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

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You have full text access to this OnlineOpen articleLack of recombinant factor VIII B-domain induces phospholipid vesicle aggregation: implications for the immunogenicity of factor VIIIK. Grushin1, J. Miller1, D. Dalm1, E. T. Parker2, J. F. Healey2, P. Lollar2 andS. Stoilova-McPhie1,3,*Article first published online: 21 APR 2014

DOI: 10.1111/hae.12421

© 2014 The Authors. Haemophilia Published by John Wiley & Sons Ltd.

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

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HaemophiliaEarly View (Online Version of Record published before inclusion in an issue)

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How to CiteGrushin, K., Miller, J., Dalm, D., Parker, E. T., Healey, J. F., Lollar, P. and Stoilova-McPhie, S. (2014), Lack of recombinant factor VIII B-domain induces phospholipid vesicle aggregation: implications for the immunogenicity of factor VIII. Haemophilia. doi: 10.1111/hae.12421

Author Information1

Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, TX, USA

2

Aflac Cancer and Blood Disorders Center, Children's Healthcare of Atlanta, The Department of Pediatrics, Emory University, Atlanta, GA, USA

3

Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX, USA

* Correspondence: Stoilova-McPhie Svetla, PhD, Department of Neuroscience and Cell Biology; Scientist, Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch at Galveston, 301 University Boulevard, Galveston, TX 77555-0620, USA.
Tel.: 409 747 2159; fax: 409 747 2200;
e-mail: svmcphie@utmb.edu

Publication HistoryArticle first published online: 21 APR 2014Manuscript Accepted: 26 FEB 2014Funded byAmerican Heart Association. Grant Number: 10SDG3500034UTMBSSMNational Institutes of Health. Grant Numbers: U54 HL112309, R01 HL082609, R01 HL040921Hemophilia of Georgia, Inc. SEARCH Search Scope All contentPublication titlesIn this journalIn this issue Search String Advanced >Saved Searches > SEARCH BY CITATION Volume: Issue: Page: ARTICLE TOOLSGet PDF (643K)Save to My ProfileE-mail Link to this ArticleExport Citation for this ArticleGet Citation AlertsRequest Permissions AbstractArticleReferencesCited By View Full Article (HTML) Enhanced Article (HTML) Get PDF (643K) Keywords:coagulation factor VIII;cryo-electron microscopy;haemophilia A;immunogenicity;protein-induced vesicle aggregationSummary

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.

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