Seventy-nine amino acids from FVIII mediate Cpep-reversible aggregation upon restoration of Glc metabolism. (A) Schematic shows structure of the FVIII 79 aa inserted in frame upstream of the human proinsulin C peptide (Cpep) with a Myc tag to generate wtFVIII-CMy. Cys329 at the carboxy terminus of the 79 aa in the FVIII A1 domain was mutated to Gly to prevent aberrant disulfide bond formation (supplemental Figure 6A). (B-C) Seventy-nine amino acids from FVIII cause Cpep aggregation. 293T cells were transfected with indicated expression plasmids. (B) At 48 hours posttransfection, culture media was analyzed by Cpep ELISA and results presented secreted Cpep as mean plus or minus SD on log₁₀ scale. Statistical analysis of secretion compared with wtFVIII-Cpep was performed by 1-way ANOVA using GraphPad Prism software. ****P < .0001. (C) At 48 hours posttransfection, cell lysates were analyzed for retention on CA membranes by probing with Cpep antibody. Data shown are from a representative experiment with biological triplicates. eGFP-CMy and FV-CMy, constructed as similar as the FVIII-CMy chimera, transfection was included as another control. (D) wtFVIII-Cpep forms in HMW complexes. Cell lysates from 293T expressing wtFVIII-CMy, F309S FVIII-CMy, and eGFP-CMy were analyzed by 5% to 20% sucrose gradient sedimentation as in Figure 3H. Protein samples from 1 to 10 fractions were analyzed by western blotting using Cpep antibody. Sucrose gradient sedimentation of cell lysate from wtFVIII-transfected cells was performed in parallel. (E) The proportion of protein in each fraction is indicated as a percentage of total. Red arrows in panels D and E indicate migration of soluble forms of F309S FVIII-CMy. (F) wtFVIII-CMy aggregation is mediated by hydrophobic interactions but not disulfide cross-links. Sucrose gradient fractions 3 and 10 were treated with/without 1% SDS or with reducing agents (10 mM DTT or 10 mM β-ME) before CA filtration and probing with Cpep antibody. Blue bars represent technical triplicates. The percentage of FVIII on CA membranes is shown as relative to untreated (UT). (G) Aggregation of wtFVIII-CMy is reversible and requires Glc. 293T cells expressing either CMy, wtFVIII-CMy, FV-CMy, or eGFP-CMy were untreated or treated with Glc-free Dulbecco's Modified Eagles Medium (DMEM) containing 20 mM 2DG and 10 mM NaN₃ for 2.5 hours. For repletion, the 2DG and NaN₃–containing medium was replaced with DMEM (25 mM glucose) and cells were cultured 4 hours. Finally, cell lysates were filtered through CA and NC membranes and probed with Cpep antibody. Blue bars = technical triplicates. The percentage of aggregated wtFVIII-CMy is relative to untreated. (H) wtFVIII-CMy specifically interacts with BDD and wtFVIII. Cell lysates from cells cotransfected with wtFVIII-CMy and wtFVIII or BDD-FVIII were immunoprecipitated with Cpep antibody. Both input and Cpep-immunoprecipitated protein were analyzed by western blot probed by FVIII antibody (Baxter) and Cpep antibody. EV, empty vector; Frxn, fraction; SP, proinsulin signal peptide.