Topology of factor VIII bound to phosphatidylserine-containing model membranes

Abstract

Factor VIII (FVIII), a plasma glycoprotein, is an essential cofactor in the blood coagulation cascade. It is a multidomain protein, known to bind to phosphatidylserine (PS)-containing membranes. Based on X-ray and electron crystallography data, binding of FVIII to PS-containing membranes has been proposed to occur only via the C2 domain. Based on these models, the molecular topology of membrane-bound FVIII can be envisioned as one in which only a small fraction of the protein interacts with the membrane, whereas the majority of the molecule is exposed to an aqueous milieu. We have investigated the topology of the membrane-bound FVIII using biophysical and biochemical techniques. Circular dichroism (CD) and fluorescence studies indicate no significant changes in the secondary and tertiary structure of FVIII associated with the membranes. Acrylamide quenching studies show that the protein is predominantly present on the surface of the membrane, exposed to the aqueous milieu. The light scattering and electron microscopy studies indicate the absence of vesicle aggregation and fusion. Binding studies with antibodies directed against specific epitopes in the A1, A2 and C2 domains suggest that FVIII binds to the membrane primarily via C2 domain including the specific phospholipid binding epitope (2303-2332) and may involve subtle conformational changes in this epitope region.

Phosphatidylinositol Containing Lipidic Particles Reduces Immunogenicity and Catabolism of Factor VIII in Hemophilia A Mice

“…2). In contrast, model membranes containing PS only bind to the previously characterized lipid binding region (2303-2332) of FVIII (19,50,52). One hypothesis to explain this may be differences in lipid organization between PS and PI containing lipidic vesicles.…”

Role of Glycosylation in Conformational Stability, Activity, Macromolecular Interaction and Immunogenicity of Recombinant Human Factor VIII

“…The ability of FVIII glycoforms to bind to artificial lipidic membranes and the extent of those interactions were evaluated based on the ability of liposomes to compete with antibodies against the C2 domain of rFVIII in a sandwich enzyme-linked immunosorbent assay (ELISA) as described previously (31). Briefly, 96-well plates were coated overnight with ESH4 or ESH8 as capture antibodies, nonspecific binding was blocked with bovine serum albumin, liposomalrFVIII complexes were incubated in the wells, a mixture of rat anti-human rFVIII polyclonal and goat anti-rat IgGalkaline phosphatase-conjugated antibodies was added, and optical density of the p-NPP substrate was read at 405 nm with a SpectraMax plate reader (Molecular Device Corporation, Sunnyvale, CA).…”

Phosphatidylserine Containing Liposomes Reduce Immunogenicity of Recombinant Human Factor VIII (rFVIII) in a Murine Model of Hemophilia A**Karthik Ramani and Razvan D. Miclea contributed equally to the manuscript.

“…23 The size distribution of the liposomes was determined using a Nicomp Model CW 380 particle size analyzer (Particle Sizing Systems, Santa Barbara, CA) as described previously. 24 Liposomes were associated with the appropriate amount of rFVIII by incubating at 378C for $30 min with gentle swirling. The protein to lipid molar ratio was maintained at 1:10000 for all the experiments, unless stated otherwise.…”