Anti-ADAMTS13 Antibodies and a Novel Heterozygous p.R1177Q Mutation in a Case of Pregnancy-Onset Immune-Mediated Thrombotic Thrombocytopenic Purpura

Abstract

In this study, we investigated a case of pregnancy-onset thrombotic thrombocytopenic purpura (TTP). The patient had severely decreased ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 motif, member 13) activity levels during acute phase and the presence of inhibitory anti-ADAMTS13 autoantibodies was demonstrated, which led to the diagnosis of immune-mediated TTP. However, ADAMTS13 activity was only mildly restored during remission, although inhibitory anti-ADAMTS13 antibodies were no longer detected. We hypothesized that genetic abnormalities could account for this discrepancy between ADAMTS13 activity and antigen. Genetic analysis revealed the presence of two heterozygous substitutions on the same allele: a single nucleotide polymorphism (SNP) c.2699C > T (p.A900V), located in the beginning of the T5 domain, and a mutation c.3530G > A (p.R1177Q) located in the third linker region of ADAMTS13. In vitro testing of those substitutions by expression of recombinant proteins revealed a normal secretion but a reduced ADAMTS13 activity by the novel p.R1177Q mutation, which could partially explain the subnormal activity levels found during remission. Although changes in the linker region might induce conformational changes in ADAMTS13, the p.R1177Q mutation in the third linker region of ADAMTS13 did not expose a cryptic epitope in the metalloprotease domain. In conclusion, we report on an immune-mediated pregnancy-onset TTP patient who had inhibitory anti-ADAMTS13 autoantibodies during acute phase, but not during remission. Genetic analysis confirmed the diagnosis of immune-mediated TTP and revealed the novel p.R1177Q mutation which mildly impaired ADAMTS13 activity.

Anti‐ADAMTS13 autoantibodies in immune‐mediated thrombotic thrombocytopenic purpura do not hamper ELISA‐based quantification of ADAMTS13 antigen

“…Our original in‐house developed mAb‐based ADAMTS13 antigen ELISA has been successfully used in different studies . As the anti‐ADAMTS13 antibody 2G3 producing hybridoma cell line was no longer viable, a new ADAMTS13 antigen ELISA was developed by selecting alternative in‐house developed mouse anti‐ADAMTS13 antibodies . The antibodies used in the revised ELISA included the capturing mAb 3H9 (directed against the metalloprotease [M] domain) and a combination of biotinylated mAbs 19H4 and 17G2 as detecting antibodies (directed against the thrombospondin type‐1 repeat [T] 8 and CUB1 domain, respectively; Figure ).…”

“…The antibodies used in the revised ELISA included the capturing mAb 3H9 (directed against the metalloprotease [M] domain) and a combination of biotinylated mAbs 19H4 and 17G2 as detecting antibodies (directed against the thrombospondin type‐1 repeat [T] 8 and CUB1 domain, respectively; Figure ). This ADAMTS13 antigen ELISA was validated in‐house, but the validation was not published at that time . Different clinical laboratories used our ADAMTS13 antigen ELISA in the meantime and validated the assay in their laboratories .…”

“…ADAMTS13 antigen levels were determined using our in house‐developed ADAMTS13 antigen ELISA, which is a modification of the originally published assay . Briefly, microtiter plates were coated overnight with the in‐house developed anti‐human ADAMTS13 mAb 3H9 (5 µg/mL in carbonate/bicarbonate buffer).…”

“…5,9,19,20 As the anti-ADAMTS13 antibody 2G3 producing hybridoma cell line was no longer viable, a new ADAMTS13 antigen ELISA was developed by selecting alternative in-house developed mouse anti-ADAMTS13 antibodies. 10,12 The antibodies used in the revised ELISA included the S T 2 T 3 T4 T 5 T 6 T7 T8 CUB1 17G2 19H4 CUB2 at that time. 10,12 Different clinical laboratories used our ADAMTS13 antigen ELISA in the meantime and validated the assay in their laboratories.…”

“…10,12 The antibodies used in the revised ELISA included the S T 2 T 3 T4 T 5 T 6 T7 T8 CUB1 17G2 19H4 CUB2 at that time. 10,12 Different clinical laboratories used our ADAMTS13 antigen ELISA in the meantime and validated the assay in their laboratories. 7,15 In the current paper we report the in-house validation of this ADAMTS13 antigen ELISA to document the reliability of our assay.…”