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  • Identification of amino acid residues responsible for von Willebrand factor binding to sulfatide by charged-to-alanine-scanning mutagenesis.

Identification of amino acid residues responsible for von Willebrand factor binding to sulfatide by charged-to-alanine-scanning mutagenesis.

International journal of hematology (2008-03-29)
Takayuki Nakayama, Tadashi Matsushita, Koji Yamamoto, Noriko Mutsuga, Tetsuhito Kojima, Akira Katsumi, Norihiko Nakao, J Evan Sadler, Tomoki Naoe, Hidehiko Saito
ABSTRACT

von Willebrand factor (VWF) performs its hemostatic functions through binding to various proteins. The A1 domain of VWF contains binding sites of not only physiologically important ligands, but also exogenous modulators that induce VWF-platelet aggregation. Sulfatides, 3-sulfated galactosyl ceramides, that are expressed on oligodendrocytes, renal tubular cells, certain tumor cells and platelets, have been suggested to interact with VWF under some pathological conditions. The binding of VWF to sulfatide requires the A1 domain, but its binding sites have not been precisely identified. Here, we report that alanine mutations at Arg1392, Arg1395, Arg1399 and Lys1423 led to decreased VWF-sulfatide binding. These sites have been reported to be the binding sites for platelet membrane glycoprotein (GP) Ib and/or snake venom botrocetin, and, interestingly, are identical to the monoclonal antibody (mAb) NMC4 epitope previously reported to inhibit the VWF-GPIb interaction. We observed that NMC4 also inhibited VWF interaction with sulfatides in a dose-dependent manner. Thus, we conclude that VWF binding sites of sulfatide overlap those of platelet GPIb and botrocetin.

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