Defects in secretion and pseudo Weibel-Palade body formation of recombinant M1304R VWF. (A) Expression of recombinant mutant or wild-type VWF. HEK293T cells were transfected with cDNAs for either wild-type or mutant VWF and the concentrations of recombinant VWF were assessed in both conditioned media (supernatant) and cell lysate by enzyme-linked immunosorbent assay. The VWF concentration in the media (supernatant) was markedly lower for the mutant than for the wild-type, although the intracellular concentrations were similar (lysate). The concentration of wild-type VWF was normalized to 100% and that of the mutant VWF was presented relative to this value. Each bar represents the mean ± standard deviation of at least 3 separate experiments. VWF inside the transfected cells was examined by immuno-staining and confocal microscopy (B,C). (B) Cells transfected with M1304R VWF showed diffuse intracellular VWF staining indicating that the mutant was defective in forming storage granules. (C) Cells transfected with wild-type VWF showed punctate staining of VWF localized to granules resembling Weibel-Palade bodies.