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Multicenter Study
. 2010 Sep;95(9):1555-62.
doi: 10.3324/haematol.2009.019299. Epub 2010 Apr 7.

Multiple domains of ADAMTS13 are targeted by autoantibodies against ADAMTS13 in patients with acquired idiopathic thrombotic thrombocytopenic purpura

X Long Zheng  1 , Haifeng M Wu, Dezhi Shang, Erica Falls, Christopher G Skipwith, Spero R Cataland, Charles L Bennett, Hau C Kwaan
Affiliations
Multicenter Study

Multiple domains of ADAMTS13 are targeted by autoantibodies against ADAMTS13 in patients with acquired idiopathic thrombotic thrombocytopenic purpura

X Long Zheng et al. Haematologica. 2010 Sep.

Abstract

Background: Type G immunoglobulins against ADAMTS13 are the primary cause of acquired (idiopathic) thrombotic thrombocytopenic purpura. However, the domains of ADAMTS13 which the type G anti-ADAMT13 immunoglobulins target have not been investigated in a large cohort of patients with thrombotic thrombocytopenic purpura.

Design and methods: Sixty-seven patients with acquired idiopathic thrombotic thrombocytopenic purpura were prospectively collected from three major U.S. centers. An enzyme-linked immunosorbent assay determined plasma concentrations of anti-ADAMTS13 type G immunoglobulins, whereas immunoprecipitation plus western blotting determined the binding domains of these type G immunoglobulins.

Results: Plasma anti-ADAMTS13 type G immunoglobulins from 67 patients all bound full-length ADAMTS13 and a variant truncated after the eighth TSP1 repeat (delCUB). Approximately 97% (65/67) of patients harbored type G immunoglobulins targeted against a variant truncated after the spacer domain (MDTCS). However, only 12% of patients' samples reacted with a variant lacking the Cys-rich and spacer domains (MDT). In addition, approximately 37%, 31%, and 46% of patients' type G immunoglobulins interacted with the ADAMTS13 fragment containing TSP1 2-8 repeats (T2-8), CUB domains, and TSP1 5-8 repeats plus CUB domains (T5-8CUB), respectively. The presence of type G immunoglobulins targeted against the T2-8 and/or CUB domains was inversely correlated with the patients' platelet counts on admission.

Conclusions: This multicenter study further demonstrated that the multiple domains of ADAMTS13, particularly the Cys-rich and spacer domains, are frequently targeted by anti-ADAMTS13 type G immunoglobulins in patients with acquired (idiopathic) thrombotic thrombocytopenic purpura. Our data shed more light on the pathogenesis of acquired thrombotic thrombocytopenic purpura and provide further rationales for adjunctive immunotherapy.

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Figures

Figure 1.
Figure 1.
Identification of anti-ADAMTS13 IgG auto-antibodies by immunoprecipitation and western blotting. (A) The schematic domain representation of the constructs of recombinant ADAMTS13 and variants used in the study. M: metalloprotease domain; D: disintegrin domain; 1–8: 1–8th thrombospondin type 1 repeats; C and S: cys-rich and spacer domains; and CUB: two CUB domains. (B) Purified and partially purified ADAMTS13 and variants (~20–50 ng/lane) were detected by SDS-PAGE and western blotting with anti-V5 (1:5,000) and IDye800-labeled anti-mouse IgG. (C-J) eight representative binding patterns of plasma anti-ADAMTS13 IgG from patients 1, 8, 9, 10, 13, 14, 50 and 52, respectively. In these experiments, ~200 ng of recombinant ADAMTS13 and variants were incubated at 4ºC overnight with 5–10 μL of patients’ plasma. After being washed three times with TBS, the bound ADAMTS13/variant-IgG complexes were pulled down by the mixture of protein-G/protein A Sepharose 4B, and determined by western blot with anti-V5 (1:5,000), followed by either anti-mouse IgG, peroxidase conjugated (1:5,000) and chemiluminescent reagents (C-H) or by IDye800-labeled anti-mouse IgG (1:12,500) and Odyssey imaging detection (panels I and J). The star on the left right side of panels E and H indicates an unknown non-specific protein contaminated in the protein A-Sepharose 4B beads used at this particular time.
Figure 2.
Figure 2.
Correlations between the C-terminal antibody binding and patients’ platelet counts or autoantibody titers. Panel A and panel B show the platelet counts and plasma autoantibody titers, respectively, in one group with anti-ADAMTS13 IgG against the middle and distal C-terminal domains (C-terminal binding) and in another group with IgG binding only the N-terminal fragments up to the spacer domain of ADATMS13 (no C-terminal binding). The differences between the two groups were determined by a two-tailed Mann-Whitney test. P values less than 0.05 were considered to be statistically significant.
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