Models depict mechanism for reversible FVIII amyloid formation and for impact of F309S mutation on FVIII structure. (A) BiP regulates reversible FVIII amyloidogenesis in the ER lumen. Once nascent FVIII (A1-A2-B-A3-C1-C2) translocates into the ER lumen, it undergoes N-linked core oligosaccharide addition and disulfide bond formation. Glycosylated FVIII subsequently interacts with the lectin chaperones CANX/CRT and the peptide-binding protein BiP. BiP binds FVIII within a short sequence aggregating motif (Aggron) in the A1 domain to facilitate folding by preventing aggregation and stabilizing soluble misfolded, but folding-competent, FVIII. Upon release from CANX/CRT correctly folded FVIII (soluble) traffics to the Golgi compartment. Glc supplementation produces ATP by glycolysis to activate the BiP ATPase cycle (client binding and release57 ) that stimulates FVIII disaggregation and BiP release for FVIII folding and secretion as a functional clotting factor. Under proteostatic stress, such as increased FVIII expression, metabolic collapse or BiP depletion, FVIII misfolds and forms amyloid-like fibrils due to antiparallel β-sheet polymerization initiated by the Aggron (aa 253-331) in the FVIII A1 domain. The Aggron- and BiP-binding site in the A1 domain overlap is indicated by the red circle. Red lines depict pathways of aggregation; green lines depict productive folding. (B) F309S reduces amyloidogenesis as well as Cu+ interaction with C310. The 3-dimensional FVIII structure (A1-A2-A3-C1-C2 domains)16,17 is shown where the 79 aa (residues 253-331) in the A1 domain are colored cyan also showing the phenylalanine (F, white) residue 309 and cysteine (C, red) residue 310 in the A1 domain. The Cu+ ions in the A1 and A3 domains are colored yellow. This predicted FVIII crystal structure was processed using Jsmol software. We propose that the hydrophobic patch aa 253-331 enforces tight Cu+ interaction with C310. Mutation of F309S (S being the homologus residue in FV that does not ligand copper ion) reduces FVIII aggregation and increases secretion. From the FVIII crystal structure, mutant F309S replaces a large hydrophobic side chain with a smaller polar one. Therefore, F309 FVIII would bind copper ion more avidly than S309 FVIII, although at the same time increasing aggregation propensity. ADP, adenosine 5′-diphosphate; HS/SH, thiols; Pi, phosphate.