Disintegrin-like and Cys-rich domains mediate substrate-dependent regulation of ADAMTS13

Background: The metalloprotease ADAMTS13 cleavesthe highly hemostatic active von Willebrand factor (VWF) to prevent VWF-induced platelet aggregation. ADAMTS13 is a multidomain protein with metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C) and spacer (S) domains, followed by seven T domains and two CUB domains. VWF cleavage by ADAMTS13 is regulated by blood shear stress and substrate-induced ADAMTS13 activation. However, these regulatory mechanisms cannot account for variability in the cleavage of VWF by ADAMTS13 from diverse species. ADAMTS13 and VWF have not evolved to be optimal enzyme-substrate pairs; however, its allosteric regulation is evolutionarily conserved. We sought to characterize the substrate-dependent regulation of ADAMTS13.

Methods: Multiplex FRET ADAMTS13 assays were performed using 10 novel fluorogenic substrates. Substrates P3-P10 are chimeric substrates between C-71 and H-71; species-specific amino acid residues were swapped sequentially. Plasmas and recombinant proteins from various vertebrates have been characterized and reported before. Assays were performed as previously described and mean results are reported.

Results: The chimeric substrates (P3-P10) were designed to substitute wild-type,species-specific amino acid residues. Compared to C-71 (P2), only P3 and P6 exhibited reduced rates of cleavage by all vertebrate plasma containing ADAMTS13. The P3 substrate substituted the D binding residues from human VWF71 into C-71, whereas P6 substituted the C binding residues from human VWF71 into C-71. For P3, all ADAMTS13 assayed showed ~30-100% reduction in the rate of cleavage except for armadillo and dog plasma ADAMTS13. Likewise, P6 was cleaved ~0% to 60% slowly relative to P2 by all ADAMTS13. Substitutions occurring outside D and C binding sites had no effects, consistent with previous studies. Interestingly, there was no major change in cleavage rates when D- and C- VWF binding sites were of the same species, i.e., P9 ~ P1 and P10 ~ P2, irrespective of the peptide backbone.

Conclusions: We found allosteric regulation of ADAMTS13 had no effect on the cleavage of any of the substrates, implying substrate-dependent regulation is independent of the allosteric activation of ADAMTS13. Thus, coupling the disintegrin-like domain to the Cys-rich domain may prevent the activation of ADAMTS13 by off-target substrates. Genetic mutations of either domain may therefore contribute to an imbalance in the cleavage of VWF multimers in vivo.