Lupus (2014) 23, 1320–1323 http://lup.sagepub.com

SPECIAL ARTICLE

Domain I: the hidden face of antiphospholipid syndrome C Pericleous and A Rahman Centre for Rheumatology Research, Division of Medicine, University College London, UK

IgG antiphospholipid antibodies (aPL) against b2-glycoprotein I (b2GPI) can target any of its five domains; however, aPL against the N-terminal domain (anti-DI, aDI) are considered the most clinically relevant in the antiphospholipid syndrome (APS). Circulating levels of aDI are elevated in patients with APS compared with disease and healthy controls, and crucially aDI are prothrombotic in in vivo and in vitro models. In addition, human recombinant DI has been shown to abrogate aPL-induced thrombosis in vivo. Therefore, although the potential of utilizing DI for management of APS is not yet fully defined, there is promise that DI could prove valuable both as a diagnostic and therapeutic tool. Lupus (2014) 23, 1320–1323. Key words: Anti-domain I antibodies; thrombosis; pregnancy morbidity

Over the last 25 years, a plethora of studies have demonstrated that antiphospholipid antibodies (aPL) against the phospholipid-binding protein b2-glycoprotein I (b2GPI), and particularly those of the IgG isotype, are the most closely associated with thrombosis in antiphospholipid syndrome (APS). b2GPI, comprised of five homologous domains (DI–V), exists primarily in a biochemically reduced state,1 resulting in DI interacting with DV to form a closed circular b2GPI structure. Upon binding to anionic phospholipids on cell membranes via DV, b2GPI opens up to its ‘active’ fishhook structure, exposing DI (Figure 1(a)). Iverson and colleagues first demonstrated the importance of DI in APS, utilizing a panel of b2GPI domain-deletion mutants (DDM) in an ELISA system2 and surface plasmon resonance experiments3 to show that affinity-purified aPL from patients with APS could only bind DDM containing DI. They subsequently identified an immunodominant region within DI, spanning Gly40-Arg43,4 later verified and extended to Arg39-Arg43 by Ioannou et al.5 Crucially, this region is hidden under physiological conditions and becomes accessible to antibody via the conformational change that results from b2GPI binding anionic membranes.6,7 Not all IgG antibodies to b2GPI (ab2GPI) target DI; multiple lines of evidence, however,

Correspondence to: Charis Pericleous, Centre for Rheumatology Research, Division of Medicine, University College London, Rayne Institute, 5 University Street, London, WC1E 6JF, United Kingdom. Email: [email protected]

demonstrate that IgG antibodies to DI (aDI) are the key pathogenic ab2GPI population in APS. Retrospective serological studies have demonstrated that aDI are highly prevalent in APS8–10 and are associated with both thrombotic and obstetric manifestations in ab2GPI-positive adults.8,9 Patient-derived plasma with aDI activity has been reported to have lupus anticoagulant (LA) activity,8 while in a separate study b2GPI-dependent LA positivity correlated with increased annexin A5 resistance, a phenomenon by which aPL disrupt the anticoagulant shield that annexin A5 forms around damaged cells. In LA-positive samples, aDI activity also correlated with annexin A5 resistance,11,12 further pointing towards a link between aDI and LA, indicating a possible mechanism of aDI-driven hypercoagulability.

Are aDI pathogenic in APS? In the Pierangeli mouse model of APS, both affinity-purified aDI from APS serum13 and a human monoclonal aPL that binds DI (IS4)14 were prothrombotic,15,16 and recombinant human DI abrogated aPL-induced thrombosis.17 In two different in vivo models, a human synthetic monoclonal aDI caused complement-mediated thrombosis and fetal loss in naı¨ ve mice treated with LPS.18 In addition, IgG recognizing a peptide derived from the human DI sequence induced monocyte and endothelial cell activation, and bound toll-like receptor

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10.1177/0961203314540354

Domain I: the hidden face of antiphospholipid syndrome C Pericleous and A Rahman

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4 (TLR4) in vitro. This DI-derived peptide shared sequence homology with an epitope on TLR4.19 Both complement and TLR4 are implicated in aPL-stimulated pathogenesis, and these two studies are the first to suggest that aDI can drive cell damage via TLR4 and/or complement activation (the aforementioned aDI-induced biological effects are summarized in Figure 1(b–e)). Finally, mice immunized with human or murine b2GPI in the presence of cardiolipin vesicles, or with human DI, developed ab2GPI and aDI; immunizing with

human DII-V or b2GPI alone did not induce production of these antibodies.20

Would measuring IgG aDI levels be valuable in management of APS? There is increasing worldwide interest in developing a reliable assay for detecting aDI.21–23 Recombinant human DI has been expressed in

Figure 1 Pathogenicity of anti-DI antiphospholipid antibodies. (A) Circulating b2GPI primarily exists in a closed circular form, opening upon encounter with anionic phospholipids on damaged cellular surfaces, and exposing DI.6,7 Antiphospholipid antibodies against DI (aDI) can now bind b2GPI, eliciting the following (known thus far) biological responses: (B) may disrupt the anticoagulant annexin A5 shield that surrounds damaged cells;11,12 (C) promote tissue factor (TF) activity13 and tumour necrosis factor-a (TNFa)19 release by monocytes/macrophages and (D) TF activity13 and vascular adhesion molecule 1 (VCAM-1) expression by endothelial cells;19 (E) aDI-b2GPI complexes activate the complement cascade.18 In vivo, aDI have been shown to directly cause thrombosis13,18 and pregnancy morbidity.18 Lupus Downloaded from lup.sagepub.com at UNIV OF CALIFORNIA SANTA CRUZ on April 1, 2015

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baculovirus,2 bacteria24 and synthetically.25 DI proteins from all these sources are conformationally correct,2,14,25 and have been used in fluid-phase ELISA (inhibiting binding of aPL to whole b2GPI) and/or solid-phase ELISA, with DI as the coating antigen.2,4,5,25 Iverson and Ioannou used nickel plates for their solid-phase assays, with DI bound to the plate by aid of a hexahistidine tag.2,4,5 De Laat and colleagues used the baculovirusexpressed DI to design a two-plate assay where DI is coated on both hydrophobic and hydrophilic plates. The immunodominant region Gly40-Arg43 is exposed on hydrophobic but not hydrophilic plates; thus APS sera would test positive on hydrophobic plates while non-APS sera would not. Results from this assay are expressed as a ratio of the value obtained from hydrophobic versus hydrophilic plates, where a ratio of >2 denotes a sample as aDI positive.8,9 Using this assay in large retrospective studies, de Laat et al. reported strong associations of aDI with both thrombotic8,9 and obstetric9 APS. In the largest study of aDI in APS published to date, they tested for IgG aDI in 420 IgG ab2GPI-positive adults, of which 87% were APS – 60% of those were IgG aDI positive, suggesting a high prevalence of IgG aDI in APS.9 Similarly, Andreoli et al. observed 67% positivity for IgG aDI in 64 patients with APS.10 Using a simple solid-phase ELISA adapted from our published nickel plate assay,5 we recently tested 119 patients with APS, of which 79% had IgG aPL; 51% of these were IgG aDIpositive. In comparison, aDI were detected in fewer than 5% of >300 disease (patients with systemic lupus erythematosus (SLE) but not APS) and healthy controls (unpublished data). Only one other published serological study measured IgG aDI in patients with SLE but found no association between these aPL and thrombosis. It is worth noting, however, that of the 164 patients with SLE and thrombosis included in this study, only 18 were positive for IgG ab2GPI and 11/164 had aDI.26 Baculovirus-expressed DI has also been used by INOVA Diagnostics Inc. to design an aDI ELISA, to date utilized by two published studies.10,26 INOVA have also created an innovative chemiluminescent immunoassay (CIA) for aDI, reporting a sensitivity of 85% in a cohort of 144 patients with APS, compared with 0.5% and 14% for 200 healthy and 72 infectious disease controls, respectively (unpublished data).27 One small published study of 39 patients with APS and 77 disease and healthy controls, utilizing INOVA CIA for IgG ab2GPI and aDI, reports sensitivities of 46% and 36% for APS, respectively.28 Numerous as yet

unpublished studies have also used the INOVA aDI ELISA or CIA, summarized in a recent comprehensive review.23 Comparison of our adapted ELISA5 and the INOVA CIA in the same samples shows good agreement between the two assays.23 Evidence so far suggests that IgG aDI is found less commonly in children than adults: approximately 25% of a population of 183 children with SLE were reported to have IgG aDI; however, only seven children had thrombosis and only one of those had IgG aDI.12 In addition, only 16% of children born to mothers with APS and 27% of children with atopic dermatitis tested positive for IgG aDI; in these children, IgG ab2GPI positivity correlated with the presence of aPL targeting DIV–V but not DI.10 Prospective studies are imperative to determine the prognostic power of aDI, while longitudinal tests should help establish if aDI remain constant or if levels change before, near to and after a clinical event has occurred.

Could DI be a therapeutic agent in APS? DI is also an attractive candidate for therapeutic application in APS. Long-term anticoagulation with warfarin is the mainstay of treatment for thrombotic APS, but can potentially cause major adverse effects and is not always effective. In fact, none of the treatments available or currently being evaluated in clinical trials (e.g. rivaroxaban, a new generation oral anticoagulant) actually target aPL. Designing a drug based on DI could provide the first of such targeted therapies; results from studies reporting in vivo inhibition of aPL-mediated thrombosis17 and in vitro inhibition of ab2GPI activity by recombinant human DI are encouraging.2,4,5,25

Conclusions Undoubtedly, IgG aDI can drive APS pathogenesis; it is imperative to note, however, that some patients with APS have antibodies against other domains and thus detecting aPL against whole b2GPI should not be abandoned. Considering the promising results amassed from various serological and functional studies, developing and validating a simple assay to measure aDI in the clinical setting is of great importance, while the additional potential application of DI as a novel theraupetic should be further explored.

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Funding Research at UCL is funded by Arthritis Research UK Programme Grant 19423.

Conflict of interest statement The authors have no conflicts of interest to declare.

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Domain I: the hidden face of antiphospholipid syndrome.

IgG antiphospholipid antibodies (aPL) against β2-glycoprotein I (β2GPI) can target any of its five domains; however, aPL against the N-terminal domain...
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