Cellular receptors involved in the uptake of coagulation factors

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. LDL receptor-related protein (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 surprisingly revealed 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 individual lysines in the interaction between FVIII and LRP. To systematically address this issue, we constructed a library of more than 40 FVIII variants carrying lysine to arginine or alanine substitutions. The interaction of the variants with LRP cluster II was evaluated using Surface Plasmon Resonance (SPR) analysis. Our SPR analysis showed that multiple lysines in the A3C1 domains contributed to the interaction. However, none of the substitutions completely abolished LRP cluster II binding. Of the various lysines within the A3C1 domains, the C1 residues K2065 and K2092 proved the most important. The finding that all variants still displayed substantial residual binding suggests that also other surface exposed residues in the FVIII light chain contribute to LRP interaction. Crystal structure analysis and docking studies revealed that all the putative ‘hot spot’ lysines may spatially align with the acidic binding pockets of the LDL receptor. We therefore propose that LRP cluster II interacts with the FVIII light chain via an extended surface that starts at the bottom of the C1 domain and extends to the top of the A3 domain.

LDL receptor (blue) in interaction with factor VIII (yellow). Indicated in red are lysine residues of factor VIII that interact with the CR domains of LDL receptor. The calcium ions critical for correct folding of a CR domain are shown in green.