American Society of Hematology

Figure 3.

$A short amino acid motif in the A1 domain seeds FVIII aggregation. (A) Homology between human FVIII and FV is shown between aa 291 and 310, with FVIII-F309 highlighted in red. (B-C) Mutations in the FVIII A1 domain exhibit different secretion efficiencies. COS-1 cells were transfected with expression vectors encoding single or multiple FVIII A1 mutations. 7LF>A has 7 Phe or Leu mutations to Ala underlined in panel A. At 48 hours after transfection, intracellular and secreted FVIII antigen were measured by ELISA. (B) Secreted FVIII relative to wtFVIII is shown as mean plus or minus standard deviation (SD) from biological triplicates. The results represented are from 1 experiment where similar results were reproducible in HepG2 cells (supplemental Figure 5). Statistical differences compared with wtFVIII were quantified by 1-way ANOVA using GraphPad Prism software: ****P < .0001; ***P < .001. Black bars represent biological replicates. (C) Lysates from transfected cells (the same as in panel B) were filtered through CA membranes to measure FVIII retention. (D-G) Seventy-nine amino acid seed FVIII aggregation. (D) Schematic structure is shown for FVIII-eGFP-CMy chimeras. The FVIII 79 aa were inserted in frame downstream of eGFP and upstream of the human proinsulin C peptide (Cpep) with a Myc tag (eGFP-CMy). The Myc tag is highlighted in purple and human Cpep is in yellow. (E) wtFVIII-eGFP-CMy–transfected cells were analyzed by fluorescence microscopy (fluorescence from enhanced green fluorescence protein; scale bar, 200 μm). (F-G) Mutations within 79 aa exhibit different FVIII-eGFP-CMy secretion and aggregation. (F) Intracellular and secreted FVIII-eGFP-CMy from 293T cells expressing wtFVIII-eGFP-CMy or different mutants (wt, 7LF>A, F309S, ES, VES, TES, and F306W) was analyzed by western blotting using Cpep antibody. The same membranes were probed with BiP and vinculin antibodies. Relative protein secretion vs intracellular protein is shown from 1 experiment with biological triplicates. (G) The protein samples in panel F were analyzed by filtration on CA membranes and probing with Cpep antibody. Similar results were observed in HepG2-transfected cells (supplemental Figure 7). (H-I) wtFVIII-eGFP-CMy forms HMW complexes. Cell lysates from 293T cells expressing wtFVIII-eGFP-CMy or mutant FVIII-eGFP-CMy were subjected to 5% to 20% sucrose gradient sedimentation. Proteins from fractions 1 to 10 were analyzed by western blot using Cpep antibody. The membrane was probed with vinculin antibody for loading control. The proportion of protein in each fraction is indicated as a percentage of total and plotted in panel I. Red arrows indicate migration of soluble forms of F309S FVIII-eGFP-CMy and Cys310Glu mutation of FVIII-eGFP-CMy (C310E FVIII-eGFP-CMy). A similar finding was observed in HepG2-transfected cells (supplemental Figure 9). Frxn, fraction; nd, not determined; SP, the proinsulin signal peptide; WCL, whole-cell lysate.$

A short amino acid motif in the A1 domain seeds FVIII aggregation. (A) Homology between human FVIII and FV is shown between aa 291 and 310, with FVIII-F309 highlighted in red. (B-C) Mutations in the FVIII A1 domain exhibit different secretion efficiencies. COS-1 cells were transfected with expression vectors encoding single or multiple FVIII A1 mutations. 7LF>A has 7 Phe or Leu mutations to Ala underlined in panel A. At 48 hours after transfection, intracellular and secreted FVIII antigen were measured by ELISA. (B) Secreted FVIII relative to wtFVIII is shown as mean plus or minus standard deviation (SD) from biological triplicates. The results represented are from 1 experiment where similar results were reproducible in HepG2 cells (supplemental Figure 5). Statistical differences compared with wtFVIII were quantified by 1-way ANOVA using GraphPad Prism software: ****P < .0001; ***P < .001. Black bars represent biological replicates. (C) Lysates from transfected cells (the same as in panel B) were filtered through CA membranes to measure FVIII retention. (D-G) Seventy-nine amino acid seed FVIII aggregation. (D) Schematic structure is shown for FVIII-eGFP-CMy chimeras. The FVIII 79 aa were inserted in frame downstream of eGFP and upstream of the human proinsulin C peptide (Cpep) with a Myc tag (eGFP-CMy). The Myc tag is highlighted in purple and human Cpep is in yellow. (E) wtFVIII-eGFP-CMy–transfected cells were analyzed by fluorescence microscopy (fluorescence from enhanced green fluorescence protein; scale bar, 200 μm). (F-G) Mutations within 79 aa exhibit different FVIII-eGFP-CMy secretion and aggregation. (F) Intracellular and secreted FVIII-eGFP-CMy from 293T cells expressing wtFVIII-eGFP-CMy or different mutants (wt, 7LF>A, F309S, ES, VES, TES, and F306W) was analyzed by western blotting using Cpep antibody. The same membranes were probed with BiP and vinculin antibodies. Relative protein secretion vs intracellular protein is shown from 1 experiment with biological triplicates. (G) The protein samples in panel F were analyzed by filtration on CA membranes and probing with Cpep antibody. Similar results were observed in HepG2-transfected cells (supplemental Figure 7). (H-I) wtFVIII-eGFP-CMy forms HMW complexes. Cell lysates from 293T cells expressing wtFVIII-eGFP-CMy or mutant FVIII-eGFP-CMy were subjected to 5% to 20% sucrose gradient sedimentation. Proteins from fractions 1 to 10 were analyzed by western blot using Cpep antibody. The membrane was probed with vinculin antibody for loading control. The proportion of protein in each fraction is indicated as a percentage of total and plotted in panel I. Red arrows indicate migration of soluble forms of F309S FVIII-eGFP-CMy and Cys310Glu mutation of FVIII-eGFP-CMy (C310E FVIII-eGFP-CMy). A similar finding was observed in HepG2-transfected cells (supplemental Figure 9). Frxn, fraction; nd, not determined; SP, the proinsulin signal peptide; WCL, whole-cell lysate.

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