Cleavage of denatured VWF substrate by leukocyte proteases. Purified VWF (Humate P) was incubated with elastase (138 nM; A-B), cathepsin G (8.6 nM; C), and PR3 (172 nM; D) for the times indicated in each panel at 37°C in 0.005 M Tris buffer, pH 8.0, with (A,C-D) or without (B) 1.5 M urea. Proteolytic cleavage products were analyzed by 5% SDS–polyacrylamide gel electrophoresis under denaturing and reducing conditions, and stained with Coomassie blue. The positions of molecular mass standards are marked. ━ indicates the intact VWF polypeptide (250 kDa), whereas the indicates the cleavage products of various sizes under denaturing and reducing conditions.