THE DEFECTIVE INTERACTION BETWEEN VON-WILLEBRAND-FACTOR AND FACTOR-VIII IN A PATIENT WITH TYPE-1 VON-WILLEBRAND DISEASE IS CAUSED BY SUBSTITUTION OF ARG19 AND HIS54 IN MATURE VON-WILLEBRAND-FACTOR

In this report we describe the further investigation of the von Willeb rand factor (VWF)/FVIII interaction in a type 1 von Willebrand disease patient characterized by discrepant VIII:C levels as determined by on e-stage and two-stage VIII:C assays. A solid-phase binding assay shows that this patient's plasma vWF is moderately defective in capturing r ecombinant FVIII. Sequence analysis of the FVIII-binding domain encode d by the vWF mRNA of the affected individual identified mutations in b oth vWF alleles. In allele A, the mutations C2344T and T2451A result i n the substitution of Trp for Arg19 (R19W) and of Gln for His54 (H54Q) in mature vWF, respectively. This allele also contains a reported pol ymorphism (A2365G, Thr26Ala). Allele B, which is underexpressed at the RNA level, contains a one-nucleotide deletion in the FVIII-binding do main (Delta G2515) that results in the premature termination of transl ation, Analysis of the binding of FVIII by full-length vWF transiently expressed in COS-7 cells confirms that the combined R19W and H54Q sub stitutions are the cause of the defective vWF/FVIII interaction in thi s patient. The FVIII-binding defect of vWF containing either mutation alone is approximately half that of the double mutant, which suggests that the effect of these mutations is additive. The mutant proteins ar e recognized equally well by vWF monoclonal antibodies MBC105.4, 32B12 , and 31H3, which block the binding of FVIII by vWF, indicating that a mino acids Arg19, Thr26, and His54 are not critical residues in the ep itopes of these antibodies. (C) 1996 by The American Society of Hemato logy.