Modification of interdomain interfaces within the A3C1C2 subunit of factor VIII affects its stability and activity.

Factor (F) VIII, a plasma protein that is decreased or defective in individuals with hemophilia A, is expressed as both single chain and heterodimer forms. The latter consists of a heavy chain (HC) comprised of A1(a1)A2(a2)B domains and a light chain (LC) comprised of (a3)A3C1C2 domains, with the lower case a representing short (~30-40 residue) segments rich in acidic residues (see Ref.1 for review). FVIII is activated by proteolytic cleavages at the a1A2, a2B and a3A3 junctions catalyzed by thrombin or factor Xa. The resulting product, FVIIIa, is a heterotrimer comprised of subunits designated A1, A2, and A3C1C2 that functions as a cofactor for the serine protease FIXa in the membrane-dependent conversion of zymogen FX to the serine protease, FXa (see Ref.1 for review). Reconstitution studies have shown that the FVIII heterodimeric structure is supported by both electrostatic and hydrophobic interactions2. Metal ions also contribute to the inter-chain affinity and activity parameters3. Occupancy of a calcium site in the A1 domain (110-126) is required to yield the active FVIII conformation4. Copper ions facilitate the association of HC and LC to form the heterodimer, increasing the inter-chain affinity by several-fold at physiologic pH5,6. Intermediate resolution X-ray structures of FVIII7,8 showed occupancy of a calcium site in the A1 domain and the two type 1 copper ion sites within the A1 and A3 domains. The instability of FVIIIa results from weak electrostatic interactions between the A2 subunit and the A1/A3C1C2 dimer9,10 and its dissociation leads to dampening of FXase activity11,12. Examination of hydrogen-bonding interactions at the A1-A2 and A2-A3 interfaces following mutation of selected charged/polar residues spatially separated by <2.8 A showed loss of function, as judged by increased rates of FVIII decay at 55°C and/or rates for FVIIIa decay relative to WT, in approximately half of the 30 residues tested13, suggesting that multiple residues at the A1-A2 and A2-A3 domain interfaces contribute to the stabilization of FVIII. Mutations to increase buried hydrophobic area and/or reduce the buried hydrophilic area often results in enhanced protein stability14. Earlier studies showed that acidic residues localized to hydrophobic pockets at the A1-A2 interface (Asp519) and A2-A3 interface (Glu665 and Glu1984) when mutated to hydrophobic residues (Ala or Val), yielded favorable effects on FVIII/FVIIIa stability and/or activity15,16. In addition, replacing Ala108 that localized to a hydrophobic pocket at the A1-C2 domain interface with the more bulky Ile also showed a marked increase in FVIII stability17. Furthermore, introducing nascent disulfide bridges between FVIII subunits by double Cys mutation at A2-A3 or A1-C2 interfaces yielded FVIII variants with enhanced FVIII/FVIIIa stability17-19. Although the A3C1C2 domains are covalently linked in FVIII light chain, the FVIII X-ray crystal structure suggests the A3-C1 and C1-C2 domain interfaces are maintained by non-covalent interactions7,8. In this study we examined the effect of generating additional covalent and non-covalent interactions at the A3-C1 and C1-C2 interfaces. Results show that a selected A3-C1 interface modification is favorable for FVIII stability and C1-C2 interface modification enhanced cofactor function likely by reducing the Km of FXase for substrate factor X. In addition these result reveal a role for C1 residue Arg2116 in maintaining FVIII stability.