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).