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).