Two Methods to Measure Residual Activity of ADAMTS13 Mutants under Flow Conditions

Introduction: Prothrombotic high molecular weight multimers of von Willebrand factor (VWF) are size-regulated by the metalloprotease ADAMTS13 by shear force-activated proteolytic cleavage. A lot of effort has previously been put into the characterization of ADAMTS13 mutants but they mostly have been investigated with respect to defects in biosynthesis or secretion. State-of-theart diagnostic tests further measure residual activity employing unphysiological static approaches. We have developed two shear flow assays to provide methods for assessing ADAMTS13 activity under near-physiological flow conditions.

Material and Methods: In a cell-based shear flow assay, cleavage of surface-bound VWF strings is mimicked by histamine-induced VWF string formation by HUVEC cells under unidirectional flow, and VWF strings are detected employing the VWF binding peptide of platelet glycoprotein GPIbα coupled to latex beads. VWF strings are then used as substrate for kinetic studies of recombinant ADAMTS13 mutants identified in patients with congenital Thrombotic Thrombocytopenic Purpura (cTTP or Upshaw-Schulman syndrome (USS)). Analysis of time-lapse images allows quantification of VWF cleavage. To simulate degradation of VWF-plateletaggregates by ADAMTS13 in the circulation, we further established a protocol using Light Transmission Aggregometry (LTA).

Results: We measured the activity of ADAMTS13 mutants, which exhibit residual secretion upon expression in HEK293 cells and found that all eight of them exhibit significant residual proteolytic activity.

Conclusions: There is considerable phenotypical variation in patients with USS. It has previously been shown that some ADAMTS13 mutations result in low protein expression levels of mutants without residual activity under static conditions. We have shown here that most of these mutants do exhibit residual activity under physiological flow conditions. Our data might explain a milder phenotype in patients carrying such mutations and indicate that disrupted synthesis and/or secretion of otherwise active mutants are the major mechanisms underlying USS.