Cleavage of multimeric VWF by leukocyte proteases under fluid shear stress. Purified plasma VWF (pVWF; 37.5 μg/mL) was incubated for 15 minutes and subjected to no vortexing (shear −) or constant vortexing at 2500 rpm for 5 minutes (shear +) in the presence of various concentrations of elastase (A), cathepsin G (B), MMP9 (C), or PR3 (D) as indicated in each panel. The reaction was performed in a polymerase chain reaction tube with a total volume of 20 μL in buffer containing 50 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM CaCl₂, and 1 mg/mL BSA. The proteolytic cleavage products were determined by 5% SDS–polyacrylamide gel under denaturing and nonreducing conditions. Western blotting was performed with anti-VWF IgG and infrared fluorescent dye labeled anti–rabbit IgG as described in “Cleavage of multimeric VWF under fluid shear stress.” → (left borders) indicate positions of a 250-kDa molecular weight marker. (right borders) indicates positions of cleavage products. The asterisk in panel C denotes 20 mM EDTA added to the reaction mixture.