Cluster III of Low-Density Lipoprotein Receptor-Related Protein 1 Binds Activated Blood Coagulation Factor VIII

Biochemistry ◽  
2014 ◽  
Vol 54 (2) ◽  
pp. 481-489 ◽  
Author(s):  
James H. Kurasawa ◽  
Svetlana A. Shestopal ◽  
Samuel A. Woodle ◽  
Mikhail V. Ovanesov ◽  
Timothy K. Lee ◽  
...  
Keyword(s):  
Factor Viii ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Coagulation Factor ◽  
Low Density ◽  
Lipoprotein Receptor ◽  
Related Protein ◽  
Density Lipoprotein Receptor ◽  
Lipoprotein Receptor Related Protein ◽  
Blood Coagulation Factor Viii

Fragments of Activated Coagulation Factor VIII Bind to Multiple Sites within Low-Density Lipoprotein Receptor-Related Protein.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1019-1019
Author(s):  
Andrey G. Sarafanov ◽  
Evgeny M. Makogonenko ◽  
Olav M. Andersen ◽  
Alexey V. Khrenov ◽  
Irina A. Mikhailenko ◽  
...  
Keyword(s):  
Factor Viii ◽  
Binding Sites ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Coagulation Factor ◽  
Low Density ◽  
Lipoprotein Receptor ◽  
Related Protein ◽  
Density Lipoprotein Receptor ◽  
Lipoprotein Receptor Related Protein

Abstract Catabolism of coagulation factor VIII (fVIII) is mediated by the hepatic multiligand receptor low-density lipoprotein receptor-related protein (LRP). The ligand-binding sites of LRP are formed by complement-type repeats (CRs) organized in four clusters, among which clusters II and IV bind most of LRP ligands. In turn, fVIII contains two major LRP-binding sites, located in A2 and A3 domains (Saenko et al, JBC 1999; Bovenschen et al, JBC 2003). In present work, we characterized binding sites in LRP for A2 domain (A2) and heterodimer A1/A3-C1-C2 (HD), the products of dissociation of activated fVIII. Using a baculovirus expression system, we generated CR clusters II, III and IV, along with eight overlapping CR triplets encompassing clusters II and IV. Surface plasmon resonance-based assays demonstrated that both A2 and HD bind to clusters II and IV, and to the same sets of their CR triplets with similar affinities (KDs 25–50 nM). The same kinetic parameters of interaction of both A2 and HD were observed for several CR doublets from cluster II, shown previously to be minimal binding sites for a classical ligand of LRP, receptor associated protein (RAP) (Andersen et al, JBC 2000). The specificity of A2 and HD interactions with all tested fragments of LRP was confirmed by the ability of RAP to inhibit these interactions, and by the ability of these fragments to inhibit binding of 125I-A2 and 125I-HD to immobilized LRP in a solid-phase assay, and LRP-mediated catabolism of 125I-A2 and 125I-HD in cell culture. Notably, some mutations of the LRP-binding site in A2 resulted in significant reduction or abolishment of its binding to certain fragments of LRP, while the binding to other LRP fragments was less affected. In summary, we demonstrated that i) A2 and HD interact with LRP via its multiple binding sites spanning CRs 3–8 in cluster II and CRs 24–29 in cluster IV, and ii) the elementary binding unit of LRP is formed by at least two adjacent CRs, similar to that shown for RAP. The above data also suggest that besides regulating fVIII levels, LRP also plays a role in clearance of the products of dissociation of activated fVIII.

Characterization of Molecular Interaction of Blood Coagulation Factor VIII and Its Clearance Receptor, Low-Density Lipoprotein Receptor.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2206-2206
Author(s):  
James H Kurasawa ◽  
Svetlana A Shestopal ◽  
Elena Karnaukhova ◽  
Evi B Struble ◽  
Timothy K Lee ◽  
...  
Keyword(s):  
Factor Viii ◽  
Blood Coagulation ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Coagulation Factor ◽  
Low Density ◽  
Lipoprotein Receptor ◽  
Density Lipoprotein Receptor ◽  
Clearance Receptor ◽  
Blood Coagulation Factor Viii

Abstract Abstract 2206 Background. Clearance of blood coagulation factor VIII (FVIII) is mediated by several receptors (Saenko E. et al, 1999; Lenting P. et al, 1999 and Sarafanov A. et al, 2001) and one of them was proposed to be low-density lipoprotein receptor (LDLR) (Bovenschen N. et al, 2005). Besides FVIII, the major ligands of LDLR are plasma lipoproteins. The ligand-binding portion of LDLR is represented by a cluster of seven complement-type repeats. Each repeat forms a structurally autonomous domain, which are connected with flexible linkers. Two and five adjacent CRs were shown to form sites for the lipoproteins ApoE and ApoB, respectively, and the site for FVIII has not been determined. Objective. As a part of elucidation of mechanisms of FVIII clearance, our goal was to identify the FVIII-binding region on LDLR. Experimental Approach. Our experimental strategy was based on generation of recombinant CR-doublets that systematically overlap the CR cluster in LDLR (Fig. 1) and testing their interactions with FVIII. The proteins were expressed in insect cells using a baculovirus system and tested for structural integrity by circular dichroism. The functional properties of the fragments were assessed by their binding with alpha-2-macroglobulin receptor-associated protein (RAP), a universal ligand of a family of receptors that LDLR belongs to. The binding studies were performed using surface plasmon resonance technique. Using this assay, the LDLR fragments were further tested for interaction with different FVIII preparations, such as plasma-derived FVIII and recombinant FVIII: Advate (full-size FVIII) and Xyntha (B-domain deleted FVIII). The specificity of these interactions was tested in a competitive binding assay using an anti-FVIII single-chain variable antibody fragment KM33 known to inhibit binding FVIII to LDLR (Limburg V. et al, 2005). Also, the specificity of the interaction of LDLR fragments and FVIII was tested upon mutating the fragments by targeting conservative tryptophanes, one residue per selected CR. Upon expressing the mutants, their structural adequacy to the respective wild-type forms was confirmed by circular dichroism. Results. Three overlapping CR doublets were found active for binding with all FVIII preparations (Fig. 2). In presence of increased concentrations of KM33 and upon site-directed mutagenesis of the LDLR fragments, this binding was diminished confirming its specificity. Conclusions. The binding site of LDLR for FVIII is formed by a region spanned by the complement-type repeats from second to fifth. We proposed a tentative model of the interaction (Fig. 3), in which this region of the receptor contacts the A3 and C1 domains of FVIII. These portions of FVIII were previously shown to be involved in interaction with another FVIII clearance receptor known as LDLR-related protein (Bovenschen N. et al, 2003 and Meems H. et al, 2011). Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽  
2003 ◽  
Vol 101 (10) ◽  
pp. 3933-3939 ◽  
Author(s):  
Niels Bovenschen ◽  
Joachim Herz ◽  
Jos M. Grimbergen ◽  
Peter J. Lenting ◽  
Louis M. Havekes ◽  
...  
Keyword(s):  
Factor Viii ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Low Density ◽  
Lipoprotein Receptor ◽  
Related Protein ◽  
Elevated Plasma ◽  
Density Lipoprotein Receptor ◽  
Lipoprotein Receptor Related Protein

Abstract It has been established that low-density lipoprotein receptor-related protein (LRP) is involved in the cellular uptake and degradation of coagulation factor VIII (FVIII) in vitro. To address the physiologic role of LRP in regulating plasma FVIII in vivo, we used cre/loxP–mediated conditional LRP- deficient mice (MX1cre+LRPflox/flox). Upon inactivation of the LRP gene, MX1cre+LRPflox/flox mice had significantly higher plasma FVIII as compared with control LRPflox/floxmice (3.4 and 2.0 U/mL, respectively; P < .001). Elevated plasma FVIII levels in MX1cre+LRPflox/flox mice coincided with increased plasma von Willebrand factor (VWF) (2.0 and 1.6 U/mL for MX1cre+LRPflox/flox and control LRPflox/flox mice, respectively; P < .05). Elevation of plasma FVIII and VWF persisted for at least 6 weeks after inactivation of the LRP gene. Upon comparing plasma FVIII and VWF in individual mice, we observed an increase of the FVIII/VWF ratio in MX1cre+LRPflox/flox mice as compared with control LRPflox/flox mice. Administration of either a vasopressin analog or an endotoxin resulted in increased plasma VWF, but not FVIII. In clearance experiments, MX1cre+LRPflox/flox mice displayed a 1.5-fold prolongation of FVIII mean residence time. Adenovirus-mediated overexpression of the 39-kDa receptor–associated protein (RAP) in normal mice resulted in a 3.5-fold increase of plasma FVIII. These data confirm that the regulation of plasma FVIII in vivo involves a RAP-sensitive mechanism. Surprisingly, plasma FVIII in MX1cre+LRPflox/flox mice increased 2-fold after RAP gene transfer. We propose that RAP-sensitive determinants other than hepatic LRP contribute to the regulation of plasma FVIII in vivo.

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

2013 ◽  
Vol 288 (30) ◽  
pp. 22033-22041 ◽  
Author(s):  
James H. Kurasawa ◽  
Svetlana A. Shestopal ◽  
Elena Karnaukhova ◽  
Evi B. Struble ◽  
Timothy K. Lee ◽  
...  
Keyword(s):  
Factor Viii ◽  
Blood Coagulation ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Coagulation Factor ◽  
Low Density ◽  
Lipoprotein Receptor ◽  
Binding Region ◽  
Density Lipoprotein Receptor ◽  
Blood Coagulation Factor Viii
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