Lack of recombinant factor VIII B-domain induces phospholipid vesicle aggregation: implications for the immunogenicity of factor VIII.
Haemophilia
; 20(5): 723-31, 2014 Sep.
Article
en En
| MEDLINE
| ID: mdl-24750465
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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Fosfatidilcolinas
/
Fosfolípidos
/
Factor VIII
/
Liposomas
/
Fusión de Membrana
Tipo de estudio:
Prognostic_studies
Límite:
Humans
Idioma:
En
Revista:
Haemophilia
Asunto de la revista:
HEMATOLOGIA
Año:
2014
Tipo del documento:
Article
País de afiliación:
Estados Unidos
Pais de publicación:
Reino Unido