Evidence for a role of anti-ADAMTS13 autoantibodies despite normal ADAMTS13 activity in recurrent thrombotic thrombocytopenic purpura

Abstract

Background: Severe ADAMTS13 deficiency is a critical component of the pathogenesis of idiopathic thrombotic thrombocytopenic purpura but is found only in about 60% of patients clinically diagnosed with this disease.

Design and methods: Over a period of 8 years and six episodes of thrombotic thrombocytopenic purpura we studied the evolution of the anti-ADAMTS13 antibody response in a patient using different ADAMTS13 assays and epitope mapping.

Results: Anti-ADAMTS13 autoantibodies were found in all episodes but were inhibitory only in the last two episodes. In a flow-based assay, normal ADAMTS13 activity was found only during the first disease episode, while ADAMTS13 activity was normal using a static assay in episodes 1 and 3, and severely deficient in the last two episodes. Fluorescence evolution in a modified fluorescence resonance energy transfer assay using a von Willebrand factor A2 domain peptide substrate was linear in episodes 1, 5 and 6, but increased exponentially in episodes 3 and 4. Despite the variable functional characteristics of the anti-ADAMTS13 autoantibodies, their principal epitope was the ADAMTS13 spacer domain in all episodes.

Conclusions: The patient is unique as he displayed features of maturation or shaping of the anti-ADAMTS13 autoantibody response during the course of multiple episodes of thrombotic thrombocytopenic purpura. Anti-ADAMTS13 autoantibodies may be important in vivo despite normal ADAMTS13 activity in routine assays. Consequently, treatment decisions should not be based solely on activity assay results.

Figure 1.

Fluorescence evolution over time in the modified FRETS-VWF73 assay for patient‘s serum samples withdrawn at the onset of the 1^st, 3^rd, 4^th and 5^th acute TTP episodes. Dotted lines represent the fluorescence evolution in standard curve samples (defined mixtures of normal human plasma and heat-inactivated normal human plasma, see Design and Methods section for details) run in the same assay.

Figure 2.

In vitro characterization of anti-ADAMTS13 autoantibodies. (A) Dot immunobinding assay. Recombinant ADAMTS13 was spotted at different concentrations onto nitrocellulose membranes and 1:100 (v:v) diluted patient’s serum samples from different acute TTP episodes (1^st – 5^th) was used as the source of the primary antibody. A 1:100 diluted normal human plasma pool (NHP) and the secondary alkaline phosphatase-conjugated rabbit anti-human IgG antibody used as primary antibody sources served as controls. (B) Epitope mapping of anti-ADAMTS13 autoantibodies by immunoprecipitation, and a schematic overview of ADAMTS13 domain structure and of different ADAMTS13 constructs used for analysis (PMDTCS-13, consisting of ADAMTS13 propeptide – metalloprotease – disintegrin – thrombospondin type I repeat number 1 – cys-rich and spacer domain; PMDTCS-1, the same as PMDTCS-13, however with replacement of ADAMTS13 spacer by the spacer domain of ADAMTS1 (highlighted); T 2–8, ADAMTS13 thrombospondin type I repeats number 2 – 8; CUB 1–2, ADAMTS13 CUB domains 1–2). ADAMTS13 constructs all containing a carboxy-terminal V5 tag,, were used as antigen. A normal human plasma pool served as a negative control, and a commercially available monocloncal anti-V5 antibody as a positive control.

Figure 3.

Adsorption of the patient’s serum samples on protein G sepharose. Antibodies and immune complexes were removed by protein G sepharose from the patient’s serum samples taken during the 1^st, 3^rd, 4^th, 5^th and 6^th episodes. ADAMTS13 activity was determined in serum before (gray columns) and after IgG-depletion using protein G sepharose (white columns).