Inhibitory autoantibodies against ADAMTS-13 in patients with thrombotic thrombocytopenic purpura bind ADAMTS-13 protease and may accelerate its clearance in vivo

Abstract

Background: Many patients with acquired thrombotic thrombocytopenic purpura (TTP) harbor autoantibodies that may bind and/or inhibit ADAMTS-13 proteolytic activity and accelerate its clearance in vivo.

Methods: To test this hypothesis, we determined ADAMTS-13 activity and antigen levels in parallel plasma samples from patients clinically diagnosed with TTP. Collagen binding, GST-VWF73 and FRETS-VWF73 assays were used to determine ADAMTS-13 activity and to detect inhibitory autoantibodies. Enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation plus Western blotting (IP/WB) were used to detect total anti-ADAMTS-13 IgG (inhibitory and non-inhibitory).

Results: Among 40 patients with TTP (21 idiopathic and 19 non-idiopathic), inhibitory autoantibodies were detected (by FRETS-VWF73) in 52% of idiopathic and 0% of non-idiopathic TTP patients. In contrast, non-inhibitory IgG autoantibodies were detected in 29% of idiopathic and 50% of non-idiopathic TTP patients. The concentration of inhibitory IgG autoantibody in idiopathic TTP patients was significantly higher than that of non-inhibitory IgG in either idiopathic or non-idiopathic TTP patients. Idiopathic TTP patients demonstrated significantly reduced ADAMTS-13 activity compared with non-idiopathic patients, but only slightly lower ADAMTS-13 antigen levels. Interestingly, patients with inhibitory autoantibodies exhibited significantly lower ADAMTS-13 antigen levels than those with only non-inhibitory IgG autoantibodies or no autoantibody. Serial plasma exchanges increased levels of ADAMTS-13 activity and antigen concurrently in patients with inhibitory autoantibodies.

Conclusion: The identification of severe ADAMTS-13 deficiency and autoantibodies or inhibitors appears to be assay-dependent; the inhibitory IgG autoantibodies, in addition to binding and inhibiting ADAMTS-13 proteolytic activity, may accelerate ADAMTS-13 clearance in vivo.

Fig. 1

Comparison of ADAMTS-13 activity in parallel samples assayed by three different methodologies. Pearson correlation coefficients were determined between collagen binding and GST-VWF73 (A, r = 0.62); between GST-VWF73 and FRETS-VWF73 (B, r = 0.73); between collagen binding and FRETS-VWF73 (C, r = 0.83). The intra- and inter-assay coefficients of variation (CV) for GST-VWF73, collagen binding and FRETS-VWF73 were all< 10% and 20%, respectively.

Fig. 2

The likelihood of three different assays to detect severe ADAMTS-13 deficiency in patients with thrombotic thrombocytopenic purpura (TTP). The percentage of patients with severe deficiency (< 10%), moderate deficiency (10%–50%) and normal (> 50%) levels of ADAMTS-13 activity among all patients with TTP (A, n = 40), patients with idiopathic TTP (B, n = 21) and non-idiopathic TTP (C, n = 19) by the collagen-binding assay (CBA); GST-VWF73 (GST) and FRETS-VWF73 (FRETS) assays. The number inside bars represents the percentage of patients in each category.

Fig. 3

The ability of the different assays to identify autoantibodies in patients with thrombotic thrombocytopenic purpura (TTP). Percentage of patients having anti-ADAMTS-13 IgG or inhibitory autoantibodies in all patients (white bars; n = 40), subgrouped by patients with< 10% (black bars; n = 17) or > 10% (gray bars; n = 23) of ADAMTS-13 activity (black bars; n = 17) (A) was detected by the FRETS-VWF73 (FRETS), enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation plus Western blotting (IP/WB) assays. FRETS-VWF73 detects only inhibitory autoantibodies, whereas the immunoassays (ELISA and IP/WB) identify both inhibitory and non-inhibitory anti-ADAMTS-13 IgG (total). The representative IP/WB signals are shown in (B), in which anti-ADAMTS-13 IgG in patient plasma was designated negative (lanes 1, 2, 4, and 5) or positive (lanes 3 and 6), based on whether a 190 kDa band of recombinant ADAMTS-13-V5-His was detected or not by Western blotting with anti-V5 IgG (arrowheads).

Fig. 4

The prevalence and concentrations of the inhibitory and the non-inhibitory IgG autoantibodies in patients with thrombotic thrombocytopenic purpura (TTP). Samples were classified as inhibitory autoantibodies or non-inhibitory anti-ADAMTS13 IgG according to the definitions in Table 2. The numbers in the bars represent the percentage of patients in each category of the autoantibodies (A). The concentration of anti-ADAMTS13 IgG (B) in idiopathic TTP with inhibitory autoantibodies (group I) was significantly higher than that in idiopathic TTP (n = 21) with non-inhibitory anti-ADAMTS-13 IgG (group II) or non-idiopathic TTP (n = 19) with non-inhibitory anti-ADAMTS-13 IgG (group III) (Kruskall–Wallis statistic = 18.01, followed by Dunn’s multiple comparison, *P < 0.05). None of the non-idiopathic group’s samples demonstrated inhibitory anti-ADAMTS-13 IgG. There was no statistically significant difference between groups II and III. The horizontal line represents the median of anti-ADAMTS-13 IgG concentration.

Fig. 5

ADAMTS-13 activity and antigen levels in patients with thrombotic thrombocytopenic purpura (TTP). A shows the ADAMTS-13 activity (open bars) and antigen (closed bars) levels in idiopathic and non-idiopathic TTP patients as indicated. B and C show the ADAMTS13 activity and antigen levels in patients with idiopathic TTP harboring inhibitory autoantibodies (black bars), non-inhibitory anti-ADAMTS-13 IgG (gray bars) and no inhibitor or autoantibody (white bars), respectively. ADAMTS-13 antigen in pooled citrated normal human plasma is 540 ± 190 ng mL⁻¹ (see Materials and methods). The statistical analysis was performed using one-factor ANOVA, followed by Bonferroni tests (*P < 0.05 and **P < 0.01) (compared with the lowest value in each panel). A two-way ANOVA to discern the interaction of TTP-type (idiopathic vs. non-idiopathic) and antibody type (inhibitory, non-inhibitory, no antibody or inhibitor) was not performed because the non-idiopathic group did not contain inhibitory autoantibody.

Fig. 6

Serial laboratory parameters from two idiopathic thrombotic thrombocytopenic purpura (TTP) patients with inhibitory autoantibodies. Patient platelet counts (A and B), ADAMTS-13 activity and antigen levels (C and D), and anti-ADAMTS-13 IgG levels (E and F) were shown in two patients during the plasma exchange therapy and follow-up. The x-axis indicates the days after initial admission. Both patients received daily plasma exchange therapy, starting from day 1 for 30 days (patient 1) and 12 days (patient 2), after which the plasma exchange was tapered o.. The ADAMTS-13 activity and inhibitor were determined by FRETS-VWF73 assay, whereas anti-ADAMTS-13 IgG was determined by enzyme-linked immunosorbent assay.