About this item:

136 Views | 137 Downloads

Author Notes:

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

KG performed all biochemical and biophysical experiments and helped with the Cryo-EM samples preparation.

JM collected all the Cryo-EM data.

DD prepared the pFVIII-BDD homology model.

ETP and JFH helped with expression and purification of the FVIII-BDD forms and functional assays.

PL critically read the manuscript and helped with the functional data interpretation.

SSM designed the work and finalized the manuscript.

The authors acknowledge the Cryo-EM and solution biophysics facilities at the Sealy Center for Structural Biology UTMB for support, as well as Ms. Christie Shumate for preliminary work on the project.

The authors stated that they had no interests which might be perceived as posing a conflict or bias.


Research Funding:

This work is supported by a National Scientist Development grant from the American Heart Association (10SDG3500034) and UTMB start-up funds to SSM and National Institutes of Health grants U54 HL112309, R01 HL082609 and R01 HL040921 and Hemophilia of Georgia, Inc. to PL.


  • Science & Technology
  • Life Sciences & Biomedicine
  • Hematology
  • coagulation factor VIII
  • cryo-electron microscopy
  • haemophilia A
  • immunogenicity
  • protein-induced vesicle aggregation

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


Journal Title:



Volume 20, Number 5


, Pages 723-731

Type of Work:

Article | Final Publisher PDF


Haemophilia Published by John Wiley & Sons Ltd. 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.

Copyright information:

© 2014 The Authors.

This is an Open Access work distributed under the terms of the Creative Commons Attribution-Noncommercial 3.0 Unported License (http://creativecommons.org/licenses/by-nc/3.0/).

Creative Commons License

Export to EndNote