Impact of VWFpp mutations on VWF expression and release
To verify the impact of the detected candidate variants on basal VWF
secretion, VWF:Ag levels in both supernatant and lysates of the
transfected HEK293T cells were measured by a particle-based
turbidimetric assay. The mean value of the constitutively secreted wt
rVWF was 42.06 ± 1.13 IU/dL. The mutant rVWF levels are here presented
as a percentage of the wt rVWF levels (Figure 1A). Four of the mutants
(p.Gly55Glu, p.Val86Glu, p.Trp191Arg, and p.Cys608Trp) demonstrated
severely impaired secretion. Their VWF:Ag levels in the medium were
significantly lower compared to the wt, ranging from 9,8 to 10,9% of wt
(corresponding to 4.12 ± 0.08 IU/dL to 4.58 ± 0.20 IU/dL) (Figure 1A,
left, dark gray columns). Nonetheless, the amount of their intracellular
VWF:Ag levels were not significantly different from wt (for p.Gly55Glu,
p.Val86Glu, and p.Cys608Trp mutants; p>0.05) or it was even
higher than that of the wt (for p.Trp191Arg), indicating intracellular
retention of these mutants (Figure 1A, left, light gray columns).
However, the total VWF production (sum of medium and lysate) of all
these four mutants was significantly reduced. To mimic heterozygosity,
co-expressions of these mutants and wt were performed. The co-expression
resulted in the correction of the expression phenotype but was still
<50% of wt. The co-expressed secreted rVWF of p.Gly55Glu/wt,
p.Val86Glu/wt, p.Trp191Arg/wt, and p.Cys608Trp/wt was reduced by 65%,
68%, 65%, and 76%, respectively, relative to the secretion of wt
(Figure 1A, right).
Moreover, the variants p.Asn211Asp and Gly334Glu showed normal basal
secretion compared with wt in both single and co-expression experiments
(p>0.05), indicating no impact on the biosynthesis of VWF
(Figure 1A).