Identification of Regions in Low-Density Lipoprotein Receptor-Related Protein Responsible for Interaction with A2 and A1/A3-C1-C2 Portions of Coagulation Factor VIII

Abstract number: OR248

Sarafanov¹ A, Makogonenko¹ E, Andersen² O, Khrenov¹ A, Mikhailenko¹ I, Strickland¹ D, Saenko¹ E

¹¹University of Maryland School of Medicine, Rockville, MD, USA ²²Max-Delbrück-Center for Molecular Medicine, Berlin, Germany

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 represented by complement-type repeats (CRs) organized in four clusters, among which the clusters II and IV appear to bind most of LRP ligands (Strickland, Thromb. Haemost. 2003). In turn, fVIII contains two major LRP-binding sites (Saenko, JBC 1999; Lenting, JBC 1999), located in A2 domain (A2) and in heterodimer A1/A3-C1-C2 (HD), the products of dissociation of activated fVIII. In present work we found that each of these portions of fVIII binds to multiple sites within the clusters II and IV. Using a baculovirus expression system, we generated the intact clusters II, III and IV along with eight overlapping fragments (3 × CR) encompassing clusters II and IV. Surface plasmon resonance-based assays demonstrated that both A2 and HD bind to the same sets of LRP fragments with similar affinities (K_Ds 10–50 nM), and that the minimal binding unit of LRP is formed by at least a pair of CRs, similarly to that shown previously for another ligand of LRP, receptor associated protein (RAP) (Andersen, JBC 2000). Notably, some mutations of the LRP-binding site in A2 resulted in abolishment or significant reduction of its binding to certain fragments of LRP, while the binding to other LRP fragments was less affected. The specificity of A2 and HD for binding to the short fragments of LRP was confirmed by the ability of RAP to inhibit these interactions, and by the ability of these fragments to inhibit LRP-mediated catabolism of ¹²⁵I-A2 and ¹²⁵I-HD in cell culture. In summary, we demonstrated that the binding sites for A2 and HD in LRP are represented by at least two adjacent CRs spanning CR3–CR8 in the cluster II, and are likely to be organized similarly within CR24–CR29 in the cluster IV. The above data also suggest that besides regulating fVIII levels, LRP also plays an important role in clearance of the remnants of activated fVIII.