OC 33.5 - Structural characterization of coagulation factor VIII

Monday, June 26, 2023

11:45 – 12:00 ET

Room: 710B

Session Available As: On-Demand

Presenting Author(s)

Samhitha Urs Ramaraje Urs, MSc

PhD Student
Institute of Experimental Haematology and Transfusion Medicine, University Clinic Bonn
Bonn, Nordrhein-Westfalen, Germany

Co-Author(s)

SS

Sneha Singh, PhD (she/her/hers)

Postdoctoral researcher
Institute of Experimental Hematology and Transfusion Medicine, University Clinic Bonn
Bonn, Nordrhein-Westfalen, Germany
Haroon Javed, MSc

PhD student
Institute of Experimental Haematology and Transfusion Medicine, University Hospital Bonn
Bonn, Nordrhein-Westfalen, Germany
MI

MD Mubassherul Islam

PhD student
Institute of Experimental Haematology and Transfusion Medicine, University Clinic Bonn
Bonn, Nordrhein-Westfalen, Germany
DU

Deniz Ugurlar

Sales Development Representative
Materials and Structural Analysis Division, Thermo Fisher Scientific, Achtseweg Noord, 5651 GG Eindhoven, the Netherlands.
Eindhoven, Noord-Brabant, Netherlands
BM

Boxue Ma

Cryo-EM Application Specialist
Materials and Structural Analysis Division, Thermo Fisher Scientific, Achtseweg Noord, 5651 GG Eindhoven, the Netherlands
Eindhoven, Noord-Brabant, Netherlands
JP

Jean Luc Pellequer

Group leader
Institut de Biologie Structurale, Methods and Electron Microscopy group, Grenoble, France
Grenoble, Rhone-Alpes, France
JT

Jean-Marie Teulon

AFM specialist
Institut de Biologie Structurale, Methods and Electron Microscopy group, Grenoble, France
Grenoble, Rhone-Alpes, France
DF

Daphna Fenel

AFM specialist
Institut de Biologie Structurale, Methods and Electron Microscopy group, Grenoble, France
Grenoble, Rhone-Alpes, France
AP

Anna Pepanian

PhD student
University of Bonn
Bonn, Nordrhein-Westfalen, Germany
DI

Diana Imhof

Professor
University of Bonn
Bonn, Nordrhein-Westfalen, Germany
Johannes Oldenburg, MD (he/him/his)

Professor, Chairman and Director
University Clinic Bonn AöR, Institute of Experimental Haematology and Transfusion Medicine, Bonn, Germany
Bonn, Nordrhein-Westfalen, Germany
Arijit Biswas, PD Dr (Biol)

Scientist
Institute of Experimental Haematology and Transfusion Medicine, University Clinic Bonn
Bonn, Nordrhein-Westfalen, Germany

Background: The coagulation factor VIII protein (FVIII) is structurally well characterized but the current structural information is skewed towards B-domain deleted FVIII (BDD-FVIII) hence, most of the B-domain is structurally unresolved.

Aims: To understand the structural importance of FVIII´s large glycosylated B-domain by applying biophysical and bioanalytical methods to full-length (FL-FVIII) and BDD-FVIII and correlating the experimental observations to computational studies performed on structural models of FVIII.

Methods: FL-FVIII purified from commercial sources i.e. recombinant FL-FVIII (rFL-FVIII) (Kovaltry) and plasma-derived FL-FVIII (pFL-FVIII) (Hemoctin) were subjected to cryo-EM (cryo-electron microscopy) and AFM (atomic force microscopy) studies. Comparative CD spectrometry for rFL-FVIII and rFVIII-BDD was performed. Atomic MD simulations were run for structural models of FL-FVIII and BDD-FVIII obtained from the alpha-fold database.

Results: The globular regions of FL-FVIII fitted well within low-resolution cryo-EM maps obtained for rFL-FVIII and pFL-FVIII. The B-domain was still missing from these maps but appeared to be part of scattered density surrounding the central core. Similar observations of B-domain wrapping around the core globular domains were made in the AFM images. The structural models of FL-FVIII post-simulation showed a similar orientation of B-domain. The secondary structure spectra comparison between FL-FVIII and BDD-FVIII showed qualitative and quantitative differences (higher distortion in helices of BDD-FVIII) in secondary structure that is reflected in structural simulation studies as well.

Conclusion(s): Although the B-domain is disordered and highly flexible, its extensive glycosylation stabilizes it in a unique orientation w.r.t the globular FVIII domains. Quantitative and qualitative differences in the secondary structure content between FL-FVIII and FVIII-BDD suggests that the presence of B-domain influences the relative surface exposure of the other FVIII domains and has a role in stabilizing their secondary structure. This further indicates that the binding of FVIII interacting partners (vWF, Thrombin) or neutralizing antibodies would structurally, and functionally differ between FL-FVIII and FVIII-BDD proteins.

Learning Objectives:

Using multiple biophysical and computational techniques to characterize the highly disordered coagulation FVIII B domain
Structural orientation of FVIII B domain within the Full-length coagulation FVIII protein
Structure-functional implications of the presence of coagulation FVIII B domain