As the first step in hemostasis, the binding of von Willebrand factor (vWF) to the platelet membrane glycoprotein (GP) Ib-IX complex is essential for platelet adhesion at high-shear blood flow. This interaction in vivo requires the prior binding of vWF to the subendothelial matrix, a process which exposes a normally cryptic binding site on vWF for the GP Ib-IX complex. This process can be mimicked in vitro by modulators such as ristocetin or the snake venom protein botrocetin or by desialation of vWF. We have previously localized the GP lb binding site on vWF to a monomeric dispase fragment which extends from Leu-480/Val-481 to Gly-718 in the primary sequence of mature vWF [Andrews, R.K., Gorman, J.J., Booth, W.J., Corino, G.L., Castaldi, P.A., & Berndt, M.C. (1989) Biochemistry 28, 8326-8336]. This fragment also contains a distinct binding site for botrocetin. Analysis of synthetic peptides corresponding to hydrophilic stretches of sequence within this fragment indicated that the sequence Asp-514-Glu-542 represents a major adhesive sequence involved in receptor recognition. This peptide inhibited both the ristocetin-and botrocetin-mediated binding of vWF to either platelets or purified GP Ib-IX complex (IC50~ 50-200 µM) as well as the asialo-vWF-and bovine vWF-dependent agglutination of platelets. Both the N-and C-terminal halves of the peptide were inhibitory but less so than the intact peptide. This peptide also inhibited botrocetin binding to vWF, suggesting that botrocetin modulates vWF-GP lb interaction by binding in close proximity to the vWF adhesion sequence. Two proline-rich peptides, Cys-474-Pro-488 and Leu-694-Pro-708, only inhibited the ristocetin-mediated binding of vWF to receptor, suggesting that they encompass a ristocetin modulation site. In support of this, irrelevant peptides containing proline-rich repeats were also effective inhibitors of ristocetin-dependent vWF receptor interaction and together with the proline-rich v WF peptides also inhibited the ristocetin-dependent flocculation of fibrinogen. Interaction of the proline-rich peptides with ristocetin was confirmed by ultraviolet difference spectroscopy. The combined results are consistent with an electrostatic model for vWF activation which involves interaction with a modulator exposing an adjacent GP Ib-IX complex binding domain encompassing the sequence Asp-514-Glu-542 ofvWF.