Abstract
Hemophilia A is a bleeding disorder caused by a deficiency in coagulation factor VIII (fVIII) that affects 1 in 5,000 males. Current prophylactic replacement therapy, although effective, is difficult to maintain due to the cost and frequency of injections. Hepatic clearance of fVIII is mediated by the LDL receptor-related protein 1 (LRP1), a member of the LDL receptor family. Although it is well established that fVIII binds LRP1, the molecular details of this interaction are unclear as most of the studies have been performed using fragments of fVIII and LRP1. In the current investigation, we examine the binding of intact fVIII to full-length LRP1 to gain insight into the molecular interaction. Chemical modification studies confirm the requirement for lysine residues in the interaction of fVIII with LRP1. Examination of the ionic strength dependence of the interaction of fVIII with LRP1 resulted in a Debye-Hückel plot with a slope of 1.8 ± 0.5, suggesting the involvement of two critical charged residues in the interaction of fVIII with LRP1. Kinetic studies utilizing surface plasmon resonance techniques reveal that the high affinity of fVIII for LRP1 results from avidity effects mediated by the interactions of two sites in fVIII with complementary sites on LRP1 to form a bivalent fVIII·LRP1 complex. Furthermore, although fVIII bound avidly to soluble forms of clusters II and IV from 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 in LRP1.
Highlights
Hemophilia A is a bleeding disorder caused by a deficiency in coagulation factor VIII that affects 1 in 5,000 males
The results (Fig. 1C) reveal that chemical modification of the BDD-factor VIII (fVIII) lysine residues with sulfo-NHS-biotin substantially reduced its binding to LDL receptor-related protein 1 (LRP1), suggesting a critical role for these residues in the binding interaction of BDD-fVIII to full-length LRP1
First we demonstrated that modification of lysine residues in fVIII by reaction of the molecule with sulfo-NHS-biotin dramatically impacted its recognition by full-length LRP1
Summary
Hemophilia A is a bleeding disorder caused by a deficiency in coagulation factor VIII (fVIII) that affects 1 in 5,000 males. The identification of LRP1 as a receptor involved in fVIII catabolism was originally reported by Saenko et al [11] and Lenting et al [7] when they noted a direct interaction between fVIII and LRP1 and observed that LRP1-expressing cells, but not LRP1-deficient cells, were able to mediate the internalization of 125I-labeled fVIII [11] These studies established that LRP1 binds fVIII in a process inhibited by receptor-associated protein (RAP). Ligand binding by this family of receptors appears to involve the docking of two or more lysine residues into acidic pockets located within the CR modules of the receptor [27] This has been clearly demonstrated for RAP, which contains two binding sites for LRP1. Our data reveal that cluster IV on LRP1 represents the major fVIII binding site on this receptor
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