Anti-Phosphatidylserine/Prothrombin Antibodies Are Associated with Adverse Pregnancy Outcomes.

Objective. To determine the prevalence and clinical association of anti-phosphatidylserine/prothrombin antibodies (aPS/PT) in patients with a history of pregnancy complications relevant to antiphospholipid syndrome (APS). Materials and Methods. Two hundred and eleven patients with a history of (a) three or more consecutive miscarriages before 10th week of gestation (WG) (n = 64), (b) death of a morphologically normal fetus beyond 10th WG (n = 72), (c) premature birth of a morphologically normal neonate before 34th WG due to eclampsia, preeclamsia and placental insufficiency (n = 33), and (d) less than three unexplained consecutive miscarriages before 10th WG (n = 42). Subjects sera were analyzed for lupus anticoagulant (LA), anti-cardiolipin (aCL), anti-ß 2-glycoprotein I (anti-ß 2GPI), and aPS/PT antibodies. Results. 41/169 (24.3%) of patients were positive for at least one measured aPL. The highest prevalence was found for aPS/PT and aCL (13.0% and 12.4%, resp.) followed by LA (7.7%) and anti-ß 2GPI (7.1%). 11/169 with APS-related obstetric manifestations had only aPS/PT. 17.8% of patients were positive for LA or aCL and/or anti-ß 2GPI; however when adding the aPS/PT results, an additional 7% of patients could be evaluated for APS. Conclusion. aPS/PT are associated with recurrent early or late abortions and with premature delivery irrespective of other aPL.

Anti-ß2-glycoprotein I paratopes and ß2-glycoprotein I epitopes characterization using random peptide libraries.

Studies concerning interactions between anti-ß2-glycoprotein I antibodies (anti-ß2GPI) and ß2-glycoprotein I (ß2GPI) suggest relevance of charge interactions and hydrogen bonds. However, paratope of diagnostically and clinically relevant anti-ß2GPI and epitope characteristics of ß2GPI, still remain unclear. The aim of our study was to determine paratope characteristics of various anti-ß2GPI antibodies and epitope characteristics of ß2GPI using phage display. Monoclonal IgG anti-ß2GPI, purified polyclonal high avidity and low avidity IgG anti-ß2GPI derived from plasma of APS patients were used to screen phage display libraries. The affinity and competition ability of selected clones were evaluated. Various heptapeptides presenting putative paratopes of anti-ß2GPI and specific heptapeptides presenting putative epitopes of ß2GPI were determined. Epitope presenting peptides bind to the respective anti-ß2GPI and consequently interrupt antibody-antigen interaction. The amino acid composition of selected peptides confirmed the importance of hydrogen bonds and charge interactions in the binding of anti-ß2GPI to the antigen. Epitopes recognized by high avidity anti-ß2GPI predominately contain hydrogen bond forming side chains, while in low avidity anti-ß2GPI epitope the charged side chains prevail. The alignment of selected sequences to three-dimensional antigen structure revealed that polyclonal high avidity anti-ß2GPI recognize native epitopes that are accessible regardless of ß2GPI's conformation whereas the epitope recognized by low avidity anti-ß2GPI is cryptic and cannot be accessed when ß2GPI takes the closed plasma conformation.

Detection of antiphosphatidylserine/prothrombin antibodies and their potential diagnostic value.

Antiprothrombin antibodies, measured with phosphatidylserine/prothrombin complex (aPS/PT) ELISA, have been reported to be associated with antiphospholipid syndrome (APS). They are currently being evaluated as a potential classification criterion for this autoimmune disease, characterized by thromboses and obstetric complications. Given the present lack of clinically useful tests for the accurate diagnosis of APS, we aimed to evaluate in-house and commercial assays for determination of aPS/PT as a potential serological marker for APS. We screened 156 patients with systemic autoimmune diseases for antibodies against PS/PT, ß2-glycoprotein I, cardiolipin and for lupus anticoagulant activity. We demonstrated a high degree of concordance between the concentrations of aPS/PT measured with the in-house and commercial assays. Both assays performed comparably relating to the clinical manifestations of APS, such as arterial and venous thromboses and obstetric complications. IgG aPS/PT represented the strongest independent risk factor for the presence of obstetric complications, among all tested aPL. Both IgG and IgM aPS/PT were associated with venous thrombosis, but not with arterial thrombosis. Most importantly, the association between the presence of IgG/IgM aPS/PT and lupus anticoagulant activity was highly significant. Taken together, aPS/PT antibodies detected with the in-house or commercial ELISA represent a promising serological marker for APS and its subsets.

International cohort study of 73 anti-Ku-positive patients: association of p70/p80 anti-Ku antibodies with joint/bone features and differentiation of disease populations by using principal-components analysis.

INTRODUCTION: An international cohort study of 73 anti-Ku-positive patients with different connective tissue diseases was conducted to differentiate the anti-Ku-positive populations of patients based on their autoantibody profile and clinical signs/symptoms and to establish possible correlations between antibodies against Ku p70 and Ku p80 with autoimmune diseases. METHODS: Sera of anti-Ku-positive patients were collected from six European centers and were all secondarily tested (in the reference center); 73 were confirmed as positive. Anti-Ku antibodies were detected with counter-immunoelectrophoresis (CIE), line immunoassay (LIA), and immunoblot analyses. All clinical and laboratory data were follow-up cumulative data, except for anti-Ku antibodies. Statistical analyses were performed by using R (V 2.12.1). The Fisher Exact test was used to evaluate the association between anti-Ku antibodies and diagnosis, gender, clinical signs, and other observed antibodies. The P values were adjusted for multiple testing. Separation of disease populations based on the presence of antibodies and clinical signs was investigated by principal-components analysis, which was performed by using thr// R's prcomp function with standard parameters. RESULTS: A 16% higher prevalence of anti-Ku p70 was found over anti-Ku p80 antibodies. In 41 (57%) patients, a combination of both was detected. Five (7%) patients, who were CIE and/or LIA anti-Ku positive, were negative for both subsets, as detected with the immunoblot; 31% of the patients had undifferentiated connective tissue disease (UCTD); 29% had systemic sclerosis (SSc); 18% had systemic lupus erythematosus (SLE); 11% had rheumatoid arthritis; 7% had polymyositis; and 3% had Sjögren syndrome. CONCLUSIONS: A significant positive association was found between female patients with anti-Ku p70 and joint/bone features, and a significant negative association was found between female patients with anti-Ku p80 only and joint/bone features (P = 0.05, respectively). By using the first and the third components of the principal-component analysis (PCA) with 29 parameters evaluated, we observed that the anti-Ku-positive population of UCTD patients had overlapping parameters, especially with SLE, as opposed to SSc, which could be helpful in delineating UCTD patients.

Autoimmune reactivity of IgM acquired after oxidation.

OBJECTIVES: Redox-reactive antibodies, mainly of the IgG class, gained a wide area of interest after their autoimmune reactivity was revealed following the application of chemical and physiological oxidants. In this study, we examined the susceptibility of IgMs to oxidation and evaluated their binding to the autoantigens important in some autoimmune diseases. METHODS: IgM and IgG fractions, isolated from healthy individuals' sera, were oxidized using direct electric current or physiological oxidant hemin. Specificities towards beta-2-glycoprotein I (ß(2)-GPI), cardiolipin (CL), and rheumatoid factor were evaluated with the enzyme-linked immunosorbent assays (ELISAs). Post-translational modification was investigated by 2,4-dinitrophenylhydrazine reaction. RESULTS: Electrochemically oxidized IgM fractions exhibited altered immunoreactivity - low to medium titers in anti-CL and low positive titers in anti-ß(2)-GPI ELISA but exhibited no rheumatoid factor reactivity. Oxidized IgG and IgM fractions exhibited 2.5- and 5-fold increase in the carbonyl content, respectively. DISCUSSION: An increase in the carbonyl content along with increased immunoreactivity after oxidation suggests modifications of the IgM paratopes. These results point towards possible modifications of native IgMs to their autoimmune state despite the fact that IgMs were less susceptible to oxidation than IgGs. The importance of an individual's redox status in maintenance of autoimmune reactions was emphasized by in vitro diagnostic tests.

Comparison and evaluation of different methodologies and tests for detection of anti-dsDNA antibodies on 889 Slovenian patients' and blood donors' sera.

AIM: To evaluate four different commercially available assays for anti-double stranded DNA (dsDNA) detection and compare them with the in-house radioimmunoassay according to Farr (FARR-RIA) in order to select the optimal primary method for use in combination with FARR-RIA. METHODS: Sera from 583 consecutive patients sent to our laboratory for routine diagnosis, 156 selected patients with autoimmune diseases (76 systemic lupus erythematosus [SLE] patients and 80 patients with other autoimmune diseases), and 150 blood donors were tested for anti-dsDNA antibodies with two enzyme-linked immunoassays (ELISA), two Crithidia luciliae immunofluorescence tests (CLIFT), and FARR-RIA. The specificities and sensitivities of the tests were calculated and compared. RESULTS: FARR-RIA and CLIFT 2 showed the highest specificity for SLE (100%), with CLIFT 2 showing higher sensitivity (33% vs 47%). Both ELISAs showed higher sensitivities (>53%) than FARR-RIA but lower specificities (<93%), whereas CLIFT 1 showed the lowest overall agreement with FARR-RIA. CONCLUSION: CLIFT 2 was selected as the primary test for use in combination with FARR-RIA. The use of CLIFT 2 reduced the number of sera that needed to be tested by FARR-RIA, the time needed to report the results, and environmental toxicity, cancerogenicity, and radioactivity.

Antiphospholipid antibodies and atherosclerosis: insights from rheumatoid arthritis--a five-year follow-up study.

OBJECTIVES: Life expectancy in rheumatoid arthritis (RA) patients is reduced by 3-10 years, probably due to cerebrovascular and cardiovascular diseases associated with atherosclerosis. In the present study, we wanted to verify if previously reported IgA anti-beta 2-glycoprotein I (2GPI) antibodies possibly represented an independent risk factor for atherosclerosis in RA patients during a longer period of follow up. METHODS: The follow-up study (after 5.5 years) comprised all initially included patients and controls (premenopausal women, non-diabetic, normotensive at the start of the study), except for two RA patients (one died and one not available). The same clinical, laboratory and ultrasound assessments were performed. RESULTS: Patients and controls were divided into three categories: Intima-media thickness (IMT) progressors, plaque progressors, IMT and plaque progressors. In controls, 55% represented IMT progressors and 5% IMT and plaque progressors. No statistically significant differences were detected comparing the progressors with delta (Δ=difference between follow-up and baseline study for each group in a time span of 5.5 years) LDL cholesterol, homocysteine and IgA anti-ß2GPI. In patients, there were 48.5% IMT progressors, 5.8% plaque progressors and 19.1% IMT and plaque progressors. The progression was statistically significant associated with the levels of Δ homocysteine and Δ apolipoprotein B but not with LDL cholesterol and IgA anti-ß2GPI. CONCLUSIONS: The follow-up study showed advanced atherosclerosis in RA patients compared to sex and age matched controls. However, we were not able to confirm our initial impression that IgA anti-ß2GPI might represent an independent risk factor for atherosclerosis.

Immunochemical properties and pathological relevance of anti-ß2-glycoprotein I antibodies of different avidity.

Despite available treatment, there is still significant morbidity and mortality present among patients with the autoimmune thrombophilic condition termed 'antiphospholipid syndrome' (Espinosa, G. and Cervera, R. 2009. Morbidity and mortality in the antiphospholipid syndrome. Curr. Opin. Pulm. Med. 15:413.). High-avidity (HAv) anti-ß(2)-glycoprotein I (anti-ß(2)GPI) antibodies, shown to correlate with thrombotic events in patients, could represent the much needed improved prognostic marker. By studying their effect on crystalline annexin A5 shield on phospholipid surfaces (one of proposed pathogenic mechanisms), with the use of atomic force microscopy, the pathogenic potential of HAv anti-ß(2)GPI antibodies was confirmed. Furthermore, by using surface plasmon resonance and enzyme-linked immunosorbent assays, unique binding characteristics of HAv antibodies in comparison with low avidity antibodies were established. HAv anti-ß(2)GPI were confirmed to (i) recognize ß(2)-glycoprotein I in a solution, (ii) interact predominantly monovalently (much lower dependency on the antigen density) and (iii) form more stable complexes with the antigen. Since enzyme-linked immunosorbent assays currently used in routine diagnostics detect anti-ß(2)GPI antibodies of unknown avidity, our observations are potentially useful for the development of improved diagnostic tests capable of detecting clinically relevant antibodies.

Modified phosphatidylserine-dependent antiprothrombin [corrected] ELISA enables identification of patients negative for other antiphospholipid antibodies and also detects low avidity antibodies.

BACKGROUND: Two approaches for detecting anti-prothrombin antibodies have been described. The first detects antibodies against prothrombin alone and the second, phos-phatidylserine-dependent antiprothrombin antibodies. The latter more often correlate with clinical manifestations of antiphospholipid syndrome and with lupus anticoagulant activity. METHODS: In order to increase the capacity of antibody binding, we modified the previously described phosphatidylser-ine-dependent antiprothrombin ELISA and determined their avidity. We examined 203 patients with systemic autoimmune diseases and 222 blood donors. RESULTS: Our modification resulted in a greater intensity of antibody binding to prothrombin on phosphatidylserine-coated plate surfaces compared to the previously described method. By changing ELISA conditions, we were able to detect with one assay the two, presumably different, populations of antiprothrombin antibodies. Diagnostic specificities of both ELISAs for antiphospholipid syndrome were similar (92.5% vs. 93.1%), while the sensitivity of the modified phosphatidylserine-dependent antiprothrombin ELISA was significantly higher than the anti-prothrombin alone ELISA (59% vs. 25%). Low avidity antiprothrombin antibodies were only detected in the modified phosphatidylserine-dependent antiprothrombin ELISA. Four percent of patients with positive phosphatidylserine-dependent antiprothrombin antibodies, showing clinical manifestations of antiphospholipid syndrome, were negative for all other antiphospholipid antibodies. The risk for antiphospholipid syndrome increased with the number of antiphospholipid antibody positivity. CONCLUSIONS: We conclude that antibodies detected with a modified phosphatidylserine-dependent antiprothrombin ELISA could improve the diagnosis of antiphospholipid syndrome by offering additional information on the risk for thrombosis, especially in patients negative for other antiphospholipid antibodies.

Significance of K(L/V)WX(I/L/V)P Epitope of the B2Gpi in Its (Patho)Physiologic Function.

ß2-glycoprotein I (ß2GPI) is a major autoantigen of autoimmune thrombophilia, known as the antiphospholipid syndrome. The exact mechanism underlying the ß2GPI's involvement in the disease is not fully elucidated, as it is not its physiological role. We used random phage peptide library to identify sequences binding to ß2GPI. Obtained K(L/V)WX(I/L/V)P motif, primarily designated as target unrelated, was confirmed as the selective binder of ß2GPI. Based on this motif we confirmed the previously suggested role of polar residues in ß2GPI interactions, and identified some already known and some new putative ß2GPI binding proteins. The latter can help to further elucidate ß2GPI's (patho)physiological role.

Alteration of antibody specificity during isolation and storage.

Isolation buffer characteristics and storage conditions could partially transform natural antibodies. 50 IgG factions were isolated from seven healthy donors' sera using various protein G columns and buffers. PAGE revealed no major antibody cleavages; purity of IgGs eluted at pH 2.4-3.5 was close to homogeneity, independent of buffer composition. Although eluting at pH 2.4 resulted in 3.5- or 17-fold higher antibody yield compared to pH 3.0 or 3.5, respectively, it distorted the antibody molecule. IgGs eluted at pH 2.4 acquired reactivity against diagnostically important autoantigens, confirmed by standardized ELISAs. Preserved antibodies' natural activity is important in further experiments with oxidatively-induced autoantibodies.

Rational Laboratory Diagnostics of Antiphospholipid Antibodies: Anti-Cardiolipin, Anti-ß2-Glycoprotein I, Anti-Prothrombin and Anti-Annexin V Antibodies.

A possible co-appearance of anticardiolipin (aCL), anti-ß2-glycoprotein I (anti-ß2-GPI), anti-prothrombin (aPT) and anti-annexin V (aANXV) antibodies of IgG, IgM and IgA class were studied in 58 patients with SLE alone and 32 patients APS in the view of rational laboratory diagnostics. The presence of anti-phospholipid antibodies (aPL) were defined by our in-house ELISA methods. Out of 17 aCL negative SLE patients 6 had other antigenically defined aPL antibodies. In 13 patients only IgA but not IgG and IgM anti-ß2GPI were detected. Different combinations of aPL subsets were equally distributed in APS and SLE groups. The prevalence of aANXV were similar in APS and SLE patients which was not the case with other aPL. Our findings support the idea of measuring additional subsets of aPL (aPT and aANXV) in unclear cases. IgA (either aCL or anti-ß2-GPI) improved neither the diagnostic specificity nor diagnostic sensitivity, but only increased the frequency of the total anti-ß2-GPI.

Autoimmune reactions after electro-oxidation of IgG from healthy persons: relevance of electric current and antioxidants.

Proteins, including immunoglobulins, can be modified by oxidation. Extensive oxidation of immunoglobulins leads to denaturation and loss of biological activity, while initial steps of oxidation may change their specificity due to chemical alteration of the paratope. Electro-oxidation of the IgG fraction from healthy persons progress to auto-immunoreactivity, as shown for several autoantibodies including anti-beta2-glycoprotein I. Changes in immunoreactivity of IgG due to oxidative reactions highly depend on electric current and levels of serum antioxidants. Autoimmune reactions, leading to certain autoimmune diseases, may be partially a consequence of unbalanced antioxidative status of an individual.

Autoantibodies in nonautoimmune individuals during infections.

Infections can act as environmental triggers inducing or promoting autoimmune disease in genetically predisposed individuals. Identification of microbial peptides similar to self-tissues may by molecular mimicry, provide the inducing mechanism for an immune response. The aim of this study was to identify autoantibodies (autoAbs) in nonautoimmune individuals during acute bacterial, viral, or parasitic infections. Specific Abs or specific infections with an increased autoAb load may shed insight into the mechanisms of autoimmune disease. Sera from 88 patients with acute infections (41 bacterial, 23 viral, 17 parasitic, and 7 rickettsial) were tested by the ELISA method for antinuclear antibodies (ANA) 8 Pro, and Abs to thyroid peroxidase (TPO), thyroglobulin, phospholipids, annexin-V, laminin, anti-Saccharomyces cervisiae (ASCA), and prothrombin, along with 80 normal controls. Elevated titers of Abs to annexin-V and prothrombin were the most prevalent in viral, parasitic, and rickettsial infections and to laminin in viral and parasitic infections. Elevated titers of ASCA and ANA were found in viral and bacterial infections. Antiphospholipid Abs were found in parasitic and Q-fever infections. Thirty-four individuals harbored elevated titers of at least two Abs. An autoAb burden was detected in individuals with hepatitis A, hepatitis B, toxoplasma or Q-fever infections. In nonautoimmune individuals with various (bacterial, viral, parasitic, and rickettsial) infections, elevated titers of Abs to annexin-V, prothrombin, laminin, ASCA, ANA, and phospholipids were most frequently detected.

Anti-beta 2-glycoprotein I antibodies of IgM class are linked to thrombotic disorders in young women without autoimmune disease.

Antiphospholipid autoantibodies particularly antibodies against beta2-glycoprotein I (anti-beta2GPI) are casually associated with thromboses in patients with autoimmune diseases. However, their exact prevalence and role in the pathogenesis of thromboses in the absence of autoimmune disease is still inconclusive. They might be particularly important when other risk factors of thrombosis are absent. We investigated antiphospholipid antibodies in 68 young women (aged <45yr at onset of the event, without autoimmune disease and with an otherwise low risk of thrombosis) in the stable period following myocardial infarction (MI), lacunar cerebral infarction (LACI) or deep vein thrombosis (DVT) and in 37 healthy age-matched controls. Patients had increased IgM anti-beta2GPI compared to controls (36.0, 11.5-49.5 vs. 17.50, 3.50-30.0 arbitrary units (AU), p<0.001), whereas no difference was obtained in other measured antibodies (anticardiolipin and antiphosphatidylserine (aPS) antibodies of IgG and IgM). IgM anti-beta2GPI positively correlated with some markers of increased coagulation potential and negatively with BMI (r=-039, p<0.005) and other parameters of the metabolic syndrome. In conclusion, we found that levels of IgM anti-beta2GPI are increased in young women suffering arterial or venous thromboses in the absence of other known autoimmune diseases and also in the absence of pronounced classical risk factors. We found that IgM anti-beta2GPI positively correlated with some markers of increased coagulation potential and negatively with parameters of the metabolic syndrome. Thus, it appears that elevated levels of IgM anti-beta2GPI are linked to thrombotic disorders in young women (without autoimmune disease) particularly when classical risk factors or the metabolic syndrome are absent.

Antiphospholipid antibodies as a possible risk factor for atherosclerosis in patients with rheumatoid arthritis.

Atherosclerosis shares many similarities with inflammatory and autoimmune diseases, among them rheumatoid arthritis (RA). Anticardiolipin antibodies (aCL) and antibodies against beta2-glycoprotein I (anti-beta2GPI) have been detected in sera of RA patients in several studies. We demonstrated aCL and anti-beta2GPI in a selected group of 70 patients with RA (premenopausal women, non-diabetic, non-hypertensive) and compared them with age- and sex-matched controls. There was a significant higher internal carotid artery intima-media thickness and number of plaques in RA patients compared to controls. aCL of IgG and IgM classes were present in 15.7% of RA patients as compared to 5% in the control group. Thirty percent of RA patients had anti-beta2GPI of IgG, IgM and IgA classes compared to 7.5% in controls. Major differences were seen in IgG and IgA classes. Our results support the idea that aCL and anti-beta2GPI represent an important risk factor for atherosclerosis in RA patients. Elevated levels of phosphatidylserine-dependent antiprothrombin antibodies did not contribute significantly to the general prevalence of antiphospholipid antibodies.

Endothelial function is impaired in patients with primary antiphospholipid syndrome.

INTRODUCTION: The aim of this study was to evaluate endothelial function in patients with primary antiphospholipid syndrome (PAPS). PATIENTS AND METHODS: Flow mediated (FMD) and glyceryl trinitrate (GTN) induced dilation of the right brachial artery were studied in 25 patients with PAPS and 25 controls matched by age, sex and conventional risk factors for atherosclerosis. Fibrinogen, D-dimer, adhesion molecules, tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) antigens and activities were measured. RESULTS: Mean (SD) FMD was significantly lower in PAPS than in controls (8+/-5% vs. 15+/-6%, P<0.001); GTN-induced dilation did not differ between the groups. There was a correlation between the baseline diameter of the brachial artery and duration of the disease (-0.56, P<0.05) and between GTN induced dilation and duration of the disease (0.51, P<0.05). Concentrations of vascular cell adhesion molecule-1 (P<0.001), intracellular adhesion molecule-1 (P<0.001) and fibrinogen (P<0.05) were higher in patients than in controls but no differences were observed for D-dimer, t-PA and PAI-1 antigens and activities. There was correlation between concentration of vascular cell adhesion molecule-1 and FMD (-0.35, P<0.05) and between intracellular adhesion molecule-1 and FMD (-0.41, P<0.05). CONCLUSIONS: This study shows that endothelial function is impaired in patients with primary APS, possibly contributing to accelerated atherosclerosis and thromboembolic complications in these patients.

Antibodies against annexin A5: detection pitfalls and clinical associations.

Antiphospholipid syndrome (APS) has been defined as a clinical and laboratory entity. Laboratory criteria include the presence of anticardiolipin antibodies (aCL) and/or lupus anticoagulant (LA), collectively termed as antiphospholipid antibodies (aPL). However, there has been a rising interest in antibodies against so-called protein cofactors, particularly in beta(2)-glycoprotein I. In the early 90s, annexins were considered as target antigens for aPL, but at present the exact role of antibodies against annexins (aANX) remains puzzling. This review is concerned with annexin V or annexin A5 (ANXA5), a widespread member of the annexin family, and antibodies directed towards it. We have endeavoured to summarise essential information about the detection of anti-annexin V antibodies (aANXA5) and their clinical relevance. This review has also brought together some relevant published data concerning the structure, physiological role and therapeutic potential of ANXA5.

Budding, vesiculation and permeabilization of phospholipid membranes-evidence for a feasible physiologic role of beta2-glycoprotein I and pathogenic actions of anti-beta2-glycoprotein I antibodies.

The in vivo physiologic role of beta2-glycoprotein I (beta2GPI) is presumed to be related to its interactions with negatively charged phospholipid membranes. Increased quantities of procoagulant microparticles derived by the vesiculation of blood cells have been detected in patients with antiphospholipid syndrome (APS) frequently associated with antibodies against beta2GPI (anti-beta2GPI). We investigated the influence of beta2GPI and anti-beta2GPI on giant phospholipid vesicles (GPVs). GPVs composed of phosphatidylserine and phosphatidylcholine were formed in an aqueous medium and individually transferred to a compartment containing either beta2GPI, anti-beta2GPI, or beta2GPI along with anti-beta2GPI. Shape changes of a single GPV were observed by a phase contrast microscope. Most GPVs transferred to the solution containing only beta2GPI budded moderately. Upon the transfer of GPVs to the solution containing beta2GPI and anti-beta2GPI either from patient with APS or mouse monoclonal anti-beta2GPI Cof-22, the budding was much more pronounced, generating also daughter vesicles. No such effects were seen when GPV was transferred to the solution containing anti-beta2GPI without beta2GPI. Our results suggest a significant physiologic role of beta2GPI in the budding of phospholipid membranes, which may be explained by the insertion of the C-terminal loop of beta2GPI into membranes, thus increasing the surface of the outer layer of a phospholipid bilayer. Anti-beta2GPI, recognizing domains I to IV of beta2GPI, enhanced the budding and vesiculation of GPVs in the presence of beta2GPI. This might be a novel pathogenic mechanism of anti-beta2GPI, promoting in vivo the expression of proadhesive and procoagulant phospholipid surfaces in APS.