title: 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.
authors.affiliation: The Department of Pharmaceutical Sciences, University at Buffalo, State University of New York, 541 Cooke Hall, Amherst, NY 14260-1200, USA.
authors.forename: Vivek S
authors.initials: VS
authors.lastname: Purohit
authors.forename: Karthik
authors.initials: K
authors.lastname: Ramani
authors.forename: Ramesh S
authors.initials: RS
authors.lastname: Kashi
authors.forename: Manzer J
authors.initials: MJ
authors.lastname: Durrani
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Kreiger
authors.forename: Sathyamangalam V
authors.initials: SV
authors.lastname: Balasubramanian
mesh_terms: D020106:Acrylamide / Q000737:chemistry; D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry*; D005169:Factor VIII / Q000737:chemistry* / Q000276:immunology; D008051:Lipid Bilayers / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry*; D008561:Membrane Fusion; D010718:Phosphatidylserines / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
chemical_list: D000906:Antibodies; D008051:Lipid Bilayers; D008081:Liposomes; D010718:Phosphatidylserines; D020106:Acrylamide; D005169:Factor VIII; D002118:Calcium; D004134:Dimyristoylphosphatidylcholine