Fragments of Activated Coagulation Factor VIII Bind to Multiple Sites within Low-Density Lipoprotein Receptor-Related Protein.

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 (K_Ds 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.