Expression of recombinant VWF mutants. Recombinant wt and mutant VWF were transiently expressed in HEK293T cells, and VWF ELISA was used to determine the concentration of secreted VWF (A) and VWF retained within the cell lysate (B). Error bars represent mean ± SD of 3 separate experiments each performed in duplicate. (C) The multimer composition of recombinant VWF was analyzed in 1.3% agarose gels and visualized with HRP-conjugated anti-VWF polyclonal antibodies. RP indicates reference plasma. Lane 1 indicates wt-rVWF; lane 2, S1731T; lane 3, W1745C; lane 4, W1745A; lane 5, wt/W1745C; lane 6, S1783A. All mutants are expressed at similar levels to wild-type rVWF, with similar supernatant to lysate ratios and normal multimer distribution.