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

Abstract

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 antibodies.

Evidence for a role of anti-ADAMTS13 autoantibodies despite normal ADAMTS13 activity in recurrent thrombotic thrombocytopenic purpura

“…The main antigenic epitope recognized by these anti-ADAMTS13 autoantibodies was found to reside in the ADAMTS13 spacer domain (Figure 2B), which has been reported to contain the primary antigenic epitope of inhibitory anti-ADAMTS13 antibodies. 19,[21][22][23] This suggests a pathophysiological role of the anti-ADAMTS13 autoantibodies present in low titers during the first acute episode despite normal in vitro ADAMTS13 activity documented by three different assays.…”

“…(B) Epitope mapping of anti-ADAMTS13 autoantibodies by immunoprecipitation, and a schematic overview of ADAMTS13 domain structure and of different ADAMTS13 constructs used for analysis (PMDTCS-13, consisting of ADAMTS13 propeptide -metalloprotease -disintegrin -thrombospondin type I repeat number 1 -cys-rich and spacer domain; PMDTCS-1, the same as PMDTCS-13, however with replacement of ADAMTS13 spacer by the spacer domain of ADAMTS1 (highlighted); T 2-8, ADAMTS13 thrombospondin type I repeats number 2 -8; CUB 1-2, ADAMTS13 CUB domains 1-2). ADAMTS13 constructs all containing a carboxy-terminal V5 tag, 19,20 were used as antigen. A normal human plasma pool served as a negative control, and a commercially available monocloncal anti-V5 antibody as a positive control.…”

“…19,20 All of the above mentioned recombinant truncated ADAMTS13 proteins contained a C-terminal V5 tag. Normal human plasma served as an autoantibody-negative control and a commercially available mono clonal anti-V5 antibody (Invitrogen, Carlsbad, CA, USA) as a positive control.…”

Residues Arg568 and Phe592 contribute to an antigenic surface for anti-ADAMTS13 antibodies in the spacer domain

“…[15][16][17][18][19] Anti-ADAMTS13 antibodies present in the plasma of patients with acquired TTP target an antigenic surface including residues Arg660, Tyr661 and Tyr665. 14 However in three out of six patients' analyzed it was seen that there was residual binding to an MDTCS variant in which Arg660, Tyr661 and Tyr665 were replaced by an alanine.…”

Massively parallel enzyme kinetics reveals the substrate recognition landscape of the metalloprotease ADAMTS13

“…Such methods include engineering deletion mutants (3), use of competitive ligands (4,5), and site-directed mutagenesis (6,7). In contrast to these techniques, substrate phage display is a highthroughput, unbiased approach to studying protease substrate specificity (8)(9)(10).…”