Analysis of the Structure and Function of the von Willebrand Factor A1 Domain Using Targeted Deletions and Alanine-Scanning Mutagenesis

von Willebrand factor (vWF) mediates the primary adhesion of platelets to sites of vascular damage through interaction with glycoprotein Ib (GPIb) of the platelet GPIb/IX complex. To investigate the vWF/GPIb interaction we introduced both in-frame deletions and substitutions into the vWF A1 domain. The introduction of nine sequential 20-amino acid deletions within the Cys509-Cys695 loop of the A1 domain caused the defective secretion of vWF from mammalian cells, and resulted in multimeric vWF without platelet-binding activity. In other experiments we substituted alanine for charged amino acids (residues 524, 534, 549, 552, 569−573, and 642−645) in proposed functional domains within the Cys509-Cys695 loop. All six substitution mutants showed normal secretion from transfected mammalian cells and bound to fixed platelets in the presence of botrocetin. In contrast, only mutants vWF-R524A and vWF-K549A showed significant binding to platelets in the presence of ristocetin. Mutant vWF-K549A showed increased platelet-binding at suboptimal concentrations of both botrocetin and ristocetin. These results suggest that the substituted amino acids do not play a critical role in the activation of vWF by botrocetin or in the direct interaction of vWF with the GPIb/IX complex. However, the charged amino acids at positions 534, 552, 569−573, and 642−645 do play an important role in the ristocetin-induced binding of vWF to platelets. The interaction of vWF with heparin was significantly reduced by substitution of Lys residues 642−645, indicating that these residues may form part of a heparin-binding domain in the carboxy-terminal half of the Cys509-Cys695 loop.