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

K. Grushin, J. Miller, D. Dalm, E. T. Parker, J. F. Healey, P. Lollar, S. Stoilova-McPhie

Research output: Contribution to journalArticle

12 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)723-731
Number of pages9
JournalHaemophilia : the official journal of the World Federation of Hemophilia
Volume20
Issue number5
DOIs
StatePublished - Sep 1 2014

Fingerprint

Factor VIII
Phospholipids
Hemophilia A
Cryoelectron Microscopy
Membranes
cancer procoagulant
Swine
Factor VIIIa
Factor IXa
Therapeutics
Serine Proteases
Circular Dichroism
Intravenous Administration
Suspensions
Glycoproteins
Proteins
Blood Platelets
Hemorrhage
human F8 protein

Keywords

  • coagulation factor VIII
  • cryo-electron microscopy
  • haemophilia A
  • immunogenicity
  • protein-induced vesicle aggregation

ASJC Scopus subject areas

  • Hematology
  • Genetics(clinical)

Cite this

Lack of recombinant factor VIII B-domain induces phospholipid vesicle aggregation : implications for the immunogenicity of factor VIII. / Grushin, K.; Miller, J.; Dalm, D.; Parker, E. T.; Healey, J. F.; Lollar, P.; Stoilova-McPhie, S.

In: Haemophilia : the official journal of the World Federation of Hemophilia, Vol. 20, No. 5, 01.09.2014, p. 723-731.

Research output: Contribution to journalArticle

Grushin, K. ; Miller, J. ; Dalm, D. ; Parker, E. T. ; Healey, J. F. ; Lollar, P. ; Stoilova-McPhie, S. / Lack of recombinant factor VIII B-domain induces phospholipid vesicle aggregation : implications for the immunogenicity of factor VIII. In: Haemophilia : the official journal of the World Federation of Hemophilia. 2014 ; Vol. 20, No. 5. pp. 723-731.
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T2 - implications for the immunogenicity of factor VIII

AU - Grushin, K.

AU - Miller, J.

AU - Dalm, D.

AU - Parker, E. T.

AU - Healey, J. F.

AU - Lollar, P.

AU - Stoilova-McPhie, S.

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AB - 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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