Identification of Residues Contributing to A2 Domain-dependent Structural Stability in Factor VIII and Factor VIIIa

Abstract

Factor VIII circulates as a heterodimer composed of heavy (A1A2B domains) and light (A3C1C2 domains) chains, whereas the contiguous A1A2 domains are separate subunits in the active cofactor, factor VIIIa. Whereas the A1 subunit maintains a stable interaction with the A3C1C2 subunit, the A2 subunit is weakly associated in factor VIIIa and its dissociation accounts for the labile activity of the cofactor. In examining the ceruloplasmin-based factor VIII A domain model, potential hydrogen bonding based upon spatial separations of <2.8 Å were found between side chains of 14 A2 domain residues and 7 and 9 residues in the A1 and A3 domains, respectively. These residues were individually replaced with Ala, except Tyr residues were replaced with Phe, and proteins stably expressed to examine the contribution of each residue to protein stability. Factor VIII stability at 55°C and factor VIIIa activity were monitored using factor Xa generation assays. Fourteen of 30 factor VIII mutants showed >2-fold increases in either or both decay rates compared with wild type; whereas, 7 mutants showed >2-fold increased rates in factor VIIIa decay compared with factor VIII decay. These results suggested that multiple residues at the A1-A2 and A2-A3 domain interfaces contribute to stabilizing the protein. Furthermore, these data discriminate residues that stabilize interactions in the procofactor from those in the cofactor, where hydrogen bonding in the latter appears to contribute more significantly to stability. This observation is consistent with an altered conformation involving new inter-subunit interactions involving A2 domain following procofactor activation.Factor VIII, a plasma protein that participates in the blood coagulation cascade, is decreased or defective in individuals with hemophilia A. Factor VIII circulates as a non-covalent, metal ion-dependent heterodimer consisting of a heavy chain comprised of A1(a1)A2(a2)B 2 domains and a light chain comprised of (a3)A3C1C2 domains (see Ref. 1 for review). The factor VIII procofactor is activated by limited proteolysis catalyzed by thrombin or factor Xa to yield the cofactor, factor VIIIa, following cleavages at the a1A2, a2B, and a3A3 junctions. Thus, factor VIIIa is a heterotrimer of subunits designated A1, A2, and A3C1C2. Factor VIIIa 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 (see Ref. 1 for review). The role of the cofactor in the intrinsic factor Xase complex is to increase the catalytic efficiency of this reaction by several orders of magnitude. Factor Xase is self-dampening as a result of instability in the factor VIIIa cofactor due to weak electrostatic interactions between the A2 subunit and the A1/A3C1C2 dimer (2, 3). Limited information is available regarding the association of the A2 subunit in factor VIIIa. Fluorescence energy transfer results (4) and activity assays following swapping the human A1 domain for the porcine homolog (5) suggest that interactions o...

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

“…Thus, chemical denaturation appears to represent a more global effect on FVIII structure and a less specific effect for chain dissociation. We reported earlier that several residues at the A2-A3 interface (Tyr-1792, Tyr-1786, and Asp-666) possibly contributed to the binding energy only in the active FVIIIa form (21). Thus, interactions between the A2 domain of the heavy chain and A3C1C2 domains of the light chain may be minimal in the procofactor.…”

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

“…This provides an explanation for the observation that K1967I is associated with hemophilia A. The same explanation has previously been proposed for other hemophilia A FVIII variants (21,22 …”

“…It has been demonstrated that residues that control the dissociation rate of the A2-a2 domain from A1-a1/A3-C1-C2 dimer, and as such affect the stability of FVIIIa, are of critical importance for cofactor func-tion (20,21). 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).…”

“…It has been demonstrated that residues that control the dissociation rate of the A2-a2 domain from A1-a1/A3-C1-C2 dimer, and as such affect the stability of FVIIIa, are of critical importance for cofactor func-tion (20,21). 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).…”

From Genotype to Phenotype - When the Parents Ask the Question

“…Missense mutations may produce a variable phenotype, depending on the site where the mutation has occurred. Generally, mutations in the sequence coding for the A2 domain of the Factor VIII gene result in CRM -positive haemophilia with varying severity (Wakabayashi & Fay, 2008) as the A2 domain is responsible for the stability of the protein. Deletions of exons usually produces severe CRM-negative haemophilia A because of reading frame disruption except for deletion of exon 22 which results in an in-frame loss of 156 bp coding sequence (Youssouffian et al, 1987).…”