Functional duplication of ligand-binding domains within low-density lipoprotein receptor-related protein for interaction with receptor associated protein, alpha2-macroglobulin, factor IXa and factor VIII.

The low-density lipoprotein receptor-related protein (LRP) binds a range of proteins including receptor associated protein (RAP), activated alpha2-macroglobulin (alpha2M*), factor IXa (FIXa), and factor VIII (FVIII) light chain. The binding is mediated by the complement-type repeats, which are clustered in four distinct regions within LRP. Cluster II of 8 repeats (CR3-10) and cluster IV of 11 repeats (CR21-31) have been implicated in ligand-binding. Previous studies have aimed to identify the cluster II repeats involved in binding alpha2M* and RAP. We now evaluated the binding to RAP, alpha2M*, FIXa and FVIII light chain of triplicate repeat-fragments of not only clusters II but also of cluster IV. Employing surface plasmon resonance analysis, we found that most efficient ligand-binding was displayed by the repeats within region CR4-8 of cluster II and within region CR24-28 of cluster IV. Whereas the binding to RAP could be attributed to two consecutive repeats (CR5-6, CR26-27), combinations of three repeats showed most efficient binding to FIXa (CR6-8, CR26-28), FVIII light chain (CR5-7, CR6-8, CR24-26), and alpha2M* (CR4-6, CR24-26). The results imply that there is an internal functional duplication of complement-type repeats within LRP resulting in two clusters that bind the same ligands.

Low density lipoprotein receptor-related protein and factor IXa share structural requirements for binding to the A3 domain of coagulation factor VIII.

Low-density lipoprotein receptor-related protein (LRP) is an endocytic receptor that binds multiple distinct ligands, including blood coagulation factor VIII (FVIII). FVIII is a heterodimeric multidomain protein that consists of a heavy chain (domains A1, a1, A2, a2, and B) and a light chain (domains a3, A3, C1, and C2). Both chains contribute to high-affinity interaction with LRP. One LRP-interactive region has previously been located in the C2 domain, but its affinity is low in comparison with that of the entire FVIII light chain. We now have compared a variety of FVIII light chain derivatives with the light chain of its homolog FVa for LRP binding. In surface plasmon resonance studies employing LRP cluster II, the FVa and FVIII light chains proved different in that only FVIII displayed high-affinity binding. Because the FVIII a3-A3-C1 fragment was effective in associating with LRP, this region was explored for structural elements that are exposed but not conserved in FV. Competition studies using synthetic peptides suggested that LRP binding involves the FVIII-specific region Lys(1804)-Ala(1834) in the A3 domain. In line with this observation, LRP binding was inhibited by a recombinant antibody fragment that specifically binds to the FVIII sequence Glu(1811)-Lys(1818). The role of this sequence in LRP binding was further tested using a FVIII/FV chimera in which sequence Glu(1811)-Lys(1818) was replaced with the corresponding sequence of FV. Although this chimera still displayed residual binding to LRP cluster II, its affinity was reduced. This suggests that multiple sites in FVIII contribute to high-affinity LRP binding, one of which is the FVIII A3 domain region Glu(1811)-Lys(1818). This suggests that LRP binding to the FVIII A3 domain involves the same structural elements that also contribute to the assembly of FVIII with FIXa.

Residues Phe342-Asn346 of activated coagulation factor IX contribute to the interaction with low density lipoprotein receptor-related protein.

When blood coagulation factor IX is converted to activated factor IX (factor IXa), it develops enzymatic activity and exposes the binding sites for both activated factor VIII and the endocytic receptor low density lipoprotein receptor-related protein (LRP). In the present study we investigated the interaction between factor IXa and LRP in more detail, using an affinity-purified soluble form of LRP (sLRP). Purified sLRP and full-length LRP displayed similar binding to factor IXa. An anti-factor IX monoclonal antibody CLB-FIX 13 inhibited factor IXa.sLRP complex formation. Both the antibody and a soluble recombinant fragment of LRP (i.e. cluster IV) interfered with factor IXa amidolytic activity, suggesting that the antibody and LRP share similar binding regions near the active site of factor IXa. Next, a panel of recombinant factor IXa variants with amino acid replacements in the surface loops bordering the active site was tested for binding to antibody CLB-FIX 13 and sLRP in a solid phase binding assay. Factor IXa variants with mutations in the region Phe(342)-Asn(346), located between the active site of factor IXa and factor VIII binding helix, showed reduced binding to both antibody CLB-FIX 13 and sLRP. Surface plasmon resonance analysis revealed that the variant with Asn(346) replaced by Asp displayed slower association to sLRP, whereas the variant with residues Phe(342)-Tyr(345) replaced by the corresponding residues of thrombin showed faster dissociation. Recombinant soluble LRP fragment cluster IV inhibited factor IXa-mediated activation of factor X with IC(50) values of 5 and 40 nm in the presence and absence of factor VIII, respectively. This inhibition thus seems to occur via two mechanisms: by interference with factor IXa.factor VIIIa complex assembly and by direct inhibition of factor IXa enzymatic activity. Accordingly, we propose that LRP may function as a regulator of blood coagulation.