Save
Characterization of Factor VIII Binding Sites on the Low-density Lipoprotein Receptor-related Protein 1 (LRP) Suggests a Dynamic Bivalent Mode of this Interaction
ISTH Academy. Sarafanov A. Jul 10, 2019; 273815; OC 76.3
Dr. Andrey Sarafanov
Dr. Andrey Sarafanov
Access to Reserved content is available to attendees of the Congress until the end of the year and always available for full ISTH members.

Click here to join ISTH or renew your membership.

Abstract
Discussion Forum (0)
Rate & Comment (0)

OC 76.3

Characterization of Factor VIII Binding Sites on the Low-density Lipoprotein Receptor-related Protein 1 (LRP) Suggests a Dynamic Bivalent Mode of this Interaction

H. Chun, J. Kurasawa, G. Uceda Cortez, S. Shestopal, E. Karnaukhova, T. Lee, A. Sarafanov
U.S. Food and Drug Administration, CBER, Silver Spring, United States

Main Topic: Coagulation and Anticoagulation
Category: FVIII/IX

Background: Current replacement therapy of Hemophilia A is limited by a relatively short plasma half-life of Factor VIII (FVIII) or its modified variants (12-19 h). Previous studies demonstrated the involvement of hepatic low-density lipoprotein receptor-related protein 1 (LRP) in FVIII clearance. Such studies also characterized FVIII-binding regions on LRP (Meijer et al, 2007) and proposed a bivalent mode for this interaction (Young et al, 2016). In the present study, we further investigated these mechanisms.
Aims: To map the LRP site(s) for binding FVIII using several in vitro approaches.
Methods: Recombinant LRP fragments were produced in a baculovirus expression system and purified by chromatography. The fragments were tested for interactions with FVIII by surface plasmon resonance (SPR) and cell-based flow cytometry assay for LRP-mediated uptake of FVIII by human hepatic Huh-7 cells. Correctness of the overall folding of selected LRP fragments was verified by circular dichroism spectroscopy.
Results: Several LRP fragments comprising specific complement-type repeats (CRs) were found to bind FVIII with affinity similar to that of LRP (KD = 50-100 nM) by SPR. The same fragments consistently inhibited LRP-mediated internalization of FVIII in the tissue culture model. Specificity of these interactions was confirmed by silencing LRP expression and using, as competitors,
(i) an anti-FVIII antibody fragment,
(ii) receptor-associated protein (RAP) and
(iii) mutated variants of the LRP fragments.
Conclusions: We identified specific CR-doublets of LRP forming primary sites for interaction with a specific region on FVIII. These results support the bivalent nature of interaction between FVIII and LRP, and indicate that the bivalent sites of the receptor interact with FVIII alternatively in a dynamic mode. These data further contribute to understanding FVIII clearance mechanisms and may be useful for generation of new FVIII variants with extended half-life in the circulation.

Code of conduct/disclaimer available in General Terms & Conditions
Anonymous User Privacy Preferences

Strictly Necessary Cookies (Always Active)

MULTILEARNING platforms and tools hereinafter referred as “MLG SOFTWARE” are provided to you as pure educational platforms/services requiring cookies to operate. In the case of the MLG SOFTWARE, cookies are essential for the Platform to function properly for the provision of education. If these cookies are disabled, a large subset of the functionality provided by the Platform will either be unavailable or cease to work as expected. The MLG SOFTWARE do not capture non-essential activities such as menu items and listings you click on or pages viewed.


Performance Cookies

Performance cookies are used to analyse how visitors use a website in order to provide a better user experience.



Google Analytics is used for user behavior tracking/reporting. Google Analytics works in parallel and independently from MLG’s features. Google Analytics relies on cookies and these cookies can be used by Google to track users across different platforms/services.


Save Settings