Background/Purpose:  

β₂GPI represents the major antigenic target for antiphospholipid antibodies (aPL), the hallmark of antiphospholipid syndrome (APS). β₂GPI contains five homologous domains, with domain I (DI) being identified as the main antigenic epitope for pathogenic anti-β₂GPI. Antibodies targeting β₂GPI-DI (anti-DI) represent a key pathogenic sub-population of aPL, thus their detection may allow the identification of patients at highest clinical risk. It has been reported that β₂GPI in vivo exists in two interconvertible biochemical redox dependent configurations. The reduced form has a circular configuration maintained by interaction between the first and fifth domain thus potentially hiding the epitope on DI, whilst the oxidised protein has an open and linear shape in which the DI is exposed. It is known that the proportion of β₂GPI in the oxidised form is elevated in patients with APS. Thus, one hypothesis is that the oxidative state of β₂GPI might alter the exposure of neo-epitopes on DI thus providing an antigenic drive for anti-DI antibodies production. In this study we investigated the association between circulating oxidised form of β₂GPI and presence of anti-DI in APS patients.

Methods:

Serum samples from 44 patients who fulfilled the revised classification criteria for APS were tested (38 primary and 6 secondary APS). The samples were screened for IgG anti-DI, anti-β₂GPI and anti-cardiolipin (anti-CL). A sandwich ELISA for quantifying total β₂GPI levels within serum was performed for all samples. Biochemically reduced β₂GPI was detected via labelling free thiols within serum proteins with a biotinylated free-thiol binding reagent and then, via coating on a streptavidin plate, detecting the presence of labelled β₂GPI with an anti-β₂GPI antibody, based on published methods. For the in vivo studies, male Sprague Dawley rats were injected intravenously with 1 mg of IgG purified pooled from 4 healthy individuals or 2 APS anti-DI positive patients (n=6 rats per IgG pool). Blood samples were collected 24-hour after IgG injection and assayed for reduced β₂GPI.   

Results:

A negative correlation was found between the reduced proportion of β₂GPI and IgG anti-DI levels (Spearman r = -0.535, p = 0.0002). Furthermore, the proportion of reduced β₂GPI was lower in anti-DI positive than anti-DI negative APS patients (p = 0.0178). The relative amount of reduced β₂GPI was no different between patients who were positive or negative for anti-β₂GPI (p=0.757) or anti-CL (p=0.062). The reduced proportion of β₂GPI was no different within serum samples collected from healthy rats injected with purified anti-DI positive IgG from APS patients versus healthy controls (p=1.000).

Conclusion:

This study demonstrates that IgG anti-DI are strongly associated with the oxidative state of β₂GPI as compared to anti-CL and anti-β₂GPI antibodies. Furthermore, the in vivo injection of APS derived IgG did not affect the redox state of β₂GPI in the experimental rat-model employed. Though not proof, this study further supports the hypothesis that epitope exposure on DI of β₂GPI defined by the redox state of the protein might affect the antigenic drive of pathogenic anti-DI antibodies.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/oxidation-of-beta2-glycoprotein-i-beta2-gpi-associates-with-the-presence-of-antibodies-to-domain-i-of-beta2-gpi-in-patients-with-the-antiphospholipid-syndrome-but-is-not-affected-by-the-antibod/