Localization of Binding Sites for Low-Density Lipoprotein Receptor in Coagulation Factor VIII.

Clearance of coagulation factor VIII (fVIII) is mediated by a hepatic receptor low-density lipoprotein receptor-related protein (LRP), a member of low-density lipoprotein receptor (LDLR) family. It has been recently discovered that LDLR acts in concert with LRP in regulating fVIII level. FVIII has the domain structure A1-A2-B-A3-C1-C2, and to identify the portions providing the interaction with LDLR, in surface plasmon resonance-based assay we studied the binding of fVIII and its fragments to immobilized recombinant ligand-binding domain of LDLR (residues 1-292). The affinity values were determined from the families of binding signals obtained for five concentrations (10–150 nM) of each analyte. The binding signals for full-length fVIII, and its portions A3-C1–C2 (or light chain, LCh) and A1/A3-C1–C2 heterodimer (derived from activated fVIII) were best fitted to a two-site model, with equilibrium dissociation constants K_D(1) ~1, 4, 14 nM and K_D(2) ~14, 45 and 37 nM, respectively. Noteworthy, we did not observe any significant binding for the isolated C2 domain (at 300 nM). This suggests that the LDLR-binding site within LCh is likely located within the A3 domain, similar to that found previously for LCh-LRP interaction. The binding signals for A1-A2-B (heavy chain, HCh) were best fitted to a one-site model, with K_D ~60 nM. We registered a dose-dependent, high-affinity binding of the isolated A2 domain to LDLR, with K_D ~14 nM whereas the A1 domain showed no appreciable binding. This suggests that within HCh, A2 domain bears the LDLR-binding site. Von Willebrand factor did not significantly block the binding of fVIII to LDLR as compared to a 3-fold inhibition of fVIII binding to LRP. This indicates that within fVIII/vWf complex, the A2 binding site for LDLR is more available than that for LRP. Anti-A2 monoclonal antibody 413 (epitope 484–509) inhibited the A2 binding to LDLR in a dose-dependent manner, similarly to that demonstrated for fVIII-LRP interaction. A number of A2 point mutants with substitutions of the residues critical for A2 binding to LRP, megalin and VLDL were found to have significantly reduced affinity also for LDLR. The obtained data indicate that fVIII interacts with LDLR preferentially via the binding sites located within the A2 domain of HCh and within the A3 domain of LCh, and that the A2 site is likely to be universal for the interactions with four tested members of LDL receptor family.