The Light Chain of Factor VIII Comprises a Binding Site for Low Density Lipoprotein Receptor-related Protein

Abstract

In the present study, the interaction between the endocytic receptor low density lipoprotein receptor-related protein (LRP) and coagulation factor VIII (FVIII) was investigated. Using purified components, FVIII was found to bind to LRP in a reversible and dose-dependent manner (K d Ϸ 60 nM). The interaction appeared to be specific because the LRP antagonist receptor-associated protein readily inhibited binding of FVIII to LRP (IC 50 ; K d Ϸ 50 nM). Furthermore, experiments using recombinant FVIII C2 domain showed that this domain contributes to the interaction with LRP. In contrast, no association of FVIII heavy chain to LRP could be detected under the same experimental conditions. Collectively, our data demonstrate that in vitro LRP is able to bind FVIII at the cell surface and to mediate its transport to the intracellular degradation pathway. FVIII-LRP interaction involves the FVIII light chain, and FVIII-vWF complex formation plays a regulatory role in LRP binding. Our findings may explain the beneficial effect of vWF on the in vivo survival of FVIII.Low density lipoprotein receptor-related protein (LRP), 1 also known as ␣2-macroglobulin receptor, is a member of the low density lipoprotein receptor family of endocytic receptors (for a review, see Refs. 1 and 2). It consists of a heavy chain and a light chain, which are associated in a noncovalent manner. The 85-kDa light chain comprises the transmembrane and cytoplasmic domains, whereas the ligand binding regions are located within the 515-kDa heavy chain (3). LRP is abundantly present in various tissues such as the liver, placenta, lung, and brain (4) and is expressed in an array of cell types: parenchymal cells, neurons and astrocytes, Leydig cells, smooth muscle cells, monocytes, and fibroblasts (4). Commonly used cell lines such as monkey kidney COS cells and Chinese hamster ovary (CHO) cells also express LRP (5, 6). The function of LRP is to mediate the binding and transport of ligands from the cell surface to the endosomal degradation pathway (1, 2). Binding and internalization of ligands is antagonized by a 39-kDa chaperone protein designated receptor-associated protein (RAP) (7,8). Currently, a wide spectrum of structurally and functionally unrelated ligands involved in a variety of processes such as lipoprotein metabolism, cell growth and migration, and neuronal regeneration (1, 2) has been identified. Furthermore, LRP seems to be linked to the process of blood coagulation. This is apparent from the observations that LRP recognizes thrombin/ antithrombin and factor Xa/␣2-macroglobulin complexes and the Kunitz-type inhibitor tissue factor pathway inhibitor (9 -11). In addition, LRP contributes to down-regulation of tissue factor expression at the surface of monocytes (12). Coagulation factor VIII (FVIII) is the precursor of its activated derivative, which stimulates factor IXa-mediated activation of factor X (for recent reviews, see Refs. 13 and 14). The fact that deficiency or dysfunction of FVIII is associated with severe bleeding tendencies demon...

Mapping the Binding Region on the Low Density Lipoprotein Receptor for Blood Coagulation Factor VIII

“…In a series of studies by Mertens and co-workers (2,64,65), it was shown that the CR clusters of LDLR and LRP compete for binding to FVIII. Within each of the LRP clusters II and IV, approximately four adjacent CRs form a binding site for FVIII (31,33), whereas the matching epitope of FVIII involves multiple lysines on all three domains of its light chain (65)(66)(67)(68). Consistent with this, the anti-FVIII ScFv KM33, which recognizes the C1-domain of the light chain, interferes with FVIII binding to both LDLR and LRP (46,48,69).…”

“…At the same time, the FVIII-binding site in LDLR is unknown. In vitro, the affinity of FVIII to LDLR (K D of ϳ200 nM) (14) was found to be less than to LRP (K D of ϳ80 nM) (2,5,33). Such affinities are unlikely to provide effective direct interactions of FVIII with both receptors in vivo, considering the concentration of FVIII in plasma (ϳ0.3 nM).…”

Elevated plasma factor VIII in a mouse model of low-density lipoprotein receptor–related protein deficiency

“…Investigation of the molecular basis thereof has recently started. [11][12][13] It has been demonstrated that FVIII clearance is facilitated by cell surface heparan sulfate proteoglycans. 13 These are thought to act by preconcentrating ligands on the cell surface that subsequently transfer their ligands to endocytic receptors.…”

The Second and Fourth Cluster of Class A Cysteine-rich Repeats of the Low Density Lipoprotein Receptor-related Protein Share Ligand-binding Properties

“…LRP can bind and internalize a diverse spectrum of structurally unrelated ligands in a calcium-dependent manner including apolipoproteins, lipases, proteinases, proteinase-inhibitor complexes, Kunitz-type inhibitors, matrix proteins, and other proteins such as lactoferrin, Pseudomonas exotoxin A, and malaria circumsporozoite protein (1,2). In addition, the blood coagulation factor VIII was recently identified as a ligand of LRP (3). The broad range of ligands suggests a role for the receptor in distinct physiological and pathophysiological processes, ranging from lipoprotein metabolism, cell growth and cell migration, fibrinolysis, and thrombosis to atherosclerosis and Alzheimer's disease.…”