Kinetic cleavage of rFRETS-VWF73 by leukocyte proteases. Leukocyte elastase, 10 nM (A), cathepsin G, 15 nM (B), MMP9, 10 nM (C), PR3, 10 nM (D), and recombinant ADAMTS13, 10 nM (E) were incubated with increasing concentrations (0-16 μM) of rFRETS-VWF73 peptide in 5 mM Bis-Tris, pH 6.0, 25 mM CaCl₂, 0.005% Tween-20. The proteolytic cleavage was monitored by the initial rate of fluorescent generation (v_max) at the excitation 485 nm and the emission 530 nm on a Vector 3 fluorescent microtiter reader. The data (slope vs substrate concentrations) were fitted into a Michaelis-Menten equation to determine the catalytic constant (k_cat) and Michaelis constant (k_m) using SigmaPlot software. The curves represent mean values of 2 independent experiments at the same concentration of for each enzyme. Slope refers to the change in fluorescence units per second.