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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
LDL receptor cooperates with LDL receptor–related protein in regulating
plasma levels of coagulation factor VIII in vivo
Niels Bovenschen, Koen Mertens, Lihui Hu, Louis M. Havekes, and Bart J. M. van Vlijmen
Low-density lipoprotein (LDL) receptor
(LDLR) and LDLR-related protein (LRP) are
members of the LDLR family of endocytic
receptors. LRP recognizes a wide spectrum
of structurally and functionally unrelated
ligands, including coagulation factor VIII
(FVIII). In contrast, the ligand specificity of
LDLR is restricted to apolipoproteins E and
B-100. Ligand binding to the LDLR family is
inhibited by receptor-associated protein
(RAP). We have previously reported that,
apart from LRP, other RAP-sensitive mecha-
nisms contribute to the regulation of FVIII in
vivo. In the present study, we showed that
the extracellular ligand-binding domain of
LDLR interacts with FVIII in vitro and that
binding was inhibited by RAP. The physiologic relevance of the FVIII–LDLR interaction was addressed using mouse models of
LDLR or hepatic LRP deficiency. In the absence of hepatic LRP, LDLR played a dominant role in the regulation and clearance of
FVIII in vivo. Furthermore, FVIII clearance
was accelerated after adenovirus-mediated
gene transfer of LDLR. The role of LDLR in
FVIII catabolism was not secondary to increased plasma lipoproteins or to changes
in lipoprotein profiles. We propose that LDLR
acts in concert with LRP in regulating plasma
levels of FVIII in vivo. This represents a
previously unrecognized link between LDLR
and hemostasis. (Blood. 2005;106:906-912)
© 2005 by The American Society of Hematology
Introduction
Low-density lipoprotein (LDL) receptor (LDLR) plays a pivotal
role in the metabolism of large cholesterol-rich lipoproteins from
the circulation in a process known as receptor-mediated endocytosis.1 This process is dependent on the high-affinity interaction
between LDLR and its protein ligands, apolipoprotein E (apoE)
and apoB-100, both of which are present at the surfaces of
lipoprotein particles in plasma.1,2 The importance of LDLR is
illustrated by the fact that genetic defects within the LDLR gene are
the underlying cause of familial hypercholesterolemia.3 These
patients display elevated plasma LDL cholesterol concentrations
and increased risk for atherosclerosis and coronary artery disease.
LDLR belongs to the LDLR family of cell-surface endocytic
receptors that also includes apoE-receptor-2, very low-density
lipoprotein (VLDL) receptor, megalin, and LDLR-related protein (LRP).2,4 The extracellular ligand-binding domains of these
receptors are composed of 1 to 4 homolog clusters of complement-type repeats. The chaperone receptor–associated protein
(RAP) blocks all ligand binding to these clusters.5 Disruption of
the LRP gene in LDLR knockout mice accumulates apoE-rich
VLDL/LDL-sized lipoproteins in plasma, indicating that LDLR
works in concert with LRP in lipoprotein metabolism in vivo.6
Unlike LDLR, however, LRP also recognizes a broad spectrum
of lipoprotein-unrelated ligands.4
In the past few years, LRP has been established to interact
with coagulation factor VIII (FVIII) and to mediate its cellular
uptake into the lysosomal degradation pathway.7,8 Within the
blood coagulation cascade, the cofactor activity of FVIII is
indispensable for appropriate hemostasis.9 Deficiency or dysfunction of FVIII is associated with the bleeding disorder hemophilia
A, whereas elevated plasma FVIII markedly increases the risk
for arterial and venous thrombosis.9-11 The cofactor circulates in
plasma in complex with its carrier protein von Willebrand factor
(VWF).12
Using conditional hepatic LRP-deficient mice, we recently
demonstrated that LRP contributes to the removal of FVIII from
the circulation.13 These mice display not only elevated plasma
FVIII levels but also prolonged FVIII half-life. Adenovirusmediated overexpression of RAP in LRP-deficient mice further
increases plasma FVIII, indicating that RAP-sensitive determinants other than hepatic LRP also contribute to the regulation of
plasma FVIII.13 FVIII has been reported to comprise multiple
binding sites for the RAP-sensitive VLDL receptor in vitro.14
However, we have recently demonstrated that VLDL receptor
does not cooperate with LRP in FVIII clearance in vivo.15 In the
present study, we investigated the physiologic role of LDLR in
the catabolism of FVIII. We took advantage of unique conditional knockout mouse models for LDLR, LRP, and the combination thereof. By this approach, we identified a novel role for
LDLR as a receptor that contributes to the catabolism of
coagulation FVIII in vivo.
From the Department of Plasma Proteins, Sanquin Research at CLB,
Amsterdam, The Netherlands; TNO Prevention and Health, Gaubius
Laboratory, Leiden, The Netherlands; Utrecht Institute for Pharmaceutical
Sciences, Utrecht University, Utrecht, The Netherlands; Departments of
Internal Medicine, Cardiology, and Hematology, Leiden University Medical
Center, Leiden, The Netherlands.
Blood Transfusion Research (K.M., B.J.M.v.V.).
Submitted November 5, 2004; accepted March 11, 2005. Prepublished online
as Blood First Edition Paper, April 19, 2005; DOI 10.1182/blood-2004-11-4230.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Supported by a fellowship from the Royal Netherlands Academy of Art and
Sciences (B.J.M.v.V.) and by grant 0104 from the Landsteiner Foundation for
906
An Inside Blood analysis of this article appears in the front of this issue.
Reprints: Koen Mertens, Department of Plasma Proteins, Sanquin Research
at CLB, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands; e-mail:
[email protected].
© 2005 by The American Society of Hematology
BLOOD, 1 AUGUST 2005 䡠 VOLUME 106, NUMBER 3
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BLOOD, 1 AUGUST 2005 䡠 VOLUME 106, NUMBER 3
Materials and methods
Materials
CNBr-Sepharose 4B and Mono Q were from Amersham Pharmacia Biotech
(Uppsala, Sweden). Microtiter plates (Microlon, Greiner bio-one), cell
culture flasks, OptiMem I medium, penicillin, and streptomycin were from
Life Technologies (Breda, The Netherlands).
Proteins
Purification of the anti-FVIII,16 anti-VWF,17 and anti-FIX18 monoclonal
antibodies CLB-CAg 12, CLB-CAg 117, CLB-CAg 69, CLB-RAg 56,
CLB-FIX 11, plasma-derived FVIII heterodimer,19 and FIX18 have been
described previously. Human VWF was purified from human VWF
concentrate (von Willebrand factor-SD; Regional Center of Blood Transfusion, Lille, France). This concentrate was first depleted for FVIII by
immunoaffinity chromatography using antibody CLB-CAg 117. VWF was
further purified by Mono Q chromatography, using 100 mM NaCl and 50
mM Tris (tris(hydroxymethyl)aminomethane) (pH 7.4) for washing and a
salt gradient from 100 mM to 1 M NaCl in the same buffer for elution. The
VWF preparation was dialyzed to 150 mM NaCl, 5 mM CaCl2, and 50 mM
Tris (pH 7.4) and was stored at 4°C. An estimated average molecular mass
of 200 kDa was used to calculate the concentration of FVIII heterodimer.
The concentration of VWF refers to the concentration of VWF monomers
(Mr ⫽ 220 kDa). RAP was expressed in Escherichia coli strain DH5␣ and
was purified using glutathione–Sepharose, as described.20 LRP cluster 2
was purified by affinity chromatography using RAP-coupled CNBrSepharose 4B.18 LDL (1.019 g/mL ⬍ d ⬍ 1.063 g/mL) and high-density
lipoprotein (HDL) (1.063 g/mL ⬍ d ⬍ 1.120 g/mL) fractions were isolated
from fresh human plasma, as described,21 and were dialyzed to 150 mM
NaCl, 5 mM CaCl2, and 50 mM Tris (pH 7.4). Protein was quantified using
the Bradford method22 with human serum albumin (HSA) (Sanquin Plasma
Products, Amsterdam, The Netherlands) as a standard.
DUAL-RECEPTOR SYSTEM REGULATES PLASMA FVIII
907
30 minutes in phosphate-buffered saline (pH 7.4), 0.1% (vol/vol) Tween 20,
and 4% (wt/vol) milk powder; this was followed by a 90-minute incubation
of a rabbit polyclonal antibody directed against CR4 of human LDLR
(Research Diagnostics, Flanders, New Jersey) in the same buffer. Bound
IgG was detected with peroxidase-conjugated goat antirabbit antibody
followed by staining with 3,3-diaminobenzidine.
Solid-phase binding assay
Purified recombinant sLDLRCR1-7 or LRP cluster 2 was adsorbed onto
microtiter wells (0.5 ␮g/well) in 50 mM NaHCO3 (pH 9.8), in a volume of
50 ␮L for 16 hours at 4°C. Wells were blocked with 150 mM NaCl, 5 mM
CaCl2, 2.5% (wt/vol) HSA, and 50 mM Tris (pH 7.4) in a volume of 200 ␮L
for 2 hours at 37°C. Subsequently, purified human FVIII, VWF, or FIX was
incubated in 150 mM NaCl, 5 mM CaCl2, 1% (wt/vol) HSA, 0.1% (vol/vol)
Tween 20, and 50 mM Tris (pH 7.4), in a volume of 50 ␮L for 2 hours at
37°C. Bound ligand was detected by incubation with peroxidaseconjugated monoclonal antibody CLB-CAg 12, CLB-RAg 56, or CLB-FIX
11, respectively, in the same buffer for 10 minutes at 37°C. In competition
experiments, immobilized sLDLRCR1-7 or immobilized LRP cluster 2 was
incubated with purified FVIII at 150 nM or 20 nM, respectively, in the
absence or presence of serial dilutions of competitor. For RAP-, LDL-, and
HDL-binding experiments, these proteins were adsorbed onto microtiter
wells (1 ␮g/well) in 150 mM NaCl, 5 mM CaCl2, and 50 mM Tris (pH 7.4)
in a volume of 100 ␮L for 16 hours at 4°C. Wells were blocked with 150
mM NaCl, 5 mM CaCl2, 2.5% (wt/vol) HSA, and 50 mM Tris (pH 7.4) in a
volume of 200 ␮L for 2 hours at 37°C. Subsequently, sLDLRCR1-7 (250 nM)
was incubated in 150 mM NaCl, 5 mM CaCl2, 1% (wt/vol) HSA, and 50
mM Tris (pH 7.4), in a volume of 100 ␮L for 2 hours at 37°C. Bound ligand
was detected by incubation with peroxidase-conjugated monoclonal antibody CLB-CAg 69 in the same buffer for 10 minutes at 37°C. All data were
corrected for binding to control microtiter wells lacking immobilized
sLDLRCR1-7, RAP, LDL, or HDL, which was less than 10% relative to
binding to wells containing these immobilized proteins.
Construction, expression, and purification of sLDLRCR1-7
Construction of the adenoviral-plasmid containing full-length human LDLR
cDNA has been described previously23,24 and was used as a template to
amplify the cDNA of the entire extracellular ligand-binding domain of
LDLR (sLDLRCR1-7), comprising amino acids 22 to 317, by polymerase
chain reaction. Oligonucleotide primers 5⬘-ATTCTCGAGGCAGTGGGCGACAGATGT-3⬘ (sense) and 5⬘-TAAACTAGTTTCGTTGGTCCCGCACTC-5⬘ (antisense) were used. This cDNA fragment was purified,
digested with XhoI and SpeI, ligated into the XhoI/SpeI-digested pZEN
vector,25 and sequenced to verify the construct. The resultant plasmid
contains a 16-amino acid tag (KKEDFDIYDEDENQSP) for purification,
which represents the epitope of monoclonal antibody CLB-CAg 69,16
followed by the sLDLRCR1-7 coding sequence. The construct was transfected into baby hamster kidney cells as described.25 Stable cell lines that
express sLDLRCR1-7 were obtained by limiting dilution, and protein was
expressed in OptiMem I medium (Life Technologies) supplemented with
100 U/mL penicillin and 100 ␮g/mL streptomycin. After harvesting of the
medium, benzamidine (10 mM) and CaCl2 (10 mM) were added. Purification of sLDLRCR1-7 was performed by affinity-chromatography using a
column of CLB-CAg 69 coupled to CNBr-Sepharose 4B as affinity matrix.
This matrix was washed with 100 mM NaCl, 5 mM CaCl2, and 50 mM Tris
(pH 7.4) and was eluted with 2 M NaCl, 5 mM CaCl2, and 50 mM Tris (pH
7.4). The obtained sLDLRCR1-7 protein was subsequently concentrated by
Mono Q chromatography using 100 mM NaCl, 5 mM CaCl2, and 50 mM
Tris (pH 7.4) for washing and 2 M NaCl, 5 mM CaCl2, and 50 mM Tris (pH
7.4) for elution. The preparation was dialyzed to 150 mM NaCl, 5 mM
CaCl2, 50% (vol/vol) glycerol, and 20 mM Tris (pH 7.4) and was stored at
⫺20°C. Purified sLDLRCR1-7 was subjected to 10% (vol/vol) sodium
dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (5 ␮g
protein/lane) under reducing conditions and was stained with Coomassie
brilliant blue. For immunoblotting, SDS-PAGE–separated sLDLRCR1-7 (0.5
␮g protein/lane) was transferred to a nitrocellulose membrane, blocked for
Transgenic animals
Mice carrying loxP sites within the LRP gene (LRPflox/flox), LDLR-deficient
mice (LDLR⫺/⫺), VLDLR-deficient mice (VLDLR⫺/⫺), and apoE-deficient
mice (ApoE⫺/⫺) were generated by homologous recombination in embryonic stem cells, as described previously.23,26-29 Mice transgenic for the
MX1cre expression construct (MX1Cre⫹) were generated by pronuclear
injection of hybrid (SJL X C57BL/6J) mice, as described.6 Combining
MX1Cre⫹, LRPflox/flox, LDLR⫺/⫺, VLDLR⫺/⫺, and ApoE⫺/⫺ genotypes
resulted in the following 7 genetically distinct knockout strains used in this
study: LRPflox/flox (control), MX1Cre⫹LRPflox/flox (LRP⫺), LDLR⫺/⫺
LRPflox/flox (LDLR⫺/⫺), LDLR⫺/⫺MX1Cre⫹LRPflox/flox (LDLR⫺/⫺LRP⫺),
VLDLR⫺/⫺LDLR⫺/⫺MX1Cre⫹LRPflox/flox (VLDLR⫺/⫺LDLR⫺/⫺LRP⫺),
ApoE⫺/⫺LDLR⫺/⫺LRPflox/flox (ApoE⫺/⫺LDLR⫺/⫺), and ApoE⫺/⫺LDLR⫺/⫺
MX1Cre⫹LRPflox/flox (ApoE⫺/⫺LDLR⫺/⫺LRP⫺). All mice were genotyped
for LRPflox/flox, MX1cre, LDLR, VLDLR, or apoE status by polymerase
chain reaction, as described.6,28,29 Mice were injected with polyinosinic:
polycytidylic-ribonucleic acid (pI:pC), as described.13 Noninduced
MX1Cre⫹LRPflox/flox, noninduced LRPflox/flox, and pI:pC-induced LRPflox/flox
control animals displayed similar plasma levels of FVIII, VWF, FV, FIX,
tissue-type plasminogen activator (t-PA), total cholesterol, and triglycerides, indicating that neither MX1Cre status nor pI:pC alone affected these
plasma protein levels in our mice. C57BL6/J mice homozygous for the
human APOCI transgene (huAPOCI) and nontransgenic littermate controls
were generated, as described.30 For experiments, male mice 8 to 12 weeks
of age were used (each weighing 20-25 g). Mice were housed under
standard conditions in conventional cages and given free access to food
(standard rodent chow diet; Hope Farms, Woerden, The Netherlands) and
water. The institutional committees on animal welfare of TNO Prevention
and Health approved all animal experiments.
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908
BLOOD, 1 AUGUST 2005 䡠 VOLUME 106, NUMBER 3
BOVENSCHEN et al
Statistical analysis
In vitro data were represented as mean plus or minus standard deviation
(SD). In vivo data were represented as geometric means and 68%
confidence intervals (CIs), which represented 1 SD from the geometric
mean if a log normal distribution was assumed. Data were analyzed by
means of the Mann-Whitney U test. P less than .05 was regarded as
statistically significant.
Results
Role of LDLR in regulating plasma FVIII in vivo
Figure 1. Plasma FVIII levels in mice that lack LRP, LDLR, or both. Four weeks
after the final pI:pC injection, blood was drawn from control (n ⫽ 12), LRP⫺ (n ⫽ 12),
LDLR⫺/⫺(n ⫽ 9), and LDLR⫺/⫺LRP⫺ mice (n ⫽ 12). Plasma samples were analyzed
for FVIII and are plotted as individual values.
Quantification of mouse plasma FVIII, FV, FIX, VWF, t-PA,
cholesterol, and triglycerides
Blood was obtained by tail bleeding into polypropylene Eppendorf tubes
containing 0.1 vol of 3.2% (wt/vol) trisodium citrate. Plasma was prepared
by centrifugation of blood at 2000g for 10 minutes at 4°C, immediately
snap-frozen in liquid nitrogen, and stored at ⫺80°C before analysis. Mouse
plasma FVIII activity, FV activity, FIX zymogen activity, VWF antigen,
t-PA activity, total cholesterol, and triglyceride levels were determined as
described previously.13,14 Plasma FVIII, FV, FIX, VWF, and t-PA levels
were expressed in murine plasma-equivalent units per milliliter. Unless
stated otherwise, control LRPflox/flox pooled plasma was used as a reference.
Lipoprotein distribution was determined by fast performance liquid chromatography (FPLC) size fractionation.28
Clearance of human FVIII
Human immunopurified plasma-derived FVIII (Aafact; Sanquin Plasma
Products, Amsterdam, The Netherlands) (20 IU in 200 ␮L) was injected
into the tail veins of weight-matched male mice. At indicated time points,
individual mice were serially monitored for human plasma FVIII antigen
levels, as described previously.13 The amount of FVIII recovered in the
plasma 1 minute after injection was 90% to 100%. Values are expressed as
the percentage of FVIII remaining in the circulation, considering the
amount of FVIII present at 1 minute after injection as 100%. Pharmacokinetic parameters were calculated using a model-independent (noncompartmental) approach.31 A double-exponential fit was used to calculate the
standard parameters area under the curve and mean residence time (MRT).
Recombinant adenovirus transduction
Recombinant adenovirus containing the human LDLR cDNA (Ad-CMVLDLR) or ␤-galactosidase cDNA (Ad-CMV-␤-Gal) under the control of the
cytomegalovirus (CMV) promotor was generated and purified, as described
previously.23,32 Normal male C57BL/6J, LDLR⫺/⫺ or LDLR⫺/⫺LRP⫺ mice
received 2 ⫻ 109 plaque-forming units of Ad-CMV-LDLR or Ad-CMV-␤Gal in 200 ␮L physiological saline into the tail vein. Blood samples (100
␮L) were withdrawn by tail bleeding before and 5 days after adenovirus
injection. FVIII levels were assayed using pooled C57BL/6J plasma as a
reference. Five days after adenovirus administration, normal C57BL/6J or
LDLR⫺/⫺LRP⫺ mice were intravenously injected with human FVIII (20
IU), and the removal of FVIII from plasma was monitored as described.
Detection of LDLR in mouse liver
Mouse liver membrane extracts13 (50 ␮g/lane) were subjected to nonreducing 4% to 15% (vol/vol) SDS-PAGE analysis and transferred to nitrocellulose membranes, and LDLR was detected as described, with the exception
that bound immunoglobulin G (IgG) was stained using the enhanced
chemiluminescence (ECL) system (Amersham Pharmacia Biotech, Uppsala, Sweden).
To address the physiologic role of LDLR in regulating plasma
FVIII, we measured plasma FVIII levels and its carrier protein,
VWF, in control, LRP⫺, LDLR⫺/⫺, and LDLR⫺/⫺LRP⫺ doubleknockout mice (Figure 1; Table 1). Plasma FVIII and, to a lesser
extent, VWF levels were increased in LRP⫺ mice approximately
1.6-fold and 1.3-fold, respectively, which is in agreement with
our previous findings.13 In contrast, LDLR⫺/⫺ mice that expressed functional LRP had normal plasma FVIII and VWF
levels. Interestingly, LDLR⫺/⫺LRP⫺ double-knockout mice demonstrated a 4.2-fold increase of plasma FVIII, which was more
pronounced, as expected, from plasma FVIII in mice that lacked
LRP or LDLR alone. This synergistic effect of plasma FVIII in
mice that lacked LRP and LDLR coincided with a 3.3-fold
increase in plasma VWF level. In contrast to FVIII and VWF,
disruption of LRP, LDLR, or both did not affect the plasma
levels of the other coagulation-related plasma proteins, FV and
FIX (Table 1). For the LRP ligand t-PA,26 we found that the
induction of hepatic LRP deficiency resulted in an increase of its
plasma level. However, this LRP-dependent increase of plasma
t-PA was not affected by LDLR status (Table 1). These data
demonstrate that LDLR knockout mice have normal plasma
FVIII and VWF levels. In contrast, in the absence of hepatic
LRP, LDLR deficiency further increased plasma FVIII and
VWF levels.
FVIII interacts with LDLR in vitro
It has been well established that LRP comprises multiple binding
sites for FVIII.25,33 We now investigated whether LDLR also
recognizes FVIII as a ligand. To this end, we constructed a soluble
recombinant LDLR fragment that contains the entire extracellular
ligand-binding domain, comprising complement-type repeats 1 to 7
(CR 1-7) (amino acid residues 22-317) (Figure 2A). Using
Table 1. Plasma parameters in mice that lack LRP, LDLR, or both
Plasma
component
Control
LRPⴚ
LDLRⴚ/ⴚ
LDLRⴚ/ⴚLRPⴚ
FVIII, U/mL
1.1 (0.9-1.2)
1.8 (1.6-2.0)*
0.9 (0.7-1.2)
4.6 (3.7-5.6)†
VWF, U/mL
1.0 (0.9-1.3)
1.3 (1.1-1.5)*
0.9 (0.7-1.2)
3.3 (2.7-4.1)†
FV, U/mL
1.0 (0.9-1.1)
1.1 (1.0-1.3)
1.1 (1.0-1.2)
1.1 (1.0-1.2)
FIX, U/mL
1.1 (0.9-1.2)
1.0 (0.9-1.1)
1.1 (0.9-1.3)
1.1 (1.0-1.3)
TPA, U/mL
1.8 (1.5-2.0)
2.4 (1.9-3.1)*
2.0 (1.8-2.1)
Cholesterol, mM
1.7 (1.5-1.9)
1.7 (1.5-1.9)
7.0 (6.1-8.1)*
19.5 (17.2-22.3)†
Triglycerides, mM
0.2 (0.1-0.3)
0.2 (0.1-0.2)
0.8 (0.7-1.0)*
3.0 (2.5-3.7)†
2.3 (1.9-2.7)
At 4 weeks after the final pI:pC injection, blood was drawn from control, LRP⫺,
LDLR⫺/⫺, and LDLR⫺/⫺LRP⫺ mice. Plasma samples were analyzed for FVIII, VWF,
FV, FIX, t-PA cholesterol, and triglycerides, as described in “Materials and methods.”
Data represent geometric mean values with 68% confidence intervals in parentheses. n ⫽ 12 for all groups except LDLR⫺/⫺ (n ⫽ 9).
*P ⬍ .05, significantly different from control mice; Mann-Whitney U test.
†P ⬍ .05, significantly different from LRP⫺, LDLR⫺/⫺, and control mice; MannWhitney U test.
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BLOOD, 1 AUGUST 2005 䡠 VOLUME 106, NUMBER 3
Figure 2. Characterization of recombinant sLDLRCR1-7. (A) Domain organization
of full-length LDLR (top) and of the recombinant soluble ligand-binding fragment
sLDLRCR1-7 (bottom). Ligand-binding domain that harbors complement-type repeats
1 to 7, EGF precursor (EGFP) domain that contains EGF-like modules (A-C) and a
beta-propeller domain comprising 6 YWTD motifs (Y), O-linked sugar domain,
transmembrane domain, cytoplasmic tail, immunopurification tag, and amino acid
residue numbers are indicated. (B) Coomassie brilliant blue–stained 10% (vol/vol)
SDS-PAGE analysis of the purified recombinant sLDLRCR1-7 (lane 1). Immunoblotting
of the purified recombinant sLDLRCR1-7 using an anti–human LDLR antibody followed
by 3,3-diaminobenzidine staining (lane 2). Molecular weight standard is indicated.
(C) Purified sLDLRCR1-7 (250 nM) was incubated with immobilized isolated human
LDL, HDL, and recombinant RAP (1 ␮g/well). Binding was detected using the
peroxidase-conjugated monoclonal antibody CLB-CAg 69. Data represent the
mean ⫾ SD of 3 separate experiments.
SDS-PAGE and immunoblotting with an antibody directed against
CR4 of human LDLR, this fragment (referred to as sLDLRCR1-7)
migrated at a single band with the expected molecular mass34
(Figure 2B). Purified sLDLRCR1-7 efficiently bound to the archetypal LDLR ligand apoB-100 containing LDL1,2 and the universal
LDLR family antagonist RAP, whereas binding to immobilized
HDL was only minor (Figure 2C). A dose-dependent increase in
binding occurred on incubation of increasing concentrations of
FVIII with immobilized sLDLRCR1-7, with half-maximum binding
at a FVIII concentration of 156 ⫾ 6 nM (Figure 3A). In contrast,
Figure 3. Binding of FVIII, VWF, and FIX to recombinant sLDLRCR1-7. (A)
Immobilized purified recombinant sLDLRCR1-7 (0.5 ␮g/well) was incubated with
purified FVIII (F) (0-500 nM), VWF (E) (0-500 nM), or FIX (f) (0-500 nM). Bound
FVIII, VWF, or FIX was detected by incubation with peroxidase-conjugated monoclonal antibodies CLB-CAg 12, CLB-RAg 56, and CLB-FIX 11, respectively. (B)
Immobilized purified recombinant sLDLRCR1-7 (0.5 ␮g/well) was incubated with
purified FVIII (150 nM) in the absence or presence of increasing concentrations of
recombinant RAP (0-3 ␮M). (C) Immobilized purified recombinant LRP cluster 2 (0.5
␮g/well) was incubated with FVIII (20 nM) in the absence or presence of increasing
concentrations of LRP cluster 2 (E) (0-0.5 ␮M) or sLDLRCR1-7 (F) (0-4 ␮M). Residual
FVIII binding was detected as described. Data represent the mean ⫾ SD of 3
separate experiments.
DUAL-RECEPTOR SYSTEM REGULATES PLASMA FVIII
909
FIX and the FVIII carrier protein VWF did not show any
interaction with immobilized sLDLRCR1-7 (Figure 3A). The
specificity of FVIII binding to LDLR was addressed using the
LDLR-antagonist RAP. Indeed, FVIII binding to sLDLRCR1-7
decreased in a dose-dependent manner in the presence of
increasing concentrations of RAP (Figure 3B). Half-maximum
inhibition (Ki) occurred at a RAP concentration of approximately 0.9 ␮M, which is close to the affinity of RAP for
full-length LDLR.35 Previously, we demonstrated that FVIII
interacts with the ligand-binding cluster 2 of LRP.33 Binding of
FVIII to immobilized purified recombinant LRP cluster 2 was
observed with half-maximum binding at a FVIII concentration
of 20 plus or minus 2 nM (data not shown). To address the
affinity of FVIII for LDLR relative to LRP, we used competition
experiments between sLDLRCR1-7 and LRP cluster 2 in the
interaction with FVIII. Both sLDLRCR1-7 and LRP cluster 2
competed for binding of FVIII to immobilized LRP cluster 2,
with Ki values of 0.51 ⫾ 0.10 ␮M and 0.15 ⫾ 0.01 ␮M,
respectively (Figure 3C). This indicates that FVIII binds to LRP
with approximately 3.4-fold higher affinity compared with the
FVIII-LDLR complex assembly. Taken together, these data
demonstrate that FVIII is a ligand of LDLR in vitro.
Adenovirus-mediated overexpression of human LDLR in mice
accelerates FVIII clearance
The ability of FVIII to bind to LDLR in vitro (Figure 3) prompts
the question whether LDLR is able to clear FVIII from the
circulation in vivo. To this end, we used adenovirus-mediated gene
transfer to induce hepatic overexpression of human LDLR. We
injected an adenovirus containing the human LDLR cDNA (AdCMV-LDLR) or control adenovirus encoding the ␤-galactosidase
cDNA (Ad-CMV-␤-Gal). It was verified that human LDLR was
indeed expressed in the livers of mice that received Ad-CMVLDLR (Figure 4A). As expected,23 Ad-CMV-LDLR completely
reversed the hypercholesterolemic effects in LDLR⫺/⫺ mice
(P ⫽ .002) (Figure 4B). Five days after adenovirus administration,
plasma FVIII levels were 1.0 U/mL (68% CI, 0.8-1.2 U/mL) and
0.9 U/mL (68% CI, 0.6-1.3 U/mL) in Ad-CMV-␤-Gal– and
Figure 4. FVIII clearance after adenovirus-mediated overexpression of human
LDLR in mice. (A) Liver membrane extracts of wild-type mice that received
Ad-CMV-␤-Gal (n ⫽ 2) (lanes 1-2) or Ad-CMV-LDLR (n ⫽ 2) (lanes 3-4) were
subjected to 4% to 15% SDS-PAGE analysis under nonreducing conditions. Human
LDLR expression was detected by immunoblotting, using a rabbit anti–human LDLR
antibody and the ECL system. (B) LDLR⫺/⫺ mice were intravenously injected with
2 ⫻ 109 plaque-forming units Ad-CMV-␤-Gal (n ⫽ 5) or Ad-CMV-LDLR (n ⫽ 5). Five
days after adenovirus injection, blood was drawn and plasma was analyzed for
plasma cholesterol. (C) Wild-type C57BL/6J mice were intravenously injected with
2 ⫻ 109 plaque-forming units Ad-CMV-␤-Gal (F) (n ⫽ 4) or Ad-CMV-LDLR (E)
(n ⫽ 6). Five days after adenovirus injection, animals were intravenously injected
with purified human FVIII (20 IU), and its plasma removal was monitored at indicated
time points. Data represent geometric mean values and 68% confidence intervals.
*P ⬍ .05, significantly different from that of control Ad-CMV-␤-Gal–treated mice;
Mann-Whitney U test.
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910
BLOOD, 1 AUGUST 2005 䡠 VOLUME 106, NUMBER 3
BOVENSCHEN et al
pattern in LRP-deficient mice (Figure 5C). These data indicate
that, in the absence of hepatic LRP, LDLR plays a predominant
role in the clearance of FVIII from the circulation.
No relation between plasma FVIII and cholesterol levels in mice
Figure 5. FVIII clearance in mice that lack LRP, LDLR, or both. At 4 weeks after
the final pI:pC injection, purified human FVIII (20 IU) was intravenously administered
into (A) control mice (‚) (n ⫽ 5) and LDLR⫺/⫺ mice (Œ) (n ⫽ 5) and into (B) LRP⫺
mice (E) (n ⫽ 5) and LDLR⫺/⫺ LRP⫺ mice (F) (n ⫽ 5). Human FVIII removal from
plasma was monitored at indicated time points. (C) LDLR⫺/⫺LRP⫺ mice were
intravenously injected with 2 ⫻ 109 plaque-forming units Ad-CMV-␤-Gal (f) (n ⫽ 8)
or Ad-CMV-LDLR (䡺) (n ⫽ 8). Five days after adenovirus injection, animals were
intravenously injected with purified human FVIII (20 IU), and its plasma removal was
monitored at indicated time points. *P ⬍ .01, significantly different from that of control
Ad-CMV-␤-Gal–treated mice; Mann-Whitney U test. Data represent geometric mean
values and 68% confidence intervals.
Ad-CMV-LDLR–treated C57BL/6J mice, respectively
(P ⫽ 1.000). Subsequently, the same mice received intravenous
bolus injections of human FVIII, and its clearance rate from
plasma was monitored. The plasma removal of FVIII in
Ad-CMV-LDLR–treated mice was slightly accelerated compared with removal in control animals that received Ad-CMV-␤Gal (Figure 4C). The MRT of FVIII was calculated to be 205
minutes in control Ad-CMV-␤-Gal–treated mice and 141 minutes in mice that received Ad-CMV-LDLR, with 68% CIs of 194
to 217 and 109 to 183 minutes, respectively (P ⫽ .01). These
data indicate that LDLR has the potential to contribute to the
clearance of FVIII from plasma in vivo.
FVIII clearance in LDLR and LRP knockout mice
To investigate whether the increased plasma FVIII levels in
LDLR⫺/⫺LRP⫺ mice resulted from a slower rate of FVIII
elimination from the circulation, we studied the plasma disappearance of intravenously injected human FVIII in control,
LRP⫺, LDLR⫺/⫺, and LDLR⫺/⫺LRP⫺ double-knockout mice.
FVIII clearance was slightly, but not statistically significantly,
slower in LDLR⫺/⫺ mice (1.25-fold) than in control mice
(Figure 5A). The MRT of FVIII was calculated to be 160
minutes in control mice and 200 minutes in LDLR⫺/⫺ mice, with
68% CIs of 117 to 218 and 154 to 259 minutes, respectively
(P ⫽ .222). The MRT of infused FVIII in LRP⫺ mice was
prolonged approximately 1.6-fold from 160 minutes (68% CI,
117-218 minutes) in control mice to 263 minutes (68% CI,
206-336 minutes) in LRP⫺ mice (P ⫽ .032) (Figure 5B). In
contrast, combined receptor deficiency resulted in an approximately 4.8-fold prolongation of the MRT compared with control
mice (P ⫽ .008) (Figure 5B). The MRT in LDLR⫺/⫺LRP⫺ mice
was calculated to be 760 minutes, with a 68% CI of 691 to 836
minutes. This is compatible with the observed FVIII plasma
levels in the different genotypes (Figure 1; Table 1). If LDLR
contributes to FVIII catabolism, one would expect that the
delayed clearance of FVIII in LDLR⫺/⫺LRP⫺ mice is rescued
after reintroduction of LDLR by adenovirus-mediated gene
transfer. Indeed, whereas FVIII clearance in LDLR⫺/⫺LRP⫺
mice was not affected by Ad-CMV-␤-Gal, administration of
Ad-CMV-LDLR to these mice significantly accelerated FVIII
clearance, almost resembling the observed FVIII clearance
It has been well established that LDLR and LRP are of major
physiologic importance in plasma lipoprotein metabolism.6 This is
illustrated by increased plasma cholesterol and triglyceride levels
in mice that lack LDLR, which becomes more evident when LRP
and LDLR are absent in combination (Table 1), leading to the
question whether the increase in plasma FVIII levels in LDLR⫺/⫺
LRP⫺ double-knockout mice is secondary to increased lipoprotein
plasma levels. To address this possibility, we used well-described
mice models of hyperlipidemia. These include mice that genetically lack apoE, either on the LDLR⫺/⫺ or the LDLR⫺/⫺
LRP⫺ background,36,37 and mice that constitutively overexpress
human apoC1.30 The hyperlipidemic lipoprotein distribution profiles of ApoE⫺/⫺LDLR⫺/⫺ and ApoE⫺/⫺LDLR⫺/⫺LRP⫺ mice are
comparable in that both genotypes display elevated VLDL and
LDL cholesterol (Figure 6A).37 Plasma cholesterol and triglyceride
levels were clearly increased in ApoE⫺/⫺LDLR⫺/⫺LRP⫺ and ApoE⫺/⫺
LDLR⫺/⫺ mice (Table 2). In contrast, plasma FVIII was elevated in
ApoE⫺/⫺LDLR⫺/⫺LRP⫺ mice but not in the ApoE⫺/⫺LDLR⫺/⫺ mice
(Table 2). Additionally, plasma FVIII levels in ApoE⫺/⫺LDLR⫺/⫺LRP⫺
mice (Table 2) were not different (P ⫽ .699) from those of
LDLR⫺/⫺LRP⫺ mice (Table 1), indicating that the apoE status does
not affect FVIII levels in plasma. To further investigate the relation
between FVIII and lipoproteins, we used human apoC1 transgenic
mice (huAPOCI) that have increased plasma levels of cholesterol
and triglyceride-rich VLDL (Figure 6B).30 Although huAPOCI
mice had increased cholesterol and triglyceride levels, plasma
FVIII levels were lower in huAPOCI mice than in nontransgenic
controls (Table 2). Taken together, these data support our observation that LDLR⫺/⫺ mice showed elevated plasma levels of mainly
apoB/E-containing LDL cholesterol and triglycerides,6,23 whereas
FVIII levels remained unaffected (Table 1). We conclude that
elevated plasma FVIII in LDLR⫺/⫺LRP⫺ double-knockout mice is
not secondary to increases in apoE, apoB, cholesterol, or triglyceride levels or to changes in lipoprotein distribution but that it is
directly attributed to LDLR deficiency.
Discussion
In the present study, we show that the level of coagulation FVIII
in the circulation is regulated by a dual-receptor system that
involves not only LRP but also LDLR. This is compatible with
Figure 6. Lipoprotein distribution. Blood was drawn from (A) ApoE⫺/⫺LDLR⫺/⫺LRP⫺
(F) and ApoE⫺/⫺LDLR⫺/⫺ (E) mice and from (B) huAPOCI transgenic (‚) and
nontransgenic control (Œ) mice. Lipoproteins were size fractionated by FPLC.
Individual fractions were analyzed for cholesterol.
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BLOOD, 1 AUGUST 2005 䡠 VOLUME 106, NUMBER 3
DUAL-RECEPTOR SYSTEM REGULATES PLASMA FVIII
911
Table 2. No relation between plasma FVIII and cholesterol or triglyceride levels in mice
Mice
ApoE⫺/⫺LDLR⫺/⫺LRP⫺
ApoE⫺/⫺LDLR⫺/⫺
No.
Cholesterol, mean mM
(68% CI)
Triglycerides, mean mM
(68% CI)
FVIII, mean U/mL
(68% CI)
8
16.2 (11.5-22.9)
1.0 (0.7-1.6)
4.2 (3.3-5.5)
10
23.3 (17.6-31.0)*
2.1 (1.4-3.1)*
1.6 (1.4-1.8)*
0.3 (0.3-0.4)
1.0 (0.9-1.1)
Nontransgenic controls
6
HuAPOCI
6
2.1 (1.9-2.2)
19.5 (16.4-23.2)†
36.9 (27.4-45.4)†
0.5 (0.3-0.8)†
Blood was drawn from ApoE⫺/⫺LDLR⫺/⫺LRP⫺, ApoE⫺/⫺LDLR⫺/⫺, huAPOCI transgenic, and nontransgenic control mice. Plasma samples were analyzed for cholesterol,
triglycerides, and FVIII, as described in “Materials and methods.” FVIII levels of huAPOCI transgenic and nontransgenic control mice were assayed using pooled C57BL/6J
plasma as a reference, whereas those of ApoE⫺/⫺LDLR⫺/⫺LRP⫺ and ApoE⫺/⫺LDLR⫺/⫺ mice were expressed against pooled LRPflox/flox plasma. Data represent geometric
mean values with 68% confidence intervals in parentheses.
*P ⬍ .05, significantly different from ApoE⫺/⫺LDLR⫺/⫺LRP⫺ mice; Mann-Whitney U test.
†P ⬍ .05, significantly different from nontransgenic controls; Mann-Whitney U test.
our previous observation that RAP-sensitive determinants other
than hepatic LRP also contribute to the catabolism of FVIII in
vivo.13 Our findings that LDLR interacts with FVIII in vitro
(Figure 3) and that LDLR plays a role in the clearance of FVIII
from the circulation (Figures 4, 5) are remarkable because thus
far only 2 ligands in plasma have been identified for this
receptor, apoE and apoB-100.1,2 This is in marked contrast to
LRP, for which more than 35 ligands have been identified to
date.2,4 The identification of FVIII as a novel ligand of LDLR
suggests that in addition to its well-established function in
lipoprotein metabolism, this receptor may play a previously
unrecognized role in modulating hemostasis.
The importance of LDLR and LRP in FVIII catabolism is
illustrated by our observation that combined deficiency of this
dual-receptor system in mice resulted in an approximately
4.2-fold increase of FVIII levels in plasma (Table 1). The
relative contribution of both receptors to FVIII catabolism
remains unclear. Strikingly, mice that lack LDLR or LRP alone
displayed FVIII levels that were normal or that were elevated no
more than 1.6-fold, respectively (Table 1). This implies that
LDLR can functionally compensate for LRP deficiency and vice
versa. The same view emerges from the pharmacokinetic
experiments in these mice (Figure 5). Whereas the MRT of
intravenously administered FVIII was 4.8-fold prolonged in
LDLR⫺/⫺LRP⫺ double-knockout mice, the effects of singlereceptor deficiencies were relatively minor, providing additional
evidence that the elimination of FVIII from the circulation
involves the concerted action of LRP and LDLR.
The in vivo catabolism of FVIII shows some intriguing
dissimilarities compared with that of lipoproteins.6,23,32 First,
LRP-deficient mice have slightly elevated plasma FVIII levels
but normal cholesterol-rich lipoprotein levels, whereas LDLR
knockout mice display the opposite (Table 1). Second, though
VLDLR plays a major role in the metabolism of postprandial
lipoproteins in vivo,38 this FVIII-binding receptor does not
contribute to the clearance of FVIII from the circulation alone15
or in concert with LDLR and LRP. This latter conclusion is
based on our observations that LDLR⫺/⫺LRP⫺ mice (Table 1;
Figure 5) display similar plasma FVIII and FVIII clearance rates
compared with VLDLR⫺/⫺LDLR⫺/⫺LRP⫺ mice. These tripleknockout mice had FVIII plasma levels of 4.6 U/mL (68% CI,
3.7-5.7 U/mL) (n ⫽ 12) and a FVIII MRT of 674 minutes (68%
CI, 587-774 minutes) (n ⫽ 5). Apparently, members of the
LDLR family of endocytic receptors display differential specificity for their ligands in vivo. FVIII catabolism involves a
complex multistage process in which circulating FVIII is first
captured by heparan sulfate proteoglycans (HSPGs) onto the
cell surface.39 This is compatible with a model in which
HSPG-mediated enrichment of FVIII on the plasma membrane
results in its subsequent internalization by LRP and LDLR. A
similar catabolic route has been proposed for apoE.40 Possibly,
differences in affinity of FVIII, apoE, and apoB for LDLR, LRP,
VLDLR, and HSPG constitute an important determinant for the
specificity of LDLR receptor family members in vivo.7,8,39,41
This might further be related to the markedly distinct endocytosis rates reported for LDLR family members (LRP greater than
LDLR greater than VLDLR).42
Another new observation from this study is that elevated
plasma FVIII levels in LRP-deficient and LDLR⫺/⫺LRP⫺ doubleknockout mice coincide with increased levels of its carrier
protein VWF (Table 1). Because VWF has been reported not to
be a ligand for LRP7,8 or LDLR (Figure 3) in vitro, it remains
difficult to explain why VWF is increased in these mice.
Although in humans an increase of the VWF concentration in
plasma often results in a concomitant increase of FVIII, this is
not the case in mice.13,43,44 One might conclude that the observed
increases in VWF levels are the direct result of elevated plasma
FVIII levels. However, further research is required to elucidate
the molecular mechanism behind the elevated levels of plasma
VWF in mice that lack hepatic LRP and LDLR.
High levels of FVIII in plasma (greater than 1.5 U/mL)
constitute a major risk factor for arterial and venous thrombosis
in humans.10,11 Our observation that the up-regulation of hepatic
LDLR protein expression in mice by gene transfer accelerated
FVIII clearance from the circulation (Figures 4 and 5) may be of
therapeutic interest for patients who have elevated plasma FVIII
levels. In humans, the up-regulation of LDLR protein is
achieved by treatment with 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, also called statins.3
Statins are widely recognized in the treatment of hypercholesterolemia in humans.3 It would be interesting to study whether
statins have the potential to lower the elevated levels of plasma
FVIII in humans, with the goal of reducing the risk for
thrombotic events.
Acknowledgments
We are indebted to Mrs M. Voskuilen, Dr S.M.S. Espirito Santo,
and Dr J.F.P. Berbée for technical assistance. We thank Dr K.
Willems van Dijk for providing the adenovirus encoding human
LDLR and Dr P.C.N. Rensen for access to the huAPOCI transgenic
mouse model.
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912
BLOOD, 1 AUGUST 2005 䡠 VOLUME 106, NUMBER 3
BOVENSCHEN et al
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2005 106: 906-912
doi:10.1182/blood-2004-11-4230 originally published online
April 19, 2005
LDL receptor cooperates with LDL receptor−related protein in regulating
plasma levels of coagulation factor VIII in vivo
Niels Bovenschen, Koen Mertens, Lihui Hu, Louis M. Havekes and Bart J. M. van Vlijmen
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