The physiological role of the interaction of VWF-GPIb : Amino acid residues of the platelet GPIb to bind VWF and the generation of knock-in mice mutated at Lys599 to A1a.

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At the site of vascular injury, von Willebrand factor (VWF) mediates platelet adhesion to subendothelial connective tissue through binding to N-terminal domain of the a chain of platelet glycoprotein Ib (GPIba). We have found the loss-of-function mutations generated in soluble fragment containing the N-terminal 287 amino acids of GPIbα. Mutations at Glu128, Glu172 and Asp175 specifically decreased both ristocetin-and botrocetin-induced VWF binding, suggesting that these sites are important for VWF binding of platelet GPIb. Monoclonal antibody 6D1 inhibited ristocetin-and botrocetin-induced VWF binding and a mutation at Glu125 specifically reduced the binding to 6D1. In contrast, antibody HPL7 had no effect for VWF binding and mutant E121A reduced the HPL7 binding. Mutations at His12 and Glu14 decreased the ristocetin-induced VWF binding with normal botrocetin-induced binding. Crystallographic modeling of the VWF-GPIba complex indicated that Glu128 and Asp175 form VWF binding sites and … More the binding of 6D1 to Glu125 interrupts the VWF binding of Glu128 but HPL7 binding to Glu121 has no effect on VWF binding. Moreover, His12 and Glu14 contact with Glu613 and Arg571 of VWF A1 domain whose mutations had shown similar phenotype. Using targeted gene disruption and Cre-loxP gene excision technique, we investigated the role of VWF-GPIb in normal hemostasis. Oligonucleotide primer were used to obtain a partial cDNA of mouse VWF in from mRNA of C57BL/6J mice using RT-PCR. The resulting PCR product was used as a probe to isolate a genomic clone containing a segment of mouse VWF gene from a 129SVJ lambda FIX II genomic library. A targeting vector was constructed for homologous recombination in embryonic stem cells. It was based on a vector pBS/MC1DTpA+loxpGKneoW and the VWF gene fragments. The generation of targeted D3 embryonic stem (ES) cells and blastocyst injection were prepared to perform the knock-in strategies. Obtained findings indicates the novel binding sites required for VWF binding of human GPIbα Less