Antiphospholipid syndrome is characterized by oxidative stress and systemic inflammation [44, 45]. The overproduction of reactive oxygen species (ROS) results in an oxidative microenvironment that exacerbates inflammation, inducing cell death and tissue damage, compromising antioxidant defence mechanisms [46]. Patients with APS have high levels of circulating pro-inflammatory cytokines interleukin-2 (IL-2), interleukin-6 (IL-6), and tumour necrosis factor (TNF), together with markers of oxidative stress and inflammation such as serum amyloid A (SAA), C-reactive protein (CRP), 8-isoprostane, and prostaglandin E2 (PGE2) [47, 48].

In vivo and under normal physiological conditions, β₂GPI is produced in the liver and exists predominately in circulation in its free thiol form, which is less immunogenic than the oxidized form (Fig. 2.2). The precise role of β₂GPI and its different forms are complex [49]; it is thought to act as a natural anticoagulant that mediates a range of functions including the clearance of liposomes, apoptotic bodies and microparticles [49–52].

Oxidized β₂GPI level in APS has been proposed as a biomarker of thrombotic risk for APS. Levels of oxidized β₂GPI in patients with APS are higher than in healthy subjects and patients with other autoimmune disease or non-aPL disease controls with thrombosis [53]. Free thiol β₂GPI may play a protective role in APS, since free thiol β₂GPI protects human umbilical vein endothelial cells (HUVEC) against hydrogen peroxide-induced cell injury [54]. Decreased plasma free thiol β₂GPI may thus lower the physiological buffer against oxidative stress-induced injury. Free thiol β₂GPI also protects human retinal pigment epithelium and the subretinal endothelial cell against oxidative, hydrogen peroxide stress-induced, cell death [55].

A multicentre, cross-sectional, international study using prospectively acquired samples has demonstrated that the redox status of β₂GPI differs between healthy individuals and patients with thrombotic APS [53]. In the former it exists predominately with free thiols; APS patients have higher levels of total and oxidized β₂GPI compared to both healthy subjects and patients with other autoimmune disease [53, 56].

Anticardiolipin antibodies, aβ₂GPI, and LA test serve as diagnostic markers in APS. The predominant isotypes of aPL in APS patients are IgG aCL and IgG aβ₂GPI [57, 58]. Although, non-criteria or non-classical aPL such as antiphosphatidylserine, antiphosphatidylethanolamine, and antiphosphatidylglycerol have been reported, only the three classical aPL tests are used in diagnosis of APS [59]. The LA assay identifies autoantibodies against either prothrombin and/or β₂GPI, whereas the aCL assay detects the aCL and/or aβ₂GPI antibodies. The aβ₂GPI assay detects only antibodies against β₂GPI. Concomitant triple positivity for aCL, β₂GPI and LA may indicate severe APS and high recurrence risk [60], a point that is still controversial [61]. Lupus anticoagulant correlates much better with the clinical manifestations of APS than the detection of the autoantibodies with an ELISA [62, 63], and a positive LA assay due to aβ₂GPI has a stronger correlation for thrombotic risk than due to antiprothrombin autoantibodies [64].

In a clinical setting, it is important to stratify risk for development of clinical events in APS and in asymptomatic, aPL-positive individuals. Delayed or inadequate treatment can result in damage and impaired quality of life [65, 66]. Although β₂GPI levels are not routinely measured in patients with APS, considering the specificity of high levels of oxidized β₂GPI, measuring their levels may assist in diagnosis and prognosis. The level of oxidized β₂GPI is calculated by subtracting the concentration of free thiol from total β₂GPI. Using an ELISA to measure post-translational redox modifications of β₂GPI (including total and free thiol β₂GPI) [53] (Fig. 2.3) and β₂GPI plasma levels in 359 patients (identified through an international multicentre initiative) who had either APS or other autoimmune diseases or non-APS vascular thrombosis, Ioannou et al. found that the redox state of β₂GPI and its concentration in APS patients had a profile distinct from that in the various control groups.

Evidence from both clinical and animal studies supports the concept that β₂GPI is the main autoantigen in APS. Understanding of the pathophysiology of APS and the involvement of β₂GPI and its post-translational modified forms remains incomplete; understanding the relevance of oxidized β₂GPI in APS will be important. Although #aPL are a defining, hallmark feature of APS, their presence does not exclusively indicate APS nor do they stratify individuals for risk of thrombosis.

Current methods for diagnosing APS patients do not incorporate quantitation of total and free thiol β₂GPI. Specific ELISAs for quantifying these parameters may enhance our diagnostic and prognostic capabilities. Prospective studies may validate measurement of oxidatively modified forms of β₂GPI as biomarkers. The AntiPhospholipid Syndrome Alliance For Clinical Trials and InternatiOnal Networking (APS ACTION), an international research network that collects patient samples from 25 centres around the world [67], may allow a longitudinal study that measures oxidized β₂GPI.