Increasing Hydrophobicity or Disulfide Bridging at the Factor VIII A1 and C2 Domain Interface Enhances Procofactor Stability

Abstract

Factor VIII (FVIII) consists of a heavy (A1A2B domains) and light chain (A3C1C2 domains), whereas the contiguous A1A2 domains are separate subunits in the cofactor, FVIIIa. FVIII x-ray structures show close contacts between A1 and C2 domains. To explore the role of this region in FVIII(a) stability, we generated a variant containing a disulfide bond between A1 and C2 domains by mutating Arg-121 and Leu-2302 to Cys (R121C/L2302C) and a second variant with a bulkier hydrophobic group (A108I) to better occupy a cavity between A1 and C2 domains. Disulfide bonding in the R121C/L2302C variant was >90% efficient as judged by Western blots. Binding affinity between the A108I A1 and A3C1C2 subunits was increased ϳ3.7-fold in the variant as compared with WT as judged by changes in fluorescence of acrylodan-labeled A1 subunits. FVIII thermal and chemical stability were monitored following rates of loss of FVIII activity at 57°C or in guanidinium by factor Xa generation assays. The rate of decay of FVIIIa activity was monitored at 23°C following activation by thrombin. Both R121C/ L2302C and A108I variants showed up to ϳ4-fold increases in thermal stability but minimal improvements in chemical stability. The purified A1 subunit of A108I reconstituted with the A3C1C2 subunit showed an ϳ4.6-fold increase in thermal stability, whereas reconstitution of the variant A1 with a truncated A3C1 subunit showed similar stability values as compared with WT A1. Together, these results suggest that altering contacts at this A1-C2 junction by covalent modification or increasing hydrophobicity increases inter-chain affinity and functionally enhances FVIII stability. Factor F (FVIII)2 is a plasma protein that is decreased or defective in individuals with hemophilia A. FVIII is expressed as both single chain and heterodimer forms in heterologous cells. The latter consists of a heavy chain composed of A1(a1)A2(a2)B domains and a light chain composed of (a3)A3C1C2 domains, with the lowercase "a" representing short segments rich in acidic residues (see Ref. 1 for review). FVIII is activated following limited proteolysis catalyzed by thrombin or FXa at Arg-372 (a1A2 junction), Arg-740 (a2B junction), and Arg-1689 (a3A3 junction). The resulting product, FVIIIa, is a heterotrimer composed of subunits designated A1, A2, and A3C1C2 that functions as a cofactor for FIXa in the membrane-dependent activation of FX to FXa (see Ref 1 for review).Earlier structural studies identified the C2 domain as making a primary contribution to anionic phospholipid membrane binding through a combination of electrostatic and hydrophobic interactions (2). More recently, the intermediate resolution x-ray structures of FVIII (3, 4) showed that the C1 and C2 domains are aligned such that both domains may interact with the phospholipid membrane surface. In addition, these structures show close contact between A1 (heavy chain) and C2 (light chain) domains. We recently reported that an FVIII variant lacking the C2 domain retained the capacity to bind phospholipid membranes, ...

Combining mutations that modulate inter-subunit interactions and proteolytic inactivation enhance the stability of factor VIIIa

“…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 stability (9). Furthermore, introducing nascent disulfide bridges between FVIII subunits by double Cys mutation at A2-A3 or A1-C2 interfaces yielded selected FVIII variants with enhanced FVIII/FVIIIa stability (9)(10)(11).…”

“…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 activity (7,8). 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 stability (9). Furthermore, introducing nascent disulfide bridges between FVIII subunits by double Cys mutation at A2-A3 or A1-C2 interfaces yielded selected FVIII variants with enhanced FVIII/FVIIIa stability (9)(10)(11).…”

Cofactor Activity in Factor VIIIa of the Blood Clotting Pathway Is Stabilized by an Interdomain Bond between His281 and Ser524 Formed in Factor VIII

“…FVIII Subunit Preparation-FVIIIa A1 (WT and H281S) and A3C1C2 subunit (WT) were prepared by the methods as described previously (10,11). A2 subunits (WT and S524H) were prepared using the Bac-to-Bac expression system and expressed in High Five cells (Invitrogen) as described previously (12,13).…”

Modification of Interdomain Interfaces within the A3C1C2 Subunit of Factor VIII Affects Its Stability and Activity

“…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 stability 17 . 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 stability 17-19 .…”