Thrombotic risk assessment in antiphospholipid syndrome: do noncriteria antibodies contribute?

BACKGROUND/AIM: In this cross-sectional study, it was aimed to test the predictive value of noncriteria antiphospholipid antibodies (aPL) in addition to the global antiphospholipid syndrome score (GAPSS) in predicting vascular thrombosis (VT) in a cohort of patients with APS and aPL (+) systemic lupus erythematosus (SLE). MATERIAL AND METHODS: This study included 50 patients with primary APS, 68 with SLE/APS, and 52 with aPL (+) SLE who were classified according to VT as VT ± pregnancy morbidity (PM), PM only or aPL (+) SLE. Antiphospholipid serology consisting of lupus anticoagulant (LA), anticardiolipin (aCL) immunoglobulin G (IgG)/IgM/IgA, antibeta2 glycoprotein I (aβ2GPI) IgG/IgM/IgA, antiphosphatidylserine/prothrombin (aPS/PT) IgG/IgM and antidomain-I (aDI) IgG was determined for each patient. The GAPSS and adjusted GAPSS (aGAPSS) were calculated for each patient, as previously defined. Logistic regression analysis was carried out with thrombosis as the dependent variable and high GAPSS, aCL IgA, aβ2GPI IgA, and aDI IgG as independent variables. RESULTS: The mean GAPSS and aGAPSS of the study population were 11.6 ± 4.4 and 9.6 ± 3.8. Both the VT ± PM APS (n = 105) and PM only APS (n = 13) groups had significantly higher GAPSS and aGAPSS values compared to the aPL (+) SLE (n = 52) group. The patients with recurrent thrombosis had higher aGAPSS but not GAPSS than those with a single thrombotic event. The computed area under the receiver operating characteristic curve demonstrated that a GAPSS ≥13 and aGAPSS ≥10 had the best predictive values for thrombosis. Logistic regression analysis including a GAPSS ≥13, aCL IgA, aβ2GPI IgA, and aDI IgG showed that none of the factors other than a GAPSS ≥13 could predict thrombosis. CONCLUSION: Both the GAPSS and aGAPSS successfully predict the thrombotic risk in aPL (+) patients and aCL IgA, aβ2GPI IgA, and aDI IgG do not contribute to high a GAPSS or aGAPSS

Anti-factor Xa antibodies in patients with antiphospholipid syndrome and their effects upon coagulation assays

- Introduction: The aim of this study was to examine the prevalence and functional effects of antibodies directed against Factor (F)Xa and other serine proteases (SP) in patients with antiphospholipid syndrome (APS). - Methods: Serum from patients with APS (n = 59), systemic lupus erythematosus (SLE; n = 106), other autoimmune rheumatic disease (ARD; n = 63) and 40 healthy controls (HC) were tested for IgG activity against thrombin (Thr), FXa, FVIIa, phosphatidylserine (PS)/FXa and antithrombin (AT)-III by enzyme-linked immunosorbent assay (ELISA). Anti-FXa positive IgG were purified to measure their avidity by chaotropic ELISA and functional effects upon clotting time (FXa-ACT) and FXa enzymatic activity (± AT-III). - Results: Anti-FXa IgG were found in patients with SLE (49.1%) and APS (33.9%) (P <0.05) but not in ARD controls and HC. In contrast, anti-Thr and anti-PS/FXa IgG were identified in other ARD and anti-FVIIa IgG were low in all groups. The avidity of APS-IgG to FXa was significantly higher than SLE-IgG (P <0.05). Greatest prolongation of FXa-ACT was observed with APS-IgG and greatest inhibitory effect upon FXa enzymatic activity was found with APS-IgG followed by SLE-IgG compared to HC-IgG. ATIII inhibition of FXa was significantly reduced by APS-IgG compared with HC and SLE (P <0.05) and did not correlate with binding to AT-III. - Conclusion: APS anti-FXa IgG have higher avidity to FXa and greater effects upon the enzymatic and coagulant activity of FXa compared with SLE anti-FXa IgG. Further studies of anti-FXa antibodies in APS, SLE and other non-autoimmune thrombotic disease cohorts are now required to evaluate whether targeting FXa with selective inhibitors in patients bearing anti-FXa antibodies may be an effective treatment strategy

Domain I of beta 2GPI is capable of blocking serum IgA antiphospholipid antibodies binding in vitro: an effect enhanced by PEGylation

Objectives This study aims to inhibit antiphospholipid syndrome (APS) serum derived IgA anti-beta-2-glycoprotein I (aβ2GPI) binding using Domain I (DI). Methods Serum from 13 APS patients was tested for IgA aβ2GPI and Anti-Domain I. Whole IgA was purified by peptide M affinity chromatography from positive serum samples. Serum was tested for IgA aβ2GPI binding in the presence and absence of either DI or of two biochemically modified variants containing either 20 kDa of poly(ethylene glycol) (PEG) or 40 kDa of PEG. Results Significant inhibition with DI was possible with average inhibition of 23% (N = 13). Further inhibitions using 20 kDa PEG-DI and 40 kDa PEG-DI variants showed significant inhibition (p = 0.0001) with both the 40 kDa PEG-DI and 20 kDa PEG-DI variants showing increased inhibition compared with DI alone (p = 0.0001 and p = 0.001, n = 10). Conclusions Inhibition of IgA aβ2GPI by DI is possible and can be enhanced by biochemical modification in a subset of patients

Domain I of β2GPI is capable of blocking serum IgA antiphospholipid antibodies binding in vitro: an effect enhanced by PEGylation

OBJECTIVES: This study aims to inhibit antiphospholipid syndrome (APS) serum derived IgA anti-beta-2-glycoprotein I (aβ2GPI) binding using Domain I (DI). METHODS: Serum from 13 APS patients was tested for IgA aβ2GPI and Anti-Domain I. Whole IgA was purified by peptide M affinity chromatography from positive serum samples. Serum was tested for IgA aβ2GPI binding in the presence and absence of either DI or of two biochemically modified variants containing either 20 kDa of poly(ethylene glycol) (PEG) or 40 kDa of PEG. RESULTS: Significant inhibition with DI was possible with average inhibition of 23% ( N = 13). Further inhibitions using 20 kDa PEG-DI and 40 kDa PEG-DI variants showed significant inhibition ( p = 0.0001) with both the 40 kDa PEG-DI and 20 kDa PEG-DI variants showing increased inhibition compared with DI alone ( p = 0.0001 and p = 0.001, n = 10). CONCLUSIONS: Inhibition of IgA aβ2GPI by DI is possible and can be enhanced by biochemical modification in a subset of patients

Antiphospholipid Antibodies to Domain I of Beta-2-Glycoprotein I Show Different Subclass Predominance in Comparison to Antibodies to Whole Beta-2-glycoprotein I

Antiphospholipid antibodies (aPL), the serological hallmark of antiphospholipid syndrome (APS), are a heterogeneous group of autoantibodies raised against circulating blood proteins. Of these proteins, the phospholipid-binding b2-glycoprotein I (β2GPI) is considered to be the main autoantigen in APS. Indeed, IgG antibodies targeting b2GPI (ab2GPI) directly cause both thrombosis and pregnancy morbidity in several mouse models. While antibodies raised against all five domains of b2GPI have been reported, a subgroup of IgG ab2GPI raised against the first domain (DI) of b2GPI (aDI), strongly correlate with thrombotic APS, and drive thrombosis and pregnancy loss in vivo. Few studies have focused on determining the type of IgG subclass(es) for aPL. The subclass of an antibody is important as this dictates the potential activity of an antibody; for example, IgG1 and IgG3 can fix complement better and are able to cross the placenta compared to IgG2 and IgG4. It is unknown what subclass IgG aDI are, and whether they are the same as ab2GPI. To determine IgG subclass distribution for ab2GPI and aDI, we purified total IgG from the serum of 19 APS patients with known ab2GPI and aDI activity. Using subclass-specific conjugated antibodies, we modified our established in-house ab2GPI and aDI ELISAs to individually measure IgG1, IgG2, IgG3, and IgG4. We found that while IgG1, IgG2, and IgG3 ab2GPI levels were similar, a marked difference was seen in IgG subclass aDI levels. Specifically, significantly higher levels of IgG3 aDI were detected compared to IgG1, IgG2, or IgG4 (p < 0.05 for all comparisons). Correlation analysis of subclass-specific ab2GPI vs. aDI demonstrated that IgG3 showed the weakest correlation (r = 0.45, p = 0.0023) compared to IgG1 (r = 0.61, p = 0.0001) and IgG2 (r = 0.81, p = 0.0001). Importantly, total subclass levels in IgG purified from APS and healthy serum (n = 10 HC n = 12 APS) did not differ, suggesting that the increased IgG3 aDI signal seen in APS-derived IgG is antigen-specific. To conclude, our data suggests that aDI show a different McDonnell et al. Subclass Differences Between aβ2GPI and aDI in APS IgG subclass distribution to ab2GPI. Our results highlight the importance of aDI testing for patient stratification and may point toward differential underlying aPL-driven pathogenic processes that may be subclass restricted

Anti–Neutrophil Extracellular Trap Antibodies in Antiphospholipid Antibody–Positive Patients: Results From the Antiphospholipid Syndrome Alliance for Clinical Trials and InternatiOnal Networking Clinical Database and Repository

OBJECTIVE: This study aimed to elucidate the presence, antigen specificities, and potential clinical associations of anti–neutrophil extracellular trap (anti-NET) antibodies in a multinational cohort of antiphospholipid (aPL) antibody–positive patients who did not have lupus. METHODS: Anti-NET IgG/IgM levels were measured in serum samples from 389 aPL-positive patients; 308 patients met the classification criteria for antiphospholipid syndrome. Multivariate logistic regression with best variable model selection was used to determine clinical associations. For a subset of the patients (n = 214), we profiled autoantibodies using an autoantigen microarray platform. RESULTS: We found elevated levels of anti-NET IgG and/or IgM in 45% of the aPL-positive patients. High anti-NET antibody levels are associated with more circulating myeloperoxidase (MPO)–DNA complexes, which are a biomarker of NETs. When considering clinical manifestations, positive anti-NET IgG was associated with lesions affecting the white matter of the brain, even after adjusting for demographic variables and aPL profiles. Anti-NET IgM tracked with complement consumption after controlling for aPL profiles; furthermore, patient serum samples containing high levels of anti-NET IgM efficiently deposited complement C3d on NETs. As determined by autoantigen microarray, positive testing for anti-NET IgG was significantly associated with several autoantibodies, including those recognizing citrullinated histones, heparan sulfate proteoglycan, laminin, MPO–DNA complexes, and nucleosomes. Anti-NET IgM positivity was associated with autoantibodies targeting single-stranded DNA, double-stranded DNA, and proliferating cell nuclear antigen. CONCLUSION: These data reveal high levels of anti-NET antibodies in 45% of aPL-positive patients, where they potentially activate the complement cascade. While anti-NET IgM may especially recognize DNA in NETs, anti-NET IgG species appear to be more likely to target NET-associated protein antigens