ADAMTS-13, a plasma reprolysin-like metalloprotease, cleaves von Willebrand factor (VWF). Severe deficiency of plasma ADAMTS-13 activity results in thrombotic thrombocytopenic purpura (TTP), while mild to moderate deficiencies of plasma ADAMTS-13 activity are emerging risk factors for developing myocardial and cerebral infarction, pre-eclampsia, and malignant malaria. Moreover, Adamts13−/− mice develop more severe inflammatory responses, leading to increased ischemia/perfusion injury and formation of atherosclerosis. Structure–function studies demonstrate that the N-terminal portion of ADAMTS-13 (MDTCS) is necessary and sufficient for proteolytic cleavage of VWF under various conditions and attenuation of arterial/venous thrombosis after oxidative injury. The more distal portion of ADAMTS-13 (TSP1 2–8 repeats and CUB domains) may function as a disulfide bond reductase to prevent an elongation of ultra-large VWF strings on activated endothelial cells and inhibit platelet adhesion/aggregation on collagen surface under flow. Remarkably, the proteolytic cleavage of VWF by ADAMTS-13 is accelerated by FVIII and platelets under fluid shear stress. A disruption of the interactions between FVIII (or platelet glycoprotein 1bα) and VWF dramatically impairs ADAMTS-13-dependent proteolysis of VWF in vitro and in vivo. These results suggest that FVIII and platelets may be physiological cofactors regulating VWF proteolysis. Finally, the structure–function and autoantibody mapping studies allow us to identify an ADAMTS-13 variant with increased specific activity but reduced inhibition by autoantibodies in patients with acquired TTP. Together, these findings provide novel insight into the mechanism of VWF proteolysis and tools for the therapy of acquired TTP and perhaps other arterial thrombotic disorders.