The first step of platelet adhesion to areas of vessel injury is carried out by the platelet glycoprotein (GP) Ib-IX-V complex, which binds von Willebrand factor affixed to the subendothelial matrix. The GP Ib-IX-V complex comprises four polypeptides, each belonging to the leucine-rich repeat (LRR) family of proteins. Glycoprotein Ibα is the largest of these and contains the VWF binding site within a ~300-amino acid domain at its N-terminus that contains 8 tandem LRRs. In this study, we investigated the role in VWF binding of charged residues in this region of GP Ibα that are conserved across three species: humans, dogs and mice. Each of eleven charged residues was converted to Ala: His37, Glu40, Asp83, His86, Asp106, Glu128, Lys137, Lys152, Glu162, Asp175 and Lys189. All of the GP Ibα mutants expressed normally on the surface of CHO bIX cells, which stably express GP Ibb and GP IX. GP Iba expression was detected by the monoclonal antibody WM23, which binds a region uninvolved by the mutations. In three mutants, His86Ala, Glu128Ala, and Asp175Ala, the mutations appeared to induce large conformational changes, as they markedly altered the binding of the antibodies AN51 and SZ2, with epitopes located N-terminal and C-terminal to the LRRs, respectively. As a result, cells expressing the mutants Glu128Ala and Asp175Ala were unable to adhere to the surface when perfused over immobilized VWF. By contrast, cells expressing mutant His86Ala adhered to the surface and rolled on it much more slowly than did cells expressing wild-type GP Ibα. The His86Ala cells also displayed a left-shifted VWF binding curve in the presence of ristocetin compared to the wild-type cells. We investigated this residue further by converting the His to Glu, a residue of opposite charge. Cells expressing the His86Glu mutant rolled even more slowly over a VWF surface than the His86Ala cells at 20 dynes/cm2, but not at 2.5 dynes/cm2. Previously, mutations of GP Ibα causing gain of VWF-binding function have been confined to the disulfide loop formed by a bond between Cys209 and Cys248, which lies immediately C-terminal to the LRR region of GP Ibα. This is the first report of a gain-of-function phenotype due to a mutation in the LRR region of GP Ibα. Since His86 is more than 20 Å away from the major region of GP Ibα in direct contact with VWF defined by published crystal structures, our data suggests that the leucine-rich repeats regulate the affinity for VWF through long-range allosteric alterations of GP Ibα structure.