Intracellular retention of recombinant mutant VWF. (A) Relative concentrations of the wild-type G1180, mutant R1180, and heterozygous G1180R proteins in COS-7 supernatants and cell lysates (mean ± SEM) (n = 3). (B) The concentration of rVWF protein secreted into the media by COS-7 cells was decreased in transfections using the pCIneodel26 plasmid (black bar) when compared with wild-type transfections. As the quantity of rVWF secreted into the media decreased with increased concentrations of mutant plasmid, the amount of protein found retained within the cell increased (hatched dark bar). Likewise, the concentration of rVWF protein secreted into the media from transfections using the pCIneodel23/26 plasmid was also decreased (white bar); this double mutation showed a trend toward increased intracellular retention (hatched white bar) when compared with the single mutation at the 75:25 and 50:50 transfection ratios. When 100% pCIneodel23/26 was transfected into COS-7 cells, no protein was secreted into the media (mean ± SEM) (n = 3). The values from the heterozygous and homozygous mutant ELISAs are normalized to those of wild-type (setting wild-type levels at 1).