Description of the function and multimeric configuration of the
VWF variants
The multimer analysis revealed a range of deficiencies in the multimer
structure of the secreted recombinant variants (Figure 1B). Single
transfections of p.Gly55Glu, p.Val86Glu, p.Trp191Arg, and p.Cys608Trp
exhibited only dimers or as well as a faint trail of tetramers (Figure
1B). The multimeric defects were only partially restored by
co-transfection with wt rVWF. Multimer analysis of co-expression of wt
with mutants p.Gly55Glu and p.Trp191Arg revealed a complete lack of high
molecular weight multimers (HMWM), emphasizing the destructive impact of
these variations on multimer assembly. Similarly, heterozygously
expressed mutations p.Val86Glu/wt and Cys608Trp/wt illustrated
deficiency in multimer profile but to a severer extent (loss of most of
the intermediate multimers and lack of HMWM) (Figure 1B).
Functional assessment is presented as ratios of GPIb binding and VWF:CB
to VWF:Ag of secreted rVWF (Figure 1C). VWF activity assays were only
performed where the VWF levels in media of expressed variants were
quantitatively adequate for reliable quantification evaluations.
Functional assay results were in the concordance of the multimer
structure pattern. Co-expressions of the p.Gly55Glu/wt, p.Val86Glu/wt,
and p.Trp191Arg/wt showed a decrease in ratios GPIb binding and CB to
VWF:Ag compared with those of wt (p<0.05), attributed to the
loss of large/and intermediate multimers.
The secreted mutants p.Asn211Asp and Gly334Glu exhibited a complete
multimer profile and normal binding activities in both single and
co-expression conditions (Figure 1B and C).