The 1.7 Å X-Ray Crystal Structure of the Porcine Factor VIII C2 Domain and Binding Analysis to Anti-Human C2 Domain Antibodies and Phospholipid Surfaces

Abstract

The factor VIII C2 domain is essential for binding to activated platelet surfaces as well as the cofactor activity of factor VIII in blood coagulation. Inhibitory antibodies against the C2 domain commonly develop following factor VIII replacement therapy for hemophilia A patients, or they may spontaneously arise in cases of acquired hemophilia. Porcine factor VIII is an effective therapeutic for hemophilia patients with inhibitor due to its low cross-reactivity; however, the molecular basis for this behavior is poorly understood. In this study, the X-ray crystal structure of the porcine factor VIII C2 domain was determined, and superposition of the human and porcine C2 domains demonstrates that most surface-exposed differences cluster on the face harboring the “non-classical” antibody epitopes. Furthermore, antibody-binding results illustrate that the “classical” 3E6 antibody can bind both the human and porcine C2 domains, although the inhibitory titer to human factor VIII is 41 Bethesda Units (BU)/mg IgG versus 0.8 BU/mg IgG to porcine factor VIII, while the non-classical G99 antibody does not bind to the porcine C2 domain nor inhibit porcine factor VIII activity. Further structural analysis of differences between the electrostatic surface potentials suggest that the C2 domain binds to the negatively charged phospholipid surfaces of activated platelets primarily through the 3E6 epitope region. In contrast, the G99 face, which contains residue 2227, should be distal to the membrane surface. Phospholipid binding assays indicate that both porcine and human factor VIII C2 domains bind with comparable affinities, and the human K2227A and K2227E mutants bind to phospholipid surfaces with similar affinities as well. Lastly, the G99 IgG bound to PS-immobilized factor VIII C2 domain with an apparent dissociation constant of 15.5 nM, whereas 3E6 antibody binding to PS-bound C2 domain was not observed.

Flow-Cytometry Platform for Intracellular Detection of FVIII in Blood Cells: A New Tool to Assess Gene Therapy Efficiency for Hemophilia A

“…Intriguingly, the rate of FVIII double fluorescence was lower than the expected cumulative value, as visualized by single staining. Steric hindrance of Abs is unlikely to explain this decrease, since simultaneous binding of multiple anti-fVIII mAbs (mAbs) to the C2 domain of FVIII was shown in previous studies 23 , 24 . Either the presence of incomplete FVIII fragments, or still a margin of staining unspecificity despite the extensive controls, might explain this finding (Figure 4D).

Figure 4Double Staining of A2 and C1 Domains of FVIIIGMA® 8012 IgG1 anti A2 domain and GMA® 8011 IgG2a anti C1 domain were used with IgG1 and IgG2a as controls for a comparison of single and double staining on HECV un-transduced cells (A), HECV cells transduced at MOI 30 (B) and 40 (C) with PGK-FVIII-LVV.

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Structure of the Human Factor VIII C2 Domain in Complex with the 3E6 Inhibitory Antibody

“…Lastly, it is notable that the Thr2197-Ala2201 loop also possesses lower B factor ratios relative to the isolated C2 domain structure. While this loop does not make direct interactions with either the 3E6 or G99 epitope, it is a β–hairpin loop that presents solvent-exposed hydrophobic residues that bridges both 3E6 and G99 epitopes 29 , is a major component of the BO2C11 epitope 27 , and is hypothesized to be the site of membrane binding 38 39 40 . Moreover, previous H/D exchange data indicate that the 2197–2201 loop has increased protection factors upon 3E6 binding 41 .…”

“…While these data seem convincing that the cooperativity is a dynamic effect, they do not completely rule out electrostatic contributions. It should be noted that the 3E6 binding site sequesters the region of the fVIII C2 domain with the highest density of positive charge 28 38 . Given that both the 3E6 and G99 antibodies recognize regions of positive charge within significant portions of their respective epitopes, sequestering one binding site may allow for electrostatic steering for the second antibody to bind.…”

SAXS analysis of intrinsic tenase complex bound to lipid nanodisc highlights intermolecular contacts between factors VIIIa/IXa

“…28,29,33,34 The fVIIIa C2 domain carries a putative binding site for the fIXa Gla domain, 30,31 both of which contribute to lipid membrane binding. 7,10,18,68 Lastly, interdomain contacts between EGF-2 and the catalytic domain of fIXa are critical for formation of the Xase complex and prevent dissociation between the fIXa heavy and light chains. 69 Our results support multiple points of contact between fVIIIa and fIXa.…”

“…1,2 Proteolytic cleavage by thrombin releases vWf and generates the activated fVIII (fVIIIa) A1/A2/A3-C1-C2 heterotrimer 3,4 which binds to activated platelet surfaces through several hydrophobic loops in the C1 and C2 domains. [5][6][7] Binding to activated factor IX (fIXa), a trypsin-like serine protease, forms the intrinsic tenase (Xase) complex, which catalyzes the activation of factor X (fX), enhancing thrombin turnover and clot formation. 8 FIXa is a heterodimer of its light chain, composed of an N-terminal γ-carboxyglutamic acid (Gla) domain 9,10 and two epidermal growth factor-like (EGF-1 and EGF-2) domains, and heavy chain, which carries the catalytic domain.…”