Linker regions and flexibility around the metalloprotease domain account for conformational activation of ADAMTS‐13

Abstract

Background Recently, conformational activation of ADAMTS13 was identified. This mechanism showed the evolution from a condensed and inhibited conformation, in which the proximal MDTCS and distal T2-CUB2 domains are in close contact with each other, to an activated structure due to ding with the von Willebrand factor (VWF). Objectives Identification of cryptic epitope/exosite exposure after conformational activation and of sites of flexibility in ADAMTS13. Methods The activating effect of 25 anti-T2-CUB2 antibodies was studied in the FRETS-VWF73 and the vortex assay. Cryptic epitope/exosite exposure was determined in ELISA and VWF binding assay. The molecular basis for flexibility was hypothesized through RADAR analysis, tested in ELISA using deletion variants and visualized using electron microscopy. Results Eleven activating anti-ADAMTS13 antibodies, directed against the T5-CUB2 domains, were identified in the FRETS-VWF73 assay. RADAR analysis identified three linker regions in the distal domains. Interestingly, identification of an antibody recognizing a cryptic epitope in the metalloprotease domain confirmed the contribution of these linker regions to conformational activation of the enzyme. The proof of flexibility around both the T2 and metalloprotease domains by electron microscopy furthermore supported this contribution. In addition, cryptic epitope exposure was identified in the distal domains, as activating anti-T2-CUB2 antibodies increased the binding to folded VWF up to ~3-fold. Conclusion Conformational activation of ADAMTS13 leads to cryptic epitope/exosite exposure in both proximal and distal domains, subsequently inducing increased activity. Furthermore, three linker regions in the distal domains are responsible for flexibility and enable the interaction between the proximal and the T8-CUB2 domains.

Influences of ABO blood group, age and gender on plasma coagulation factor VIII, fibrinogen, von Willebrand factor and ADAMTS13 levels in a Chinese population

“…The analysis of genotype uncovered that the A1 and B alleles were positively and significantly associated with VWF:Ag, whereas O and A2 alleles were negatively associated (Desch et al, 2013; Song et al, 2015). Using a phage display system, Desch and colleagues (2015) revealed mutations in VWF gene (at least in VWF73, D1596–R1668) also resulted in reduced susceptibility to ADAMTS13 cleavage, which challenged the hypothesis that specific ABO glycosylation patterns affect proteolysis of VWF (Deforche et al, 2015). ABO-related variations in FVIII levels are primarily mediated through VWF, because VWF is a carrier of FVIII, which protects FVIII from clearance.…”

ADAMTS‐13 glycans and conformation‐dependent activity

“…A recent study has demonstrated that ADAMTS-13 has three linker regions, with two of these being located between (i) TSP1/2 and TSP1-3, and (ii) TSP1-4 and TSP1-5. It was shown that these linker regions provide flexibility to the ADAMTS-13 molecule, allowing it to adopt its closed conformation, and that their deletion resulted in ADAMTS-13 molecules favoring the more open active conformation [30]. On the basis of the primary amino acid sequence, N707 and N828 lie close to these linker regions, and their bulky configurations may therefore provide a degree of rigidity to the surrounding region.…”

“…Proteins were analyzed by SDS-PAGE, followed by western blotting and probing with anti-myc-horseradish peroxidase antibodies to detect the myc-tagged CUB1/2 domain. Binding of ADAMTS-13 to the 6A6 antibody was performed as previously described [30]. Statistical analysis was performed with GRAPHPAD PRISM Student's t-test.…”

Anti‐ADAMTS13 autoantibodies in immune‐mediated thrombotic thrombocytopenic purpura do not hamper ELISA‐based quantification of ADAMTS13 antigen

“…After blocking, plasma (with starting dilution of 1/100 or 1/12.5 for healthy donor and acute TTP samples, respectively) was added in a 1.5 over 2.5 dilution series. Captured ADAMTS13 was detected using a mixture of two in‐house developed biotinylated anti‐ADAMTS13 mAbs 17G2 and 19H4 (1.5 µg/mL each) followed by horseradish peroxidase (HRP)‐labeled streptavidin (1/10 000; Roche Diagnostics). The colorimetric reaction was initiated by addition of o‐phenylenediamine (OPD) and H 2 O 2 , stopped with 4 mol/L sulfuric acid, and the absorbance was measured at 490 nm.…”