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.
Kroner
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Blood Research Institute, Blood Center of Southeastern Wisconsin, Milwaukee 53201-2178, USA.
Frey
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D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites / Q000235:genetics; D001792:Blood Platelets / Q000502:physiology; D019556:COS Cells; D000939:Epitopes / Q000737:chemistry / Q000235:genetics; D006493:Heparin / Q000378:metabolism; D006801:Humans; D007120:Immunochemistry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection; D014841:von Willebrand Factor / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D019038:Platelet Glycoprotein GPIb-IX Complex; D014841:von Willebrand Factor; D006493:Heparin; D000409:Alanine