Antibodies against oxidized phospholipids in laboratory tests exploring lupus anti-coagulant activity

Abstract

Lupus anti-coagulants (LA) are a variety of anti-phospholipid antibodies characterized by their capacity to interfere with phospholipid-dependent coagulation assays. LA are increasingly recognized as important predictors of thrombosis. However, the antigen specificity of LA is still poorly characterized. Growing evidence indicates that oxidized phospholipids are among the targets of anti-phospholipid antibodies. This prompted us to investigate the role of IgG directed against different oxidized phospholipids in 164 subjects without clotting factor defects that were tested for the presence of LA using a LA-sensitive activate partial thromboplastin time (aPTT-FSL) and a screening/confirmation assay based on diluted Russell's viper venom test (dRVVT-PL). The response to aPTT-FSL was significantly (P < 0.0005) associated with high titres of IgG against oxidized phosphatidylserine, phosphatidylethanolamine and phosphatidylinositol, whereas positivity to dRVVT-PL was associated with the elevation of IgG against oxidized phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine (P < 0.0005) and phosphatidylinositol (P < 0.01). No difference in reactivity against oxidized cardiolipin was evident between the different groups. Positivity to the dRVVT-PL test was also associated significantly (P < 0.005) with the elevation of anti-cardiolipin and anti-beta(2)-glycoprotein-1 IgG. However, stepwise logistic regression demonstrated that IgG recognizing oxidized phosphatidylethanolamine and oxidized phosphatidylcholine were the only independent predictors of the response to dRVVT-PL assay, while IgG recognizing oxidized phosphatidylethanolamine and oxidized phosphatidylinositol were independent predictors of the response to aPTT-FSL test. In conclusion, autoantibodies against defined oxidized phospholipids are independent predictors of LA detection by aPTT-FSL or dRVVT-PL assays and might contribute to the variability often observed in the responses to the functional tests detecting LA.

Fig. 1

Distribution of anti-cardiolipin (anti-CL) and anti-β₂-glycoprotein-1 (β₂GP-1) IgG in 164 patients without evidence of clotting defects evaluated for lupus anti-coagulant (LA) activity by both standardized activated partial thromboplastin time (aPTT-FSL) and a combination of screening and confirmation assays based on the diluted Russell's viper venom test plus phospholipid supplementation (dRVVT-PL). Abnormal aPTT-FSL was found in 74 subjects, while 48 had an abnormal dRVVT-PL test. The dotted lines represent the cut-off value for anti-cardiolipin and anti-β₂GP-1 IgG. Values in parentheses are the percentage of subjects with anti-cardiolipin and anti-β₂GP-1 IgG above the threshold. Differences of proportions calculated by Newcombe's method were: *35% (95% CI 17–51%) for anti-cardiolipin IgG; #21% (95% CI 9–37%) for anti-β₂GP-1 IgG.

Fig. 2

IgG reactivity against oxidized (filled bars) and native (empty bars) forms of cardiolipin (CL), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC) and phosphatidylinositol (PI) in sera of 10 randomly selected sera showing high titres of antibodies against the different oxidized phospholipids. Statistical significance was estimated by Wilcoxon's matched-pairs tests: *P < 0·005, # P < 0·05.

Fig. 3

IgG reactivity against oxidized phosphatidylserine (a), phosphatidylethanolamine (b), phosphatidylcholine (c) and phosphatidylinositol (d) in the sera of subjects positive (empty bars) or negative (filled bars) for, respectively, activated partial thromboplastin time (aPTT-FSL) or diluted Russell's viper venom test plus phospholipid supplementation (dRVVT-PL) tests. Among the 164 subjects investigated 74 had abnormal aPTT-FSL, while 48 has abnormal dRVVT-PL values. The sera were tested at 1 : 50 dilution in microplate enzyme-linked immunosorbent assay plates coated with the different antigens and revealed with peroxidase-linked goat anti-human IgG anti-serum. The results are expressed as optical density (OD) at 490 nm after subtracting the background reactivity of each serum. Boxes include the values within 25th and 75th percentiles and the horizontal bars represent the medians. Eighty per cent of the values are comprised between the extremities of the vertical bars (10th−90th percentiles). The extreme values are represented by individual points. Statistical significance was evaluated by non-parametric Mann–Whitney U-test.