Frail older adults show a distinct plasma microvesicle profile suggesting a prothrombotic and proinflammatory phenotype.

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.

Uremia and Inadequate Oxygen Supply Induce Eryptosis and Intracellular Hypoxia in Red Blood Cells.

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 Ca2+ 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 Ca2+ (Fluo-3/AM) and intracellular oxygen (Hypoxia Green) measurements, at baseline and after incubation with uremic serum and/or hypoxia (5% O2), 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% O2 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.

Enhanced Eryptosis in Glucose-6-Phosphate Dehydrogenase Deficiency.

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.

Effect of hemodialysis on extracellular vesicles and circulating submicron particles.

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.

Platelet-derived microparticles regulates thrombin generation via phophatidylserine in abdominal sepsis.

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.

Association of silent infarcts in sickle cell anemia with decreased annexin A5 resistance.

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-ß2GP1, anti-ß2GP1, 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.

Cardiolipin-mediated procoagulant activity of mitochondria contributes to traumatic brain injury-associated coagulopathy in mice.

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.

A method for red blood cell biotinylation in a closed system.

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.

Validation of a Gravitational Model to Study Local Endogenous Biomarkers in Chronic Venous Insufficiency.

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 (C4a-b) and 14 volunteers (C0-1) 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.

Cancer-associated circulating large extracellular vesicles in cholangiocarcinoma and hepatocellular carcinoma.

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.

Normalized levels of red blood cells expressing phosphatidylserine, their microparticles, and activated platelets in young patients with ß-thalassemia following bone marrow transplantation.

Bone marrow transplantation (BMT) serves as the only curative treatment for patients with ß-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 ß-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 ß-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.

Response to letter to the editor: annexin A5 levels or circulating microparticles: what we see depends mainly on what we look for.

Click here to view the article Letter to the Editor by S. Shetty et al.

Annexin A5 levels or circulating microparticles: what we see depends mainly on what we look for.

Click here to view the article Letter to the Editor by C. Reutelingsperger et al.

Circulating annexin A5 predicts mortality in patients with heart failure.

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.

The IgG and IgM isotypes of anti-annexin A5 antibodies: relevance for primary antiphospholipid syndrome.

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-ß2 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.

Annexin A5 promotes macrophage activation and contributes to pulmonary fibrosis induced by silica particles.

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 ß1 (TGF-ß1), 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-ß1 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-ß1, 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-ß1, 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.

Examining the prevalence of non-criteria anti-phospholipid antibodies in patients with anti-phospholipid syndrome: a systematic review.

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-ß2-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-ß2GPI 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 TEST OF GENERATION OF THROMBIN IN DYNAMICS IN PATIENTS AFTER TRANSCUTANEOUS CORONARY INTERVENTION].

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.

Young women with polycystic ovary syndrome have raised levels of circulating annexin V-positive platelet microparticles.

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.

Plasma annexin A5, anti-annexin A5 antibodies and annexin A5 polymorphism in Egyptian female patients with systemic lupus erythematosus and antiphospholipid syndrome.

BACKGROUND: Annexin A5 exhibits anticoagulant properties that appear to be defective in patients with antiphospholipid syndrome (APS) resulting in repeated thrombosis and recurrent pregnancy loss (RPL). APS occurs frequently in association with systemic lupus erythematosus (SLE). The present study aimed to find out a possible relationship between annexin A5 (gene polymorphism, antibodies or plasma level) and the pathophysiology of SLE, APS and RPL. METHODS: 47 female patients divided into 3 groups (SLE, APS and RPL) and 20 healthy controls are included in the study. Detection of annexin A5 (-1C/T) gene polymorphism was done by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) assay. Anti-annexin A5 antibodies (IgG and IgM) and annexin A5 plasma level were measured by Enzyme Linked Immunosorbent Assay (ELISA). RESULTS: The frequency of annexin A5 (-1C/T) polymorphism was significantly higher in SLE related groups (p = 0.02), but it did not correlate with RPL (p = 0.57) or annexin A5 level (p = 0.5). Anti-annexin A5 IgM level was significantly higher among APS patients and was associated with RPL (p = 0.005, odds ratio = 23.75, 95% confidence interval = 2.15 - 262.48). CONCLUSIONS: Annexin A5 (-1C/T) gene mutation may play a role in the pathophysiology of SLE. Anti-annexin A5 IgM was the antibody associated with RPL in this group of APS patients. Annexin A5 plasma levels are not affected by the presence of annexin A5 (-1C/T) polymorphism.