ADAMTS13 (A Disintegrin And Metalloprotease with ThromboSpondin type1 repeats-13) is an enzyme that is mainly synthesized in the liver and secreted into the blood stream. In plasma, ADAMTS13 cleaves ultra large (UL) von Willebrand factor (VWF) newly released from stimulated and/or injured endothelial cells. It also cleaves soluble UL-VWF and VWF in flowing blood or at the site of growing thrombi under high shear stress. The basic enzymology and structure-function of the ADAMTS13-VWF interaction have presented many interesting questions, particularly in the context of physiological cofactors and fluid shear stress. The relatively poor understanding of this system reflects the complex assessment of VWF proteolysis, as VWF in solution exhibits a conformation that does not actively engage with ADAMTS13. It has been unclear whether force-induced unfolding of VWF is the only mechanism to enhance proteolysis by ADAMTS13, or if interactions of ADAMTS13 and VWF with cofactors ideally position ADAMTS13 on VWF multimers for enhanced cleavage. We demonstrated that coagulation factor VIII (FVIII) and blood platelets cooperatively accelerate proteolytic cleavage of soluble VWF by ADAMTS13 through an alteration of VWF substrate conformation under physiologically relevant fluid shear stresses. In addition, we have established the critical role of the VWF propeptide and FVIII- and platelet-binding domains of VWF in regulating proteolysis of VWF by ADAMTS13 under physiologically relevant shear stress. Through site-directed mutagenesis, kinetic analyses, and peptide inhibition assays we have identified a substrate-binding exosite containing ADAMTS13 residues Arg659, Arg660 and Tyr661 that exhibits an important role in proteolytic cleavage of VWF under both non-physiological and physiological conditions. In addition, modification of this exosite region of ADAMTS13 yielded ADAMTS13 variants with reduced inhibition by autoantibodies and enhanced specific activity. Finally, we have demonstrated that infusion of ADAMTS13 and a truncated variant into ADAMTS13-/- mice can restore the thrombus composition and kinetics of fibrin and platelet accumulation in an arterial thrombosis model. Together, these results suggest an important physiological role of cofactor binding to VWF and VWF interactions with ADAMTS13 exosites in regulating ADAMTS13 function in hemostasis.