Optical-Tweezer Single-Molecule Assay for ADAMTS13-Mediated Proteolysis of the von Willebrand Factor A2 Domain

Optical tweezers enable real-time visualization of mechanosensitive enzyme-substrate reactions at the level of single protein molecules. We present a protocol that quantifies the force-dependent cleavage of the von Willebrand factor (VWF) A2 domain by the metalloprotease ADAMTS13, in the absence or presence of its physiological cofactor factor VIII (FVIII). Recombinant A2 domains are site-specifically conjugated to double-stranded DNA handles via SpyTag-SpyCatcher chemistry and tethered between two beads. Controlled stretching by optical tweezers unfolds the A2 substrate, exposing the Tyr1605-Met1606 bond for enzymatic cleavage, whereas sequential extension-relaxation cycles in an ADAMTS13-containing buffer reveal stepwise tether-length reductions that correspond to individual cleavage events. Dwell-time analysis across multiple enzyme concentrations yields single-molecule Michaelis-Menten parameters (k_cat and K_M) and demonstrates that FVIII accelerates catalysis under tensile load. The assay consumes picomolar quantities of substrate, resolves turnover events with millisecond resolution, and is readily adaptable to other mechanoenzymes, providing a versatile platform for dissecting force-regulated proteolysis in hemostasis and beyond.