Cellular receptors involved in the uptake of coagulation factors

Principal investigators: Sander Meijer PhD and prof Koen Mertens PhD

LDL receptor-related protein (LRP) contributes to the clearance of coagulation factor VIII (FVIII) from the circulation. Ligand binding of the low-density lipoprotein (LDL) receptor family is mediated by clusters of small complement-type repeats (CR). It has been proposed that at least two CRs are required for high-affinity interaction by utilizing two spatially distinct lysine residues on the ligand surface. LRP mediates the cellular uptake of a multitude of ligands, some of which bind LRP with a relatively low affinity suggesting a suboptimal positioning of the two critical lysines. We have recently addressed the role of the two critical lysines not only for LRP binding but also for endocytosis, initially by employing Receptor Associated Protein (RAP) as a model ligand. Variants of the third domain (D3) of RAP were constructed in which lysines were replaced by alanine or arginine at the putative contact residues K253, K256 and K270. Surface Plasmon Resonance revealed that replacement of K253 has no effect on high-affinity LRP binding at all whereas replacement of either K256 or K270 markedly reduced the binding affinity. The interaction was completely abolished when both lysines were replaced. Substitution by either alanine or arginine exerted an almost identical effect on LRP binding, suggesting arginine residues do not support receptor binding. Confocal microscopy and flow cytometry studies revealed surprisingly that the single mutants were still internalized by cells. We therefore propose that the presence of only one critical lysine is sufficient to drive endocytosis.

We next set out to identify the contribution of lysines in the interaction between FVIII and LRP. We have established that antibody fragment KM33 inhibits the co-factor function of FVIII by interacting with the membrane binding region K2092-F2093 of the C1 domain. As KM33 also blocks the interaction between LRP and FVIII, we now assessed the role of K2092 for LRP-dependent endocytosis. For this purpose, we employed FVIII-YFP 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 internalized effectively 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 K2092-F2093 are of major importance for FVIII endocytosis by contributing to cell surface binding and receptor binding. 

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Last edited on: 18 February 2016