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Multiple B‐cell clones producing antibodies directed to the spacer and disintegrin/thrombospondin type‐1 repeat 1 (TSP1) of ADAMTS13 in a patient with acquired thrombotic thrombocytopenic purpura
Author(s) -
LUKEN B. M.,
KAIJEN P. H. P.,
TURENHOUT E. A. M.,
KREMER HOVINGA J. A.,
VAN MOURIK J. A.,
FIJNHEER R.,
VOORBERG J.
Publication year - 2006
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/j.1538-7836.2006.02164.x
Subject(s) - adamts13 , disintegrin , thrombospondin , antibody , microbiology and biotechnology , thrombotic thrombocytopenic purpura , chemistry , epitope , immunoglobulin light chain , monoclonal antibody , phage display , biology , metalloproteinase , biochemistry , immunology , platelet , enzyme
Summary. Background: The cysteine‐rich/spacer domains of ADAMTS13 contain a major binding site for antibodies in patients with acquired thrombotic thrombocytopenic purpura (TTP). Objective: To study the heterogeneity of the antibody response towards these domains an immunoglobulin V‐gene phage‐display library was constructed to isolate monoclonal anti‐ADAMTS13 antibodies from the immunoglobulin repertoire of a patient with acquired TTP. Methods: Combined variable heavy chain (VH) and variable light chain (VL) segments, expressed as single‐chain Fv fragments (scFv), were selected for binding to an ADAMTS13 fragment consisting of the disintegrin/thrombospondin type‐1 repeat 1 (TSP1)/cysteine‐rich/spacer domains. Results: Seven different scFv antibody clones were identified that were assigned to four groups based on their homology to VH germline gene segments. Epitope‐mapping revealed that scFv I‐9 (VH1‐69), I‐26 (VH1‐02), and I‐41 (VH3‐09) bind to an overlapping binding site in the ADAMTS13 spacer domain, whereas scFv I‐16 (VH3‐07) binds to the disintegrin/TSP1 domains. The affinity of scFv for the disintegrin/TSP1/cysteine‐rich/spacer domain was determined by surface plasmon resonance analysis and the dissociation constants ranged from 3 to 254 n m . The scFv partially inhibited ADAMTS13 activity. However, full‐length IgG prepared from the variable domains of scFv I‐9 inhibited ADAMTS13 activity more profoundly. Plasma of six patients with acquired TTP competed for binding of scFv I‐9 to ADAMTS13. Conclusion: Our data indicate that multiple B‐cell clones producing antibodies directed against the spacer domain are present in the patient analyzed in this study. Our findings also suggest that antibodies with a similar epitope specificity as scFv I‐9 are present in plasma of other patients with acquired TTP.