Altered disulfide connectivity because of loss of the p.(Cys1060)-p.(Cys1084) disulfide bond causes reduced VWF secretion. (A) Transient transfection of HEK293 cells with the expression vector encoding p.(Cys1060Ala) caused a significant reduction in VWF secretion. Transfection of cells with the expression vector coding for the p.(Cys1060Ala;Cys1084Tyr) double variant did not rescue VWF secretion. (B) Similar results were observed for p.(Cys1060Ser) and the p.(Cys1060Ser;Cys1084Tyr) double variant. Data are represented as mean ± SEM, ****P < .0001. (C) Three-dimensional quantification of the number of pseudo-WPB/cell revealed no significant difference in the number of storage organelles formed/cell by each variant. (D) Immunofluorescent imaging of WT-VWF (i), p.(Cys1060Ala;Cys1084Tyr) (ii), and p.(Cys1060Ser;Cys1084Tyr) (iii). VWF is depicted in green, and blue represents 4′,6-diamidino-2-phenylindole. Elongated rod-shaped WPBs are shown (inset, top). Areas of diffuse VWF staining for p.(Cys1060Ala;Cys1084Tyr) and p.(Cys1060Ser;Cys1084Tyr) indicating impaired regulated storage are also shown (inset, bottom). Images are representative of n = 3 independent experiments.