The spacer domain of ADAMTS13 contains a major binding site for antibodies in patients with thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP) is a microangiopathy often associated with a severely decreased activity of ADAMTS13. In plasma of the majority of patients with TTP, antibodies are present that inhibit the von Willebrand factor (VWF) processing activity of ADAMTS13. We describe a sensitive assay that monitors binding of recombinant ADAMTS13 to immobilized IgG derived from patient plasma. Analysis of fifteen patients with TTP and severely reduced ADAMTS13 activity revealed that in all patients antibodies directed to ADAMTS13 were present. Levels of anti-ADAMTS13 antibodies varied considerably among patients, specific antibody levels in plasma range from less than 100 ng/ml to over 1 microg/ml. Longitudinal analysis in three patients revealed that anti-ADAMTS13 antibody levels declined with different kinetics. For further characterization of anti-ADAMTS13 antibodies, we prepared a series of recombinant fragments corresponding to the various ADAMTS13 domains. All seven TTP plasma samples tested, showed reactivity of antibodies towards a fragment consisting of the disintegrin/TSR1/cysteine-rich/spacer domains. In one patient, we also observed reactivity towards the TSR2-8 repeats. No binding of antibodies to propeptide, metalloprotease and CUB domains was detected. To further delineate the binding site in the disintegrin/TSR1/cysteine-rich/spacer region, we prepared additional ADAMTS13 fragments. Antibodies directed towards the cysteine-rich/spacer fragment were found in all plasma samples analyzed. No antibodies reacting with the disintegrin/TSR1 domains were detected. A recombinant fragment comprising the spacer domain was recognized by all patients samples analyzed, suggesting that the 130-amino-acid spacer domain harbors a major binding site for anti-ADAMTS-13 antibodie

Amino acid regions 572-579 and 657-666 of the spacer domain of ADAMTS13 provide a common antigenic core required for binding of antibodies in patients with acquired TTP

Antibodies directed against ADAMTS13 have been detected in the majority of patients with acquired thrombotic thrombocytopenic purpura (TTP). We have previously localized a major antigenic determinant within the spacer domain of ADAMTS13. To identify the amino acid residues of the spacer domain that are involved in binding of anti-ADAMTS13 antibodies, we constructed a series of fifteen hybrids (designated A-O) in which 5-10 amino acids of the spacer domain were exchanged for the corresponding region of ADAMTS1. Plasma from six patients with antibodies directed against the spacer domain was analyzed for reactivity with the ADAMTS13/ADAMTS1 chimeras. Exchange of amino acid residues 572-579 (hybrid C) and 657-666 (hybrid M) completely abolished the binding of antibodies from all six patients analyzed. Regions 580-587 (D), 602-620 (G, H), 629-638 (J), and 667-767 (N) contributed to binding of antibodies from patients 2, 4, and 5 (epitope present within regions CDGHJMN). Antibodies derived from patient 1 required region 602-620 (G, H) for binding (CGHM-epitope). For antibodies of patient 3, residues 564-571 (B), 580-587 (D), and 629-638 (J) were required (BCDJM-epitope), whereas replacement of residues 602-610 (G) and 629-638 (J) greatly diminished binding of antibodies from patient 6 (CGJM-epitope). Despite the presumably polyclonal origin of the antibodies present in plasma of patients, our results suggest that residues 572-579 (C) and 657-666 (M) comprise a common antigenic core region that is crucial for binding of anti-ADAMTS13 antibodies. Other regions that spatially surround this antigenic core further modulate binding of antibodies to the spacer domai

Factor VIII inhibitor in a patient with mild haemophilia A and an Asn618-->Ser mutation responsive to immune tolerance induction and cyclophosphamide

We describe a patient with mild haemophilia A (original value of factor VIII activity 0.30 U/ml) who developed an inhibitor (36.1 Bethesda U/ml) which cross-reacted with his endogenous factor VIII. This caused a decline in basal factor VIII level ( Ser in the A2 domain of factor VIII. Immunoprecipitation analysis showed that the antibodies present in the patient's plasma reacted with metabolically labelled A2 domain and, to a lesser extent, with factor VIII light chain. Inhibitory antibodies were completely neutralized by recombinant A2 domain, whereas no neutralization was observed after the addition of factor VIII light chain (A3-C1-C2) and C2 domain. More detailed analysis showed that the majority of inhibitory antibodies were directed against residues Arg484-Ile508, a previously identified binding site for factor VIII inhibitors. Our findings suggest that immune tolerance therapy and cyclophosphamide were successful in eradicating inhibitory antibodies against a common epitope on factor VII

Two classes of germline genes both derived from the V(H)1 family direct the formation of human antibodies that recognize distinct antigenic sites in the C2 domain of factor VIII

Most plasmas from patients with inhibitors contain antibodies that are reactive with the C2 domain of factor VIII. Previously, we have shown that the variable heavy chain (V(H)) regions of antibodies to the C2 domain are encoded by the closely related germline gene segments DP-10, DP-14, and DP-88, which all belong to the V(H)1 gene family. Here, we report on the isolation and characterization of additional anti-C2 antibodies that are derived from V(H) gene segments DP-88 and DP-5. Competition experiments using murine monoclonal antibodies CLB-CAg 117 and ESH4 demonstrated that antibodies derived from DP-5 and DP-88 bound to different sites within the C2 domain. Epitope mapping studies using a series of factor VIII/factor V hybrids revealed that residues 2223 to 2332 of factor VIII are required for binding of the DP-10-, DP-14-, and DP-88-encoded antibodies. In contrast, binding of the DP-5-encoded antibodies required residues in both the amino- and carboxy-terminus of the C2 domain. Inspection of the reactivity of the antibodies with a series of human/porcine hybrids yielded similar data. Binding of antibodies derived from germline gene segments DP-10, DP-14, and DP-88 was unaffected by replacement of residues 2181 to 2243 of human factor VIII for the porcine sequence, whereas binding of the DP-5-encoded antibodies was abrogated by this replacement. Together these data indicate that antibodies assembled from V(H) gene segments DP-5 and the closely related germline gene segments DP-10, DP-14, and DP-88 target 2 distinct antigenic sites in the C2 domain of factor VII