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

Abstract

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.

Proteolytic cleavage of factor VIII heavy chain is required to expose the binding‐site for low‐density lipoprotein receptor‐related protein within the A2 domain

“…Indeed, previous studies from our laboratory have established that FVIII light chain displays high‐affinity LRP binding [14,20]. Recently, the same finding has also been confirmed by Sarafanov et al [39]. Nevertheless it remains an open question whether or not this high‐affinity interaction drives the clearance of non‐activated FVIII from the circulation.…”

Identification of Coagulation Factor VIII A2 Domain Residues Forming the Binding Epitope for Low-Density Lipoprotein Receptor-Related Protein,

“…This can be explained by our most recent finding that within LRP clusters II and IV, the sites for A2 are multiple. Each elementary site is formed by two adjacent CRs interacting with A2 likely in a nonidentical way, which is evident from different effects of mutations on the A2 affinity for short fragments overlapping clusters II and IV (67). Therefore, it is likely that each A2 mutation affects the binding to certain but not all elementary sites in LRP, and the determined affinities correspond to the persisting interactions.…”

“…In this work, we also demonstrated the existence of a mechanism for removal of A2 from the circulation via LRP (and possibly other members of the LDLR family) that is consistent with a higher affinity of A2 for LRP in comparison with that for fVIII. Notably, the other product of fVIIIa dissociation, the A1/A3-C1-C2 heterodimer, also has a higher affinity for LRP than fVIII, can be internalized via LRP in cell culture (67), and can be cleared via LRP from mice circulation (A. Sarafanov, unpublished data). These results indicate that LRP may be involved in clearance of both products of dissociated fVIIIa, though the bulk of the A1/A3-C1-C2 moiety is likely to remain associated with platelets at the sites of clot formation.…”