title: Evidence That Factor VIII Forms a Bivalent Complex with the Low Density Lipoprotein (LDL) Receptor-related Protein 1 (LRP1): IDENTIFICATION OF CLUSTER IV ON LRP1 AS THE MAJOR BINDING SITE.
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.
authors: From the Center for Vascular and Inflammatory Disease and the Departments of Surgery and Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201.
authors: Patricia A
authors: PA
authors: Young
authors: From the Center for Vascular and Inflammatory Disease and the Departments of Surgery and Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201.
authors: Mary
authors: M
authors: Migliorini
authors: From the Center for Vascular and Inflammatory Disease and the Departments of Surgery and Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201 dstrickland@som.umaryland.edu.
authors: Dudley K
authors: DK
authors: Strickland
mesh_terms: D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D006224:Cricetinae; D005169:Factor VIII / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D026503:Low Density Lipoprotein Receptor-Related Protein-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008297:Male; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020349:Surface Plasmon Resonance
chemical_list: C475476:LRP1 protein, human; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D046912:Multiprotein Complexes; C078147:F8 protein, human; D005169:Factor VIII
keywords: LRP; blood coagulation; endocytosis; factor VIII (FVIII); ligand; lipoprotein receptor; lipoprotein receptor-related protein (LPR); lysine; surface plasmon resonance (SPR)