Antiphospholipid syndrome: diagnostic aspects of lupus anticoagulants

Antiphospholipid syndrome (APS) is an autoimmune disorder in which antiphospholipid antibodies (aPL) are thought to be involved in the development of venous and/or arterial thrombosis. APL found in this syndrome are antibodies directed against a variety of phospholipid (PL) binding-proteins of which beta3-glycoprotein I (beta2GPI) and prothrombin are considered to be the major antigens. Some of these antibodies prolong PL-dependent clotting reactions and are termed lupus anticoagulants (LA). Autoimmune aPL which bind through beta2GPI to cardiolipin are called anticardiolipin antibodies (aCL). Clinical studies indicate that LA is a stronger risk factor for thrombosis than aCL. The production of monoclonal antibodies against beta2GPI and prothrombin has enabled us to understand the mechanism by which LA prolong coagulation in vitro. LA form bivalent antigen-antibody complexes with increased affinity for PL which compete with coagulation factors for the same catalytic surface. These LA positive monoclonal antibodies may be helpful in further improving the laboratory diagnosis of LA.     

Some patients with antiphospholipid syndrome express hitherto undescribed antibodies to cardiolipin-binding proteins

Contrary to infective anticardiolipin (aCL) antibodies, autoimmune aCL antibodies react with phospholipids (PL) mainly via binding to the plasma glycoprotein cofactor beta2-Glycoprotein I (beta2GPI). While there is a well-documented link between the risk of thrombosis and the presence of beta2GPI-dependent anticardiolipin antibodies, the pathological impact of other antiphospholipid antibodies is less clear. By means of cardiolipin affinity-chromatography, we isolated and identified 3 CL-binding proteins, complement component C4, complement factor H and a kallikrein-sensitive glycoprotein, and tested for the presence of autoantibodies against these proteins in patients with antiphospholipid syndrome (APS), systemic lupus erythematosus (SLE) and other autoimmune diseases. High titers of autoantibodies to C4 as compared to age- and sex-matched healthy controls were present in 3 of 26 patients with APS, and weak titers were found in 2 of 26 patients with SLE and in none of 26 patients with other autoimmune diseases. Autoantibodies to complement factor H were found in 4 APS, 3 SLE and none of the other autoimmune patients. Autoantibodies to kallikrein-sensitive glycoprotein were detected in 6 APS patients, 1 SLE patient, and 1 patient with another autoimmune disease. A close relationship between these antibodies was found, suggesting their origin from a common macromolecular complex. However, no relationship with anti-beta2GPI antibodies was found, with the three patients with higher levels of autoantibodies having a low titer of anti-beta2GPI antibodies. In conclusion, some patients with APS harbor circulating antibodies to other CL-binding proteins which might be useful to further characterize these patients.     

Evaluation of a new set of automated chemiluminescense assays for anticardiolipin and anti-beta2-glycoprotein I antibodies in the laboratory diagnosis of the antiphospholipid syndrome

Introduction

The laboratory diagnosis of antiphospholipid syndrome (APS) requires the demonstration of antiphospholipid antibodies (aPL): lupus anticoagulant (LAC) measured through coagulation assays, anticardiolipin IgG or IgM antibodies (aCL) and/or anti-β2glycoprotein I IgG or IgM antibodies (aβ2GPI), usually detected by ELISA

Materials and methods

We evaluated the diagnostic value of aCL and aβ2GPI measured by a new automated system using the chemiluminescence principle, the immunoanalyzer Zenit RA (Menarini).

Results

Results of aCL and aβ2GPI were correlated with the clinical background of the patients and with results of ELISA (n = 314). Correlated to the clinical background sensitivity/specificity ranged for aCL IgG between 7.5-45.2% / 54.2-98.8%, for aCL IgM 3.4-5.5% / 89.9-94%, for aβ2GPI IgG 5.5-25.3% / 75.6-100% and aβ2GPI IgM 3.4-4.8% / 89.9-92.3%, depending on the cut-off used. Sensitivity with manufacturer's cut-offs was comparable to ELISA, except for aβ2GPI IgG with a significantly lower sensitivity compared to ELISA (5.5% vs 11.6%).In the APS patient population (n = 30) sensitivity of aCL IgG and aβ2GPI IgG was higher measured by ELISA compared to Zenit RA (46.7% vs 30.0%, and 46.7% vs 26.7%, respectively).Agreement between Zenit RA results and ELISA results for the four parameters was moderate (Kappa-values ranging 0.509-0.565). Sensitivity was 38.5%, 53.3%, 40% and 69.2% for aCL IgG, aCL IgM, aβ2GPI IgG and aβ2GPI IgM, respectively, applying the highest cut-off value for Zenit RA, raising towards 64.3%, 100%, 57.1%, for aCL IgG, aCL IgM, aβ2GPI IgG, respectively, in a APS patient population.

Conclusions

The new technology of chemiluminescense for measuring aPL showed good performance characteristics. Interpretation of results with a cut-off value associated with a good discrimination for disease, resulted in a lower sensitivity for the diagnosis of APS for aβ2GPI IgG measured by Zenit RA assays compared to ELISA; sensitivity for aCL IgG was comparable to ELISA. Specificity for all parameters was high and comparable for aCL and aβ2GPI.     

Antiprothrombin antibodies--are they worth assaying?

According to the preliminary classification criteria of the antiphospholipid syndrome (APS) (Sapporo Criteria), β2-glycoprotein I (β2GPI)-dependent anticardiolipin antibodies (aCL) and lupus anticoagulant (LA) are the only laboratory tests considered as criteria for the classification of the APS. Recently, antibodies against phosphatidylserine–prothrombin complex (aPS/PT) have been detected and these antibodies, rather than antibodies against prothrombin alone, are closely associated with APS and LA. We assessed the sensitivity and specificity of aPS/PT for the diagnosis of APS in our population of patients with a variety of autoimmune disorders and investigated whether aPS/PT could be used as diagnostic test in patients suspected of having APS. The study population comprised 219 patients with autoimmune diseases including 82 patients with APS and 137 without APS (55 systemic lupus erythematosus, 32 rheumatoid arthritis, 10 primary Sjogren's syndrome, 8 scleroderma, 5 Behcet's disease and 27 other rheumatic diseases). IgG/M aPS/PT were measured by ELISA using phosphatidylserine–prothrombin complex as antigen immobilized on ELISA plates in the presence of CaCl₂. IgG/M aCL were measured by standard methods and LA was detected by clotting assays. aPS/PT, aCL and LA were more frequently found in patients with APS (47, 46 and 69, respectively) than in those without APS (11, 19 and 29, respectively) (OR 95% [CI]; 15.4 [7.2–32.7], 7.9 [4.1–15.2, 19.8 [9.6–40.6], respectively]. The sensitivity of each assay for the diagnosis of APS was 57%, 56% and 86% with a specificity of 92%, 86% and 79%, respectively. aPS/PT and aCL have similar diagnostic value for APS, therefore, we propose that aPS/PT should be further explored, not only for research purposes, but also as a candidate of one of the laboratory criteria for the classification of the APS.     

Antibodies to oxidized LDL/beta2-glycoprotein I in antiphospholipid syndrome patients with venous and arterial thromboembolism

It has been reported that IgG to oxidized LDL/beta2-glycoprotein I (oxLDL/beta2GPI) complexes are associated with arterial thromboembolism (TE) in patients with antiphospholipid syndrome (APS). How these antibodies behave in arterial as compared to venous TE in APS is unknown. The aim of the present study was to evaluate the association of IgG anti-oxLDL/beta2GPI with clinical manifestations in category I APS patients. Fifty-seven APS patients with triple positivity (Lupus Anticoagulant (LAC), anti cardiolipin (aCL) and anti-beta2-glycoprotein I (abeta2GPI) antibodies), 28 with arterial and 29 with venous thromboembolism, were included in the study. There were no differences in the dRVVT ratio, IgG/IgM aCL and IgG/IgM abeta2GPI titers in the two patient groups. There were no differences in the IgG (78.5 U+/-59.8 vs. 112.2 U+/-92.3) and IgM (16.3 U+/-15.9 vs. 21.1 U+/-14.3) anti-oxLDL/beta2GPI mean values. A significant correlation was found between IgG anti-oxLDL/beta2GPI and IgG anti-beta2GPI titers in the whole group of APS patients. Patients in the arterial group were older and had more risk factors for atherosclerosis. Data from this study do not support the hypothesis that IgG anti-oxLDL/beta2GPI are specifically associated to arterial TE in Category I APS patients.     

APS--controversies in diagnosis and management, critical overview of current guidelines

Because the diagnosis of antiphospholipid syndrome is based on laboratory data, performance and interpretation of such tests is essential. Positivity in a single test among Lupus Anticoagulant (LA), anti cardiolipin (aCL) and anti β2-glycoprotein I (aβ2GPI) antibodies calls APS diagnosis into question. Conversely, triple positivity is strongly associated to thrombosis and pregnancy morbidity. A new era in studying APS should take into account different antiphospholipid antibodies profiles. Clinical studies on groups of patients that are homogeneous in terms of laboratory tests will provide information on the real risk of patients and on the real effect of therapeutic interventions.     

Extra-criteria antiphospholipid antibodies in patients with small vessel brain lesions and clinical manifestations associated with antiphospholipid syndrome

ObjectivesNeurological manifestations compatible with small vessel brain lesions (SVBL), such as migraine, cognitive impairment, seizures, and transverse myelitis, may be related to antiphospholipid syndrome (APS) and patients could need APS therapies even though they do not fit into thrombosis or obstetric morbidity. Furthermore, extra-criteria antiphospholipid antibodies (aPL) provide an increase in sensitivity in patients with clinical manifestations related to APS but negative for IgG/IgM anticardiolipin (aCL), anti-β2 glycoprotein I (aβ2GPI), and lupus anticoagulant, which are the antibodies included in the classification criteria for APS.MethodsWe determined extra-criteria aPL in 65 SVBL patients with neurological traits and Magnetic Resonance Imaging suggestive of APS but negative for APS classification criteria, 47 of whom were prospectively followed and tested over three years. A group of 95 patients with autoimmune diseases (AD) but without clinical traits of APS was also studied.ResultsA persistent presence of extra-criteria aPL was detected in 27.7% of patients: 12.77% IgM anti- prothrombin (PT), 6.38% IgG anti-PT, 6.38% IgM anti-phosphatidylethanolamine (PE), 4.26% IgA aβ2GPI, 2.13% IgG anti-phosphatidylserine/prothrombin (PS/PT) and 2.13% IgM anti-PS/PT.There was a tendency towards a higher prevalence of these aPL in SVBL patients than in AD – especially for IgA aβ2GPI – and a lack of IgG aPS/PT positivity in the AD group. We found no SVBL patient positive for IgA aCL, IgG anti-PE, annexin V, or aβ2GPI domain I.ConclusionsExtra-criteria aPL can improve sensitivity for APS diagnosis in patients with SVBL, especially IgA aβ2GPI and IgG anti-PS/PT antibodies.     

Platelet activation rather than endothelial injury identifies risk of thrombosis in subjects positive for antiphospholipid antibodies

BACKGROUND: Anti-phospholipid antibodies (APLA) are often associated with thrombosis, defining the antiphospholipid syndrome (APS) but it remains unclear why many subjects who are positive for APLA chiefly anti-cardiolipin (aCL) or anti-beta2GPI (abeta2GPI) do not develop thrombosis. A related question addressed in this study is whether the target of cellular injury in APS is predominately platelets or endothelial cells (EC). METHODS: aCL and abeta2GPI were determined by ELISA in 88 patients, 60 of whom were thrombotic and 28 non-thrombotic. Platelet activation was measured by CD62P and by concentration of platelet microparticles (PMP) and EC activation was assessed by endothelial microparticles (EMP), both by flow cytometry. Lupus anticoagulant (LAC) was measured in the hospital laboratory. RESULTS: There was no difference in frequency of aCL or abeta2GPI, neither IgG or IgM, between the thrombotic and non-thrombotic groups. Both groups showed elevated EMP compared to controls but this did not differ between thrombotic and non-thrombotic groups. In contrast, PMP were not significantly elevated in non-thrombotic but were elevated in thrombotic compared to non-thrombotic (p=0.03) and controls. CD62P, an independent marker of platelet activation, was also elevated in thrombotic vs. non-thrombotic. There was a trend for increased LAC in the thrombotic group but not significant. CONCLUSION: Although all subjects had evidence of endothelial activation, only platelet activation differed between thrombotic and non-thrombotic. This supports the hypothesis that platelet activation predisposes to thrombosis in the presence of chronic EC activation. These data also raise the possibility of distinguishing risk-prone APLA-positive individuals.     

Distinguishing features of anti-beta2 glycoprotein I antibodies between patients with leprosy and the antiphospholipid syndrome

Anticardiolipin (ACA), anti-beta2 glycoprotein I (beta2GPI), and antiprothrombin antibodies of IgG and IgM classes were quantitated by enzyme-linked immunosorbent assays in 176 untreated leprosy patients across the histopathological spectrum. Positivity rates ranged from 21% (IgG ACA) to 30% (IgM anti-prothrombin) versus 4% in healthy controls (p <10(-2) to 10(-3)). Levels of IgM anti-beta2GPI and IgG ACA were significantly higher in lepromatous leprosy and multibacillary patient subgroups. IgG3 was the most common subclass reactive to both beta2GPI and prothrombin in selected high-titer leprosy sera, unlike antibodies from patients with the antiphospholipid syndrome (APS) largely restricted to IgG2. In leprosy patients, but not in the APS control group, there was no statistical correlation between ACA and anti-beta2GPI antibody levels. Likewise, a large fraction of anti-beta2GPI positive sera (36/45 and 28/44 for IgG and IgM, respectively) were unreactive in the standard ACA assay. Most assayed anti-beta2GPI antibodies from leprosy patients showed (i) ability to recognize both human and bovine beta2GPI immobilized on non-irradiated polystyrene plates, (ii) concentration-dependent inhibition of binding by cardiolipin, and (iii) relatively high avidity binding to fluid-phase beta2GPI, thereby differing from those found in APS. Finally, the location of the major epitopic region on the beta2GPI molecule targeted by autoantibodies was different in leprosy and APS, as assessed by direct binding to domain I- and V-deleted mutants and competition with the mouse monoclonal antibody 8C3, directed at domain I. Thus, leprosy-related antiphospholipid antibodies comprise persistent IgG and IgM anti-beta2GPI that differ from APS-related ones with respect to IgG subclass, avidity and epitope specificity, possibly reflecting distinct pathophysiological significance.     

Libman-Sacks endocarditis associated with antiphospholipid syndrome and infection

INTRODUCTION: Antiphospholipid syndrome (APS) is a systemic autoimmune disease, associated not only with a hypercoagulable state and recurrent fetal loss but with many diverse clinical manifestations including heart involvement, neurological manifestations, as well as skin, kidney and hematologic abnormalities. Cardiac manifestations include coronary by-pass graft and angioplasty occlusions, cardiomyopathy, cyanotic congenital heart disease, intracardiac thrombus and complications of cardiovascular surgery. The valvular heart disease was defined as Libman-Sacks nonbacterial endocarditis. Previously, we have shown a linear subendothelial deposition of anti-cardiolipin/beta2 glycoprotein I (beta2GPI) antibodies in the valve specimens derived from APS patients. The involvement of complement C3c in the pathogenesis was documented. We assessed the beta2GPI-related target epitope recognized by the anti-beta2GPI Abs on the valves. MATERIALS AND METHODS: In order to find the beta2GPI-related target epitopes recognized by the anti-beta2GPI antibodies on the valves, we used beta2GPI-related synthetic peptides. The presence of anti-beta2GPI Abs on the studied valves was detected by anti-idiotypic antibody, followed by immunoperoxidase analysis. Biotin attached to the N-terminal of beta2GPI-related synthetic peptides and control peptide were used to identify the epitope addressed by the anti-beta2GPI Abs deposited on the patient's valve. The binding was probed by streptavidin-peroxidase and appropriate substrate. The specificity was confirmed by competition assays with control peptide and anti-idiotypic antibody. RESULTS: Among the beta2GPI-related synthetic peptides, two peptides were found in previous studies to mimic common pathogens either bacteriae or viruses, which raised a possible infectious origin for APS. One of these peptides, TLRVYK, is a specific target for anti-beta2GPI Abs deposited on the APS valves. This synthetic peptide was able to displace the anti-anti-beta2GPI anti-idiotypic Abs for binding the anti-beta2GPI Abs on the valve by a competition assay. CONCLUSION: We point to the possibility that Libman-Sacks nonbacterial endocarditis may have an infectious origin.     

Heterogeneity of anti-beta2-glycoprotein I antibodies. A factor of variability in test results

The aim of this study was to evaluate the heterogeneity of IgGanti-beta2-glycoprotein I antibodies (IgG-abeta2GPI) as regarding their reactivity pattern against different sources of human beta2 GPI, their avidity and their association with clinical events of antiphospholipid syndrome (APS). Three thousand six hundred and eighty-four consecutive patient sera were routinely tested for IgGabeta2 GPI over 1 year using an in-house ELISA with 2 different commercial preparations of human purified beta2GPI. Of the 340 sera found positive, all those clinically documented were included in this study; 61 were positive with only one preparation (S1) and 59 with both (S2). The results of ELISA were confirmed by Western blot. Heterogeneity was stressed by testing sera with a human recombinant protein and 3 beta2GPI-related peptides. No contribution of glycosylation in the binding to beta2GPI was found. The avidity indices for each protein were significantly higher in S1 than in S2 (p=0.0021). S2 were more associated with antiphospholipid antibodies than S1 (75% versus 21% ; p<0.0001). A similar frequency of the main clinical features of APS was found in S1 and S2 sera (69% and 71%, respectively). In conclusion, our data show a heterogeneity in the antigenic reactivity pattern of IgG- abeta2 GPI and a relationship between a binding profile and antibody avidity. This heterogeneity could represent a crucial factor of variability in test results and underlines the difficulty of getting standardisation.     

Antibody profiles for the diagnosis of antiphospholipid syndrome

Among the so called 'antiphospholipid antibodies', the presence of Lupus Anticoagulant (LA) is associated with thrombosis-related events and defines the antiphospholipid syndrome. The role of anti-cardiolipin (aCL) antibodies and anti-human beta2-glycoprotein I (abeta2GPI) antibodies is less striking. Since the problem of standardization for these tests is far from resolved, we evaluated whether the combination of results (antiphospholipid laboratory profiles) could help to better classify these patients. Over a 6-year period, 618 consecutive subjects (55% of whom had previous documented thrombosis-related events) were referred to our clinic for Antiphospholipid antibody detection. LA was detected according to internationally accepted recommendations. ACL and abeta2GPI antibodies were detected by Enzyme-Linked-Immunosorbent Assay (ELISA). Patients' records were reviewed for the presence of previous thromboembolic events or obstetric complications according to Sapporo's clinical criteria for the diagnosis of antiphospholipid syndrome (APS) and each patient underwent a physical examination. When individual tests were considered in a multivariate analysis which took into account age, gender, the presence of SLE or other autoimmune diseases and established risk factors for venous and arterial thromboembolism, LA (Odds Ratio 4.4, Confidence Interval 1.5-13.3) and abeta2GPI antibodies (Odds Ratio 2.9, Confidence Interval 1.1-7.5) but not aCL antibodies (Odds Ratio 1.2, Confidence Interval 0.5-2.7) were found to be independent risk factors for thrombosis-related events. When antiphospholipid antibody profiles instead of individual test positivity were analyzed in the above mentioned model, triple positivity resulted a strong independent risk factor (Odds Ratio 33.3, Confidence Interval 7.0-157.6), retaining its significance when the association with venous or arterial thromboembolism was considered. Double positivity with negative LA was close to significance for thrombosis-related events (Odds Ratio 2.2, Confidence Interval 1.0-5.2, p=0.056) and highly significant risk factor for obstetric complications (Odds Ratio 10.8, Confidence Interval 2.9-40.8). Other combinations did not reach statistical significance. The mean level of IgG abeta2GPI antibodies was statistically higher in triple positive profile and might account for positive LA. As compared to a single test, the analysis of a complete antiphospholipid antibody profile can better determine patients at risk.     

Clinical manifestations of chronic inflammatory demyelinating polyneuropathy with anti-cardiolipin antibodies

OBJECTIVE: Chronic inflammatory demyelinating polyneuropathy (CIDP) is an autoimmune syndrome where certain autoantibodies define clinicopathologic subgroups. In the present study, serum anti-cardiolipin antibodies (aCL) were evaluated. MATERIALS AND METHODS: We investigated aCL in sera from 21 patients diagnosed with CIDP in our hospital between 1991 and 2001. The four CIDP patients with aCL (aCL+) were compared with 17 patients without aCL (aCL-). RESULTS: All aCL+ patients displayed sensory-motor polyneuropathy, with severity and distribution of weakness resembling those in aCL- patients. Anti-nuclear antibody titer of aCL+ patients were significantly higher than those in aCL- patients. None of aCL+ patients presented clinical manifestations of primary anti-phospholipid syndrome (APS), such as thromboses or recurrent abortion. Although the aCL+ patients were older and had more complications and more severe pathologic features than aCL- patients, they responded well to steroid pulse or intravenous immunoglobulin. CONCLUSION: The aCL in CIDP apparently differ from 'autoimmune' aCL in APS, instead being among the autoantibodies pathologically involved in CIDP subgroups.     

Antibodies to beta2-glycoprotein I associated with antiphospholipid syndrome suppress the inhibitory activity of tissue factor pathway inhibitor

Anionic phospholipid membranes have a dual role in blood coagulation: they are essential for the initiation and propagation as well as for the limitation and termination of the blood coagulation process. Patients with the anti-phospholipid syndrome (APS) carrying antibodies against complexes of anionic phospholipids and plasma proteins, show in vitro inhibited phospholipid dependent coagulation reactions, whereas in vivo the presence of these antibodies is associated with an increased risk of thrombosis. In this study we focussed on the effects of these anti-phospholipid antibodies on the regulation of TF-mediated factor Xa (FXa) generation in plasma. We hypothesized that anti-phospholipid antibodies interfere with the phospholipid-dependent inhibition by tissue factor pathway inhibitor (TFPI) of TF-induced coagulation. Indeed, total-IgG, anti-cardiolipin-IgG (aCL) and anti-beta2GPI-IgG, isolated from patient plasmas, all stimulated TF-induced FXa generation in normal plasma. This enhanced FXa generation was not observed when the patient's IgG was depleted of anti-beta2GPI-IgG or when normal plasma was depleted of beta2PGPI or TFPI. Taken together, these data indicate that antibodies to beta2GPI, circulating in patients with APS, suppress TFPI-dependent inhibition of TF-induced coagulation, which results in an increased FXa generation.     

Anti-CD40 antibodies in antiphospholipid syndrome and systemic lupus erythematosus

Anti-beta2glycoprotein I (anti-beta2GPI) antibodies constitute the main autoantibody specificity in the sera of patients with antiphospholipid syndrome (APS). There is evidence that anti-beta2GPI antibodies induce the precoagulant activity of the endothelium by cross-linking the beta2 glycoprotein I (beta2GPI) on the cell surface. Since beta2GPI lacks intracellular domains, homology with other molecules such as CD40 that could initiate signaling, was extensively searched. A 86% homology between the amino acid position 239-245 of the CD40 and 7-13 of the beta2glycoprotein was found. The CD40 peptide corresponding to amino acids 239-245 of the CD40 molecule was synthesized and coupled to a multiple antigenic peptide carrier. Antibodies to CD40 peptide were found in 61.5% APS patients (n = 39), in 72.7% of systemic lupus erythematosus (SLE) positive for anti-beta2GPI antibodies (n = 11) and 31.6% of SLE negative for anti-beta2GPI antibodies (n = 19), but not in rheumatoid arthritis patients (n = 28) or controls (n = 36). Antibodies to CD40 peptide were associated with arterial thrombosis and/or brain microinfarcts. Affinity purified anti-CD40 peptide antibodies as well as affinity purified anti-beta2GPI antibodies recognized both, the beta2GPI and the CD40 peptide. The specificity of this recognition was confirmed with homologous and heterologous inhibition experiments. Confocal microscopy experiments demonstrated this cross-recognition of CD40 and beta2GPI molecules, by the purified anti-CD40 peptide antibodies, at the protein level. Thus, antibodies reacting with the beta2GPI can react and potentially activate different cells which express CD40 molecules at their surface.     

Oxidation of beta2-glycoprotein I (beta2GPI) by the hydroxyl radical alters phospholipid binding and modulates recognition by anti-beta2GPI autoantibodies.

We investigated whether beta2-glycoprotein I (beta2GPI), the key antigen in the antiphospholipid syndrome, is susceptible to oxidative modifications by the hydroxyl radical (*OH) that may influence its lipid-binding and antigenic properties. The effects on human and bovine beta2GPI of *OH free radicals generated by gamma-radiolysis of water with 137Cs were studied. Radiolytic *OH caused a dose-dependent loss of tryptophan, production of dityrosine and carbonyl groups. dimerization and/or extensive aggregation of beta2GPI. It ensued a reduction in affinity binding to cardiolipin liposomes and loss of beta2GPI-dependent autoantibody binding to immobilized cardiolipin. Patient anti-beta2GPI antibodies (n = 20) segregated into two groups based on the effect in the beta2GPI-ELISA of beta2GPI pretreatment with *OH: enhancement (group A, n = 10) or suppression (group B, n = 10) of IgG binding. The avidities of group A antibodies for fluid-phase beta2GPI were low but increased in a dose-dependent manner upon beta2GPI irradiation, in relation to protein crosslinking. Distinguishing features of group B antibodies included higher avidities for fluid-phase beta2GPI that was no longer recognized after *OH treatment, and negative anticardiolipin tests suggesting epitope location near the phospholipid binding site. The *OH scavengers thiourea and mannitol efficiently protected against all above changes. Therefore, oxidative modifications of beta2GPI via *OH attack of susceptible amino acids alter phospholipid binding, and modulate recognition by autoantibodies depending on their epitope specificities. These findings may be of clinical relevance for the generation and/or reactivity of anti-beta2GPI antibodies.     

β2-Glycoprotein I – dependent anticardiolipin antibodies in ischemic stroke

Our study was undertaken to investigate whether beta2-glycoprotein I (GPI) is relevant for enhancing anticardiolipin antibody (aCL) binding in stroke patients, in particular view of vascular risk factors and recurrence of previous cerebral ischemic events. One-hundred and twenty-one sera from patients with ischemic stroke and 154 control sera from patients with non-ischemic neurological disorders ( n = 43) and healthy subjects ( n = 111) were included in the study. All sera were tested for either GPI-independent aCL and GPI-dependent aCL. GPI-independent aCL were detected in two (1.7%) stroke patients. When GPI was added to the assay system, 13 (10.8%) sera were positive. Of the 43 sera in the neurological control group one was positive for both GPI-independent aCL and GPI-dependent aCL. Multiple linear regression in the stroke group revealed that GPI-dependent aCL are marginally associated with sex, prior TIA/strokes and embolism. More importantly, GPI-dependent aCL were significantly more frequent in stroke patients with a history of prior TIA/strokes compared with patients with a first ischemic stroke ( p = 0.029).
The present study demonstrates that aCL in stroke patients are of the GPI-dependent type and emphasizes the importance of adding GPI in the immunoassay for the optimal detection of aCL. Furthermore, it supports the recommendation of testing aCL in those patients who experience unexplained recurrent cerebral ischemic events.     

Antibody profile and clinical course in primary antiphospholipid syndrome with pregnancy morbidity

In women diagnosed as having category I primary obstetric antiphospholipid syndrome, clinical characteristics and the risk of subsequent thromboembolic events and further unsuccessful pregnancy has not been clearly documented. Women with unexplained obstetric complications and no definite autoimmune systemic diseases were tested for lupus anticoagulant (LA), IgG/IgM anticardiolipin (aCL) and IgG/IgM anti-human beta2-Glycoprotein I (abeta2GPI) antibodies and diagnosed as having primary antiphospholipid syndrome (APS) in classification category I on the basis of more than one laboratory criteria present in any combination. Characteristics at the time of diagnosis and risk factors for subsequent clinical events during a mean follow-up of 6.3 years were evaluated. Fifty-three of 600 women studied were found to fulfil obstetric criteria and had more than one positive laboratory test at the time of diagnosis. All the women were aCL and abeta2GPI positive, and 16 were also LA positive. This latter group (triple positivity) had distinct features and had more frequently experienced previous thromboembolism (OR = 122.5, 95% CI 16-957, p < 0.001). They also had an increased rate of late pregnancy loss (OR = 16.2, 95%CI 0.9-292, p = 0.01), and a higher IgG abeta2GPI titer at diagnosis (median, 25(th) and 75(th) percentile were 118, 37-962, vs. 23, 18-32, respectively, p < 0.0001). During follow-up, the rate of thromboembolic events was significantly higher in the group of women with triple positivity and/ or previous thromboembolism (OR = 57.5, 95% CI 2.7-1160, p = 0.0004) which were the only independent predictors of TE in the multivariate model. Recurrent pregnancy loss took place in seven out of 47 women who had a new pregnancy. Triple positivity and/or previous thromboembolism were again the only independent markers (OR = 34.4, 95% CI 3.5-335.1, p = 0.003) of an unsuccessful new pregnancy. In conclusion, in primary APS with pregnancy morbidity in classification category I, quite different groups of patients may be identified on the basis of laboratory tests. Triple positivity and/or a history of thromboembolism predict new TE events and new unsuccessful pregnancies.     

Lupus anticoagulant activity is frequently dependent on the presence of beta 2-glycoprotein I.

Antiphospholipid antibodies (aPL) are defined by anticardiolipin antibody (aCL) ELISA and prolongation of phospholipid dependent coagulation assays (lupus anticoagulant; LAC). For the binding of aCL to cardiolipin a cofactor, beta 2-glycoprotein I (beta 2-GPI), is necessary. We have investigated whether the same cofactor is essential for LAC activity. Plasma from 6 LAC positive patients and 3 controls was depleted from beta 2-GPI by means of affinity chromatography. From the 6 LAC positive plasmas, 4 became LAC negative (tested with dRVVT) when beta 2-GPI was depleted and became positive again when purified beta 2-GPI (200 micrograms/ml) was added. A dose response curve showed that addition of 50 micrograms/ml beta 2-GPI to beta 2-GPI deficient patient plasma, led to a positive dRVVT. Depletion of, and addition of beta 2-GPI to plasma from controls had no effect on the dRVVT. Measurement of beta 2-GPI plasma levels in 19 LAC positive patients, 40 LAC negative patients and 15 controls showed no difference in beta 2-GPI levels. These results show that a combination of aPL and beta 2-GPI is essential not only for binding to cardiolipin, but also for LAC activity and imply that low beta 2-GPI levels (less than 50 micrograms/ml) can lead to false negative LAC tests. These observations may lead to new insights in the pathophysiological complications associated with aPL.     

Elevated anticardiolipin antibodies in schizophrenic patients before and during neuroleptic medication

The objective of this study was to examine the prevalence of IgG and IgM anticardiolipin antibodies (aCL) in schizophrenic patients and to determine the relation of aCL levels with neuroleptic medication and psychotic symptoms. Twenty-three patients with acute episodes of chronic schizophrenia, drug-free for at least 3 months before entering the study, were tested for aCL at admission (T1) and 42 days (T2) after neuroleptic treatment started. Blood samples were taken from 20 healthy volunteers as well. Diagnosis was performed according to DSM-IV. Serum samples were analysed for IgG and IgM autoantibodies against beta2-glycoprotein-1-cardiolipin complex by commercially available ELISA kits (Binding-site, UK). Significantly higher levels of aCL antibodies of both isotypes were found in schizophrenic patients versus controls. In patients IgM-aCL positivity was significantly more frequent than in controls. The elevated IgM-aCL and IgG-aCL values were not associated with neuroleptic treatment and psychotic symptoms, as measured by the Positive and Negative Syndrome Scale and the Pentagonal Structural Model of Schizophrenic Symptoms. The negative correlation of IgM-aCL and IgG-aCL with the positive symptoms scale and the autistic preoccupation scale (Pentagonal Structural Model) may indicate the consumption of these antibodies in the exacerbation of the disease.