Cluster III of low-density lipoprotein receptor-related protein 1 binds activated blood coagulation factor VIII

Low-density lipoprotein receptor-related protein 1 (LRP) mediates clearance of blood coagulation factor VIII (FVIII). In LRP, FVIII binds the complement-type repeats (CRs) of clusters II and IV, which also bind a majority of other LRP ligands. No ligand is known for LRP cluster I, and only three ligands, including the LRP chaperone alpha-2 macroglobulin receptor-associated protein (RAP), bind cluster III. Using surface plasmon resonance, we found that in addition to clusters II and IV, activated FVIII (FVIIIa) binds cluster III. The specificity of this interaction was confirmed using an anti-FVIII antibody fragment, which inhibited the binding. Recombinant fragments of cluster III and its site-directed mutagenesis were used to localize the cluster's site for binding FVIIIa to CR.14-19. The interactive site of FVIIIa was localized within its A1/A3'-C1-C2 heterodimer (HDa), which is a major physiological remnant of FVIIIa. In mice, the clearance of HDa was faster than that of FVIII and prolonged in the presence of RAP, which is known to inhibit interactions of LRP with its ligands. In accordance with this, the cluster III site for RAP (CR.15-19) was found to overlap that for FVIIIa. Altogether, our findings support the involvement of LRP in FVIIIa catabolism and suggest a greater significance of the biological role of cluster III compared to that previously known.