Generation of enhanced stability factor VIII variants by replacement of charged residues at the A2 domain interface

Abstract

Factor VIII consists of a heavy chain (A1A2B domains) and light chain (A3C1C2 domains), whereas the contiguous A1A2 domains are separate subunits in the cofactor, factor VIIIa. The intrinsic instability of the cofactor results from weak affinity interactions of the A2 subunit within factor VIIIa. The charged residues Glu272, Asp519, Glu665, and Glu1984 appear buried at the interface of the A2 domain with either the A1 or A3 domain, and thus may impact protein stability. To determine the effects of these residues on procofactor/ cofactor stability, these residues were individually replaced with either Ala or Val, and stable BHK cell lines expressing the B-domainless proteins were prepared. Specific activity and thrombin generation parameters for 7 of the 8 variants were more than 80% the wild-type value. Factor VIII activity at 52°C to 60°C and the decay of factor VIIIa activity after thrombin activation were monitored. Six of the 7 variants showing wild-type-like activity demonstrated enhanced stability, with the Glu1984Val variant showing a 2-fold increase in thermostability and an approximately 4-to 8-fold increase in stability of factor VIIIa. These results indicate that replacement of buried charged residues is an effective alternative to covalent modification in increasing factor VIII (VIIIa) stability. (Blood. 2008;112:2761-2769) IntroductionFactor VIII, a plasma protein that is decreased or defective in patients with hemophilia A, circulates as a noncovalent, metal ion-dependent heterodimer. This procofactor form of the protein consists of a heavy chain (HC) composed of A1(a1)A2(a2)B domains and a light chain (LC) composed of (a3)A3C1C2 domains, with the lower case "a" representing short (ϳ 30-40 residue) segments rich in acidic residues (for review, see Fay 1 ). Factor VIII is activated by proteolytic cleavages at the a1A2, a2B, and a3A3 junctions catalyzed by thrombin or factor Xa. The product of this reaction, factor VIIIa, is a heterotrimer composed of subunits designated A1, A2, and A3C1C2 that functions as a cofactor for the serine protease factor IXa in the membrane-dependent conversion of zymogen factor X to the serine protease, factor Xa (for review, see Fay 1 ).Reconstitution studies have shown that the factor VIII heterodimeric structure is supported by both electrostatic and hydrophobic interactions, 2,3 and the interchain affinity is further strengthened by factor VIII binding von Willebrand factor. 2,4 Metal ions also contribute to the interchain affinity and activity parameters. 5 Calcium is required to yield the active factor VIII conformation. Mutagenesis studies mapped a calcium-binding site to a segment rich in acidic residues within the A1 domain (residues 110-126) and identified specific residues within this region prominent in the coordination of the ion. 6 A recent intermediate resolution X-ray structure 7 confirmed this calcium-binding site as well as suggested a second potential site within the A2 domain. This structure also showed occupancy of the 2 type 1 copper ion sites within...

Mass Spectrometry-assisted Study Reveals That Lysine Residues 1967 and 1968 Have Opposite Contribution to Stability of Activated Factor VIII

“…5). However, Fay and co-workers demonstrated that replacement of residue Glu 665 for a valine or an alanine increases the stability of FVIIIa (24). This finding is in disagreement with the suggestion that interaction between Lys 1967 and Glu 665 contributes to the binding of the A2-a2 domain to the A1-a1/A3-C1-C2 dimer.…”

“…For a number of hemophilia A variants carrying single amino acid substitutions, it has been suggested that a decreased stability of FVIIIa is the cause for the bleeding disorder (21,22). Employing molecular modeling studies on the available FVIII structures in combination with site-directed mutagenesis, Fay and co-workers have now successfully identified several amino acid residues that enhance or decrease the stability of FVIII (21,(23)(24)(25).…”

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

“…In this assay the higher thermal stability variants also showed higher stability in this plasma assay (7). Further, the thrombin generation assays represent a good indicator of FVIIIa stability following thrombin activation and are monitored in a plasma-based assay.…”

“…The amount of thrombin generated in plasma was measured by Calibrated Automated Thrombography (28) using methods previously described (7). Briefly, FVIII deficient plasma (<1% residual activity, platelet-poor) from a severe hemophilia A patient lacking FVIII inhibitor (George King Bio-Medical) was mixed at 37ºC with a final concentration of 0.13 nM FVIII, 0.5 pM rTF, 4 μM PSPCPE vesicles, 433 μM fluorogenic substrate, 13.3 mM CaCl 2 , and 105 nM thrombin calibrator.…”

“…Mutations to increase buried hydrophobic area and/or reduce the buried hydrophilic area often result in enhanced protein stability (6). 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).…”