Lack of recombinant factor VIII B-domain induces phospholipid vesicle aggregation: implications for the immunogenicity of factor VIII

• Published in: Haemophilia : the official journal of the World Federation of Hemophilia 2014-09, Vol.20 (5), p.723-731
• Main Authors: Grushin, K., Miller, J., Dalm, D., Parker, E. T., Healey, J. F., Lollar, P., Stoilova-McPhie, S.
• Format: Article
• Language: English
• Subjects: coagulation factor VIII; cryo-electron microscopy; Cryoelectron Microscopy - methods; Factor VIII - chemistry; Factor VIII - immunology; haemophilia A; Humans; immunogenicity; Liposomes - chemistry; Membrane Fusion; Original; Phosphatidylcholines - chemistry; Phospholipids - chemistry; Phospholipids - metabolism; protein-induced vesicle aggregation; Recombinant Proteins; Scattering, Radiation
• ISSN: 1351-8216; 1365-2516
• DOI: 10.1111/hae.12421

Summary 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.