The causative VWFpp mutations affect the exit of VWF from ER
Intracellular trafficking of the four rVWF mutants (p.Gly55Glu,
p.Val86Glu, p.Trp191Arg, and p.Cys608Trp), causing intracellular
retention of the rVWF, was investigated by immunofluorescence staining
after their expression in HEK293 cells. The visual observation of the
immunofluorescent images demonstrated the accumulation of the mutated
rVWF within the ER (Figure 2, as well as Supplemental Videos 1-9).
Further, the calculations of Pearson’s coefficient confirmed an
increased co-localization of mutants p.Gly55Glu, p.Val86Glu,
p.Trp191Arg, and p.Cys608Trp with ER marker PDI when is compared with
the wt (mean Pearson’s coefficient of 0.51±0.03, 0.52±0.02, 0.57±0.02,
and 0.51±0.02, respectively, vs. 0.15±0.03; P<0.001) (Figure
2A and C). Furthermore, the co-expressed VWF of p.Gly55Glu/wt,
p.Val86Glu/wt, p.Trp191Arg/wt, and p.Cys608Trp/wt showed also an
increased degree of co-localization compared with wt (mean Pearson’s
coefficient of 0.31±0.03, 0.29±0.03, 0.34±0.04, and 0.32±0.03,
respectively; P<0.05), but in lesser extent when they are
compared with homozygously expressed variants (Figure 2B and C).