Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement

Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.


2-Glycoprotein Exists in a Distinct Molecular Post-Translational Oxidative State in the Antiphospholipid Syndrome: Novel Prognostic Assays and Insights into Antigenicity.

Ioannou10,  Yiannis, Zhang8,  Jing-Yun, Qi11,  Miao, Lu12,  Gao, Qi11,  Jian Cheng, Yu7,  De-min, Lau6,  Herman

Hokkaido University, Sapporo, Japan
University College London, University of New South Wales
University of New South Wales
University of New South Wales, Tianjin Medical University
Hokkaido University, Japan
Natl Univ of Athens Med Schl, Athens, Greece
Natl Univ of Athens Med Schl
St. George Hospital, Sydney, NSW, Australia
St. George Hospital
Tianjin Medical University
Tianjin Medical University, University of New South Wales
University College London

Background:

We have recently reported the novel finding that the major autoantigen in the antiphospholipid syndrome (APS), b2-glycoprotein I (b2GPI), exists in serum in a biochemically reduced state with free thiols (1). This study aims to characterise the oxidative molecular state of b2GPI with respect to free thiol content in APS and evaluate relevance to antigenicity.

Methods:

Through a multi-centre collaborative effort (Sydney, Athens, London, Sapporo), a total of 502 patient samples were collected from APS patients and three control groups: 182 APS (93 had an additional autoimmune disease (AID), 189 AID controls (± persistent antiphospholipid antibodies (aPL) but no APS), 38 clinical event controls (vascular thrombosis, no AID) and 93 healthy controls. Each sample was assayed for total levels of b2GPI and relative amounts of b2GPI present with free thiols. This was performed as described previously (1) and based upon a sandwich ELISA system using a biotinylated free thiol binding reagent to capture proteins with free thiols on a streptavidin plate and detect b2GPI using a monoclonal anti-b2GPI antibody. Oxidised versus reduced b2GPI binding avidity to polyclonal patient anti-b2GPI antibodies was then assessed.

Results:

Total levels of b2GPI were significantly increased in the APS group (216.7 ± 79.5 mg/ml, median ± SD, n=181) as compared to all of the three control groups studied (p<=0.0001). No differences were observed between the other three groups. The relative proportion of b2GPI in the biochemically reduced form expressed as a percentage of that observed with the in-house standard was significantly less in APS patients (median ± SD, 57.15%± 23.5, n=177) as compared to healthy control (p<=0.001), AID disease control (p<=0.001) and clinical event control (aPL negative, p<=0.001) groups. Hence, b2GPI in APS patients is in an oxidised state relative to each of the other three control groups. Sub-analyses of the APS group with anti-b2GPI positivity reveals that APS patients with LA positivity harbour the lowest proportion of b2GPI with free thiols (anti-b2GPI + LA, 47.52%± 22.96 (median ± SD, n=49) versus anti-b2GPI without LA 74.93%± 17.86 (median ± SD, n=28), p<=0.001). IgG from 10 patients with APS was purified. b2GPI within plasma derived from healthy volunteers oxidised by pre-incubation with H2O2 was a significantly better inhibitor of anti-b2GPI activity than untreated serum (p<=0.001, n=10).

Conclusion:

In APS patients, a greater proportion of b2GPI circulates in an oxidised versus reduced state relative to disease and healthy control groups. The finding that oxidised b2GPI harbours a greater avidity for patient derived anti-b2GPI antibodies coupled with the observation that APS patients have a greater antigenic load supports the theory that high amounts of oxidised b2GPI may lower the threshold for breaking tolerance, driving antigenicity and hence anti-b2GPI production in autoimmune susceptible patients.

(1)Ioannou, Y et al, Naturally occurring free thiols within b2-glycoprotein I in vivo: nitrosylation, redox modification by endothelial cells and regulation of oxidative stress induced cell injury. BloodJune 2010 [Epub ahead of print]

To cite this abstract, please use the following information:
Ioannou, Yiannis, Zhang, Jing-Yun, Qi, Miao, Lu, Gao, Qi, Jian Cheng, Yu, De-min, et al; 2-Glycoprotein Exists in a Distinct Molecular Post-Translational Oxidative State in the Antiphospholipid Syndrome: Novel Prognostic Assays and Insights into Antigenicity. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :621
DOI: 10.1002/art.28389

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