Prevalence and Significance of Non-conventional Antiphospholipid Antibodies in Patients With Clinical APS Criteria

Abstract

Background: The biological diagnostics of antiphospholipid syndrome (APS) takes into account the persistent positivity for anticardiolipin and/or anti-β2GP1 antibodies and/or presence of lupus anticoagulant (LA). However, some non-conventional antiphospholipid antibodies have emerged that could help in the diagnosis of APS. Objectives: To study the potential usefulness of non-conventional antiphospholipid antibodies in clinical practice. Methods: Eighty-seven patients, aged from 15 to 92 years were included and classified in following groups: 41 patients positive for the conventional antibodies with clinical criterion of APS (31 with primary APS and 10 secondary), 17 seronegative APS (SNAPS) patients (i.e., persistent negativity for the conventional antibodies with a strong clinical suspicion of APS), 11 asymptomatic antiphospholipid antibodies carriers (i.e., persistent positivity for the conventional antibodies without clinical evidence of APS), and 18 patients presenting with a first thrombotic or obstetrical event. IgG and IgM were detected to the following antigens: phosphatidylserine/prothrombin (PS/PT) by ELISA, and phosphatidic acid, phosphatidyl-ethanolamine, phosphatidyl-glycerol, phosphatidyl-inositol, phosphatidylserine, annexin V, prothrombin by immunodot. Anti-β2GP1 IgA, and anti-β2GP1 domain 1 IgG were detected by chemiluminescence. Results: Positivity for the non-conventional antibodies was correlated with APS severity; patients with catastrophic APS (CAPS) being positive for 10.7 (Median, Range: 5-14) non-conventional antibodies. 9/17 seronegative patients were positive for at least one of non-conventional antibodies. A study of non-supervised hierarchical clustering of all markers revealed that anti-PS/PT antibodies showed high correlation with the presence of LA. All patients with APS triple positivity (highest risk profile) exhibited also persistent positivity for anti-PS/PT antibodies. Conclusions: Our data obtained from a prospective cohort constituted mainly by patients with primary APS, suggest that non-conventional APS antibodies may be useful for patients classified as SNAPS. They demonstrate the potential value of aPS/PT antibodies as a strong marker of APS. We propose that anti-PS/PT antibodies could be a surrogate APS biological marker of LA to classify in high-risk profile patients treated by direct oral anticoagulants (DOACs), in whom LA detection cannot be achieved.

Keywords: anti-phosphatidylserine-prothrombin antibodies; antiphospholipid syndrome; autoantibodies; lupus anticoagulant; thrombosis.

Figure 1

Results of anti-PS/PT antibodies. The distribution of anti-PS/PT IgG or IgM antibodies is shown in all groups of patients and in HD (A,B according to the disease group; C,D according to the clinical type of APS). The positive thresholds (30 AU/mlAU/ml) are shown by dotted lines. (A,C) aPS/PT antibodies of IgG isotype, (B, D) aPS/PT antibodies of IgM isotype. The data are presented as mean ± s.e.m. with all individual dots shown. The p-values were calculated using Mann-Whitney test. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001. HD, Healthy Donors; SNAPS, seronegative anti-phospholipid syndrome; Asympt APA, asymptomatic carriers of antiphospholipid antibodies; APS 1 BM, APS with 1 biomarker; APS 2 BM, APS with 2 biomarkers; APS 3 BM, APS with 3 biomarkers; V thromb, venous thrombosis; Art thromb, arterial thrombosis; Obst APS, obstetrical APS; CAPS, Catastrophic anti-phospholipid syndrome.

Figure 2

Results of anti-β2GP1 IgA and anti- β2GP1 D1 IgG antibodies. The distribution of IgA aβ2GP1 and aβ2GP1 D1 antibodies is shown in all groups of patients and in HD (A,B according to the disease group; C,D according to the clinical type of APS). The data are presented as mean ± s.e.m. with all individual dots shown. The p-values were calculated using Mann–Whitney test. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001. The limit of positivity for IgA aβ2GP1 (20 AU/ml) is shown by the dotted line (A,C). The limit of positivity for aβ2GP1 D1 is 20 lAU/ml (not shown) (B,D). (A) Statistical differences are significant between APS 3 BM and all other groups (p < 0.001). (B) In addition to differences shown in the graph, statistical differences are significant between APS 3 BM, and APS 2 BM (p < 0.01) and between APS 3 BM and all other groups (p < 0.001). HD, Healthy Donors; SNAPS, seronegative anti-phospholipid syndrome; Asympt APA, asymptomatic carriers of antiphospholipid antibodies; APS 1 BM, APS with 1 biomarker; APS 2 BM, APS with 2 biomarkers; APS 3 BM, APS with 3 biomarkers; V thromb, venous thrombosis; Art thromb, arterial thrombosis; Obst APS, obstetrical APS; CAPS, Catastrophic anti-phospholipid syndrome.

Figure 3

Frequency of other APS non-conventional markers in the different groups of patients and HD. Non-conventional APS markers of IgG and IgM isotypes were detected by immunodot technique in sera of different groups of patients and in HD. Y ax shows a mean of positive markers (PA, PG, PI, PS, A5, and PT of IgG and IgM isotypes) per patient in each group. HD, Healthy Donors; SNAPS, seronegative anti-phospholipid syndrome; Asympt APA, asymptomatic carriers of antiphospholipid antibodies; APS 1 BM, APS with 1 biomarker; APS 2 BM, APS with 2 biomarkers; APS 3 BM, APS with 3 biomarkers; V thromb, venous thrombosis; Art thromb, arterial thrombosis; Obst APS, obstetrical APS; CAPS, Catastrophic anti-phospholipid syndrome; Pos, positive.

Figure 4

Hierarchical clustering of APS biomarkers using Genesis software. Unsupervised hierarchical clustering was performed, using Pearson correlation and complete linkage analysis. Each column corresponds to an APS marker. Each line corresponds to a patient with a number corresponding to the “disease group”: 1, Thrombosis; 2, SNAPS; 3, asymptomatic APA carrier; 4, APS with 1 biomarker; 5, APS with 2 biomarkers; 6, APS with 3 biomarkers. Colors are correlated with the positivity of the markers from negative (in black) to highly positive (in bright red). Gray boxes correspond to absence of data. This analysis reveals the close clustering of LA with anti-PS/PT IgG and IgM among APS patients, and with anti-PS/PT IgM alone among asymptomatic APA carriers (framed in yellow).