VWF cleavage depends on shear stress, time, platelets, and ADAMTS13. (A) Reactions containing VWF (30 μg/mL) and ADAMTS13 (2.5 U/mL), without platelets (□) or with platelets (1 × 10⁶/μL) (■), were subjected to different levels of fluid shear stress for 3 minutes and analyzed for VWF cleavage by SDS-PAGE and Western blotting with polyclonal anti-VWF. Error bars indicate SD (n = 5, ■) or the range of duplicates (□). (B) Reactions containing VWF (30 μg/mL) and ADAMTS13 (2.5 U/mL), without (●, ○) or with formalin-fixed platelets (10⁶/μL) (■, □) were performed without (□, ○) or with (■, ●) 50 dyne/cm² shear stress for different times and analyzed for VWF cleavage. ■, 50 dyne/cm² shear stress and 10⁶ platelets/μL (n = 5); ●, 50 dyne/cm² and no platelets (n = 2); □, no shear stress and 10⁶ platelets/μL (n = 2); ○, no shear stress and no platelets (n = 1). Error bars indicate SD (■) or the range of duplicates (●, ○). (C) Reactions containing the indicated concentration of ADAMTS13 (U/mL) with or without platelets (10⁶/μL) were exposed to fluid shear stress (16 dyne/cm² for 5 minutes) and analyzed for VWF cleavage by SDS-PAGE and Western blotting as described in the legend to Figure 1.