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

Haematology

Journal of Blood Disorders & Transfusion

Author(s): Grushin K, Miller J, Dalm D, Parker ET, Healey JF,

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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. © 2014 The Authors. Haemophilia Published by John Wiley & Sons Ltd.
This article was published in Haemophilia and referenced in Journal of Blood Disorders & Transfusion

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