Vera M. Ripoll

Purified IgG from Patients with Obstetric but not IgG from Non-obstetric Antiphospholipid Syndrome Inhibit Trophoblast Invasion

Some patients with antiphospholipid syndrome (APS) suffer pregnancy morbidity (PM) but not vascular thrombosis (VT), whilst others suffer VT only. Therefore, we compared the effects of IgG from VT+/PM− and VT−/PM+ subjects on human first‐trimester trophoblast (HTR8) cells.

Changes in regulation of human monocyte proteins in response to IgG from patients with antiphospholipid syndrome

The effects of immunoglobulin G (IgG) from patients with the antiphospholipid syndrome (APS) upon monocyte activation have not been fully characterized. We carried out a comprehensive proteomic analysis of human monocytes treated with IgG from patients with different manifestations of the APS. Using 2-dimensional differential gel electrophoresis (2D DiGE), 4 of the most significantly regulated proteins (vimentin [VIM], zinc finger CCH domain-containing protein 18, CAP Gly domain-containing linker protein 2, and myeloperoxidase) were differentially regulated in monocytes treated with thrombotic or obstetric APS IgG, compared with healthy control (HC) IgG. These findings were confirmed by comparing monocytes isolated from APS patients and HC. Anti-VIM antibodies (AVAs) were significantly increased in 11 of 27 patients (40.7%) with APS. VIM expression on HC monocytes was stimulated more strongly by APS IgG from patients with higher-avidity serum AVA. We further characterized the proteome of thrombotic APS IgG-treated monocytes using a label-free proteomics technique. Of 12 proteins identified with the most confidence, 2 overlapped with 2D DiGE and many possessed immune response, cytoskeletal, coagulation, and signal transduction functions which are all relevant to APS and may therefore provide potential new therapeutic targets of this disease.

Antiphospholipid antibodies enhance rat neonatal cardiomyocyte apoptosis in an in vitro hypoxia/reoxygenation injury model via p38 MAPK

A significant amount of myocardial damage during a myocardial infarction (MI) occurs during the reperfusion stage, termed ischaemia/reperfusion (I/R) injury, and accounts for up to 50% of total infarcted tissue post-MI. During the reperfusion phase, a complex interplay of multiple pathways and mechanisms is activated, which ultimately leads to cell death, primarily through apoptosis. There is some evidence from a lupus mouse model that lupus IgG, specifically the antiphospholipid (aPL) antibody subset, is pathogenic in mesenteric I/R injury. Furthermore, it has previously been shown that the immunodominant epitope for the majority of circulating pathogenic aPLs resides in the N-terminal domain I (DI) of beta-2 glycoprotein I (β2GPI). This study describes the enhanced pathogenic effect of purified IgG derived from patients with lupus and/or the antiphospholipid syndrome in a cardiomyocyte H/R in vitro model. Furthermore, we have demonstrated a pathogenic role for aPL containing samples, mediated via aPL–β2GPI interactions, resulting in activation of the pro-apoptotic p38 MAPK pathway. This was shown to be inhibited using a recombinant human peptide of domain I of β2GPI in the fluid phase, suggesting that the pathogenic anti-β2GPI antibodies in this in vitro model target this domain.

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

IntroductionThe 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).MethodsSerum 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).ResultsAnti-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.ConclusionAPS 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.

Laboratory Tests for the Antiphospholipid Syndrome

Antiphospholipid syndrome (APS) is an autoimmune disorder characterized by recurrent vascular thrombosis (VT) and/or pregnancy morbidity (PM) in the presence of persistent antiphospholipid antibodies (aPL), detected by lupus anticoagulant (LA), anticardiolipin (aCL) antibody, and/or anti-β₂ glycoprotein I (aβ₂GPI) antibody assays. These aPL, considered to be diagnostic markers and pathogenic drivers of APS, are a heterogeneous group of antibodies directed against anionic phospholipids, phospholipid-binding plasma proteins, and phospholipid-protein complexes. Although APS is currently considered as a single disease, it presents with a wide range of clinical symptoms and biological characteristics. The clinical diagnosis of APS in a patient with symptoms and signs is dependent upon the presence of a persistently positive result in an aPL assay. The tests recommended for detecting aPL are the standardized enzyme-linked immunosorbent assay (ELISA) to detect aCL and aβ₂GPI and clotting assays for LA performed according to the guidelines of the International Society on Thrombosis and Haemostasis. This chapter describes the standard laboratory test for the diagnosis of APS discussing the clinical and theoretical aspects of LA, aCL, and aβ₂GPI assays.

PS5:99 Examining the modulatory effects of anti-serine protease antibodies upon factor xa, thrombin and complement interactions

Purpose Serine protease (SP) enzymes play a critical role in both the coagulation and complement cascades. Anti-SP antibodies are found in approximately 40% of patients systemic lupus erythematosus (SLE) and antiphospholipid Syndrome (APS). Complement activation is important in SLE and APS. However, little is known about the effects of anti-SP antibodies on complement activation in these diseases. We sought to investigate whether affinity-purified antibodies to the SP Factor(F)Xa and Thrombin (Thr) alter the effects of these SP on complement cleavage in the presence or absence of the physiological inhibitor anti-thrombin (AT). Methods Serum was obtained by informed consent from patients with SLE (n=10) and/or APS (n=2) under long-term follow-up at University College London Hospital. A novel method was developed to affinity purify anti-FXa and anti-Thr IgG separately. We incubated FXa (2 µM) and Thr (2.7 µM) separately with complement component C3 to determine baseline C3 cleavage. We then repeated the experiment in the presence of AT3, the appropriate anti-SP (anti-FXa or anti-Thr) or both. Finally we reviewed medical records of 40 patients with SLE to determine whether low C3 was associated with seropositivity for anti-FXa and/or anti-Thr in these patients. Results Both FXa and Thr cleaved C3 into C3a/C3 b. This was inhibited by AT, though both cleavage and inhibition by AT were stronger for Thr than FXa. Conversely anti-SP antibodies enhanced C3 cleavage by SP. Anti-FXa IgG (n=3) increased FXa-mediated cleavage of C3 by 1.3-fold. Anti-Thr IgG (n=8) increased Thr-mediated C3 cleavage by 1.8-fold. AT mediated inhibition was prevented by addition of anti-FXa IgG (n=2) but not anti-Thr IgG. Analysis of clinical serology records for the last 10 consecutive clinic visits of 28 anti-SP-positive patients showed a lower level of C3 (0.92 g/L) in the patients double positive for anti-FXa and anti-Thr than for anti-Thr alone (1.12 g/L) or anti-FXa alone (1.16 g/L). Conclusions Anti-FXa and anti-Thr enhance cleavage of C3 by FXa and Thr respectively. Presence of these antibodies in-vivo in patients with SLE and/or APS may promote increased complement activation and disease activity. This finding may have potential translational implications for future treatment of these diseases.Abstract PS5:99 Figure 1 Data showing relative C3 cleavage of Thr and FXa in the presence and absence of antibodies and inhibitors

S6D:5 Antiphospholipid antibodies differentially regulate the expression & activity of the lysosomal proteases with effects upon monocyte autophagy

Purpose Antiphospholipid antibodies (aPL) activate monocytes in antiphospholipid syndrome (APS), although the precise mechanisms by which this activation occurs are not fully understood. We have identified several novel protein targets using proteomic analysis of human monocytes treated with APS-IgG. Amongst these novel targets lysosomal proteases cathepsin B and D were identified. The balance between different cathepsins is important in protein degradation, apoptosis and autophagy. Reduced cathepsin B and D with increased cathepsin L is a phenotype suggesting reduced autophagy. Dysregulation of autophagy may be important in the pathogenesis of APS. Therefore, we aimed to determine the effect of aPL on monocyte cathepsin balance and autophagy. Methods Healthy control (HC) monocytes were treated with 200 µg/ml of IgG purified from (n=9) patients with APS-IgG or (n=9) HC-IgG for 6 hour. Cathepsin B and D expression were measured by western blot. Cathepsin D, B and L activity were measured using fluorescence-based assays. Intracellular proteolytic activity was determined using DQ-BSA. Results Consistent with our previous proteomic analysis, western blots confirmed that cathepsin B and cathepsin D were down-regulated in monocytes treated with APS-IgG compared to HC-IgG. Similarly, cathepsin B and D activities were significantly reduced in monocytes treated with APS-IgG (p=0.0188 for B, 0.0323 for D). In contrast, cathepsin L activity was increased in monocytes treated with APS-IgG (p=0.0106). We then examined cathepsin activity in monocytes from patients with APS. Cathepsin L activity was increased significantly when these monocytes were treated with APS-IgG compared to HC-IgG (p=0.0257). Lysosomal proteolysis is a key process in the late phase of autophagy. We therefore exposed HC monocytes to IgG, treated them with GM-CSF overnight and tested their intracellular proteolytic activity. APS-IgG reduced the lysosomal activity of GM-CSF-treated monocytes whereas HC-IgG had no effect. Conclusions We found that APS-IgG regulate the expression and activity of lysosomal proteases cathepsins B/D and cathepsin L in opposite directions. Consistent with this finding, APS-IgG reduced lysosomal proteolysis in monocytes. Additional experiments are now underway to increase our understanding of how modulation of cathepsin activity and autophagy may be important in the pathogenesis of APS and to provide new therapeutic targets.

Gene expression profiling identifies distinct molecular signatures in thrombotic and obstetric antiphospholipid syndrome

Antiphospholipid antibodies (aPL) cause vascular thrombosis (VT) and/or pregnancy morbidity (PM). Differential mechanisms however, underlying the pathogenesis of these different manifestations of antiphospholipid syndrome (APS) are not fully understood. Therefore, we compared the effects of aPL from patients with thrombotic or obstetric APS on monocytes to identify different molecular pathways involved in the pathogenesis of APS subtypes. VT or PM IgG induced similar numbers of differentially expressed (DE) genes in monocytes. However, gene ontology (GO) analysis of DE genes revealed disease-specific genome signatures. Compared to PM, VT-IgG showed specific up regulation of genes associated with cell response to stress, regulation of MAPK signalling pathway and cell communication. In contrast, PM-IgG regulated genes involved in cell adhesion, extracellular matrix and embryonic and skeletal development. A novel gene expression analysis based on differential variability (DV) was also applied. This analysis identified similar GO categories compared to DE analysis but also uncovered novel pathways modulated solely by PM or VT-IgG. Gene expression analysis distinguished a differential effect of VT or PM-IgG upon monocytes supporting the hypothesis that they trigger distinctive physiological mechanisms. This finding contributes to our understanding of the pathology of APS and may lead to the development of different targeted therapies for VT or PM APS.

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 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.

PEGylated Domain I of beta-2-glycoprotein I inhibits the binding, coagulopathic and thrombogenic properties of IgG from patients with the antiphospholipid syndrome

APS is an autoimmune disease in which antiphospholipid antibodies (aPL) cause vascular thrombosis and pregnancy morbidity. In patients with APS, aPL exert pathogenic actions by binding serum beta-2-glycoprotein I (β2GPI) via its N-terminal domain I (DI). We previously showed that bacterially-expressed recombinant DI inhibits biological actions of IgG derived from serum of patients with APS (APS-IgG). DI is too small (7 kDa) to be a viable therapeutic agent. Addition of polyethylene glycol (PEGylation) to small molecules enhances the serum half-life, reduces proteolytic targeting and can decrease immunogenicity. It is a common method of tailoring pharmacokinetic parameters and has been used in the production of many therapies in the clinic. However, PEGylation of molecules may reduce their biological activity, and the size of the PEG group can alter the balance between activity and half-life extension. Here we achieve production of site-specific PEGylation of recombinant DI (PEG-DI) and describe the activities in vitro and in vivo of three variants with different size PEG groups. All variants were able to inhibit APS-IgG from: binding to whole β2GPI in ELISA, altering the clotting properties of human plasma and promoting thrombosis and tissue factor expression in mice. These findings provide an important step on the path to developing DI into a first-in-class therapeutic in APS.