ADAMTS-13 R1206K variant exhibits an open conformation and an enhanced proteolytic activity toward von Willebrand...

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening thrombotic microangiopathy caused by severe deficiency of ADAMTS-13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats, 13), a plasma metalloprotease that cleaves von Willebrand factor (VWF). Recombinant ADAMTS-13 has been developed for treatment of TTP, but it requires a high dose and frequent administrations to achieve therapeutic efficacy.

The present study aims to identify more efficacious ADAMTS-13 variants for potential management of TTP.

Site-directed mutagenesis, recombinant protein production and purification, enzyme kinetic and binding assays, and microfluidic thrombus formation were all used in the study.

ADAMTS-13 R1206K variant exhibits a significantly increased activity compared with wild-type ADAMTS-13 for proteolytic cleavage of multimeric VWF and its surrogate peptide. Additionally, the R1206K is significantly more efficacious for inhibiting platelet adhesion and aggregation on a collagen-coated surface under arterial flow. Biolayer interferometry assay reveals a faster association kinetics (k_a) between R1206K and VWF73^L1603A substrate but a reduced binding affinity between R1206K and autoantibodies against ADAMTS-13, derived from immune-mediated TTP. Consistent with these results, the fluorescent energy transfers (FRETS)-VWF73 assay reveals a higher IC₅₀ for autoantibodies (IgG4 4-16 and 4-20) to inhibit the R1206K than wild-type ADAMTS-13.

These results demonstrate that ADAMTS-13 R1206K variant has an enhanced enzymatic activity, resulting from the disruption of autoinhibition that leads to an open conformation, which increases its substrate binding but reduces its autoantibody inhibition. This gain-of-function ADAMTS-13 variant may be further explored for the management of TTP and perhaps other inflammatory thrombotic disorders.