Factor VIII Lacking the C2 Domain Retains Cofactor Activity in Vitro

Abstract

Factor (F) VIII consists of a heavy chain (A1A2B domains) and light chain (A3C1C2 domains). The activated form of FVIII, FVIIIa, functions as a cofactor for FIXa in catalyzing the membrane-dependent activation of FX. Whereas the FVIII C2 domain is believed to anchor FVIIIa to the phospholipid surface, recent x-ray crystal structures of FVIII suggest that the C1 domain may also contribute to this function. We constructed a FVIII variant lacking the C2 domain (designated ⌬C2) to characterize the contributions of the C1 domain to function. Binding affinity of the ⌬C2 variant to phospholipid vesicles as measured by energy transfer was reduced ϳ14-fold. However, the activity of ⌬C2 as measured by FXa generation and one-stage clotting assays retained 76 and 36%, respectively, of the WT FVIII value. Modest reductions (ϳ4-fold) were observed in the functional affinity of ⌬C2 FVIII for FIXa and rates of thrombin activation. On the other hand, deletion of C2 resulted in significant reductions in FVIIIa stability (ϳ3.6-fold). Thrombin generation assays showed peak thrombin and endogenous thrombin potential were reduced as much as ϳ60-fold. These effects likely result from a combination of the intermolecular functional defects plus reduced protein stability. Together, these results indicate that FVIII domains other than C2, likely C1, make significant contributions to membrane-binding and membrane-dependent function. Factor (F)2 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 lowercase a representing short (ϳ30 -40 residues) segments rich in acidic residues (see Ref (1) for review). FVIII is activated by thrombin-or FXa-catalyzed cleavages at the a1A2, a2B, and a3A3 junctions. The resulting product, FVIIIa, is a heterotrimer comprised of subunits designated A1, A2, and A3C1C2 that functions as a cofactor for the serine protease factor IXa (FIXa) in the membrane-dependent conversion of zymogen FX to the serine protease, FXa (see Ref.(1) for review).Early structural studies implicated the C2 domain as making the predominant contribution to membrane binding through a combination of electrostatic and hydrophobic interactions (2). In addition, the C2 domain contains apparent secondary binding sites for von Willebrand factor (VWF) (3, 4), thrombin (5), and FIXa (6). The intermediate resolution x-ray crystal structures of FVIII (7, 8) indicate that the C1 and C2 domains are aligned such that both domains may interact with the phospholipid surface. A similar alignment was noted for factor Vai, the activated protein C-inactivated form of the homologous protein, factor Va (9). Recent results comparing isolated FVIII C2 domain with a C1C2 protein showed enhanced affinity of the latter in platelet binding suggesting direct and/or indirect roles for C1 in this interaction (10). It was also reported tha...

Factor VIII C1 Domain Spikes 2092–2093 and 2158–2159 Comprise Regions That Modulate Cofactor Function and Cellular Uptake

“…6, B and D, and 7B), suggesting that membrane binding cannot be fully mediated by the C2 domain alone under these conditions of limiting PS exposure. This supports the view that both C domains support membrane interaction in a cooperative manner (15) and is also compatible with the finding that a FVIII deletion mutant lacking the C2 domain still displays substantial FVIII cofactor activity (37).…”

From Genotype to Phenotype - When the Parents Ask the Question

“…As a rule, missense mutations cause CRM-positive haemophilia A with varying severity, depending on the mutation in question and the parts of the gene it affects. Recently, Wakabayashi et al demonstrated that Factor VIII devoid of its C2 domain remains functional, at least in vitro, but is less stable (Wakabayashi et al, 2010). In our practice, we have had two (related) patients with exon 24 missense mutation (the C2 domain) and with mild haemophilia A (40 %), supporting the notion of decreased stability of the protein.…”

Structure of the factor VIII C2 domain in a ternary complex with 2 inhibitor antibodies reveals classical and nonclassical epitopes

“…The positioning of the C1 domain relative to the C2 domain supports previous data indicating that the C1 domain contributes to membrane association and the C2 domain is not essential for function. [29][30][31] Lastly, the x-ray crystal structure of the C2 domain/3E6/G99 complex is remarkably similar to the previously modeled ternary complex from low-resolution SAXS data ( Figure 1B). 41 These structural findings suggest that using rapid, low-resolution techniques such as SAXS may be effective tools for characterizing other antibody epitopes for fVIII inhibitor antibodies as well as other antigen/antibody interactions.…”

“…[26][27][28] Recent studies also suggest that the C1 domain contributes to binding both VWF and PS, 29,30 and deletion studies of the C2 domain indicate that it is not essential for PL membrane binding, factor IXa binding, or clotting activity. 31 The C2 domain of fVIII is a major antigenic determinant for recognition by inhibitor antibodies. 32,33 Initial mechanistic investigations of anti-C2 inhibitor antibodies suggested that these antibodies There is an Inside Blood commentary on this article in this issue.…”