Recombinant spacer domain inhibits proteolytic cleavage of multimeric VWF by ADAMTS13 under fluid shear stress. (A) SDS-PAGE with Coomassie blue shows the various fractions of recombinant ADAMTS13 spacer domain before and after Ni-affinity chromatography. (B) Purified plasma-derived VWF (37.5 μg/mL) was incubated at 37°C with 0-1.0μM of purified spacer domain in the absence or presence of 20mM EDTA for 60 minutes. Then, ADAMTS13 (20nM) along with recombinant FVIII (10nM) and lyophilized platelets (∼ 200 × 103/μL) was added into the reaction (total volume, 20 μL). The reaction mixture was subjected to vortexing (2500 rpm) for 5 minutes. The proteolysis of VWF was determined by multimer analysis on 1% agarose gel electrophoresis and Western blot. HMW indicates high-molecular-weight multimer. The proteolytic cleavage product (350 kDa) is indicated with dashed frame and arrowhead. (C) Quantification of the 350-kDa cleavage product by densitometry is plotted in the y-axis against the concentrations of purified recombinant spacer domain (micromole) in x-axis.
