Fluid shear stress and platelet-VWF interactions increase VWF cleavage by ADAMTS13. Reactions containing VWF (30 μg/mL) were treated with (+) or without (−) shear stress and other components as indicated. Samples were analyzed by SDS-PAGE and Western blotting with polyclonal anti-VWF to detect the 350-kDa homodimeric cleavage product containing C-terminal fragments of VWF. The corresponding 280-kDa N-terminal homodimeric product is recognized less efficiently by anti-VWF and was visible in some reactions. (A) Cleavage of VWF by ADAMTS13 (5 U/mL) was increased slightly by fluid shear stress (50 dyne/cm² for 10 minutes, lane 7) or formalin-fixed platelets (10⁶/μL, lane 4). Maximal cleavage required both shear stress and platelets (lane 8) and was blocked by 5 mM EDTA (lane 9). (B) Monoclonal antibody 6D1 against GPIbα (30 μg/mL) blocked the cleavage of VWF exposed to fluid shear stress (50 dyne/cm² for 10 minutes) in reactions containing platelets (lane 8) but not in reactions without platelets (lane 4). Control mouse IgG1 had no effect (lanes 3 and 7). (C) Recombinant GPIbα-Ig/2V (30 μg/mL) increased the cleavage of VWF by ADAMTS13 (2.5 U/mL) in reactions exposed to fluid shear stress (16 dyne/cm² for 5 minutes) without platelets (lane 4) and partially inhibited the cleavage of VWF in reactions with platelets (lane 8). (D) The cleavage of endogenous VWF in fresh platelet-rich plasma subjected to fluid shear stress (20 dyne/cm² for 5 minutes) was markedly greater than the cleavage of VWF in platelet-poor plasma. Cleavage was prevented by 50 mM EDTA.