Characterization of the binding and clearance mechanisms of coagulation factor VIII by LRP1
AdvisorStrickland, Dudley K.
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AbstractThe LDL receptor-related protein 1 (LRP1) is a member of the LDL receptor family and mediates the endocytosis of numerous structurally unrelated ligands. Currently, it is not clear how LRP1 recognizes all these ligands, but based on a structure of RAP D3 domain with fragments from the LDL receptor, a model has been proposed. The major concern with this model is that the RAP/LDLR interaction does not occur physiologically and thus the model may not universally apply to other receptor/ligands. We hypothesized that this model, in which lysine residues on the ligand dock into acidic pockets on the receptor, may serve as a common mechanism for LRP1 ligand binding. This hypothesis was investigated in studies examining the binding of the D1D2 domains of RAP to LRP1. This work identified a critical role for lysine 60 in D1 and lysine 191 in D2 for binding to LRP1 and revealed that D1D2 forms a bivalent complex with LRP1. The binding mechanism between LRP1 and another known ligand, coagulation factor VIII (fVIII), is not well understood, as most of the studies have employed fragments of fVIII and LRP1. Through the combination of surface plasmon resonance and ELISA measurements we confirmed that, similar to D1D2 of RAP, lysine residues on fVIII interact with LRP1 through the formation of a bivalent complex. We also determined that while fVIII binds to soluble forms of cluster II and IV of LRP1, only soluble cluster IV competed with the binding of fVIII to full length LRP1, revealing that cluster IV represents the major fVIII binding site on LRP1. Through the use of an activation-resistant fVIII mutant, we determined that activation by thrombin is not required for removal of fVIII from circulation. Combined, these experiments reveal that lysine residues accessible in the non-activated form of fVIII interact with the cluster IV region of LRP1 to form a high affinity, bivalent complex. Our work may contribute to the development of approaches that will extend the half-life of fVIII in circulation, in order to make prophylactic treatment easier to maintain for Hemophilia A patients.
DescriptionUniversity of Maryland, Baltimore. Molecular Medicine. Ph.D. 2016