Abstract Number: PB0098
Meeting: ISTH 2021 Congress
Background: Extension of the plasma half-life of therapeutic factor VIII (FVIII) products is important for improving their efficacy in treatment of Hemophilia A. This requires understanding mechanisms of FVIII plasma clearance mediated by hepatic low-density lipoprotein receptor (LDLR) and LDLR-related protein 1 (LRP1), which were shown to significantly contribute to FVIII clearance in mice model (Bovenschen, 2005). Therefore, it is of interest to establish also a cell-culture based model to study these interactions.
Aims: To develop a method to test FVIII internalization mediated by LDLR/LRP1 in cell culture.
Methods: We measured the expression level of LDLR and LRP1 in four human hepatic established cell lines and evaluated relative contribution of both receptors on FVIII internalization by RNAi silencing. FVIII internalization in the presence and absence of LRP1 fragments and FVIII inhibitors were measured using flow cytometry.
Results: Huh-7 human hepatic cells showed the highest expression of LDLR and LRP1 compared to HepG2, Hep3B, and SK-HEP1 cells. Using Huh-7 cells, we observed dose- and time-dependent FVIII uptake that allowed to select the optimal assay conditions. Inhibition of LRP1 expression by RNAi or use of competitive ligands, specific to either LDLR/LRP1 or FVIII, resulted in 60~70% decrease of FVIII internalization, indicating a more prominent role of LRP1 than LDLR in FVIII uptake.
Conclusions: We developed a novel cell-based assay to measure LDLR/LRP1-mediated uptake of FVIII using Huh-7 cells. This model system can be used to study mechanisms of LDLR/LRP1-mediated clearance of FVIII. In particular, this system can be used for testing FVIII variants with extended plasma half-life to facilitate generation of more efficient therapeutic FVIII products.
Disclaimer: This is an informal communication and it represents the authors’ own best judgment. These comments do not bind or obligate FDA.
To cite this abstract in AMA style:Chun H, G Sarafanov A. Development of a Cell-based Assay to Test Factor VIII Internalization Mediated by the Low-density Lipoprotein Receptor (LDLR) and LDLR-related Protein 1 (LRP1) [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 1). https://abstracts.isth.org/abstract/development-of-a-cell-based-assay-to-test-factor-viii-internalization-mediated-by-the-low-density-lipoprotein-receptor-ldlr-and-ldlr-related-protein-1-lrp1/. Accessed September 24, 2021.
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