Catabolism of coagulation factor VIII (fVIII) is mediated by the hepatic multiligand receptor low-density lipoprotein receptor-related protein (LRP). The ligand-binding sites of LRP are formed by complement-type repeats (CRs) organized in four clusters, among which clusters II and IV bind most of LRP ligands. In turn, fVIII contains two major LRP-binding sites, located in A2 and A3 domains (Saenko et al, JBC 1999; Bovenschen et al, JBC 2003). In present work, we characterized binding sites in LRP for A2 domain (A2) and heterodimer A1/A3-C1-C2 (HD), the products of dissociation of activated fVIII. Using a baculovirus expression system, we generated CR clusters II, III and IV, along with eight overlapping CR triplets encompassing clusters II and IV. Surface plasmon resonance-based assays demonstrated that both A2 and HD bind to clusters II and IV, and to the same sets of their CR triplets with similar affinities (KDs 25–50 nM). The same kinetic parameters of interaction of both A2 and HD were observed for several CR doublets from cluster II, shown previously to be minimal binding sites for a classical ligand of LRP, receptor associated protein (RAP) (Andersen et al, JBC 2000). The specificity of A2 and HD interactions with all tested fragments of LRP was confirmed by the ability of RAP to inhibit these interactions, and by the ability of these fragments to inhibit binding of 125I-A2 and 125I-HD to immobilized LRP in a solid-phase assay, and LRP-mediated catabolism of 125I-A2 and 125I-HD in cell culture. Notably, some mutations of the LRP-binding site in A2 resulted in significant reduction or abolishment of its binding to certain fragments of LRP, while the binding to other LRP fragments was less affected. In summary, we demonstrated that i) A2 and HD interact with LRP via its multiple binding sites spanning CRs 3–8 in cluster II and CRs 24–29 in cluster IV, and ii) the elementary binding unit of LRP is formed by at least two adjacent CRs, similar to that shown for RAP. The above data also suggest that besides regulating fVIII levels, LRP also plays a role in clearance of the products of dissociation of activated fVIII.

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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 KD(1) ~1, 4, 14 nM and KD(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 KD ~60 nM. We registered a dose-dependent, high-affinity binding of the isolated A2 domain to LDLR, with KD ~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.

SummaryCatabolism of coagulation factorVIII (FVIII) is mediated by lowdensity lipoprotein receptor-related protein (LRP). The ligandbinding sites of LRP are formed by complement-type repeats (CR), and CR clusters II and IV bind most of LRP ligands. FVIII contains two major LRP-binding sites located in the A2 and A3 domains. This study was aimed to identify specific complementtype repeats of LRP involved in interaction with the A2 site and to probe their functional importance in A2 catabolism. We generated individual LRP clusters II, III and IV, along with nine overlapping CR triplets encompassing clusters II and IV in a baculovirus expression system and studied their interaction with isolated A2. In surface plasmon resonance (SPR) assay, A2 bound to clusters II and IV with KDs 22 and 39 nM, respectively, and to the majority of CR triplets with affinities in the range of KDs 25–90 nM. Similar affinities were determined for A2 interaction with a panel of CR doublets overlapping cluster II (CR 3–4, 4–5, 5–6 6–7 and 7–8). These LRP fragments inhibited the binding of 125I-A2 to LRP in solid-phase assay,LRP-mediated internalization of 125I-A2 in cell culture and 125I-A2 clearance from the mouse circulation. Point mutations of critical A2 residues of the LRPbinding site resulted in differential reduction or abolishment of its binding to LRP fragments. We conclude that A2 interacts with LRP via multiple binding sites spanning CR 3–8 in cluster II and CR 23–29 in cluster IV, and the minimal A2-binding unit of LRP is formed by two adjacent CR.

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