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
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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.

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