PB1104 - Understanding distinct conformational states of full-length coagulation Factor VIII and its complex with vWF : A comprehensive AFM analysis

Tuesday, June 25, 2024

13:45 – 14:45 ICT

Room: Exhibition Hall

Presenting Author(s)

Samhitha Urs Ramaraje Urs, M.Sc

PhD Student
Institute for Experimental Hematology and Transfusion Medicine, University Hospital Bonn
Bonn, Nordrhein-Westfalen, Germany

Co-Author(s)

JP

Jean Luc Pellequer

CEA Researcher
Univ. Grenoble Alpes, CEA, CNRS, IBS (Institut de Biologie Structurale), F-38000 Grenoble, France
Grenoble, Rhone-Alpes, France
JT

Jean Marie Teulon

CEA Engineer
Univ. Grenoble Alpes, CEA, CNRS, IBS (Institut de Biologie Structurale), F-38000 Grenoble, France
Grenoble, Rhone-Alpes, France
SS

Sneha Singh, PhD

Post Doctoral Scientist
Institute for Experimental Hematology and Transfusion Medicine, University Hospital of Bonn
Bonn, Nordrhein-Westfalen, Germany
MI

Md. Mubasherrul Islam

PhD student
Institute for Experimental Hematology and Transfusion Medicine, University Hospital Bonn
Bonn, Nordrhein-Westfalen, Germany
Johannes Oldenburg, MD

Professor, Chairman and Director
University Clinic Bonn AöR, Institute of Experimental Haematology and Transfusion Medicine
Bonn, Nordrhein-Westfalen, Germany
AB

Arijit Biswas

Staff Scientist and Group leader
Institute for Experimental Hematology and Transfusion Medicine, University Hospital of Bonn
Bonn, Nordrhein-Westfalen, Germany

Background: The structure-functional aspects of the full-length FVIII (FL-FVIII) and the disordered B-domain remain a subject of curiosity.

Aims: This study integrated atomic force microscopy (AFM) with molecular modeling, employing a distinctive AFM assembly process. We aimed to unravel the spatial orientation of the FVIII B-domain and understand the effect of the B-domain in the binding of interacting partners and inhibitory antibodies using refined FL-FVIII and BDD-FVIII structural models.

Methods: An off-resonance liquid AFM imaging was performed on highly pure recombinant and plasma-derived FL-FVIII and FVIII-vWF complex proteins. Simulation equilibrated glycosylated FL-FVIII models were refined using x-plor, and Coot. FL-FVIII-vWF model was built by superposing vWF (D’-D3) from a recent cryoEM structure of BDD-FVIII-vWF (PDBID: 7KWO) to our refined FL-FVIII model. All three refined models (FL-FVIII, BDD-FVIII, and FL-FVIII-vWF complex) were validated using MolProbity. The validated models were fit onto the topographic images using DockAFM. Comparative surface area accessibility studies (SAAS) using YASARA were performed to understand the effect of the B-domain in binding the interacting partners.

Results: AFM topographs revealed globular/spherical conformations of FL-FVIII and BDD-FVIII, whereas elongated tails of vWF connected to globular FVIII (FVIII-vWF complex) (Image 1a). An average height of 6-7nm for the FL-FVIII, FVIII-vWF complex, and 4nm for BDD-FVIII was observed. Refined models were validated with low clash scores and MolProbity scores of < 2. Diverse orientations and spatial positionings of the B-domain in the models were identified from the docks obtained (Image 1b). SAAS suggests that FL-FVIII has a decreased interaction with inhibitors compared to BDD-FVIII.

Conclusion(s): Validated models emphasize the role of glycosylation in FL-FVIII stability. Our atomic model is compatible with AFM topographs, and the height variation is attributed to FL-FVIII's B-domain. SAAS differences suggest potential distinctions in the binding of interacting partners or antibodies between FL-FVIII and BDD-FVIII.