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

• Published in: Biochemistry (Easton) 1996-10, Vol.35 (41), p.13460-13468
• Main Authors: Kroner, Philip A, Frey, Amy B
• Format: Article
• Language: English
• Subjects: Alanine - chemistry; Amino Acid Sequence; Animals; Antibodies, Monoclonal; Binding Sites - genetics; Blood Platelets - physiology; COS Cells; Epitopes - chemistry; Epitopes - genetics; Heparin - metabolism; Humans; Immunochemistry; Molecular Sequence Data; Molecular Structure; Mutagenesis, Site-Directed; Platelet Glycoprotein GPIb-IX Complex - metabolism; Sequence Deletion; Transfection; von Willebrand Factor - chemistry; von Willebrand Factor - genetics; von Willebrand Factor - physiology
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9610313

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.