Generation and validation of small ADAMTS13 fragments for epitope mapping of anti‐ADAMTS13 autoantibodies in immune‐mediated thrombotic thrombocytopenic purpura. Issue 5 (25th June 2020)

• Record Type: Journal Article
• Authors: Kangro, Kadri; Roose, Elien; Schelpe, An‐Sofie; Tellier, Edwige; Kaplanski, Gilles; Voorberg, Jan; De Meyer, Simon F.; Männik, Andres; Vanhoorelbeke, Karen
• Is Part Of: Research and practice in thrombosis and haemostasis. Volume 4:Issue 5(2020)
• Page Start: 918
• Page End: 930
• Publication Date: 2020-06-25
• Subjects: ADAMTS13 protein -- antibodies -- epitopes -- human serum albumin -- thrombotic thrombocytopenic purpura; Thrombosis -- Periodicals; Hemostasis -- Periodicals; 616.135005
• DOI: 10.1002/rth2.12379
• Languages: English
• ISSNs: 2475-0379

Abstract

Abstract: Background: In immune‐mediated thrombotic thrombocytopenic purpura (iTTP), patients develop an immune response against the multidomain enzyme ADAMTS13. ADAMTS13 consists of a metalloprotease (M) and disintegrin‐like (D) domain, 8 thrombospondin type 1 repeats (T1‐T8), a cysteine‐rich (C), a spacer (S), and 2 CUB domains (CUB1‐2). Previous epitope mapping studies have used relatively large overlapping ADAMTS13 fragments. Objectives: We aimed at developing small nonoverlapping ADAMTS13 fragments to fine map anti‐ADAMTS13 autoantibodies in iTTP patients. Methods: A library of 16 ADAMTS13 fragments, comprising several small (M, DT, C, S, T2‐T5, T6‐T8, CUB1, CUB2), and some larger fragments with overlapping domains (MDT, MDTC, DTC, CS, T2‐T8, CUB1‐2, MDTCS, T2‐C2), were generated. All fragments, and ADAMTS13, were expressed as a fusion protein with albumin domain 1, and purified. The folding of the fragments was tested using 17 anti‐ADAMTS13 monoclonal antibodies with known epitopes. An epitope mapping assay using small ADAMTS13 fragments was set up, and validated by analyzing 18 iTTP patient samples. Results: Validation with the monoclonal antibodies demonstrated that single S and CUB1 were not correctly folded, and therefore CS and CUB1‐2 fragments were selected instead of single C, S, CUB1, and CUB2 fragments. Epitope mapping of antibodies of patients with iTTP confirmed that 6 nonoverlapping ADAMTS13 fragments M, DT, CS, T2‐T5, T6‐T8, and CUB1‐2 were sufficient to accurately determine the antibody‐binding sites. Conclusion: We have developed a tool to profile patients with iTTP according to their anti‐ADAMTS13 antibodies for a better insight in their immune response.