title: C1 domain residues Lys 2092 and Phe 2093 are of major importance for the endocytic uptake of coagulation factor VIII.
abstract: Factor VIII (FVIII) catabolism has been demonstrated to involve LDL receptor-related protein (LRP). We have established that antibody fragment KM33 inhibits cofactor function of FVIII by interacting with the membrane binding region 2092-2093 of the C1 domain. As KM33 also inhibits LRP-dependent uptake of FVIII, we now assessed the role of region 2092-2093 for LRP-dependent endocytosis. For this purpose, we employed functional fluorescent FVIII-YFP or -GFP derivatives and U87MG cells which express high levels of LRP. Confocal microscopy studies and flow cytometry analysis combined with siRNA technology showed that the fluorescent FVIII derivatives are indeed effectively internalized by U87MG cells in a LRP-dependent manner. Competition experiments employing an antagonist of the LDL receptor family members revealed that there is a cell surface binding event for FVIII, which is independent of LRP. Cell surface binding proved to be less effective for the FVIII-YFP variants K2092A, F2093A and K2092A/F2093A. Surface plasmon resonance analysis showed that these substitutions affect LRP binding as well. Finally, flow cytometry analysis revealed a major reduction of endocytic uptake of these FVIII-YFP variants. Our results demonstrate that C1 domain residues 2092-2093 are of major importance for FVIII endocytosis by contributing to cell surface binding and receptor binding.
authors.affiliation: Department of Plasma Proteins, Sanquin Research, Amsterdam, The Netherlands.
authors.forename: Henriët
authors.initials: H
authors.lastname: Meems
authors.forename: Maartje
authors.initials: M
authors.lastname: van den Biggelaar
authors.forename: Mariska
authors.initials: M
authors.lastname: Rondaij
authors.forename: Carmen
authors.initials: C
authors.lastname: van der Zwaan
authors.forename: Koen
authors.initials: K
authors.lastname: Mertens
authors.forename: Alexander B
authors.initials: AB
authors.lastname: Meijer
mesh_terms: D001665:Binding Sites; D001692:Biological Transport; D045744:Cell Line, Tumor; D004705:Endocytosis; D005169:Factor VIII / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D026503:Low Density Lipoprotein Receptor-Related Protein-1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D008239:Lysine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
chemical_list: D026503:Low Density Lipoprotein Receptor-Related Protein-1; D010649:Phenylalanine; D005169:Factor VIII; D008239:Lysine