Enzymatically active ADAMTS13 variants are not inhibited by anti-ADAMTS13 autoantibodies: a novel therapeutic strategy?

Abstract

ADAMTS13 (a disintegrin and metalloprotease with thrombospondin motifs), a circulating multidomain zinc metalloprotease of the reprolysin subfamily, is critical for preventing von Willebrand factor-platelet interaction under high shear stress conditions. A deficiency of the protease, due to mutations in the ADAMTS13 gene or the presence of antibodies that inhibit the activity of the protease, causes thrombotic thrombocytopenic purpura (TTP). Plasma therapy, the conventional therapy for TTP, may cause serious adverse reactions and is ineffective in some patients. In order to develop new strategies for improving the diagnosis and treatment of TTP, we produced a series of truncated ADAMTS13 proteins in mammalian cells and analyzed their binding with and suppression by the IgG derived from the TTP patients. The results revealed that truncation of the ADAMTS13 protein at its cysteine-rich region eliminated its recognition by the antibodies without abolishing its von Willebrand factor-cleaving activity. This raises the possibility that resistant ADAMTS13 variants may be exploited to circumvent inhibitory antibodies that cause TTP.

FIGURE 1. Design and production of recombinant ADAMTS13 proteins

A, schematic depiction of the domain structure of ADAMTS13 and the forms of variant ADAMTS13 proteins used in this study. For each construct, the numbers in the parenthesis indicate the segment of amino acid residues of ADAMTS13. Sig, signal peptide; Pro, propeptide; MP, metalloprotease; Dis, disintegrin; TSP1, thrombospondin-1 motif 1; Cys, cysteine-rich region; Spa, spacer; TSP2—8, thrombospondin-1 motifs 2–8; CUB, cub domains. B, production of proteins in COS-7 cells. Cells were transiently transfected with plasmid constructs for various ADAMTS13 variants. Conditioned medium was concentrated 15-fold, whereas the cells were lysed with SDS-PAGE sample buffer. A monoclonal anti-V5 antibody was used to detect levels of the ADAMTS13 proteins in cell lysates (C) and in the culture media (M) by immunoblotting.

FIGURE 2. Immunoprecipitation of ADAMTS13 proteins with TTP IgG

A, immunoprecipitation with ADAMTS13 affinity-purified TTP IgG. Left panel, four ADAMTS13 variant proteins (AD1, AD2, AD3, and AD5) and an irrelevant protein, β-galactosidase (LacZ), as detected with anti-V5 antibody before immunoprecipitation (IPP). Middle panel, AD5 was depleted from the supernatant after the immunoprecipitation. Right panel, only AD5 was present in the pellets of immunoprecipitation. B and C, immunoprecipitation of ADAMTS13 variant proteins AD4—AD13 with TTP IgG. For each protein, the panel includes three lanes: the protein before immunoprecipitation, the protein in the pellets of normal IgG/protein A beads (Nl), and the protein in the pellets of TTP IgG/protein A beads (TTP). AD5, AD6, AD7, and AD10 were detected in the pellets of TTP IgG. None of the proteins were in the pellets of normal IgG.

FIGURE 3. Binding between the ADAMTS13 variants and TTP IgG

A and D, immunoblots of the recombinant proteins used in the binding experiments. The recombinant proteins were detected by using monoclonal anti-V5. B, C, E, and F, recombinant ADAMTS13 proteins immobilized onto monoclonal anti-V5 coated microtiter plate wells were incubated with either TTP or normal human plasma. The captured human IgG were detected chromogenically as described under “Materials and Methods.” Each symbol represents the result with one TTP or normal sample. Each protein was tested against 5–9 different TTP and 2–7 normal plasma samples. Compared with normal plasma, TTP plasma contained IgG molecules with positive binding to AD5, AD6, AD7, and AD10 but not to AD1—AD4, AD8, AD9, or AD11—AD13. To confirm the specificity of the observed binding, the TTP samples were also tested against AD7 or AD10 after incubation with untagged recombinant full-length ADAMTS13, which blocked the binding of TTP IgG to AD7 or AD10 (marked as *AD7 and *AD10).

FIGURE 4. Detection of VWF-cleaving activity in recombinant ADAMTS13 proteins and its suppression by TTP plasma

A and B, immunoblots of SDS-PAGE depicting the VWF fragments ((176kD)₂, homodimer of the 176-kDa fragment; (140kD)₂, homodimer of the 140-kDa fragment, which is barely visible) generated after digestion of the VWF substrate by the ADAMTS13 activity in either normal human plasma (NHP) or the recombinant proteins. The catalytic activity of each recombinant protein was assayed after incubation with heated normal human plasma as a control (A) or with TTP plasma (B). Compared with heat-treated normal plasma in A, the heated TTP plasma in B suppressed the catalytic activity of AD5, AD6, and AD7, but not of AD2 or AD3. C and D, immunoblots of SDS-PAGE showing the VWF-cleaving activity in mixtures of AD7 (C) or AD2 (D) and heat-treated normal plasma (NHP*) or one of nine different TTP plasma samples. Each of the TTP plasma samples (TT2—TTP10) suppressed the VWF-cleaving activity of AD7 but not AD2. To ensure the specificity of the assay, each digestion was conducted in the absence or presence of EDTA. EDTA suppressed the generation of VWF fragments observed in the absence of EDTA. Because of high concentrations, AD5—AD7 were diluted 1:20 for the analysis.

FIGURE 5. Cleavage of GST-VWF73 (Asp¹⁵⁹⁶—Arg¹⁶⁶⁸)-His by recombinant ADAMTS13 proteins and its suppression by TTP plasma

A, immunoblots of SDS-PAGE depicting the cleavage of GST-VWF73-His (38.1 kDa) to a 30.4-kDa band by the catalytic activity of normal human plasma (NHP) or of recombinant proteins AD2—AD7. Each protease was assayed after incubation with heat-treated normal human plasma as a control. Bu, Tris-NaCl buffer. B, after incubation with heat-treated TTP plasma, only AD2 and AD3 remained active in cleaving GST-VWF73-His. C, levels of GST-VWF73-His-cleaving activity as determined by ELISA. Compared with heat-treated normal human plasma (NHP*, filled bars), heat-treated TTP plasma (TTP*, open bars) suppressed the activity of AD5, AD6, and AD7 but not AD2 or AD3.

FIGURE 6. Restoration of ADAMTS13 activity in TTP plasma with the ADAMTS13 variant protein AD2

A, the course of platelet counts in a TTP patient who relapsed through plasma exchanges and died on day 19. Each diamond represents one session of plasma exchange. B, the course of ADAMTS13 inhibitor titers in the same patient. C, the addition of AD7 to each of the patient’s plasma samples raised the ADAMTS13 activity level on days 3–7 to a maximal value of 1.05 units/ml instead of the expected 10.5 units/ml. The plasma samples from day 8 and thereafter abolished the activity of AD7. D, the addition of variant AD2 (0.77 units/ml) to the patient’s plasma samples raised the ADAMTS13 activity to the expected level, irrespective of the inhibitor titers.