A novel mutation in the D3 domain of von Willebrand factor markedly decreases its ability to bind factor VIII and affects its multimerization.
In type 2N von Willebrand disease (vWD), von Willebrand factor (vWF) is characterized by normal multimeric pattern, normal platelet-dependent function, but a markedly decreased affinity for factor VIII (FVIII). In this report, we describe the case of a vWD patient who has an abnormal vWF multimers distribution associated with a markedly decreased vWF ability to bind FVIII. Sequencing analysis of patient's vWF gene showed, at heterozygous state, a G-->A transition resulting in the substitution of Asn for Asp at position 116 of the mature vWF subunit and a C-->T transition, changing the codon for Arg 896 into a stop codon. His sister who has a subnormal vWF level, but a normal FVIII/vWF interaction, was found to be heterozygous for the Arg896ter mutation only. Recombinant vWF (rvWF) containing the candidate (Asn116) missense mutation was expressed in COS-7 cells. The expression level of Asn116rvWF was significantly decreased compared with wild-type rvWF. The multimeric pattern of Asn116rvWF was greatly impaired as shown by the decrease in high molecular weight forms. The FVIII binding ability of Asn116rvWF was dramatically decreased. These data show that the Asp116Asn substitution is the cause of both the defective FVIII/vWF interaction and the impaired multimeric pattern observed in the patient's vWF. The monoclonal antibody 31H3 against D' domain of vWF (epitope aa 66-76) that partially inhibits the FVIII binding and recognizes only nonreduced vWF, showed a decreased ability to bind Asn116rvWF when used as capture-antibody in enzyme-linked immunosorbent assay (ELISA). This result suggests that a potential conformation change in the D' domain is induced by the Asp116Asn substitution, which is localized in the D3 domain.
Jorieux
S
S
Laboratoire de Recherche sur l'Hémostase, Laboratoire Français du Fractionnement et des Biotechnologies, Lille, France.
Gaucher
C
C
Goudemand
J
J
Mazurier
C
C
D019943:Amino Acid Substitution / Q000235:genetics; D001216:Asparagine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D001483:Base Sequence; D004252:DNA Mutational Analysis; D004591:Electrophoresis, Polyacrylamide Gel; D005169:Factor VIII / Q000378:metabolism*; D006579:Heterozygote; D006801:Humans; D007962:Leukocytes / Q000737:chemistry; D008297:Male; D008875:Middle Aged; D009154:Mutation; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary; D014842:von Willebrand Diseases / Q000235:genetics*; D014841:von Willebrand Factor / Q000235:genetics* / Q000378:metabolism*
D014841:von Willebrand Factor; D001224:Aspartic Acid; D001216:Asparagine; D005169:Factor VIII