von Willebrand factor (vWF) is a multimeric plasma glycoprotein with three tandem A domains. Domains A1 and A3 bind to platelet glycoprotein Ibalpha (GPIbalpha) and collagen, respectively. Domain A2 contains the Tyr-1605-Met-1606 bond that is cleaved by the metalloprotease ADAMTS13, and this reaction inhibits platelet thrombus growth. Fluid shear stress increases the rate of cleavage, suggesting that productive interaction with ADAMTS13 requires conformational changes within or near domain A2. The influence of the adjacent A1 and A3 domains was assessed by mutagenesis of a recombinant substrate consisting of domains A1A2A3. Deletion of domain A3 did not affect cleavage by ADAMTS13, whereas deletion of domain A1 increased the rate of cleavage approximately 10-fold. Similar effects were observed with plasma ADAMTS13 and recombinant ADAMTS13 truncated after the spacer domain. Digestion of A1A2A3 by plasma ADAMTS13 was enhanced to a similar extent by a recombinant mutant fragment of platelet GPIbalpha that binds with high affinity to domain A1 or by heparin. Heparin also increased the digestion of purified plasma vWF. Neither GPIbalpha nor heparin increased the cleavage of substrate A2A3 that lacks domain A1. The results suggest that vWF domain A1 inhibits the cleavage of domain A2, and that inhibition can be relieved by interaction of domain A1 with platelet GPIbalpha or certain glycosaminoglycans. Thus, binding of vWF to its major physiological ligands may promote the feedback inhibition of platelet adhesion by stimulating the cleavage of domain A2 by ADAMTS13 independent of fluid shear stress.