A CRYO-ELECTRON MICROSCOPY STUDY OF RECOMBINANT FVIII BINDING TO PS LIPOSOMES

Abstract number: P-T-034

Parmenter¹ C.D.J., Cane¹ M.C., Kemball-Cook² G., Stoilova-McPhie¹ S.

¹¹Biological Sciences, University of Warwick, Coventry ²²Clinical Sciences Centre, Faculty of Medicine, Imperial College, London, United Kingdom

How-to-cite Parmenter CDJ, Cane MC, Kemball-Cook G, Stoilova-McPhie S. A CRYO-ELECTRON MICROSCOPY STUDY OF RECOMBINANT FVIII BINDING TO PS LIPOSOMES. J Thromb Haemost 2007; 5 Supplement 2: P-T-034

Abstract

Introduction: Factor VIII (FVIII) is a plasma glycoprotein essential for blood coagulation(1). The focus of this paper is to investigate rFVIII-FL and rFVIII-BDD (Baxter Inc) binding to PS-containing liposomes by cryo-electron microscopy (cryo-EM). The results were compared with biophysical data for secondary structure changes and aggregation state of the proteins upon binding(2,3).

Methods: PC/PS liposomes were extruded through a 100nm filter. Dynamic light scattering (DLS) data were analysed with Dynamics V6 software. Circular Dichroism (CD) data were collected with a JASCO 715 and analysed with CDSSTR software(4). Digital Images from the frozen-hydrated samples were recorded on a 2x2k pixels CCD camera (Gatan Ltd)with a JEM2011 micorscope.

Results: DLS data showed strong aggregation of the vesicles only upon adding rfVIII-BDD. No aggregation of the proteins was observed. CD spectra showed a 1.3 increase in beta sheet to alpha helices ratio for rFVIII-FL. Only the 3/1alpha helix to pure alpha helix ratio increased upon protein binding: 2.5 for rFVIII-BDD and 1.4 times for rFVIII-FL. Cryo-EM confirmed aggregation only for rFVIII-BDD with vesicles.

Conclusions: Cryo-EM, DLS and CD studies showed that rFVIII-FL and rFVIII-BDD are stable in solution. Binding of both proteins to liposomes did not lead to changes in the secondary structure. Aggregation of the vesicles upon adding rFVIII-BDD suggests a stabilising role of the B domain.Cryo-EM implies strong protein-membrane and portein-protein interactions.

This work is supported by a British Heart Foundation grant: PG/04/070 and the protein was kindly provided by Baxter Inc, Vienna. We thank Prof A. Rodger and Dr. M. Hicks (Chemistry, University of Warwick, UK) for support in the CD experiments.

References:

1. Vehar GA et al (1984) Nature 254, 48-54

2. Sandberg H et al (2001) Thromb Haemost 85, 93-100

3. Purohit VS et al (2003) BBA 1617, 31-38

4. Hennessey JP and Johnson WC(1981) Biochemistry, 20, 1086-94

To cite this abstract use the following format:

Journal of Thrombosis and Haemostasis 2007; Volume 5, Supplement 2: abstract number

Session Details

Date:	01/08/2007
Time:	00:00-00:00
Session name:	XXIst ISTH Congress
Subject:
Location:	Oxford, UK
Presentation type:
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