Von Willebrand Factor A1 Domain Binds To Clusters Ii And Iv Of Low-Density Lipoprotein Receptor-Related Protein 1

Abstract Introduction. Von Willebrand factor (VWF) is a large multimeric plasma glycoprotein involved in hemostasis and thrombosis. Genetic and in vivo studies have identified low-density lipoprotein receptor-related protein 1 (LRP) as the major clearance receptor of VWF. The ligand-binding moiety of LRP is represented by complement-type repeats grouped into four clusters, among which clusters II and IV are prominent in ligand binding. In in vitro studies under shear stress conditions, VWF acquires an unfolded conformation and interacts with LRP. More than 30 mutations of VWF have been reported to associate with its increased plasma clearance and result in type 2B von Willebrand disease. The majority of these mutations are located within the VWF A1 domain (A1), which is in agreement with previous reports that isolated A1 binds to LRP. However, the interactive site of LRP has not been identified. Objective. We aim to characterize the LRP binding site for VWF by investigating the interaction of individual LRP clusters II-IV to different A1 variants. Experimental Approach. An A1-containing large N-terminal homo-dimeric proteolytic fragment of VWF (SPIII) was produced upon its proteolytic digestion by V8-protease. Alternatively, we generated three variants of recombinant A1: (i) A1(residues 1261-1478), (ii) a VWD type 2B mutant, A1(V1316M) (residues 1261-1478) and (iii) an N-terminus truncated form, (residues 1269-1478). These proteins were tested for binding to immobilized LRP, purified from human placenta, and recombinant LRP clusters II and IV by surface plasmon resonance. Results. We did not find any interaction between the SPIII proteolytic fragment and LRP, suggesting that this fragment may still preserve a folded conformation of VWF that masked its LRP-binding site. In turn, all three forms of recombinant A1 were found to bind LRP and its clusters II and IV. Notably, A1(V1316M) and A1(1269-1478) had higher affinity for LRP and its clusters than A1. This result is consistent with the enhanced clearance of the VWF(V1316M) in vivo and better exposure of the A1 site in a truncated form of A1 to the platelet receptor GpIb-alpha reported previously. Conclusions. Our data demonstrate that LRP has at least two sites for binding to VWF, which are located within clusters II and IV. This finding is in accordance with previous observations that LRP clusters II and IV can serve as “functional duplicates” for binding selected ligands. Disclosures No relevant conflicts of interest to declare.