In FVIII-binding assays, vWF-W19 and vWF-Q54 show maximal binding that is 79% and 75% of wt vWF, respectively (Fig 6). Maximal binding for FVIII by vWF.3 was 52% of wt vWF. Added together, the percent reduction in binding for vWF-W19 and vWF-Q54 is nearly equal to the percent reduction of binding shown by vWF.3 (46% v 48%). These mutations also show an additive effect on the concentration of FVIII at which half-maximal binding occurs.
ExperimentName: Factor VIII binding assays for variants exogenously expressed in COS-7 cells
NonPatientCell: COS-7 cell line (CLO:0002597, EFO:0002009)
GeneFunction: GO:0005515 (protein binding, specifically to Factor VIII)
GeneAlteration: Site-directed mutagenesis was used to introduce a mutation encoding "His54Gln" (corresponding to the p.His817Gln substitution from the variant) into the mature (cleaved) VWF protein.
FunctionalEvidence: The variant did not disrupt multimer formation (Figure 5) but reduced Factor VIII binding to 75% of wild-type levels (Figure 6). In combination with another missense mutation in cis that reduced Factor VIII binding by an additional 21-23%, and a frameshift mutation in trans predicted to cause the absence of a gene product, the variant appears likely to be contributing to pathogenicity.
FAVariants: A construct equivalent to the c.2451T>A (p.His817Gln) variant was used (ClinVar:100224), although it expressed the proteolytically processed version of the protein product and referred to the variant as "His54Gln".