Autoantibodies against dsDNA measured with nonradioactive Farr assay-an alternative for routine laboratories.

Autoantibodies against dsDNA are utilized for the diagnosis and prognosis of SLE as they are highly specific and correlate with disease activity/renal involvement. However, different detection methods are used in routine diagnostic laboratories. Farr radioimmunoassay (Farr-RIA) has been designated as the preferred method, since it provides very specific and at the same time quantitative results, enabling follow-up of level variations over time. Using intercalating fluorescent dsDNA dye would enable all the benefits of Farr-RIA without the radioactive material and organic solvents. To develop a modified fluorescent Farr method (Farr-FIA) and compare it to the classical Farr-RIA in regard to laboratory parameters, as well as clinical utility. Assays were tested on sera of 70 SLE patients, 78 other autoimmune patients, and 145 healthy blood donors. DNA for Farr-FIA was isolated from healthy donor, for Farr-RIA, 14C-labeled dsDNA from E. coli was used and mixed with sera in borate-buffered saline, followed by precipitation with saturated ammonium sulfate solution and centrifugation. The supernatant (S) was separated from the precipitate (P), and content of dsDNA was measured with PicoGreen (Invitrogen) in Farr-FIA or radioactive isotope in scintillation solution in Farr-RIA. The results were calculated as a ratio (P-S)/(P+S). Farr-FIA has a diagnostic sensitivity of 53% and diagnostic specificity of 100% (ROC AUC 0.781). Good correlation and agreement were shown between Farr-RIA and Farr-FIA. Also, there is good correlation between Farr-FIA and SLEDAI, comparable to that of Farr-RIA. Farr-FIA differs from Farr-RIA in the changed detection system yielding comparable results and thus could represent a nonradioactive replacement for Farr-RIA.

Analysis of Drug Effects on Primary Human Coronary Artery Endothelial Cells Activated by Serum Amyloid A.

BACKGROUND: RA patients have a higher incidence of cardiovascular diseases compared to the general population. Serum amyloid A (SAA) is an acute-phase protein, upregulated in sera of RA patients. AIM: To determine the effects of medications on SAA-stimulated human coronary artery endothelial cells (HCAEC). METHODS: HCAEC were preincubated for 2 h with medications from sterile ampules (dexamethasone, methotrexate, certolizumab pegol, and etanercept), dissolved in medium (captopril) or DMSO (etoricoxib, rosiglitazone, meloxicam, fluvastatin, and diclofenac). Human recombinant apo-SAA was used to stimulate HCAEC at a final 1000 nM concentration for 24 hours. IL-6, IL-8, sVCAM-1, and PAI-1 were measured by ELISA. The number of viable cells was determined colorimetrically. RESULTS: SAA-stimulated levels of released IL-6, IL-8, and sVCAM-1 from HCAEC were significantly attenuated by methotrexate, fluvastatin, and etoricoxib. Both certolizumab pegol and etanercept significantly decreased PAI-1 by an average of 43%. Rosiglitazone significantly inhibited sVCAM-1 by 58%. CONCLUSION: We observed marked influence of fluvastatin on lowering cytokine production in SAA-activated HCAEC. Methotrexate showed strong beneficial effects for lowering released Il-6, IL-8, and sVCAM-1. Interesting duality was observed for NSAIDs, with meloxicam exhibiting opposite-trend effects from diclofenac and etoricoxib. This represents unique insight into specific responsiveness of inflammatory-driven HCAEC relevant to atherosclerosis.

Consequences of proceeding with surgery after resuscitation from intra-operative anaphylaxis.

Intra-operative acute hypersensitivity reactions require a decision to be made regarding whether to proceed with or abandon the planned surgical procedure once the patient has stabilised. Using retrospective case controls, we examined all cases (223) of proven acute hypersensitivity reactions from 2005 to 2014 in Western Australia, in which the syndrome was recognised by the treating clinician before or during surgery, to determine whether recovery outcomes were adversely affected by proceeding with the planned procedure. Surgery proceeded in 104 patients (47%) and was abandoned in 119 (53%). The severity of acute hypersensitivity reactions was Société Française d'Anesthésie et de Réanimation grade 1 or 2 in 56 patients (25%), grade 3 in 128 (56%) and grade 4 in 39 (17%). Abandoning surgery was more common in patients with increasing severity of hypersensitivity. The rate of major hypersensitivity-related complications for all patients was zero for grade 1 and 2 reactions, 4.7% for grade 3 and 12.8% for grade 4. There were no deaths. Patients in whom surgery was completed were not observed to have a higher frequency of major hypersensitivity-related complications when compared with cases of similar severity in whom surgery was abandoned. For patients admitted to the intensive care unit, proceeding with surgery was not associated with an increased duration of mechanical ventilation of the lungs. Our results suggest that, once initial resuscitation has been achieved and if resuscitative efforts can be re-instituted if required, continuing with planned surgery in grade 1, 2 and 3 immediate hypersensitivity was not associated with poorer outcomes. After grade 3 reactions, there was a significant incidence of complications attributable to acute hypersensitivity regardless of whether surgery proceeded or was abandoned. Surgery was frequently abandoned in grade 4 immediate hypersensitivity and was associated with a high rate of complications.

Vitamin D receptor gene polymorphisms association with the risk of sepsis and mortality.

Association of vitamin D receptor (VDR) gene polymorphisms with sepsis risk and mortality was studied. VDR FokI CC genotype was associated with increased sepsis risk (OR = 13.396, p = .000009) compared to the TT genotype. Results suggest possible role of VDR FokI (rs2228570) as a molecular biomarker of increased sepsis risk.

Secondary hyperparathyroidism prevalence and prognostic role in elderly males with heart failure.

AIM: Evaluation of secondary hyperparathyroidism (SHPT) and its prognostic impact on all-cause mortality in elderly males with heart failure (HF). METHODS: Seventy three males (67 ± 7 years old) with systolic HF were included. Baseline PTH was measured. Patients were grouped according to PTH cut-off levels of 65 pg/ml (>65 pg/ml = SHPT vs. normal PTH). All-cause mortality was evaluated at 6-year follow-up. RESULTS: SHPT was diagnosed in 43 (59 %) patients. They were more severe compared to the patients with normal PTH regarding NYHA functional class (2.4 ± 0.5 vs. 2.1 ± 0.2, p = 0.001), quality of life score (34 ± 14 vs. 24 ± 12, p = 0.005), 6-min walking distance (378 ± 79 vs. 446 ± 73 m, p < 0.0001), left ventricular ejection fraction (27 ± 8 vs. 31 ± 7 %, p = 0.019), and NT-proBNP [2452 (3399) vs. 918 (1372) pg/ml, p < 0.0001]. No differences in age, vitamin D status, and renal function were noted between studied groups. A total of 41 (56 %) patients died within 6 years of follow-up. Kaplan-Meier survival analysis showed impaired long-term survival in patients with SHPT versus patients with normal PTH (p = 0.009). The rate of death was highest (75 %) in the group of patients with SHPT and NT-proBNP levels above median value (p = 0.003). Cox regression analysis demonstrated that NT-proBNP was the single independent predictor of all-cause mortality at 6-year follow-up [HR 3.698 (1.927-7.095), p < 0.0001]. CONCLUSION: SHPT was highly prevalent in elderly males with HF and was associated with impaired survival. HF patients with SHPT had more severe disease compared to the patients with normal serum PTH. Determination of serum PTH levels provided additional value to NT-proBNP for risk stratification in these patients.

Oxidatively altered IgG with increased immunoreactivity to ß2-glycoprotein I and its peptide clusters influence human coronary artery endothelial cells.

Oxidative stress has been shown to play a role in modifying antibodies in favor of higher auto-immunoreactivity. We studied the immunoreactivity of oxidized IgG (oxIgG) to ß2-glycoprotein I (ß2GPI), six peptide sequences corresponding to amino acid clusters on its different domains, to determine their effects on human coronary artery endothelial cells (HCAEC). Human IgG was purified from seven donors, electro-oxidized and checked for immunoreactivity and avidity to ß2GPI and to peptides by ELISA. Conformational stability and antibody-antigen complex formation of oxIgG was analyzed by fluorescence spectroscopy and dynamic light scattering. Resting and activated sub-confluent HCAEC were stimulated with oxIgG or IgG. Secreted cytokines were measured by ELISA. Immunoreactivity of seven oxIgG samples increased to 7.5-fold against ß2GPI and to 3.8-fold against six peptides as compared to IgG. oxIgG showed low avidity "properties." Conformational changes and exposure of protein hydrophobic regions were confirmed by an elevation in fluorescence (2.4- to 5.0-fold) on bis-ANS dye binding to oxIgG. oxIgG significantly elevated the release of GROα and IL-8 in resting and activated states of HCAEC. Oxidation alters IgG in favor of autoreactivity toward whole ß2GPI and corresponding peptides on different domains of ß2GPI and could lead to dysfunction of arterial endothelium by upregulation of chemokines.

Immunoreactivity and avidity of IgG anti-ß2-glycoprotein I antibodies from patients with autoimmune diseases to different peptide clusters of ß2-glycoprotein I.

The pathogenicity of antibodies against ß2-glycoprotein I (anti-ß2GPI) depends on multiple factors such as subclass type, epitope binding and avidity. Due to their large heterogeneity, their impact on antiphospholipid syndrome (APS) onset is still not fully clarified. We studied the binding characteristics of IgG anti-ß2GPI with known avidity from sera of 201 autoimmune patients (87 with APS, 67 with APS associated with systemic lupus erythematosus (SLE), 47 with only SLE) to six ß2GPI peptides corresponding to amino acid clusters on domains I-II, II, III and III-IV by indirect ELISA and evaluated their association with clinical features of APS. Peptides A (LKTPRV; domain I-II), B (KDKATF; domain IV) and C (TLRVYK; domain III) were derived from a hexapeptide phage display library previously shown to react with pathogenic monoclonal anti-ß2GPI. Peptides D (NGPANSK; domain III), E (YNPLWFV; domain II) and F (KMDGNHP; domain III-IV) represent surface amino acid clusters on ß2GPI. The percentage of patients positive for peptides were observed as follows: 30.3% for peptide D, 28.90% for B, 25.9% for C, 24.9% for E, 24.4% for F and 10.0% for A. The anti-peptide antibodies in studied serum samples were predominantly of heterogeneous avidity, followed by law avidity anti-peptide antibodies, whereas only a few were of high avidity. Positive and negative correlations were found between several anti-peptide antibodies and the rate of thrombosis. Our results indicated diverse reactivity of IgG anti-ß2GPI to different epitopes on ß2GPI. Classification of IgG anti-ß2GPI into subgroups regarding epitope specificity and avidity could represent an additional tool in understanding their pathogenicity in APS.

Avidity of anti-ß2-glycoprotein I antibodies in patients with antiphospholipid syndrome.

Antibodies against ß(2)-glycoprotein I (anti-ß(2)GPI) are one of the hallmarks of the antiphospholipid syndrome (APS). However, they are heterogenic regarding their epitope specificity, pathogenic mechanisms and their avidity. In the current study we present some outstanding issues about avidity of anti-ß(2)GPI antibodies. Our results confirmed that high avidity anti-ß(2)GPI are associated with thrombosis and APS, while in low avidity anti-ß(2)GPI group non-APS (predominantly systemic lupus erythematosus) patients prevailed.

Antibodies to phosphatidylserine/prothrombin complex as an additional diagnostic marker of APS?

Antiprothrombin antibodies can be measured by ELISA using either a prothrombin/phosphatidylserine complex (aPS/PT) or prothrombin alone (aPT) as antigen. We aimed to compare the clinical features of autoimmune patients with avidity of aPS/PT and determine the diagnostic efficiency of aPS/PT and aPT for assessing antiphospholipid syndrome (APS). aPS/PT were of low (n = 9), heterogeneous (n = 31) and high (n = 8) avidity out of 48 cases. None of the samples with low avidity were positive in aPT ELISA. Among patients with heterogeneous or high avidity aPS/PT, there was a significantly greater number of patients with APS as compared to patients with low avidity (38/39 vs. 7/9; p < 0.05). No SLE patients had high avidity antiprothrombin antibodies.

Parathyroid hormone response to vitamin D insufficiency in elderly males with chronic heart failure.

Secondary hyperparathyroidism (SHPT) may contribute to the systemic illness that accompanies chronic heart failure (CHF). Healthy elderly with vitamin D deficiency who did not develop hyperparathyroidism (functional hypoparathyroidism, FHPT) had lower mortality than those who did. This study was designed to examine determinants of the PTH response in the vitamin D insufficient CHF patients. Sixty five vitamin D insufficient males with NYHA class II and III and 20 control subjects age >/=55 years were recruited. Echocardiography, physical performance, NT-pro-BNP, PTH, 25-hydroxyvitamin D (25(OH)D), adiponectin and bone activity surrogate markers (OPG, RANKL, OC, beta-CTx) were assessed. Increased NYHA class was associated with SHPT, while physical performance was inferior compared to FHPT. SHPT was associated with lower left ventricular ejection fraction (LVEF) and flow mediated dilatation, but with higher left heart dimensions, left ventricular mass index and right ventricular systolic pressure. CHF patients with SHPT had increased NT-pro-BNP, adiponectin and bone markers, but decreased 25(OH)D compared to those with FHPT. Independent determinants for SHPT in CHF patients with vitamin D insufficiency were LVEF, adiponectin and beta-CTx, irrespective of renal function and serum vitamin D levels. In conclusion, increased PTH levels, but not low vitamin D, demonstrated close relation to CHF severity.

The avidity of anti-beta2-glycoprotein I antibodies in patients with or without antiphospholipid syndrome: a collaborative study in the frame of the European forum on antiphospholipid antibodies.

OBJECTIVE: The objective of this study was to extend the findings of the preliminary study by measuring the avidity of IgG anti-ß2-glycoprotein I antibodies (anti-ß2-GPI) on a larger group of patients with primary or secondary antiphospholipid syndrome (APS) and anti-ß2-GPI positive patients without APS in the frame of the European Forum on antiphospholipid antibodies (aPL). METHODS: Serum from 137 patients with primary APS, APS associated with autoimmune diseases, and patients with autoimmune diseases other than APS from five EU rheumatology centres were tested for anti-ß2-GPI antibodies. The 109 patients who were sera positive for anti-ß2-GPI by the in-house anti-ß2-GPI enzyme-linked immunosorbent assay (ELISA) at the Immunology Laboratory, UMC Ljubljana were selected for further testing on avidity with chaotropic anti-ß2-GPI ELISA. RESULTS: High, low and heterogeneous avidity IgG anti-ß2-GPI was found in 32/109, 17/109 and 60/109 patients respectively. Significantly more patients with APS were in the high avidity than in the low avidity anti-ß2-GPI group, while the opposite was observed for non-APS (both p < 0.001). The most common clinical feature among patients with high avidity anti-ß2-GPI was thrombosis, mainly due to venous thrombosis (p < 0.01 and p < 0.001, versus low avidity anti-ß2-GPI group). CONCLUSION: Patients with or without APS had anti-ß2-GPI of high, low or heterogeneous avidity. High avidity anti-ß2-GPI was associated with thrombosis and APS, while in the low avidity anti-ß2-GPI group non-APS (predominantly SLE) patients prevailed. Determination of anti-ß2-GPI avidity should be considered in the analytical strategies for further differentiation of patients with anti-ß2-GPI antibodies.

Impact of multidisciplinary team management in head and neck cancer patients.

BACKGROUND: We analysed the outcomes of 726 cases of primary head and neck cancer patients managed between 1996 and 2008, including those managed in the multidisciplinary clinic or team setting (MDT) and those managed outside of an MDT by individual disciplines (non-MDT) in the same institution. METHODS: Data were collected from the Hospital Based Cancer Registry and a database within the Head and Neck Cancer Clinic. Univariable comparisons and multivariable analyses were performed using a logistic regression model. Survival by staging was analysed. Comparisons of management and outcomes were made between MDT and non-MDT patients. RESULTS: 395 patients (54%) had been managed in the MDT vs 331 patients (46%) non-MDT. MDT patients were more likely to have advanced disease (likelihood ratio χ(2)=44.7, P<0.001). Stage IV MDT patients had significantly improved 5-year survival compared with non-MDT patients (hazard ratio=0.69, 95% CI=0.51-0.88, P=0.004) and more synchronous chemotherapy and radiotherapy (P=0.004), and the non-MDT group had more radiotherapy as a single modality (P=0.002). CONCLUSIONS: The improved survival of MDT-managed stage IV patients probably represents both the selection of multimodality treatment and chemotherapeutic advances that these patients received in a multidisciplinary team setting by head and neck cancer specialists as opposed to cancer generalists in a non-MDT setting.

An international multicentre-laboratory evaluation of a new assay to detect specifically lupus anticoagulants dependent on the presence of anti-beta2-glycoprotein autoantibodies.

BACKGROUND: Antiphospholipid syndrome (APS) is diagnosed by the simultaneous presence of vascular thrombosis and/or pregnancy morbidity and detection of antiphospholipid antibodies in plasma. OBJECTIVES: We have shown that prolongation of clotting time by anti-beta2-glycoprotein I (beta2GPI) antibodies correlates better with thrombosis than a positive classic lupus anticoagulant (LAC) assay in a single center study. To confirm or falsify this finding we have conducted a multicenter study. METHODS AND RESULTS: In 325 LAC-positive samples, we found that the beta2GPI-dependent LAC correlated 2.0 times better with thrombosis than the classic LAC assay. Although significant, this was a minimal improvement compared with the 'classic' LAC. It was published that calcium influences the behavior of anti-beta2GPI antibodies in coagulation assays. To investigate whether calcium plays a role in the present study, we divided the patient population into two groups: (i) blood was collected in 0.109 m sodium citrate and (ii) blood was drawn in 0.129 m sodium citrate as anticoagulant. We found that a positive result with the beta2GPI-dependent LAC assay correlated better with thrombosis [odds ratio (OR): 3.3, 95% confidence interval (CI) 1.9-5.8] when 0.109 m sodium citrate was used compared with 0.129 m sodium citrate (OR: 0.4, 95% CI 0.1-1.1). CONCLUSION: We were able to confirm in an international multicenter study that a positive result in a beta2GPI-dependent LAC assay correlates better with thrombosis than the classic LAC assay, but that the assay needs further study as it is sensitive to external factors such as the sodium citrate concentration used as anticoagulant in the test sample.

Association of increased parathyroid hormone with neuroendocrine activation and endothelial dysfunction in elderly men with heart failure.

High PTH levels have been reported in patients with chronic heart failure (CHF). Similarly, its levels increase with aging and are related to impaired survival in elderly adults. However, its relationship with neuroendocrine activation and endothelial dysfunction in CHF has not been previously studied. Seventy-three CHF males with New York Heart Association (NYHA) classes II and III and 20 control subjects aged ≥ 55 yr were recruited. PTH, 25-hydroxyvitamin D [25(OH)D], N-terminal pro-brain natriuretic peptide (NT-pro-BNP), adiponectin, and osteoprotegerin were measured. Endothelial function (brachial flow mediated dilation), echocardiography, physical performance, and quality of life were assessed, as well. CHF patients had markedly increased serum PTH (77 ± 33 vs 40 ± 11 pg/ml, p<0.0001), NT-pro-BNP [1809 (2742) vs 67 (74) pg/ml, p<0.0001], adiponectin (17 ± 9 vs 10 ± 2 µg/ml, p<0.0001), osteoprotegerin, whereas 25(OH)D levels were decreased compared to controls. Increased PTH is positively correlated with NTpro- BNP (r=0.399, p<0.0001), adiponectin (r=0.398, p<0.0001), and osteoprotegerin, whereas negatively with 25(OH)D in CHF patients. Additionally, increased serum PTH was associated with endothelial dysfunction, echocardiographic variables of heart failure progression, impaired physical performance, and deteriorated quality of life. In a multivariate linear regression analysis, increased serum PTH was independently associated with neuroendocrine activation (NT-pro-BNP, adiponectin) and endothelial dysfunction in elderly CHF men (R2=0.455). Additionally, demonstrated relations with other well-established variables of heart failure severity suggest the potential role of serum PTH in the pathogenesis and non-invasive monitoring of heart failure progression. Future studies are needed to evaluate the predictive value of serum PTH for clinical outcomes as well as beneficial potential of PTH suppression in CHF patients.

New experimental model of acute aqueductal blockage in cats: effects on cerebrospinal fluid pressure and the size of brain ventricles.

It is generally assumed that cerebrospinal fluid (CSF) is secreted in the brain ventricles, and so after an acute blockage of the aqueduct of Sylvius an increase in the ventricular CSF pressure and dilation of isolated ventricles may be expected. We have tested this hypothesis in cats. After blocking the aqueduct, we measured the CSF pressure in both isolated ventricles and the cisterna magna, and performed radiographic monitoring of the cross-sectional area of the lateral ventricle. The complete aqueductal blockage was achieved by implanting a plastic cannula into the aqueduct of Sylvius through a small tunnel in the vermis of the cerebellum in the chloralose-anesthetized cats. After the reconstitution of the occipital bone, the CSF pressure was measured in the isolated ventricles via a plastic cannula implanted in the aqueduct of Sylvius and in the cisterna magna via a stainless steel cannula. During the following 2 h, the CSF pressures in the isolated ventricles and cisterna magna were identical to those in control conditions. We also monitored the ventricular cross-sectional area by means of radiography for 2 h after the aqueductal blockage and failed to observe any significant changes. When mock CSF was infused into isolated ventricles to imitate the CSF secretion, the gradient of pressure between the ventricle and cisterna magna developed, and disappeared as soon as the infusion was terminated. However, when mock CSF was infused into the cisterna magna at various rates, the resulting increased subarachnoid CSF pressure was accurately transmitted across the brain parenchyma into the CSF of isolated ventricles. The lack of the increase in the CSF pressure and ventricular dilation during 2 h of aqueductal blockage suggests that aqueductal obstruction by itself does not lead to development of hypertensive acute hydrocephalus in cats.

Autoimmune and proinflammatory activity of oxidized immunoglobulins.

AIMS: Oxidation reactions can modify protein activity or specificity. Recently, a novel redox-reactive family of autoantibodies was described, which indicated involvement of altered antibodies (beside altered antigens) into autoimmune reactions. The aim of our study was to determine the binding capacity alterations of electro-oxidized blood donors' IgGs, and to evaluate their effects on released proinflammatory interleukin 6 in HUVEC. RESULTS: We found out that 1.) Isolated blood donor IgGs bound after electro-oxidation to beta2-glycoprotein I, cardiolipin, citrullinated cyclic peptide and protein 3 by enzyme-linked immunosorbent assay, extractable nuclear antigens by counterimmuno-electrophoresis, and cell antigens by indirect immunofluorescence; 2.) Alterations in immunoreactivity of IgGs due to oxidation highly depend on electric current, time of exposure and the presence of antioxidants, 3.) Treatment of HUVEC with oxidized IgGs resulted in changed cell morphology, accompanied by an increase in released interleukin-6. CONCLUSIONS: Our data suggest repeatable transformation of antibodies present in the blood of healthy persons and patients. Inter-individual differences in chemical stability of antibodies, patient's antioxidant status, and the microenvironmental changes at the cellular level may influence the range of antibody alterations and their involvement in pathophysiological autoimmune processes.

Vesicle self-reproduction: the involvement of membrane hydraulic and solute permeabilities.

Conditions for self-reproduction are sought for a growing vesicle with its growth defined by an exponential increase of vesicle membrane area and by adequate flow of the solution across the membrane. In the first step of the presumed vesicle self-reproduction process, the initially spherical vesicle must double its volume in the doubling time of the membrane area and, through the appropriate shape transformations, attain the shape of two equal spheres connected by an infinitesimally thin neck. The second step involves separation of the two spheres and relies on conditions that cause the neck to be broken. In this paper we consider the first step of this self-reproduction process for a vesicle suspended in a solution whose solute can permeate the vesicle membrane. It is shown that vesicle self-reproduction occurs only for certain combinations of the values of membrane hydraulic and solute permeabilities and the external solute concentration, these quantities being related to the mechanical properties of the membrane and the membrane area doubling time. The analysis includes also the relaxation of a perturbed system towards stationary self-reproduction behavior and the case where the final shape consists of two connected spheres of different radii.

Annexin A5 binding to giant phospholipid vesicles is differentially affected by anti-beta2-glycoprotein I and anti-annexin A5 antibodies.

OBJECTIVES: Anti-phospholipid antibodies have been recognized to play a role in vascular thrombosis and pregnancy morbidity. They were first thought to be directed to phospholipids, but it is now known that the majority of pathogenic antibodies recognizes epitopes on phospholipid-binding plasma proteins such as beta2-glycoprotein I (beta2GPI) or possibly also annexin A5 (ANXA5). The mechanism of their prothrombotic action is still not completely understood. The aim of the present study was to observe the effect of antibodies against ANXA5 (aANXA5) and antibodies against beta2GPI (abeta2GPI) on the binding of ANXA5 to the negatively charged phospholipid membrane. METHODS: Giant phospholipid vesicles (GPVs) were used as a simple model of the membrane surface. GPVs composed of phosphatidylserine and phosphatidylcholine were produced in an aqueous medium. A single GPV was transferred to the solution containing ANXA5 conjugated with Alexa Fluor 488 (FANXA5) and (i) aANXA5 or abeta2GPI and (ii) different concentrations of abeta2GPI together with beta2GPI. The emission of the fluorescent light from the GPV surface, as the result of FANXA5 binding, was measured. RESULTS: Beta2GPI together with abeta2GPI reduced the binding of FANXA5 to GPVs. On the contrary, aANXA5 enhanced the binding of ANXA5 to the GPV surface. CONCLUSIONS: Our results point to the competition between FANXA5 and complexes of beta2GPI-abeta2GPI for the same binding sites and therefore support the hypothesis of the disruption of the ANXA5 protective shield on procoagulant phospholipid surface. The influence of increased cell surface ANXA5 concentration in the presence of aANXA5 on coagulation needs to be further studied.