex, and has received travel support from Ferring, Merck Serono, and MSD. From October 1st 2015, Thomas D'Hooghe is Vice-President and Head of Global Medical Affairs Infertility for the Multinational Pharmaceutical company Merck Serono (Darmstadt, Germany) and continues on a part time basis his academic appointment as Professor of Reproductive Medicine at the University of Leuven (KU Leuven) in Belgium.

Aim : to evaluate the associations between signatures of apoptotic endothelial cell-derived microvesicles (MVs) with phenotypes of chronic heart failure (HF). Methods: The study cohort consisted of 388 prospectively involved subjects with HF patients with predominantly reduced left ventricular ejection fraction (HFrEF), HF with preserved ejection fraction (HFpEF) and HF with mid-range ejection fraction (HFmrEF). All biomarkers were measured at baseline. Results: The number of circulating CD31 + /annexin V + MVs in HFrEF and HFmrEF patients was similar. The number of circulating CD144 + /annexin V + MVs in HFrEF patients was significantly higher than HFmrEF and HFpEF. We determined that a combination of number of circulating CD31 + /annexin V + MVs and Gal-3 was the best predictor of HFpEF and that number of circulating CD144 + /annexin V + MVs is able to increase predictive capabilities of soluble ST2 (sST2) and Gal-3 for HFrEF. Conclusion: We found that the number of circulating CD31 + /annexin V + MVs may improve a predictive capacity for conventional HF biomarkers.

Objective/background: Certain hemostatic anomalies found in patients with thalassemia suggest the existence of a chronic hypercoagulable state. Several etiologic factors may play a role in the pathogenesis of the hypercoagulable state in those patients. One of these factors is abnormal thalassemic red blood cells (RBCs), which may provide a procoagulant. To substantiate these findings, we measured the ability of RBCs from thalassemia patients to bind annexin V with increased fraction of platelets carrying the activation marker CD62P (P-selectin). To study the expression of RBC annexin V and platelets P-selectin in study patients (those with thalassemia major, thalassemia intermedia, thalassemia minor) and control group, four-color flow cytometry was performed and the correlation between these two markers was evaluated., Methods: A case-control study was conducted on 50 β-thalassemia patients (10 patients with thalassemia minor, 30 patients with thalassemia major, and 10 patients with thalassemia intermedia, with 10 normal adult volunteers as a control) from June 2016 to March 2017. Flow cytometry was used to study the expression of anionic phospholipids (Annexin V) on the RBCs and CD62P (P-selectin) on the activated platelet., Results: The mean expression of annexin V in patients with thalassemia major and intermedia was significantly higher than that in the control group and patients with thalassemia minor. Although the mean expression was higher in patients with thalassemia intermedia than in those with thalassemia major, it was not statistically significant., Conclusion: The mean expression of platelets P-selectin in patients with thalassemia major and thalassemia intermedia was significantly higher than that in controls and patients with thalassemia minor. However, its expression was significantly higher in patients with thalassemia intermedia than in those with thalassemia major. Annexin V also showed a positive correlation with P-selectin, and both markers positively correlated with regularity of blood transfusion., (Copyright © 2018 King Faisal Specialist Hospital & Research Centre. Published by Elsevier Ltd. All rights reserved.)

Introduction: Dysregulation of apoptosis has been explored in acute myeloid leukemia (AML); yet, its correlation with clinical outcomes in pediatric AML is unknown. This study was aimed to analyze percentage of apoptosis and apoptosis mediated through the intrinsic pathway with clinical outcomes in patients with pediatric AML., Methods: This prospective study included pediatric AML patients enrolled from July 2013 to August 2016. Annexin-V (marker of total apoptosis) and caspase-9 expression (marker of intrinsic pathway) was determined in baseline bone marrow (BM) samples by flow cytometery and compared with controls (unaffected BM of solid tumors and peripheral blood [PB] of unaffected siblings). Overall survival (OS) and event-free survival (EFS) were compared using log-rank test., Results: A total of 151 AML patients were enrolled, median age 10 (range: 0.7-18 years). Annexin-V expression in blast cells was significantly high in AML patients as compared to BM of subjects with solid tumors (P = 0.01) and PB of healthy subjects (P = 0.04). Caspase-9 expression in blast cells was not significantly different. Median annexin-V expression was significantly higher in patients with WBC count ≥11 000/mm 3 (P = 0.02), poor-risk cytogenetics (P = 0.02), the absence of RUNX1-RUNX1T1 translocation (P = 0.004), and the absence of NPM1 mutation (P = 0.05). Patients with high annexin-V expression had significantly inferior OS (P = 0.05) in univariate analysis but not in multivariate analysis (P = 0.32)., Conclusion: Apoptosis as a whole was found to be activated in baseline BM samples of AML patients. High apoptosis may be associated with high-risk phenotype in this disease., (© 2018 John Wiley & Sons Ltd.)

Objective/background: Unlike most systemic chronic diseases, chronic venous insufficiency (CVI) is ideal to study using endogenous biomarkers. The stimulus causing damage can be turned on and off with gravitational positioning and venous blood samples can be taken locally. Annexin V (apoptosis) and microparticles (cell membrane debris) were used as markers of cell destruction, with matrix metalloproteinases (MMPs) as markers of tissue remodelling. The aim of this proof of concept study was to validate a gravitational model by investigating whether standing induced biochemical stress and whether recovery occurs on lying and after compression., Methods: Fourteen patients (C 4a-b ) and 14 volunteers (C 0-1 ) were tested under three supervised laboratory conditions for 1 h on separate days: (i) stationary standing on a small paper square; (ii) lying with both legs elevated 20°; (iii) compression standing using a 23-32 mmHg below knee stocking. Immediately after each condition, venous blood was withdrawn from the ankle. Commercial enzyme linked immunosorbent assay kits were used for batch analysis of the plasma samples., Results: Median (interquartile range [IQR]) values of annexin V (AU/mL) and microparticles (nM) standing were as follows: volunteers 2.9 (2 - 3.4) and 10.2 (8.8 - 13.8), and patients 2.2 (1.3 - 6) and 11.3 (7.7 - 20), respectively. Significant reductions were observed lying: volunteers 2.1 (1.5 - 2.7; p = .019) and 8.5 (7.4 - 9.4; p = .041), patients 1.7 (1.2 - 2.7; p = .004) and 8.5 (7.0 - 11.4; p = .041), respectively. Globally, all median MMP values in the patients reduced with lying and with compression versus standing (p = .004). Individually, significant reductions occurred in MMPs 2 and 13 with compression and MMPs 3, 7, 8, 9, 10, and 12 on lying. Lying was more effective at reducing MMP levels than compression., Conclusion: Annexin V and microparticle concentrations are responsive to elevation and compression after 1 h. In the patients, all the tested MMPs decreased after lying and with compression versus standing. This model provides evidence supporting gravitational protection in the treatment of CVI., (Copyright © 2018 European Society for Vascular Surgery. Published by Elsevier B.V. All rights reserved.)

Background: Cardiogenic embolism (CE) in cats is a devastating condition primarily associated with hypertrophic cardiomyopathy (HCM). Hypercoagulability may pose a risk for thrombus formation; however, no single test can predict CE development. Platelet microparticles (PMPs) released from platelet membranes are associated with thrombosis in humans., Objectives: The aims were to validate flow cytometric PMP quantification in cats analytically and, in a pilot study, evaluate the procoagulant annexin V (AnV) positive PMP concentration in healthy cats and cats with asymptomatic HCM., Methods: With CD61 as a platelet marker, CD61 + AnV + PMPs (0.3-1.0 μm) were quantified in citrated whole blood (WB) and platelet-poor plasma (PPP) using flow cytometry. Analyses were performed in 6 healthy cats and 5 cats with asymptomatic HCM. The coefficient of variation (CV) for duplicate (intra-assay) and parallel (inter-assay) analyses were calculated., Results: PMP concentrations were quantified with acceptable intra-assay CV for WB (CD61 + /AnV - ; 2.4%, 0.2%-8.4% (median, range), CD61 + /AnV + ; 3.8%, 0.1%-12.5%) and PPP (CD61 + /AnV - ; 5.0%, 0.7%-12.8%, CD61 + /AnV + ; 7.4%, 0.5%-15.3%), and acceptable inter-assay CV for WB in 10/11 cats (CD61 + /AnV - ; 6.2%, 1.4%-13.3%, CD61 + /AnV + ; 6.4%, 0.7%-17.2%), but unacceptable for PPP (CD61 + /AnV - ; 15.6%, 5.8%-42.7%, CD61 + /AnV + ; 27.8%, 8.4%-77.1%). For WB PMP concentrations, the pilot data demonstrated no differences between healthy cats and cats with asymptomatic HCM (4/5 with left ventricular outflow obstruction) for either the CD61 + /AnV - or the CD61 + /AnV + PMPs., Conclusions: Only WB PMP concentrations could be quantified reliably in cats in a clinical setting. PMP concentrations did not differ between healthy and asymptomatic HCM cats in this pilot study., (© 2018 American Society for Veterinary Clinical Pathology.)

Background: Several circumstances require the accurate measurement of red blood cell (RBC) survival and clearance, such as determination of posttransfusion recovery of stored RBCs to investigate the effect of new additive solutions. To this end, biotin as a marker of RBCs to track donor RBCs in the blood of the recipient has been used in many studies. However, so far only experimental, nonvalidated, biotin-labeled red cell concentrates (RCCs) are transfused. The goal of this study was to produce a standardized biotin-labeled RCC product in a fast, simple, and sterile manner that can be used for clinical research and for the evaluation of new blood products according to Good Practice Guidelines (GPG) for blood establishments., Study Design and Methods: RCC fractions were labeled with two different concentrations of biotinylation reagent in a closed system, to prevent bacterial contamination of the end product. Using flow cytometry, the reproducibility and robustness of the biotin labeling was assessed, as well as the stability of the biotin label on the (un-)irradiated RCC fraction. Additionally, parameters such as phosphatidylserine (PS) exposure, sodium (Na), potassium (K), free hemoglobin, adenosine triphosphate (ATP), pH, and morphology were determined prior to and after biotin labeling to rule out detrimental effects of the labeling procedure on the RCC., Results: Our data show that RCCs can be labeled under sterile conditions in a closed system with two different biotinylation reagent concentrations, without affecting the biological activity., Conclusion: An easy, rapid (<2 hr), and robust method was developed to manufacture biotin-labeled RCCs for clinical research compliant to GPG., (© 2018 AABB.)

Background: Sickle cell anemia (SCA) is characterized by abnormally shaped, adhesive RBCs that interact with white blood cells and the endothelium, leading to chronic hemolysis, vasculopathy and a prothrombotic state. About 10% of subjects with a thrombotic event in the general population will have an associated antiphospholipid (aPL) antibody. One proposed mechanism for the thrombophilic nature of aPL antibodies is the disruption of the potent anticoagulant annexin A5 or Annexin A5 resistance (A5R). We designed a pilot study assessing the presence of aPL antibodies and disruption of A5R in pediatric sickle cell subjects., Methods: 39 subjects with SCA participated in this study. A5R, DRVVT, anti-β2GP1, anti-β2GP1, anti-phosphatidylserine and anti-cardiolipin antibody assays were performed., Results: There was a high prevalence of abnormal A5R despite a low prevalence of antiphospholipid antibodies. Multivariate logistic regression analyses showed an association with silent infarcts (p=0.015), lower hemoglobin (p=0.037), older age (p=0.047) and abnormal A5R., Conclusion: We report an association between annexin A5 resistance and presence of silent infarct, low hemoglobin, and older age in a subgroup of SCA subjects. A potential role for perturbed A5R in the pathophysiology of SCA needs to be evaluated further., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Sepsis is associated with dysfunctional coagulation. Recent data suggest that platelets play a role in sepsis by promoting neutrophil accumulation. Herein, we show that cecal ligation and puncture (CLP) triggered systemic inflammation, which is characterized by formation of IL-6 and CXC chemokines as well as neutrophil accumulation in the lung. Platelet depletion decreased neutrophil accumulation, IL-6, and CXC chemokines formation in septic lungs. Depletion of platelets increased peak thrombin formation and total thrombin generation (TG) in plasma from septic animals. CLP elevated circulating levels of platelet-derived microparticles (PMPs). In vitro generated PMPs were a potent inducer of TG. Interestingly, in vitro wild-type recombinant annexin V abolished PMP-induced thrombin formation whereas a mutant annexin V protein, which does not bind to phosphatidylserine (PS), had no effect. Administration of wild-type, but not mutant annexin V, significantly inhibited thrombin formation in septic animals. Moreover, CLP-induced formation of thrombin-antithrombin complexes were reduced in platelet-depleted mice and in animals pretreated with annexin V. PMP-induced TG attenuated in FXII- and FVII-deficient plasma. These findings suggest that sepsis-induced TG is dependent on platelets. Moreover, PMPs formed in sepsis are a potent inducer of TG via PS exposure, and activation of both the intrinsic and extrinsic pathway of coagulation. In conclusion, these observations suggest that PMPs and PS play an important role in dysfunctional coagulation in abdominal sepsis., (© 2017 Wiley Periodicals, Inc.)

Background and Aim: Raynaud's phenomenon (RP) is a microvascular disorder characterized by episodic peripheral vasospasm and ischemia and is commonly found in patients with autoimmune diseases (AID). The vasomotor homoeostasis and endothelial cells damage are involved in RP. Endothelial microparticles (EMPs) may act as a biomarker for endothelial damage. The aim of this study is to investigate the correlation between the levels of microparticles (MPs) and microvasculopathy in AID with RP., Methods: Thirty-seven patients with AID and RP (RP group) and 27 patients with AID but without RP (non-RP group) were enrolled. The microvasculopathy score of RP was graded by nailfold capillary microscopy. The plasma levels of MPs were measured by flow cytometry utilizing specific labels for endothelial MPs (CD105 and CD144) and annexin V staining for phosphatidylserine bearing-MPs (annexin V+MPs)., Results: The levels of circulating EMPs (CD105+ p = 0.005, CD144+ p = 0.004), and the annexin V+ MPs (p < 0.001) were significantly elevated in the RP group compared with the non-RP group. Moreover, the high microvasculopathy scores were closely related with annexinV+ MPs levels in the RP group (p = 0.041)., Conclusions: Levels of circulating EMPs and annexin V+ MPs are elevated in AID patients with RP indicate the endothelial damage and endothelial dysfunctions. In addition, levels of annexin V+ MPs can predict the severity of microvasculopathy in AID with RP., (Copyright © 2016. Published by Elsevier B.V.)

Aim: To investigate the expression of annexin A5 in serum and tumor tissue of patients with colon cancer and to analyze its clinical significance., Methods: Ninety-three patients with colon cancer treated at our hospital between February 2013 and March 2016 were included in an observation group, and 40 healthy individuals were included in a control group. Enzyme-linked immunosorbent assay was performed to determine the serum level of annexin A5, while immunohistochemistry was performed to determine the expression of annexin A5 in cancer tissues., Results: The serum level of annexin A5 was 0.184 ± 0.043 ng/mL in the observation group, which was significantly higher than that in the control group ( P < 0.05). Annexin A5 expression was detected in 79.31% of the patients with lymph node metastasis, which was significantly higher than that in patients without lymph node metastasis ( P < 0.05). Moreover, annexin A5 expression was detected in 86.96% of the patients with stage III to IV disease, which was significantly higher than that in patients with stage I to II disease ( P < 0.05). The serum level of annexin A5 was 0.215 ± 0.044 ng/mL in patients whose tumors were positive for annexin A5 expression, which was significantly higher than that in patients whose tumors were negative for annexin A5 expression ( P < 0.05). The serum level of annexin A5 was correlated with annexin A5 expression in colon cancer tissues ( r = 0.312, P < 0.05). When a cutoff value of > 0.148 ng/mL for serum level of annexin A5 was used in the diagnosis of colon cancer, the sensitivity was 83.90%, and the specificity was 57.50%., Conclusion: For patients with colon cancer, annexin A5 expression in cancer tissues is related to lymph node metastasis and tumor grade. Serum level of annexin A5 is related to annexin A5 expression in cancer tissues and is of diagnostic relevance., Competing Interests: Conflict-of-interest statement: No conflict of interest.

Bone marrow transplantation (BMT) serves as the only curative treatment for patients with β-thalassemia major; however, hemostatic changes have been observed in these BMT patients. Aggregability of thalassemic red blood cells (RBCs) and increased red blood cell-derived microparticles (RMPs) expressing phosphatidylserine (PS) are thought to participate in thromboembolic events by initially triggering platelet activation. To our knowledge, there has been no report providing quantitation of these circulating PS-expressing RBCs and RMPs in young β-thalassemia patients after BMT. Whole blood from each subject was fluorescently labeled to detect RBC markers (CD235a) and annexin-V together with the known number TruCount™ beads. PS-expressing RBCs, RMPs, and activated platelets were identified by flow cytometry. In our randomized study, we found the decreased levels of three aforementioned factors compared to levels in patients receiving regular blood transfusion (RT). This study showed that BMT in β-thalassemia patients decreases the levels of circulating PS-expressing RBCs, their MPs, and procoagulant platelets when compared to patients who received RT. Normalized levels of these coagulation markers may provide the supportive evidence of the effectiveness of BMT for curing thalassemia.

Introduction: Preeclampsia (PE) remains to be an enigmatic puzzle for clinicians and researchers perplexing them for decades. As delivery remains only choice of treatment, early prediction of PE will offer timely therapeutic invention and hence extensive research efforts have been put in identification of biomarkers which will facilitate early prediction of PE., Methods: Serum levels of CPP, PlGF and plasma total annexin V MPs were assessed in women who subsequently developed PE (n = 33), IUGR (n = 81) and normal pregnancy outcome (n = 112) at 10-14 weeks of gestation. Comparison of biomarker levels between patients and control group was done using Mann Whitney test. Receiver operating curve (ROC) analysis and binary logistic regression analysis were used to evaluate predictive utility of combination of CPP, PlGF and total annexin V MPs for prediction of PE., Results: Women who subsequently developed PE showed significantly elevated levels of total annexin V MPs [2766.04 (2086.88-3794) versus 1090.74 (631.91-2197.16)] and CPP [440.98 (365.12-488.92) versus 217.8 (171.13-308.98)] compared to controls. Serum PlGF levels were significantly reduced in women with PE 17.68 (12.66-22.32) compared to controls 105.22 (35.02-255.1). Using logistic regression analysis, the combination of CPP, PlGF and total annexin V MPs gave high predictive value with AUC of 0.970, 93.1% sensitivity, 90.7% specificity, 77.50% Positive predictive value, 98.10% Negative predictive value, 11.69 Positive likelihood ratio and 0.07 Negative likelihood ratio for PE prediction at 10-14 weeks., Conclusion: The combination of serum markers and plasma microparticles can be used for 10-14 weeks prediction and discrimination of PE from other pregnancy complications., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Annexin A5 (anxA5) is involved in processes which are crucial in atherogenesis. However, anxA5's relationship with atherosclerotic lesion extension and plaque composition in high-risk patients with type 2 diabetes remains unclear. Thus, we characterized the association between circulating anxA5 levels with atherosclerotic burden and coronary plaque composition in diabetes mellitus patients., Methods: Intima-media thickness was determined in 96 diabetes mellitus patients with stable coronary artery disease. Furthermore, intracoronary optical coherence tomography was performed in 106 lesions to determine plaque composition., Results: AnxA5 plasma levels of patients with intima-media thickening were higher (3.49 ± 2.19 ng/mL) compared to patients with normal intima-media thickness (2.24 ± 1.67 ng/mL, p = 0.002). Furthermore, anxA5 was associated with intima-media thickening on univariable [odds ratio = 1.445 (1.106-1.889), p = 0.007] and multivariable [odds ratio = 1.643 (1.166-2.314), p = 0.005] logistic regression analysis when adjusted for multiple cardiovascular risk factors and biomarkers. Furthermore, receiver operating characteristic analysis demonstrated that anxA5 predicted intima-media thickening with low-moderate diagnostic efficiency [area under the curve = 0.700 (0.592-0.808)]. In contrast, there was no association between anxA5 levels and coronary plaque composition as assessed by optical coherence tomography including the presence of lipid, calcified, fibrous plaque or the minimal thickness of the fibrous cap overlying the necrotic lipid core ( p = ns)., Conclusion: Circulating anxA5 levels are associated with carotid intima-media thickness but not coronary plaque composition in high-risk patients with diabetes mellitus.

Background & Aims: Large extracellular vesicles, specifically AnnexinV + EpCAM + CD147 + tumour-associated microparticles (taMPs), facilitate the detection of colorectal carcinoma (CRC), non-small cell lung carcinoma (NSCLC) as well as pancreas carcinoma (PaCa). Here we assess the diagnostic value of taMPs for detection and monitoring of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). Specifically, the aim of this study was to differentiate liver taMPs from other cancer taMPs, such as CRC and NSCLC., Methods: Fluorescence-activated cell scanning (FACS) was applied to detect various taMP populations in patients' sera that were associated with the presence of a tumour (AnnexinV + EpCAM + CD147 + taMPs) or could discriminate between cirrhosis (due to HCV or HBV) and liver cancers (AnnexinV + EpCAM + ASGPR1 + taMPs). In total 172 patients with liver cancer (HCC or CCA), 54 with cirrhosis and no liver neoplasia, and 202 control subjects were enrolled., Results: The results indicate that AnnexinV + EpCAM + CD147 + taMPs were elevated in HCC and CCA. Furthermore, AnnexinV + EpCAM + ASGPR1 + CD133 + taMPs allowed the distinction of liver malignancies (HCC or CCA) and cirrhosis from tumour-free individuals and, more importantly, from patients carrying other non-liver cancers. In addition, AnnexinV + EpCAM + ASGPR1 + taMPs were increased in liver cancer-bearing patients compared to patients with cirrhosis that lacked any detectable liver malignancy. The smallest sizes of successfully detected cancers were ranging between 11-15mm. AnnexinV + EpCAM + ASGPR1 + taMPs decreased at 7days after curative R0 tumour resection suggesting close correlations with tumour presence. ROC values, sensitivity/specificity scores and positive/negative predictive values (>78%) indicated a potent diagnostic accuracy of AnnexinV + EpCAM + ASGPR1 + taMPs., Conclusion: These data provide strong evidence that AnnexinV + EpCAM + ASGPR1 + taMPs are a novel biomarker of HCC and CCA liquid biopsy that permit a non-invasive assessment of the presence and possible extent of these cancers in patients with advanced liver diseases., Lay Summary: Microparticles (MPs) are small vesicles that bleb from the membrane of every cell, including cancer cells, and are released to circulate in the bloodstream. Since their surface composition is similar to the surface of their underlying parental cell, MPs from the bloodstream can be isolated and by screening their surface components, the presence of their parental cells can be identified. This way, it was possible to detect and discriminate between patients bearing liver cancer and chronic liver cirrhosis., (Copyright © 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Microparticles (MPs) are formed from platelets (PMPs), endothelial cells (EMPs) and monocytes (MMPs), and in acute myocardial infarction (MI), there is an increase of MPs in the culprit artery. In this study MPs were evaluated in whole blood in 105 patients with MI at five time-points during a two-year follow-up (FU). Patients with non-ST-elevated MI had higher concentrations of CD41+MPs compared to ST-elevated MI patients (p=0.024). The concentrations of PMPs in whole blood increased during the time period (p<0.001), but no significant change over time was found for EMPs and MMPs. CD62P+MP counts were higher in MI patients with diabetes (p=0.020), and patients with hypertension had increased levels of CD14+MPs (p=0.004). The amount of CD62P+TF+MPs increased significantly during FU (p<0.001). Patients with atherosclerosis in three arterial beds, i. e. coronary, carotid and peripheral arteries, had lower concentrations of CD62P+TF+MPs (p=0.035) and CD144+TF+MPs (p=0.004) compared to patients with atherosclerosis in one or two arterial beds. Higher concentrations of CD62P+MPs early after MI were associated with an increased risk of cardiovascular events during FU, hazard ratio 3.32 (95 %CI1.20-9.31). Only small variations in PMP, EMP and MMP concentrations were found during long-term FU after MI and their levels seem to reflect the underlying cardiovascular disease rather than the acute MI. PMPs expressing P-selectin might be a promising biomarker for predicting future cardiovascular events, but further studies are needed to confirm these results.

It has been suggested that platelet function in chronic immune thrombocytopenic purpura (ITP) may be abnormal. Thrombopoietin mimetics used for treatment can affect it, but the data remain limited. We investigated platelet function of 20 children diagnosed with severe ITP (aged 1-16 years, 12 females and eight males). Platelet functional activity in whole blood was characterized by flow cytometry before and after stimulation with SFLLRN plus collagen-related peptide. Levels of CD42b, PAC1, and CD62P, but not CD61 or annexin V, were significantly increased (P < 0.05) in resting platelets of patients before treatment compared with healthy donors. On average, PAC1 and CD62P in patients after activation were also significantly elevated, although some patients failed to activate integrins. Romiplostim (1-15 μg/kg/week s.c.) was prescribed to seven patients, with clinical improvement in six. Interestingly, one patient had clinical improvement without platelet count increase. Eltrombopag (25-75 mg/day p.o.) was given to four patients, with positive response in one. Others switched to romiplostim, with one stable positive response, one unstable positive response, and one non-responding. Platelet quality improved with romiplostim treatment, and their parameters approached the normal values. Our results suggest that platelets in children with severe ITP are pre-activated and abnormal, but improve with treatment.

Deficient anticoagulant activity of annexin A5 and deficient profibrinolytic activity of annexin A2 have been linked to increased risk of thrombotic events. Placental dysfunction due to fibrin deposition/microthrombi has been implicated in the pathogenesis of pre-eclampsia (PE). In this study, we aimed to assess serum levels of annexin A5 and annexin A2 in a cohort of PE patients and investigate their role as biomarkers for the development of the disease. We examined 80 women in total; 40 healthy pregnant women and 40 pregnant women with PE after 20weeks of pregnancy. Women were subjected to full clinical assessment, ultrasonography, and laboratory testing including complete blood picture, liver and kidney function tests and assessment of serum and urine proteins. Annexin A5 and annexin A2 were analyzed using enzyme-linked immunosorbent assay. The study showed serum annexin A2 but not annexin A5 was significantly reduced (P=0.029) in women with PE (total and severe cases) compared to those with normal pregnancy. The ROC analysis of annexin A2 level for the prediction of development of PE showed an area under the curve of 0.64 (P=0.029), and the best cut-off value was 0.89ng/ml with a sensitivity of 70.0% and a specificity of 70.0%. Univariate analysis showed annexin A2 of <0.89ng/ml, proteinuria, lower platelet count and higher BP were associated with significantly higher risk to develop PE. Based on this pilot study, serum annexin A2 levels may be a useful biomarker for pre-eclampsia. However, a larger study is required before a final conclusion is made., (Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.)

A hallmark feature of antiphospholipid syndrome (APS) is the presence of antiphospholipid antibodies (aPLs). Few studies have addressed the clinical relevance of anti-annexin A5 antibodies (aANXA5) in Chinese patients with APS. In this study, we evaluated the clinical performance of aANXA5 in the diagnosis of APS. Sera from 313 subjects were tested, including 170 samples from patients with APS, 104 samples from patients with non-APS diseases as disease controls (DC), and 39 healthy controls (HC). Serum IgG and IgM aANXA5 were determined by ELISA. Overall, the levels of both IgG and IgM aANXA5 were significantly increased in patients with primary APS (PAPS) and APS associated to other diseases (APSAOD) compared with DC and HC. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for IgG and IgM aANXA5 in the diagnosis of APS were 33.5 and 15.3, 99.0 and 99.0, 98.3 and 96.3, and 47.7 and 41.7%, respectively. Significant associations between IgG aANXA5 and arterial thrombotic events (OR, 2.60; 95% CI, 1.44-4.71) and between IgG aANXA5 and venous thrombotic events (OR, 2.80; 95% CI, 1.55-5.06) were identified. No correlations were identified between IgG or IgM aANXA5 and obstetric complications. Our data suggest that aANXA5 could serve as a diagnosis biomarker for patients with APS. More importantly, our data highlighted a potential role of IgG aANXA5 in identifying APS patients with high risk of thrombosis.

Objective: Annexin A5 (AnxA5) has been previously linked to the presence of carotid and cardiac target organ damage (TOD) in the context of heart failure and rheumatologic patients. However, information is scant in the context of hypertension. Aim of our study was to evaluate AnxA5 in treated hypertension patients compared with normotensive controls and to determine whether it is associated with vascular and heart TOD evaluated as arterial stiffness, carotid plaque and left ventricular hypertrophy., Methods: We enrolled 123 consecutive treated hypertension and 124 normotensive controls. TOD was evaluated as pulse wave velocity (PWV, complior), left ventricular hypertrophy (echocardiography) and intima-media thickness and carotid plaque presence (ecographic methods). AnxA5 levels was dosed and compared in patients with and without hypertension and with and without TOD., Results: With similar age hypertension patients showed higher SBP, DBP and AnxA5 levels (13.9 ± 11.1 vs 10.1 ± 8.4 ng/ml, P < 0.001) compared with controls. Regarding TOD hypertension showed higher PWV (8.5 ± 1.8 vs 7.6 ± 1.5 m/s, P < 0.001) and LVMI (121.7 ± 29.3 vs 113.5 ± 21.1 g/m, P < 0.05), whereas carotid intima-media thickness was superimposable. AnxA5 correlates with PWV (r = 0.13, P < 0.05) and DBP (r = 0.15, P < 0.01), whereas it has never been found as a significant independent predictor of TOD in linear regression analysis., Conclusion: Our data have shown that AnxA5 levels are increased in treated hypertension patients. In this condition, it is probably released in the plasma as a defensive mechanism through its anti-inflammatory and anticoagulants effects. We found a significant association with arterial stiffness, but AnxA5 was not found to be a significant predictor of TOD.

Background: Serum annexin A5 (anxA5) level is significantly increased in patients with acute coronary syndrome. Hematological indices are significantly increased in patients with ischemic heart disease. This study aimed to demonstrate the changes in the distribution of blood cells and the levels of anxA5 in patients presented with significant low ejection fraction ST-elevation acute myocardial infarction (STEMI) in comparison with corresponding patients with ischemic heart disease., Methods: Patients with low ejection fraction presenting at Hospital of Diyala University of Iraq were enrolled. Electrocardiograph (ECG), echocardiograph, hematological indices, serum annexin V (anxV) levels and the determinants of the cardio-metabolic risk factors were performed. Based on clinical examination, ECG findings and laboratory tests, patients were divided into healthy subjects (n=20); patients with acute MI (n=40) and with chronic MI (n=12)., Results: Acute MI has significant high levels of serum triglyceride, uric acid and high mean value of red cell distribution width (RDW) compared with healthy subjects and chronic MI. Platelet distribution width (PDW) is significantly reduced in patients of acute MI and chronic MI compared with healthy subjects, whereas the plateletcrit (PCT) is significantly higher in acute MI compared with healthy subjects. There is an insignificant difference between the means of serum anxA5 levels of acute MI (35.6±7.2ng/ml) and chronic MI (32.4±8.9ng/ml), but significantly higher than the cutoff level of the healthy subjects (5ng/ml)., Conclusions: Measurement of serum annexin level is a useful diagnostic marker of acute or chronic MI with low ejection fraction., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Antiphospholipid syndrome (APS) is an autoimmune disease that is characterized by the presence of thromboses and/or recurrent pregnancy losses (RPL). The persistent presence of antiphospholipid antibodies (aPL Abs): IgG and/or IgM isotypes of the anticardiolipin and/or anti-β2 glycoprotein I antibodies and lupus anticoagulant is mandatory for the laboratory diagnosis of APS. Due to the heating debate on the relevance of the IgM isotype of aPL Abs as a laboratory criterion defining APS, the focus of this article was to analyze whether both the IgG and IgM isotype of anti-annexin A5 Abs have equal relevance for clinical and serological features of patients with primary APS (PAPS). The IgG isotype of anti-annexin A5 Abs is associated with RPL in PAPS patients, although it is not elucidated whether these Abs are the cause or the consequence of RPL in PAPS. No data that could substantiate the association of the IgG and/or the IgM isotypes of anti-annexin A5 Abs with the presence of arterial and/or venous thromboses and/or their main complications in PAPS is available so far. However, the presence of clinical manifestations of the PAPS is increasing with the multiple positivity for aPL Abs and the IgM isotype of anti-annexin A5 Abs. The importance of the IgM isotype of anti-annexin A5 Abs in PAPS needs further elucidation due to the facts that majority of the available articles did not differentiate between both isotypes or only investigated the IgG isotype of anti-annexin A5 Abs.

Objective: To investigate the contributions and underlying molecular mechanisms of annexin A5 toward silica-induced pulmonary fibrosis., Methods: Male C57BL/6 mice were randomly divided into three groups and instilled intratracheally with silica, saline, or air. Mice were euthanized at 3, 7, 14, or 28 days following treatment. Annexin A5 levels in serum and lung tissues were detected by enzyme-linked immunosorbant assay (ELISA) assays or Western blots. The association of annexin A5 levels with silica-induced lung fibrosis was further investigated in the macrophage cell line, RAW264.7. Following exposure of these cells to silica at a concentration of 200 μg/ml for 6 or 12 h, the expression levels of transforming growth factor β1 (TGF-β1), interleukin 1α (IL-1α), Fas ligand (FasL), and their downstream targets were evaluated by Western blots. Furthermore, annexin A5 and FasL were knocked down by small interfering ribonucleic acid (siRNA) and TGF-β1 secretion into the cell culture medium was measured by ELISA assays or Western blots., Results: Mice treated with silica demonstrated lung fibrosis at 28 days following exposure, whereas, in controls, only mild and transient inflammation was evident at day 3 and day 7 postinstillation and was not present at day 14. Furthermore, silica-exposed mice exhibited significantly (p < 0.05) elevated levels of annexin A5 in serum and lung tissues, relative to control groups. Consistent with these findings, silica exposure of RAW264.7 cells for 6 or 12 h, led to an annexin A5-dependent increase in the expression levels of TGF-β1, IL-1α, FasL, and their downstream target molecules. These silica-induced changes were reversed by siRNA-mediated knockdown of annexin A5, but downregulation of FasL led to increased annexin A5 expression and reduced levels of TGF-β1, IL-1α, and FasL downstream target molecules., Conclusions: These findings define a role of annexin A5 in promoting macrophage activation via Fas/FasL pathways in silica-induced lung fibrosis., (© The Author(s) 2015.)

Cardiolipin (CL) is an anionic phospholipid located exclusively in the mitochondrial inner membrane. Its presence in blood indicates mitochondrial damage and release from injured cells. Here, we report the detection of CL-exposed brain-derived mitochondrial microparticles (mtMPs) at 17 547 ± 2677/μL in the peripheral blood of mice subjected to fluid percussion injury to the brain. These mtMPs accounted for 55.2% ± 12.6% of all plasma annexin V-binding microparticles found in the acute phase of injury. They were also released from cultured neuronal and glial cells undergoing apoptosis. The mtMPs synergized with platelets to facilitate vascular leakage by disrupting the endothelial barrier. The disrupted endothelial barrier allowed the release of mtMPs into the systemic circulation to promote coagulation in both traumatically injured and mtMP- or CL-injected mice, leading to enhanced fibrinolysis, vascular fibrin deposition, and thrombosis. This mtMP-induced coagulation was mediated by CL transported from the inner to the outer mitochondrial membrane and was blocked by the scavenging molecule lactadherin. The mtMP-bound CL was ∼1600 times as active as purified CL in promoting coagulation. This study uncovered a novel procoagulant activity of CL and CL-exposed mitochondria that may contribute to traumatic brain injury-associated coagulopathy and identified potential pathways to block this activity., (© 2016 by The American Society of Hematology.)

Correlations between biochemical and immunological markers of programmed cell death (apoptosis), and the functional state of the thyroid gland (hyperthyroidism, euthyroidism, hypothyroidism) have been investigated in autoimmune thyroiditis (AT) (also known as chronic autoimmune thyroiditis). Annexin V, TRAIL and TNF-a, as well as DNA-hydrolyzing antibodies were used as the main markers. Increased levels of TRAIL were found in the serum of AT patients (hyperthyroidism>hypothyroidism>euthyroidism) compared with healthy individuals. The highest frequency of antibodies to denatured DNA (Abs-dDNA) had the highest frequency in AT patients (97%) compared with healthy controls. Among these patients, 75% had hyperthyroidism, 85% had hypothyroidism, and 84.7% had euthyroidism. Abs hydrolyzing activity demonstrated correlation dependence with symptoms of the thyroid dysfunction.

Background: Natriuretic peptides are currently used to predict mortality in patients with heart failure (HF). However, novel independent biomarkers are needed to improve risk stratification in these patients. We hypothesized that annexin A5 (anxA5) would be highly expressed by organs which are generally affected by HF and that circulating anxA5 levels would predict mortality in HF patients., Methods: We prospectively determined the diagnostic value of anxA5, N-terminal pro-B-type natriuretic peptide (NT-proBNP), C-reactive protein (CRP) and estimated glomerular filtration rate (eGFR) to predict mortality in 180 HF patients during a median follow-up of 3.6 years. Studies were conducted with anxA5(-/-) mice to investigate the underlying mechanisms., Results: AnxA5 levels were significantly elevated in HF patients compared to healthy control subjects. Cox regression analysis demonstrated that anxA5, NT-proBNP and eGFR all predict mortality independently. AnxA5 significantly improved the diagnostic efficiency of NT-proBNP alone (improvement of c-statistic from 0.662 to 0.705, P < 0.001) and also combined with eGFR and CRP (improvement of c-statistic from 0.675 to 0.738, P < 0.001) to predict mortality in the Cox regression model. Receiver operating characteristic curve analysis showed that anxA5 predicted 3-year survival (area under curve 0.708) with an optimal cut-off value of 2.24 ng mL(-1) . Using anxA5(-/-) mice, we demonstrated that anxA5 is highly expressed in organs that are often affected by HF including lung, kidney, liver and spleen. Lysis of these organs in vitro resulted in a marked and significant increase in anxA5 concentrations., Conclusion: AnxA5 improves the diagnostic efficiency of conventional biomarkers to predict mortality in HF patients. Whereas natriuretic peptides originate from the myocardium, high circulating anxA5 levels in patients with HF are likely to reflect peripheral organ damage secondary to HF., (© 2015 The Association for the Publication of the Journal of Internal Medicine.)

Objective: To systematically review and establish the prevalence of antibody positivity in assays not currently included in the APS classification criteria to detect antibodies directed against other phospholipids (PLs), PL binding proteins, coagulation factors and a mechanistic test for resistance of Annexin A5 (AnxA5) anticoagulant activity in APS and control populations., Methods: We searched PubMed and EMBASE using the key words APS, antiphospholipid antibodies, non-criteria, new assays, IgA anticardiolipin antibodies, lupus anticoagulant, anti-Domain I, IgA anti-β2-glycoprotein I antibodies, antiphosphatidylserine, anti-phosphatidylethanolamine, anti-phosphatidic acid, antiprothrombin, antiphosphatidylserine-prothtombin, anti-vimentin/cardiolipin complex and Annexin A5 resistance. Studies that met inclusion criteria to describe prevalence of non-criteria aPLs in APS patients (n > 10), disease and healthy control subjects were systematically examined., Results: We selected 16 retrospective studies of 1404 APS patients, 1839 disease control and 797 healthy controls. The highest prevalence of non-criteria aPLs in the largest number of patients with APS was found in IgA anti-β2GPI studies (129/229, 56.3%), AnxA5R (87/163, 53.4%) and IgG anti-Domain I (241/548, 44.0%)., Conclusion: Our finding of a significantly high prevalence of all non-criteria aPLs studied in patients with APS compared with controls was tempered by wide variation in sample size, retrospective collection, assay methodology and different determination of positivity. Therefore, prospective studies of sufficient size and appropriate methodology are required to evaluate the significance of these assays and their utility in the management of patients with APS., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The article presents results of observation of generation of thrombin in patients with ischemic heart disease in different terms after transcutaneous coronary intervention. The sampling included 37 patients with stable ischemic heart disease. The control group included 30 healthy individuals. To study system of hemostasis of this category of patients the test of generation of thrombin and its modification with added thrombomodulin were applied for evaluating anti-coagulant activity of system of protein C. The comparison of indicators of test of generation of thrombin in patients with ischemic heart disease before operation and in individuals of control group revealed no reliable differences (p > 0.05). The observation of patients with stable ischemic heart disease in various time-frame after mechanical re-vascularization of myocardium established significant increasing of generation of thrombin and decreasing of anticoagulant activity of system of protein C at 1-3 day after operation (p < 0.05). The positive correlation was established between endogenous thrombin potential and annexin 5, an early marker of dysfunction of endothelium in mentioned time-frame after operation (p = 0.0008; r = 0.57). The significant increasing of content of anti-inflammatory markers of C-reactive protein and fibrinogen was observed at 1-3 day after transcutaneous coronary intervention (p < 0.05). In that way, the study data give evidence to hyper-coagulation in patients with stable ischemic heart disease in early time-frame after operation despite normal values of activated partial thromboplastin time, prothrombin time, D-dimer and applied standard disaggregant therapy.

Objective: Annexin A5 (ANXA5) has been suggested to possess antiatherogenic properties. We investigated whether ANXA5 genetic variations and plasma ANXA5 levels were associated with carotid atherosclerosis and contributed to cardiovascular disease (CVD) risk in patients with familial hypercholesterolemia (FH)., Methods: We sequenced the promoter region and exon 2 of ANXA5 in 284 FH patients from the ASAP (Atorvastatin versus Simvastatin on Atherosclerosis Progression) trial. Common haplotypes (H) were constructed based on seven single nucleotide polymorphisms (SNPs). We studied whether plasma ANXA5 levels or ANXA5 haplotypes were associated with the extent of atherosclerosis (evaluated by carotid intima-media thickness (IMT). The association between ANXA5 haplotypes and the risk for CVD events was investigated in 1730 FH patients from the GIRaFH (Genetic Identification of Risk factors in Familial Hypercholesterolemia) study., Results: In ASAP, individuals carrying the ANXA5 haplotype H2 exhibited lower plasma ANXA5 levels, whereas H4 carriers had increased levels of circulating ANXA5 compared to non-carriers. Plasma ANXA5 levels were not associated with carotid IMT. None of the four ANXA5 haplotypes correlated with the age-related IMT progression (ASAP study) or contributed to CVD risk (GIRaFH cohort)., Conclusions: Both ANXA5 haplotypes and plasma ANXA5 levels were not associated with carotid IMT progression or CVD risk in FH patients., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Study Question: Are circulating microparticles (MPs) altered in young women with polycystic ovary syndrome (PCOS)?, Summary Answer: Women with PCOS have elevated concentrations of circulating platelet-derived MPs, which exhibit increased annexin V binding and altered microRNA (miR) profiles compared with healthy volunteers., What Is Known Already: Some studies have shown that cardiovascular risk is increased in young women with PCOS but the mechanisms by which this occurs are uncertain. Circulating MPs are elevated in patients with cardiovascular disease but the characteristics of MPs in patients with PCOS are unclear., Study Design, Size, Duration: Case-control study comprising 17 women with PCOS (mean ± SD; age 31 ± 7 years, BMI 29 ± 6 kg/m(2)) and 18 healthy volunteers (age 31 ± 6 years, BMI 30 ± 6 kg/m(2))., Participants/materials, Setting, Methods: The study was conducted in a University hospital. Nanoparticle tracking analysis (NTA) and flow cytometry (CD41 platelet, CD11b monocyte, CD144 endothelial) were used to determine MP size, concentration, cellular origin and annexin V positivity (reflecting phosphatidylserine exposure). Fatty acid analysis was performed by gas chromatography and MP miR expression profiles were compared by microarray., Main Results and the Role of Chance: PCOS subjects showed increased MP concentrations compared with healthy volunteers (mean ± SD; 11.5 ± 5 × 10(12)/ml versus 10.0 ± 4 × 10(12)/ml, respectively; P = 0.03), which correlated with the homeostasis model of insulin resistance (r = 0.53, P = 0.03). This difference was predominantly seen in MPs whose size was in the small exosomal range (<150 nm in diameter, P< 0.05). PCOS patients showed a greater percentage of annexin V(+) MPs compared with healthy volunteers (84 ± 18 versus 74 ± 24%, respectively, P = 0.05) but the cellular origin of MPs, which were predominantly platelet-derived (PCOS: 99 ± 0.9%; controls: 99 ± 2.5%), did not differ. MP fatty acid concentration and composition was similar between groups but 16 miRs were differentially expressed (P < 0.05)., Limitations, Reason for Caution: Patients with PCOS were classified by the Rotterdam criteria, which describes a less severe metabolic phenotype than other definitions of the syndrome. Our findings may thus not be generalizable to all patients with PCOS. MicroRNA expression analysis was only undertaken in an exploratory subset of the overall study population hence, validation of our findings in a larger cohort is mandatory. Furthermore, miR levels were unaltered for the highly expressed miRs and it is unclear whether differences in the lowly expressed miRs carries pathological relevance., Wider Implications of the Findings: This study suggests that women with PCOS have an altered MP profile but further studies are needed to confirm this, to explore the mechanisms by which these alterations develop and to establish whether therapies that improve insulin sensitivity are able to reduce circulating MP concentrations., Study Funding/competing Interests: The study was funded by grants from the Wales Heart Research Institute and Mrs John Nixon Scholarship. The authors have no conflicts of interest to declare., (© The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The detection of biomarkers to be applied to reveal different types of tumors is an actual demand of today. Since course of this disease can be asymptomatic these kinds of markers can be applied in widely-distributed screening procedures. Many types of biomarkers for detection of cancer exist. However all these markers are specific only for particular type of tumor and have no use in screening procedures for detection of this pathology. The Annexin 5, a Ca-depended phospholipid binding protein, was discovered in serums of all pregnant women on various stages of pregnancy. The study was implemented using method of latex agglutination. More than that, this protein was detected in serums of patients with several types of cancer. The possibility to apply annexin 5 as marker for screening of various types of cancer is considered.

Unlabelled: The coupling of polyethylene glycol (PEG) to proteins (PEGylation) has become a standard method to prolong blood circulation of imaging probes and other proteins, liposomes, and nanoparticles. However, concerns have arisen about the safety of PEG, especially with respect to its poor biodegradability and antibody formation, including new evidence about preformed anti-PEG antibodies in a quarter of healthy blood donors. Here, we apply a new hydrophilic polypeptide XTEN to extend the blood half-life of an imaging probe. As an example, we chose annexin A5 (AnxA5), a recombinant 35-kD protein extensively used for the in vitro and in vivo detection of apoptosis, that has a blood half-life of less than 7 min in mice, limiting its accumulation in target tissues and therefore limiting its utility as an imaging reagent., Methods: The sequence of XTEN was developed by Volker Schellenberger and colleagues by evolutionary in vitro optimization to yield PEG-like properties but provides several key advantages in comparison to PEG. The DNA of a 288-amino-acid version of XTEN with an additional N-terminal cysteine for site-directed coupling was fused to AnxA5 (XTEN-AnxA5). The fusion protein could be highly expressed in Escherichia coli and efficiently purified using XTEN conveniently as a purification tag. It was labeled with a thiol-reactive fluorescent dye and via a chelator with a radionuclide., Results: SPECT/CT imaging revealed a blood half-life of about 1 h in mice, markedly longer than the 7-min blood half-life for unmodified AnxA5, which should allow improved imaging of target tissues with low perfusion. In comparison to AnxA5, XTEN-AnxA5 demonstrated a substantially higher accumulation in tumors under chemotherapy in near-infrared fluorescence imaging., Conclusion: The presented method allows the expression and production of high amounts of long-circulating XTEN-AnxA5 without the necessity of PEGylation, thereby simplifying the synthesis while avoiding labeling-induced inactivation of AnxA5 and potential adverse effects of PEG. It is readily applicable to other recombinant protein or peptide-based imaging probes and allows fine-tuning of the desired blood half-life, because longer XTEN variants yield longer blood half-lives.

Background: Annexin V (AnxV) has potent anticoagulant properties and regulatory functions for apoptosis and inflammation. Antibodies against annexin V (anti-AnxVs) may inhibit AnxV functions, leading to thrombosis during autoimmune diseases. Type 1 diabetes is an autoimmune disease and related with an ongoing autoimmune inflammation and thrombotic complications. There is no study evaluating anti-AnxVs/AnxV in a disease setting., Objective: The aim of this study was to evaluate the status of AnxV and anti-AnxVs in patients with type 1 diabetes., Methods: One hundred twenty-one patients with type 1 diabetes and 92 healthy controls were included in this study. Serum levels of AnxV and anti-AnxVs and expression of the AnxV gene and its common polymorphism in Kozak sequence (-1C>T) were studied. As a functional assay, the binding capacity of AnxV to platelets was evaluated., Results: As compared with controls, type 1 diabetic patients had significantly low serum AnxV levels and AnxV gene expression. The number of anti-AnxV positivity and their serum levels were significantly higher in type 1 diabetic patients than controls. AnxV binding to platelets were significantly decreased in the type 1 diabetic patients. The frequencies of the -1C>T polymorphism of AnxV gene did not differ between groups., Conclusions: This study demonstrated the significant changes in AnxV levels and its function in type 1 diabetic patients. These results support the hypothesis that the defective AnxV system may have a role in ongoing autoimmune activity and the development of thrombotic complications in type 1 diabetes. Further studies are necessary to elucidate the clinical impact of anti-AnxVs and dysregulated AnxV function in type 1 diabetes.