The VWFpp mutations interfere with the quantity and morphology
of the pseudo-WPBs
A quantitative exploration of the 3D images (generated by arivis
Vision4D) demonstrated a significant difference in number, size, and
shape of the WPB-like vesicles within cells expressing rVWF mutants when
is compared with wt (Figure 3 A-H). Our visual surveys ruled out the
formation of any WPBs within single-transfected cells with p.Gly55Glu,
p.Val86Glu, p.Trp191Arg, and p.Cys608Trp variants, consistent with their
broad accumulation inside the ER as declared above. Approximately 60%
of cells expressing wt VWF displayed the formation of pseudo-WPBs, with
an average computed number of seven WPBs per cell, whereas only
32.6-42.9% cells co-expressing wt plus p.Gly55Glu, p.Val86Glu, or
p.Cys608Trp exhibited the formation of WPBs, with an average number of
3.5-4.0 pseudo-WPBs per cell, indicating a significant discount in
generating WPBs (p<0.0001) (Figure 3A-C). However, the cells
co-expressing wt, and p.Trp191Arg showed a significant difference in the
percentage of cells forming pseudo-WPBs (~44%,
p<0.05), but no change in an average amount of WPBs/cell
(~ 7.5, p=0.616). On the other hand, quantification of
the morphometric parameters of the 3D models of the pseudo-WPBs within
cells coexpressing p.Trp191Arg/wt demonstrated a slight decrease in
length (2.09 ± 0.04 µm) and width (1.21 ± 0.03 µm) of pseudo-WPBs
compared with wt (with an average length and width of 2.18 ± 0.09 µm and
1.28 ± 0.07 µm, respectively; p<0.05) (Figure 3A, E, and G).
Furthermore, heterozygous p.Trp191Arg/wt pseudo-WPBs displayed a
slightly increased sphericity value (0.670 ± 0.002 vs. 0.658 ± 0.011 of
wt WPBs; p< 0.0001), favoring a less elongated shape (Figure
3H). No significant changes in size (length, width, and depth) and
sphericity of the pseudo-WPBs inside cells coexpressing wt and
p.Gly55Glu, p.Val86Glu, and p.Cys608Trp mutants were observed when
compared with wt (Figure 3A, E, F, G, and H). Nonetheless, pseudo-WPBs
formed by all heterozygous mutants (with a greater extent for the
p.Trp191Arg/wt) showed a substantial decline in mean intensities values
of the green signals (VWF), indicating the impact of these mutants in
the conformation of the storage vesicles (Figure 3D).