PB0298 - Functional multiplex activity assay reveals molecular perturbations in ADAMTS13 structure and composition

Background: ADAMTS13 is composed of metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C) and spacer (S) domains, followed by seven T domains and two CUB domains. Severe ADAMTS13 deficiency causes thrombotic thrombocytopenic purpura (TTP), which is characterized by life-threatening microvascular thrombosis. We discovered that ADAMTS13 Dis- and Cys-rich exosites synchronize to regulate substrate-induced activation of ADAMTS13.

Aims: To develop an activity assay based on Dis- and Cys-rich coupling that distinguishes molecular perturbations of ADAMTS13 structure.

Methods: The ADAMTS13 multiplex FRET assay utilizes two wild-type and six chimeric fluorogenic substrates prepared from the human (H-71) and bovine (C-71) VWF A2 peptides. ADAMTS13 Dis- or Cys-rich exosite binding sites were swapped between H-71 (S1) and C-71 (S8) (Fig 1A). Mutations causing partial decreases in ADAMTS13 activity were introduced by mutagenesis and proteins transiently expressed in HEK293 cells. Plasma and recombinant ADAMTS13 were assayed, and the rates of cleavage were normalized to those of C-71.

Results: Cleavage of S1-S8 fluorogenic substrates normalized to C-71 using >10 animal plasmas yielded 7 characteristic cleavage rates for each vertebrate, as expected. Species-specific ADAMTS13 is known to differ at amino acid residues critical for its interaction with VWF. Compared to human plasma, rhADAMTS13 produced in HEK293 cells showed slight rate variations (Fig 1B). Remarkably, previously characterized single mutations in ADAMTS13 DTC region (K318A, T339R, Q448E, P475S, G525D) each showed cleavage rates overall distinct from the wild-type rhADAMTS13 (intra‐assay/inter-assay CV < 2%). ADAMTS13 variants truncated from the T8 (MDTCS, MT2, MT6, MT8) tended to discriminate less in cleaving across the S1-S7 substrates, consistent with the loss of allosteric regulation. This tendency was reinstated by the gain-of-function ADAMTS13 (GoF) which is allosterically unregulated.

Conclusion(s): This latest multiplex activity assay could be a useful tool to evaluate subtle and inherent changes in ADAMTS13 structure and thus, function. However, further validation studies are needed.