Defective secretion despite normal storage of p.(Cys1084Tyr) VWF in vitro. (A) HEK293T cells were transiently transfected with cDNA encoding WT-VWF, the p.(Cys1084Tyr) variant, or a 1:1 molar ratio of both constructs to mimic the heterozygous state. Transfection with cDNA encoding the p.(Cys1084Tyr) variant led to a significant reduction in the amount of VWF secreted into the conditioned media whether transfected alone or cotransfected with the cDNA coding for WT-VWF. The reduction in VWF secretion was more severe for the transfection mimicking the homozygous condition. Data are represented as mean ± standard error of the mean (SEM), ****P < .0001. (B) No increase in variant VWF was observed in the cellular lysate for either the heterozygous or homozygous state indicating that the p.(Cys1084Tyr) variant does not cause intracellular retention of VWF. (C) Multimer analysis of recombinant VWF showed a relative reduction in high-molecular-weight multimers for VWF produced via cotransfection and a complete absence of high- and intermediate-weight multimers for p.(Cys1084Tyr) VWF. (D) Pseudo-WPB formation was assessed via transient transfection in HEK293 cells. Transfected cells were fixed and stained for VWF (green; i, ii, iii) and resident ER protein protein disulfide isomerase (PDI) (red; iv, v, vi). Merged images (vii, viii, ix) are shown in the final column. For all images, blue indicates 4′,6-diamidino-2-phenylindole. Colocalization of VWF and PDI was observed for cotransfected cells (viii) and for cells transfected with p.(Cys1084Tyr)-VWF alone (ix). Magnified views of regions of colocalization are shown in the inset. Images are representative of n = 3 independent experiments.