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Publication

Development of a multiplex assay to characterize ADAMTS13 structure and function

Momanyi, Thomas O.
Miranda, Frida Z.
Muia, Joshua
Abstract
Introduction: The metalloprotease ADAMTS13 is composed of metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cysteine-rich (C), and spacer (S) domains, followed by seven T domains and two CUB domains. Genetic or autoimmune deficiency of ADAMTS13 causes thrombotic thrombocytopenic purpura (TTP), which is characterized by life-threatening microvascular thrombosis. We recently uncovered a cryptic mechanism by which ADAMTS13 Dis and Cys-rich VWF binding sites couple to regulate substrate-induced activation of ADAMTS13.
Aim: To develop multisubstrate-based assays to evaluate ADAMTS13 activity and function.
Method: The novel multiplex ADAMTS13 assay utilized up to 12 fluorogenic substrates prepared from human and bovine VWF A2 sequences. The substrates are chimeric substrates between Cattle VWF (C-71) and Human VWF (H-71). ADAMTS13 Dis- or Cys-rich binding sites were swapped between H-71 or C-71 peptide backgrounds. Disease-causing mutations were introduced into the wild-type ADAMTS13 by mutagenesis. Wild-type recombinant ADAMTS13 and variant proteins were expressed transiently or stably in HEK293 cells. The animal plasmas used in the current work has been described previously. Assays were performed as we described before, and mean results were reported.
Results: Ten novel chimeric FRET substrates were prepared by swapping unique amino residues between cattle VWF71 (C-71) and human VWF71 (H-71). Simultaneous cleavage of all 12 substrates by species-specific ADAMTS13 led to distinctive cleavage rates for each plasma ADAMTS13. This was expected because each species ADAMTS13 has its protein sequence and posttranslational modifications that dictate its interaction with the substrate VWF. Peptide substrates that showed stringent cleavage rates across multiple species were selected for additional validation. These substrates differed mainly between the ADAMTS13 Dis and Cys binding sites. Human pooled normal plasma (PNP), recombinant human ADAMTS13, and ADAMTS13 variants were assayed further by the multiplex ADAMTS13 activity method. Consistent with our expectation, recombinant ADAMTS13 expressed in HEK293 cells showed cleavage rates slightly different from the plasma ADAMTS13. Remarkably, the multiplex assay was able to distinguish truncated ADAMTS13 proteins and recombinant ADAMTS13 with mutations that cause partial activity decreases from the wild type.
Conclusions: The ADAMTS13 multiplex assay can be used to evaluate structural perturbations in ADAMTS13. This assay may be useful to test ADAMTS13 function in diseased states where a near-normal ADAMTS13 activity is reported by conventional methods.
Date
2024-02-16