An open conformation of ADAMTS‐13 is a hallmark of acute acquired thrombotic thrombocytopenic purpura

Abstract

Background Acquired thrombotic thrombocytopenic purpura (aTTP) is an autoimmune disorder characterized by absent ADAMTS-13 activity and the presence of anti-ADAMTS-13 autoantibodies. Recently, it was shown that ADAMTS-13 adopts a folded or an open conformation. Objectives As conformational changes in self-antigens play a role in the pathophysiology of different autoimmune diseases, we hypothesized that the conformation of ADAMTS-13 changes during acute aTTP. Methods Antibodies recognizing cryptic epitopes in the spacer domain were generated. Next, the conformation of ADAMTS-13 in 40 healthy donors (HDs), 99 aTTP patients (63 in the acute phase versus 36 in remission), 12 hemolytic-uremic syndrome (HUS) patients and 63 sepsis patients was determined with ELISA. Results The antibody 1C4 recognizes a cryptic epitope in ADAMTS-13. Therefore, we were able to discriminate between a folded and an open ADAMTS-13 conformation. We showed that ADAMTS-13 in HDs does not bind to 1C4, indicating that ADAMTS-13 circulates in a folded conformation. Similar results were obtained for HUS and sepsis patients. In contrast, ADAMTS-13 of acute aTTP patients bound to 1C4 in 92% of the cases, whereas, in most cases, this binding was abolished during remission, showing that the conformation of ADAMTS-13 is open during an acute aTTP episode. Conclusions Our study shows that, besides absent ADAMTS-13 activity and the presence of anti-ADAMTS-13 autoantibodies, an open ADAMTS-13 conformation is also a hallmark of acute aTTP. Demonstrating this altered ADAMTS-13 conformation in acute aTTP will help to further unravel the pathophysiology of aTTP and lead to improved therapy and diagnosis.

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).…”

“…Routine ADAMTS13 antigen determination could therefore facilitate improvement of the initial treatment. Furthermore, ADAMTS13 antigen assessment revealed antibody‐mediated ADAMTS13 clearance as the major pathogenic mechanism in this disease, as ADAMTS13 antigen levels are reduced in the majority of iTTP patients …”

“…Furthermore, ADAMTS13 antigen assessment revealed antibody-mediated ADAMTS13 clearance as the major pathogenic mechanism in this disease, as ADAMTS13 antigen levels are reduced in the majority of iTTP patients. [5][6][7][8][9][10] The first aim of this study was to validate our in-house developed monoclonal antibody (mAb)-based ADAMTS13 antigen enzyme-linked immunosorbent assay (ELISA) in terms of sensitivity, repeatability, and reproducibility and to determine ADAMTS13 antigen levels in a large cohort of healthy donor and iTTP patient plasma samples. The second aim of this study was to investigate whether the presence of anti-ADAMTS13 autoantibodies impede the correct determination of ADAMTS13 antigen levels in our ELISA, as three murine anti-ADAMTS13 monoclonal antibodies are used to capture and detect bound plasma ADAMTS13.…”