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).