Synthetic peptides derived from ADAMTS13 spacer domain block proteolytic cleavage of rF-VWF73 and multimeric VWF by ADAMTS13. (A) Purified rF-VWF73 (1.0μM) was incubated with various concentrations (0-200μM) of synthetic peptides as indicated for 10 minutes. Then, recombinant ADATMS13 (2.5nM) was added and proteolytic cleavage of rF-VWF73 was monitored at 37°C on a fluorescent microtiter plate reader (ex 485 nm and em 540 nm). The maximal rates of fluorescent generation per second (units/second) were determined and plotted against the log concentrations of synthetic peptides used. The data presented are means ± SD of 3 independent measurements. (B-D) Purified plasma-derived VWF (37.5 μg/mL) was incubated for 60 minutes at 37°C with various concentrations (0-200μM) of synthetic peptides RRYGEE (B), RRY (C), and GEE (D). ADAMTS13 (50nM) along with recombinant FVIII (10nM) and lyophilized platelets (210 × 10³/μL) was added into the reaction (total volume, 20μL). The reaction mixture was subjected to vortexing (2500 rpm) for 5 minutes. The proteolytic cleavage product was determined by 1% agarose gel and Western blot. (E) Quantification of the 350-kDa cleavage product (indicated by arrowheads in panels B, C, and D) by densitometry is plotted against the concentrations of synthetic peptides (micromole).