Epitope mapping of ADAMTS13 autoantibodies in acquired thrombotic thrombocytopenic purpura

Abstract

Severe deficiency of the von Willebrand factor (VWF)-cleaving protease ADAMTS13 can lead to thrombotic thrombocytopenic purpura (TTP), a disease associated with the widespread formation of platelet-rich thrombi in many organs. Autoantibodies that inactivate ADAMTS13 are the most frequent cause of acquired TTP. Little is known about epitope specificity and reactivity of anti-ADAMTS13 antibodies. In this study, a series of ADAMTS13 domains were expressed in Escherichia coli, and the reactivity of purified recombinant fragments with anti-ADAMTS13 autoantibodies from 25 patients with severe ADAMTS13 deficiency was evaluated in vitro. All TTP plasmas contained antibodies directed against the cysteine-rich spacer (cys-rich/spacer) domain of ADAMTS13. In the plasma of 3 patients, antibodies were detected that reacted exclusively with the cys-rich/spacer domain, underscoring the importance of this region for functional activity of ADAMTS13. In 64% of the plasmas, antibodies reacted with the 2 CUB domains, and in 56% they reacted with the isolated first thrombospondin type 1 (TSP-1) repeat and with the compound fragment consisting of the catalytic, the disintegrin-like, and the TSP1-1 domain. Less frequently, in 28% of the plasmas, antibodies reacted with the TSP1 repeats 2 to 8. Unexpectedly, antibodies reacted with the propeptide region in 20% of the plasmas. In conclusion, this study shows that even though anti-ADAMTS13 autoantibodies react with multiple domains of the protease, the cys-rich/spacer domain is consistently involved in antibody reactivity. (Blood. 2004;103:4514-4519)

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.…”

Proteolytic inactivation of ADAMTS13 by thrombin and plasmin

“…23,24 Moreover, certain strains of mice have been identified that express an ADAMTS13 variant that lacks the CUB domains (and also TSP1-7 and TSP1-8), 27 further suggesting that the functions of these domains are not critical in vivo. Although autoantibodies against the CUB domains have been found in patients with acquired TTP, 28 it is unclear whether these compromise ADAMTS13 function. The recent study reporting that CUB domain-derived peptides inhibit VWF-cleaving activity under flowing, but not static, conditions might, however, imply a potentially important functional role to these domains.…”

Thrombotic Thrombocytopenic Purpura: A Thrombotic Disorder Caused by ADAMTS13 Deficiency

“…The spacer domain is required for binding to a short fragment in the VWF A2 domain downstream of the cleavage site [24] and also for recognition by TTP IgG [21;25]. One study also detected interaction of TTP IgG with other domain fragments [26], although this observation remains controversial. The TSR 2-8 region is required for effective cleavage of VWF multimers but not of the VWF A2 fragment.…”