title: Lack of recombinant factor VIII B-domain induces phospholipid vesicle aggregation: implications for the immunogenicity of factor VIII.
abstract: Factor VIII (FVIII) is a multidomain blood plasma glycoprotein. Activated FVIII acts as a cofactor to the serine protease factor IXa within the membrane-bound tenase complex assembled on the activated platelet surface. Defect or deficiency in FVIII causes haemophilia A, a severe hereditary bleeding disorder. Intravenous administration of plasma-derived FVIII or recombinant FVIII concentrates restores normal coagulation in haemophilia A patients and is used as an effective therapy. In this work, we studied the biophysical properties of clinically potent recombinant FVIII forms: human FVIII full-length (FVIII-FL), human FVIII B-domain deleted (FVIII-BDD) and porcine FVIII-BDD bound to negatively charged phospholipid vesicles at near-physiological conditions. We used cryo-electron microscopy (Cryo-EM) as a direct method to evaluate the homogeneity and micro-organization of the protein-vesicle suspensions, which are important for FVIII therapeutic properties. Applying concurrent Cryo-EM, circular dichroism and dynamic light scattering studies to the three recombinant FVIII forms when bound to phospholipid vesicles revealed novel properties for their functional, membrane-bound state. The three FVIII constructs have similar activity, secondary structure distribution and bind specifically to negatively charged phospholipid membranes. Human and porcine FVIII-BDD induce strong aggregation of the vesicles, but the human FVIII-FL form does not. The proposed methodology is effective in characterizing and identifying differences in therapeutic recombinant FVIII membrane-bound forms near physiological conditions, because protein-containing aggregates are considered to be a factor in increasing the immunogenicity of protein therapeutics. This will provide better characterization and development of safer and more effective FVIII products with implications for haemophilia A treatment.
authors.affiliation: Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, TX, USA.
authors.forename: K
authors.initials: K
authors.lastname: Grushin
authors.forename: J
authors.initials: J
authors.lastname: Miller
authors.forename: D
authors.initials: D
authors.lastname: Dalm
authors.forename: E T
authors.initials: ET
authors.lastname: Parker
authors.forename: J F
authors.initials: JF
authors.lastname: Healey
authors.forename: P
authors.initials: P
authors.lastname: Lollar
authors.forename: S
authors.initials: S
authors.lastname: Stoilova-McPhie
mesh_terms: D020285:Cryoelectron Microscopy / Q000379:methods; D005169:Factor VIII / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D008081:Liposomes / Q000737:chemistry*; D008561:Membrane Fusion; D010713:Phosphatidylcholines / Q000737:chemistry*; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins; D012542:Scattering, Radiation
chemical_list: D008081:Liposomes; D010713:Phosphatidylcholines; D010743:Phospholipids; D011994:Recombinant Proteins; D005169:Factor VIII
keywords: coagulation factor VIII; cryo-electron microscopy; haemophilia A; immunogenicity; protein-induced vesicle aggregation
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