Microfluidic Shear-Based Assay for Assessing ADAMTS13 Activity Under Flow.

A microfluidic system using the flow chamber techniques has long been utilized for thrombosis research. With the advent of technology, it has become possible to evaluate the relationship between blood flow dynamics and thrombus formation with greater precision. ADAMTS13, a plasma metalloprotease, efficiently cleaves von Willebrand factor (VWF) under flow conditions. The microfluidic shear-based assay is highly relevant for investigating the biological function of ADAMTS13. In this chapter, I describe the method using a microfluidic system to investigate the interaction between platelets and VWF, which is regulated by ADAMTS13. Whole blood samples obtained from mice with various genotypes, including wild type (WT), Adamts13-deficient (Adamts13-/-), and vwf-deficient (VWF-/-), and from humans, including healthy individuals, patients with immune-mediated thrombotic thrombocytopenic purpura (iTTP), are assessed under arterial shear stress. The platelet-neutrophil interaction is visualized in real time with Rhodamine 6G. Following fixation with 4% paraformaldehyde in phosphate-buffered saline (PBS), the platelets are stained with fluorescent anti-CD41 antibody, and DNA strings are stained with SytoxGreen to assess the formation and accumulation of neutrophil extracellular traps (NETs). Furthermore, by adding DNase I, recombinant ADAMTS13, or caplacizumab, the therapeutic effects of these agents can also be evaluated within the microfluidic system. In conclusion, the microfluidic shear-based assay is an excellent method for assessing ADAMTS13 function under arterial flow.