FVIII-YFP binding to VWF-CFP type 2N variants. VWF variants include wild-type VWF-CFP (a), Arg854Gln (b), Cys1060Arg (c), Thr791Met (d), Arg816Trp (e), and Arg763Gly (f). (A) FVIII-YFP was passed over a chip to which 22 fmol/mm² VWF-CFP type 2N variants were coupled. Association and dissociation were both followed for 240 seconds. Depicted is a representative SPR experiment showing the average curve of 3 injections of 5 nM FVIII-YFP. (B) Summary of binding for all VWF-CFP type 2N variants. Percentage of binding was calculated relative to wild-type VWF-CFP after 235 seconds of association. Each value represents the mean of 9 injections (1-5 nM FVIII-YFP) and the SEM is indicated. (C) FVIII-YFP (0-3 nM) was incubated for 2 hours with recombinant VWF-CFP variants bound to monoclonal antibody CLB-RAg20. The amount of FVIII bound to VWF was measured by chromogenic assay and calculated using plasma-derived FVIII as a standard. Each value represents the mean of 3 experiments and the SEM is indicated. The curves were fitted to a standard hyperbola using nonlinear regression analysis. (D) Cell lysates containing FVIII-YFP and VWF-CFP type 2N variants were assessed on FVIII/VWF complex as described under “Intracellular FVIII/VWF type 2N binding.” *The extent of intracellular FVIII/VWF type 2N complex is expressed relative to wild-type VWF-CFP. Each value represents the mean plus or minus SD of 3 experiments.