OC 55.2 - Characterization of the Factor VIII and LRP1 Interaction Suggests a Dynamic Binding Mode with Switching of Alternative Multiple Canonical Bivalent and Non-Canonical Electrostatic Contacts

Tuesday, July 12, 2022

11:00 AM – 11:15 AM

Location: ExCel Center, ICC Capital Suite Room 8&9

Presentation Format: In-Person

Co-Author(s)

HC

Haarin Chun

Biologist
U.S. Food and Drug Administration
Silver Spring, Maryland, United States
JK

James H. Kurasawa

Scientist
AstraZeneca
Gaithersburg, Maryland, United States
PO

Philip Olivares

ORISE fellow
U.S. Food and Drug Administration
Silver Spring, Maryland, United States
EM

Ekaterina Marakasova

Scientist
Food and Drug Administration
Silver Spring, Maryland, United States
SS

Svetlana A. Shestopal

Scientist
Food and Drug Administration
Silver Spring, Maryland, United States
GH

Gabriela U. Hassink

Scientist
GSK-Rockville Center for Vaccines Research
Rockville, Maryland, United States
EK

Elena Karnaukhova

Scientist
Food and Drug Administration
Silver Spring, Maryland, United States
MM

Mary Migliorini

Scientist
University of Maryland School of Medicine
Baltimore, Maryland, United States
JO

Juliet O. Obi

Scientist
University of Maryland School of Pharmacy
Baltimore, Maryland, United States
AS

Ally K. Smith

Scientist
University of Maryland School of Pharmacy
Baltimore, Maryland, United States
PW

Patrick L. Wintrode

Professor
University of Maryland School of Pharmacy
Baltimore, Maryland, United States
PD

Prasannavenkatesh Durai

Scientist
Korea Institute of Science and Technology
Gangneung, Seoul-t'ukpyolsi, Republic of Korea
KP

Keunwan Park

Scientist
Korea Institute of Science and Technology
Gangneung, Seoul-t'ukpyolsi, Republic of Korea
DD

Daniel Deredge

Assistant Professor
University of Maryland School of Pharmacy
Baltimore, Maryland, United States
DS

Dudley K. Strickland

Professor
University of Maryland School of Medicine
Baltimore, Maryland, United States

Presenting Author(s)

Andrey G. Sarafanov, PhD

Chemist
U.S. Food and Drug Administration
Silver Spring, Maryland, United States

Background: Deficiency in blood coagulation factor VIII (FVIII) results in life-threating bleeding (Hemophilia A), which is treated by infusions of therapeutic FVIII. Understanding FVIII plasma clearance mechanisms facilitates generation of prolonged plasma lifetime FVIII to improve the disease treatment. An important FVIII clearance receptor is the hepatic low-density lipoprotein receptor‐related protein 1 (LRP1). Previous studies indicated multiplicity of FVIII binding sites for LRP1 and complexity of the molecules’ interaction, at the same time, defined to be bivalent. However, the exact mechanism of this interaction remains unknown.

Aims: To characterize the bivalent sites of LRP1 and build a model of its interaction with FVIII.

Methods: Recombinant ligand-binding complement-type repeat (CR) fragments of LRP1 and their mutant variants were generated using a baculovirus system and tested for interactions with FVIII using surface plasmon resonance, a tissue culture model of LRP1-mediated internalization of FVIII, hydrogen‐deuterium exchange mass-spectrometry, and in silico docking.

Results: We identified a series of adjacent LRP1 CR doublets providing minimal requisite sites for binding FVIII and showed an additive effect of other CR domains on FVIII-LRP1 affinity. A CR doublet was found to provide the bivalent interaction following the canonical mode of ligand recognition by the receptor in addition to the non-canonical electrostatic contributions of other CR domains. For selected CR doublets, we found numerous contact sites on FVIII.

Conclusion(s): We characterized LRP1 bivalent binding sites for FVIII, confirmed their relevance to the canonical mode, and described a real-time LRP1 interactive site. We propose that LRP1 interacts with FVIII via a string of CR domains providing both canonical bivalent and non-canonical electrostatic contacts switched over time in a dynamic mode (Fig. 1) that represents a novel mechanism of biomolecular interaction. Disclaimer: This is an informal communication, and it represents the authors’ own best judgment. These comments do not bind or obligate FDA.