A new method to determine the cleavage of von Willebrand Factor by recombinant ADAMTS13 under physiological shear-stress conditions

Introduction Regulation of von Willebrand Factor (VWF) activity by a disintegrin and metalloproteinase with a thrombospondin type 1 motif (ADAMTS13) is critical for hemostasis by controlling VWF multimer size. Demonstrating the ADAMTS13-mediated cleavage of VWF under physiological shear has proven difficult. Traditional analytical methods utilize denaturing environments, thereby poorly replicate the natural vascular environment. This research aimed to develop a sensitive methodology to visualize and quantitate VWF proteolysis by a recombinant ADAMTS13 (rADAMTS13) drug candidate under arterial shear flow in human blood.

Method A microfluidics-based approach utilizing the BioFlux 1000Z system (Fluxion Biosciences) was optimized by: coating channels with 143 μg/ml collagen type I, blood sample preheating, applying 20 dyne/cm² pulsatile shear, relying on inherent VWF levels in donor blood, inputting hematocrit based viscosity parameters, and implementing timed protocols. rADAMTS13 was added to healthy donor blood at different concentrations and the time course of platelet adhesion to immobilized collagen was determined by microscopy using fluorescent labeled platelets.

Results Optimized analytical techniques enabled visualization and quantification of VWF proteolysis based on platelet binding under shear flow. Addition of 1.875-7.5 U/ml rADAMTS13 to blood reduced the VWF-mediated platelet adhesion to collagen in a concentration dependent manner. Repeated testing validated the sensitivity. Statistical analysis quantified inter-sample variability.

Conclusion This research successfully established a powerful methodology harnessing microfluidics to gain fundamental insights into rADAMTS13 function under physiologically relevant shear flow conditions. Further enhancement of the techniques, increased biological sampling, and exploration of collagen types could build on these findings.

 

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Conflict of Interest

Autor and co-autors are full-time employees of Baxalta Innovations GmbH, Vienna, Austria

Publication History

Article published online:
26 February 2024