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Cellular uptake of coagulation factor VIII: Elusive role of the membrane-binding spikes in the C1 domain
- Source :
- International Journal of Biochemistry and Cell Biology, 89, 34. Elsevier Limited
- Publication Year :
- 2017
- Publisher :
- Elsevier BV, 2017.
-
Abstract
- Low density lipoprotein receptor-related protein 1 (LRP1) is involved in the catabolism of many ligands, including factor VIII (FVIII) and alpha-2-macroglobulin (α2M). Transfer of FVIII to LRP1 is currently believed to be preceded by pre-concentration on the cell surface, by interacting with a so far unidentified component. In the present study, we used confocal microscopy and flow cytometry to compare endocytosis of FVIII and α2M using U87MG cells. The results show that α2M is rapidly internalized and does not compete for LRP1 mediated internalization of FVIII. FVIII endocytosis did not occur in the presence of receptor-associated-protein (RAP), but FVIII remained visible as a striated fluorescent pattern at the cell borders. In the presence of Von Willebrand Factor (VWF), no FVIII was observed on or within the cells, suggesting that VWF blocks interaction with both cell surface and LRP1. The same dual inhibition has previously been observed for FVIII C1 domain directed monoclonal antibody KM33. Elimination of the KM33 epitope by replacing FVIII C1 residues 2091-2095 and 2155-2160 for the homologues from factor V (FV), however, did not impair FVIII endocytosis. These membrane spikes alone were insufficient for cellular uptake, because FV was neither internalized by U87MG cells nor capable of effectively competing for FVIII endocytosis. These results show that FVIII endocytosis is driven by interaction with LRP1, but at the same time involves the spikes in the C1 domain that have been implicated in lipid binding.
- Subjects :
- Models, Molecular
0301 basic medicine
receptor-mediated endocytosis
congenital, hereditary, and neonatal diseases and abnormalities
animal diseases
media_common.quotation_subject
Cell
Endocytosis
Biochemistry
Epitope
Cell Line
Flow cytometry
α2-macroglobulin
03 medical and health sciences
Protein Domains
Von Willebrand factor
hemic and lymphatic diseases
medicine
Humans
Internalization
low density lipoprotein receptor related protein 1
media_common
Factor VIII
biology
medicine.diagnostic_test
Chemistry
Cell Membrane
Factor V
Cell Biology
Receptor-mediated endocytosis
LRP1
Cell biology
030104 developmental biology
medicine.anatomical_structure
biology.protein
Low Density Lipoprotein Receptor-Related Protein-1
Protein Binding
Subjects
Details
- ISSN :
- 13572725
- Volume :
- 89
- Database :
- OpenAIRE
- Journal :
- The International Journal of Biochemistry & Cell Biology
- Accession number :
- edsair.doi.dedup.....c6922ed2c814eb8df4024af50c314565