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Prepublished online as a Blood First Edition Paper on January 9, 2003; DOI 10.1182/blood-2002-07-2081.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Department of Plasma Proteins, Sanquin
Research at CLB, Amsterdam, the Netherlands;
Department of Molecular Genetics, University of Texas Southwestern
Medical Center, Dallas, TX; TNO Prevention and Health, Gaubius
Laboratory, Leiden, the Netherlands; Departments of
Cardiology and Internal Medicine, Leiden University Medical Center,
Leiden, the Netherlands; and Utrecht Institute for
Pharmaceutical Sciences (UIPS), Utrecht University, Utrecht, the
Netherlands.
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/flox
mice (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.
(Blood. 2003;101:3933-3939) Coagulation factor VIII (FVIII) is a 300-kDa
glycoprotein that acts as a cofactor for activated factor IX (FIX) in
the middle phase of the coagulation cascade.1,2 The fact
that deficiency or dysfunction of FVIII is associated with the bleeding
disorder hemophilia A demonstrates that this cofactor is indispensable for appropriate hemostasis. FVIII is synthesized in various tissues, including liver, spleen, and kidney.3,4 In plasma, FVIII circulates in complex with its carrier protein von Willebrand factor
(VWF), which is produced and secreted by vascular endothelial cells.5,6 VWF serves a predominant role in stabilizing
FVIII and prevents its premature clearance.7 Upon
triggering of the coagulation cascade, FVIII is activated upon multiple
proteolytic cleavages by thrombin or factor Xa, which results in the
dissociation from VWF.6,8 After the participation of
activated FVIII in the factor X-activating complex, it is rapidly
inactivated by spontaneous subunit dissociation or via enzymatic
degradation.9,10
Of the many parameters that regulate the plasma level of FVIII in vivo,
its removal from plasma plays a central role. Investigation of the
molecular basis thereof has recently started.11-13 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.14 Recently,
it has been demonstrated that FVIII comprises multiple binding
sites that mediate the interaction with the low-density lipoprotein receptor-related protein (LRP).11,12 LRP is a large cell
surface receptor that is ubiquitously expressed in a variety of tissues and is present on a wide range of different cell types, including hepatocytes, monocytes, and smooth muscle cells.15 LRP is
a member of the low-density lipoprotein (LDL) receptor family of endocytic receptors and recognizes a wide range of structurally and
functionally distinct ligands.14,16 Among these ligands, the 39-kDa receptor-associated protein (RAP) serves a unique role as
an LRP chaperone, which blocks all ligand binding to the
receptor.17,18
It has been established that LRP contributes to the cellular uptake and
subsequent lysosomal delivery of FVIII in vitro.11,12,19 In vivo, FVIII half-life is markedly prolonged in mice in the presence
of a bolus administration of purified RAP.12 Similar effects of RAP on FVIII half-life are observed in a mouse model of VWF
deficiency.20 Moreover, administration of RAP triggers a
sustained rise in endogenous plasma FVIII in these mice.21 In addition to LRP, however, RAP is known to block ligand binding to
many other endocytic cell surface receptors, including other members of
the LDL receptor family.22-25 With the use of the
antagonist RAP, only indirect evidence is collected regarding
the physiologic role of LRP in the removal of FVIII from plasma.
Therefore, it remains inconclusive whether LRP indeed plays a role in
regulating plasma FVIII in vivo.
In this study, we took advantage of a unique mouse model of LRP
deficiency as a tool to investigate whether LRP contributes to the
regulation of plasma FVIII in vivo. Previously, this LRP-deficient mouse model proved to be successful for studying the role of
LRP in regulating plasma lipoprotein levels in vivo.26,27
In addition, other RAP-sensitive mechanisms involved in regulating
plasma FVIII in vivo were addressed with the use of
adenovirus-mediated gene transfer of RAP in LRP-deficient mice.
Transgenic animals
Quantification of mouse plasma factors VIII, V, and IX
Quantification of mouse plasma VWF Plasma VWF antigen levels were measured by means of an enzyme-linked immunosorbent assay, with the use of a rabbit antihuman VWF polyclonal antibody (DAKO, Glostrup, Denmark) for both capture and detection of VWF. We found that this antibody cross-reacts with mouse VWF. Plasma was prepared as described, diluted in 100 µL buffer containing 1% (wt/vol) BSA and 0.1% (vol/vol) Tween 20 in phosphate-buffered saline (PBS) (pH 7.4), and incubated with the immobilized rabbit antihuman VWF polyclonal antibody (3.3 pmol per well) for 2 hours at 37°C. After washing with 0.1% (vol/vol) Tween 20 in PBS (pH 7.4), 0.3 pmol peroxidase-labeled anti-VWF polyclonal antibody (DAKO) was incubated in 100 µL of the same buffer for 1 hour at 37°C. VWF antigen levels were expressed in murine plasma-equivalent units per milliliter, with the use of MF-1 normal pooled mouse plasma as a reference.Administration of DDAVP and endotoxin DDAVP (1-deamino-8-D-arginine vasopressin) was obtained from Ferring (Malmö, Sweden), and endotoxin (Escherichia coli, serotype 0111:B4) was purchased from Sigma-Aldrich. Female C57BL/6J mice received 12 µg DDAVP per kg body weight diluted in 200 µL physiologic saline by intraperitoneal injection. Female MX1cre+LRPflox/flox mice received 2 mg endotoxin per kg body weight diluted in 200 µL physiologic saline by intraperitoneal injection. Before injection and at 0.5, 1, 2, 4, and 6 hours after injection, blood samples of 50 µL were taken via tail bleeding. Plasma was prepared and analyzed for FVIII and VWF as described.Plasma clearance of human FVIII FVIII clearance studies were performed with the use of human immunoaffinity-purified FVIII concentrate (Aafact; Sanquin Plasma Products, Amsterdam, The Netherlands). This comprises homogeneous heterodimeric FVIII in a human albumin-containing formulation.29 After reconstitution, 200 µL (20 IU FVIII) was injected into the tail vein of weight-matched female MX1cre+LRPflox/flox and LRPflox/flox mice. At 1, 30, 60, 120, 180, 300, and 420 minutes after injection, blood samples of 50 µL were drawn via tail bleeding in EDTA-coated capillary tubes (Sarstedt, Nümbrecht, Germany). 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 prior to analysis. Human FVIII
antigen was measured with an immunosorbent assay, with the use of the
antihuman FVIII monoclonal antibodies CLB-CAg A and CLB-CAg 117 as
described previously.29 FVIII antigen levels were
expressed in international units per milliliter, with calibrated human
plasma used as a reference. 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, with the amount of
FVIII present at 1 minute after injection considered as 100%.
Pharmacokinetic parameters were calculated by means of a
model-independent (ie, noncompartmental) approach.30 A
double-exponential fit was used to calculate the standard parameters
area under the curve (AUC), fractional catabolic rate (FCR), and mean
residence time (MRT).
Detection of LRP in mouse livers Mouse liver membranes were prepared as described previously.27 Briefly, mouse liver membrane extracts (50 µg per lane) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis on 4% to 15% polyacrylamide gels under nonreduced conditions. Nitrocellulose membranes were blocked for 30 minutes at room temperature in 0.5% (vol/vol) Tween 20, 2% (wt/vol) BSA, 5% (wt/vol) milk powder, and PBS (pH 7.4), followed by a 60-minute incubation of a peroxidase-labeled rabbit polyclonal antibody directed against the 85-kDa subunit of LRP in the same buffer.27 Bound immunoglobulin G (IgG) was detected by means of the enhanced chemiluminescence system (Amersham Pharmacia Biotech, Uppsala, Sweden).Recombinant adenovirus transduction Recombinant adenovirus containing the rat RAP cDNA (Ad-RAP) or -galactosidase cDNA (Ad--Gal) driven by the cytomegalovirus promotor were generated, grown, and purified as described
previously.31 For in vivo adenovirus transduction, 6 weeks
after the last pI:pC injection, 2 × 109 plaque-forming
units (PFUs) in 200 µL physiologic saline was injected into the tail
vein. Blood samples (50 µL) were withdrawn via tail bleeding before
and 3 days after adenovirus injection. Expression of RAP in mouse
plasma was quantified by enzyme-linked immunosorbent assay, with the
use of polyclonal antibodies against RAP for capture and
detection.27
Statistical analysis Data are represented as geometric means and 68% confidence intervals (CI), which represent one standard deviation from the geometric mean if a log-normal distribution is assumed. Data are analyzed by means of the Mann-Whitney U test. P < .05 was regarded as statistically significant.
Induction of LRP deficiency results in accumulation of plasma FVIII and VWF Transgenic mice that both were homozygous for a loxP-flanked LRP gene and expressed cre recombinase under the control of the interferon-inducible MX1 promotor (ie, MX1cre+LRPflox/flox) were used to inactivate the LRP gene by administration of pI:pC. PI:pC-induced littermates that were MX1cre (ie, LRPflox/flox) served as
controls. We previously demonstrated that the cre/loxP recombination
system effectively achieves inducible disruption of LRP in adult mice,
thereby allowing in vivo studies on the role of LRP for at least 6 weeks after pI:pC injection.26,27 To investigate the role
of LRP in regulating plasma FVIII in vivo, both plasma FVIII and its
carrier protein VWF were measured in MX1cre+LRPflox/flox and control
LRPflox/flox mice.
Noninduced MX1cre+LRPflox/flox animals had
plasma FVIII levels (1.9 U/mL; CI, 1.5-2.2 U/mL) that were not
statistically different (P = .2) from those of noninduced
LRPflox/flox mice (1.6 U/mL; CI, 1.4-2.0 U/mL). Noninduced
MX1cre+LRPflox/flox animals had plasma VWF (1.3 U/mL; CI, 1.1-1.5 U/mL) levels similar (P = .7) to those
observed in noninduced LRPflox/flox mice (1.4 U/mL; CI,
1.1-1.6 U/mL). In addition, pI:pC-induced control
LRPflox/flox mice had plasma FVIII and VWF levels similar
to those of their noninduced littermates (Table
1). These observations indicate that
neither pI:pC nor MX1cre status alone affects plasma FVIII or
VWF.
As demonstrated in Figure 1, at 10 days
following pI:pC induction, plasma FVIII showed an
approximately 2-fold increase in MX1cre+LRPflox/flox mice as compared with
control LRPflox/flox (3.4 and 2.0 U/mL, respectively;
P < .001). Induction with pI:pC also
resulted in elevated plasma VWF in
MX1cre+LRPflox/flox mice as compared with
control LRPflox/flox mice (2.0 and 1.6 U/mL, respectively;
P < .05) (Figure 1). As shown in Table 1, both plasma
FVIII and VWF remained elevated in
MX1cre+LRPflox/flox mice for at least 6 weeks
after induction. In contrast, inactivation of the LRP gene did not
affect plasma levels of the FVIII-related cofactor FV (0.6 U/mL [CI,
0.5-0.7 U/mL], 0.5 U/mL [CI, 0.4-0.6 U/mL], and 0.7 U/mL [CI,
0.6-0.8 U/mL], for noninduced, control LRPflox/flox, and
MX1cre+LRPflox/flox,
respectively).1 Similarly, LRP deficiency did not
affect the plasma level of the FIX zymogen, which does not interact
with LRP (0.5 U/mL [CI, 0.4-0.6 U/mL], 0.5 U/mL [CI, 0.4-0.6 U/mL], and 0.5 U/mL [CI, 0.4-0.6 U/mL], for noninduced, control
LRPflox/flox, and
MX1cre+LRPflox/flox,
respectively).32 Collectively, these data
demonstrate that inactivation of the LRP gene results in an elevation
of plasma FVIII and, to a lesser extent, of plasma VWF in
vivo.
Relation between plasma VWF and plasma FVIII in mice It has been well established that VWF is a major regulator of FVIII in plasma, as VWF deficiency is associated with low FVIII levels in many species, including humans, pigs, dogs, and mice.5,33,34 In agreement with this notion, MX1cre+LRPflox/flox mice displayed an increase of both plasma FVIII and plasma VWF (Figure 1). A closer look at these data, however, revealed that inactivation of the LRP gene is associated with an increased FVIII/VWF ratio (P < .001) (Figure 1C). This demonstrates that plasma FVIII does not merely covary with plasma VWF in these mice.To further investigate the relation between plasma FVIII and VWF, we
triggered an increase in plasma VWF employing DDAVP or endotoxin.
Administration of DDAVP had only limited effect on plasma VWF in normal
C57BL/6J mice. Plasma VWF increased from 1.4 U/mL (CI, 1.2-1.7 U/mL)
before the experiment to 1.8 U/mL (CI, 1.5-2.1 U/mL) 30 minutes after
DDAVP administration. This increase was not associated with a change in
plasma FVIII (1.3 U/mL [CI, 1.2-1.4 U/mL] and 1.2 U/mL [CI, 1.2-1.3 U/mL] for 0 and 30 minutes after DDAVP administration, respectively).
As for noninduced MX1cre+LRPflox/flox mice,
DDAVP did not affect the VWF level at all (data not shown). To obtain a
more pronounced increase of plasma VWF, we administered endotoxin into
noninduced MX1cre+LRPflox/flox mice to elicit a
VWF response. As shown in Figure 2,
plasma VWF increased by about 3-fold after the administration of
endotoxin, whereas plasma FVIII did not respond to endotoxin. These
observations support previous data demonstrating that VWF, but not
FVIII, is an acute-phase protein in mice.35 We therefore
conclude that, in mice, a rise in plasma VWF is not necessarily
associated with a secondary rise in plasma FVIII.
Plasma FVIII clearance in LRP-deficient mice Several in vitro studies using LRP-deficient cells revealed that LRP contributes to the cellular degradation of FVIII.11,12,19 In vivo, a bolus administration of RAP is known to prolong FVIII half-life, suggesting that a RAP-sensitive mechanism contributes to the clearance of FVIII from plasma.12,20,21 To investigate whether increased plasma FVIII in MX1cre+LRPflox/flox mice is the result of a slower plasma FVIII elimination rate, we intravenously injected purified human FVIII into MX1cre+LRPflox/flox and control LRPflox/flox mice. As demonstrated in Figure 3, FVIII clearance was slower in MX1cre+LRPflox/flox mice than in control LRPflox/flox mice. The mean residence time was calculated to be 155 minutes in control LRPflox/flox mice and 230 minutes in MX1cre+LRPflox/flox mice, with 68% confidence intervals of 133 to 180 and 209 to 254 minutes, respectively (P = .002). As FVIII clearance in MX1cre+LRPflox/flox mice was still fairly efficient, we considered the possibility that this was due to residual hepatic LRP. Therefore, mouse liver membrane extracts were subjected to immunoblotting analysis, with the use of an antibody directed against the 85-kDa subunit of LRP. Whereas LRP was readily detectable in the livers of control LRPflox/flox mice, it was lacking in MX1cre+LRPflox/flox mice 10 days after pI:pC induction (Figure 3 inset). Collectively, these data indicate that the mean residence time of injected human FVIII is prolonged in hepatic LRP-deficient mice. This suggests that the increased plasma FVIII in MX1cre+LRPflox/flox mice is, at least in part, due to a slower clearance of FVIII from the circulation.
Effect of adenovirus-mediated gene transfer of RAP on plasma FVIII and VWF in LRP-deficient mice Previous studies have demonstrated that a bolus injection of the LRP antagonist RAP prolongs FVIII half-life in normal mice.12,20,21 In addition, RAP induces a rise in endogenous plasma FVIII in VWF-deficient mice, thus underscoring that FVIII levels are dependent on a RAP-sensitive mechanism.21 Since RAP is known to antagonize ligand binding to a variety of other cell surface receptors involved in endocytosis, including the entire LDL receptor family,22-25 other LRP-independent RAP-sensitive mechanisms that contribute to FVIII catabolism were addressed. To this end, we employed adenovirus-mediated gene transfer to overexpress the RAP protein in both MX1cre+LRPflox/flox and control LRPflox/flox mice. We injected a dose of 2 × 109 PFUs of adenovirus containing the rat RAP cDNA under the control of a cytomegalovirus promotor (Ad-RAP). As a control, mice received an adenovirus encoding the-galactosidase
gene (Ad--Gal).
To evaluate the functionality of Ad-RAP, we first verified whether this
dose effectively induced RAP expression. In LDL-receptor-deficient mice, Ad-RAP induced hypercholesterolemia (higher than 30 mM) and
hypertriglycideridemia (higher than 15 mM), which is in agreement with
our previous studies.27,31 Furthermore, RAP expression was
measured in the plasma C57BL/6J mice after transfection of either
Ad- Administration of control Ad-
FVIII requires complex assembly with its carrier protein VWF to be protected from premature clearance from the circulation.7 In the presence of VWF, FVIII half-life varies between 12 and 14 hours in humans7,36 and about 1 hour in mice.37 In VWF deficiency, however, FVIII half-life is only to 2 to 3 hours in humans38 and less than 10 minutes in mice.20 While FVIII clearance from the circulation seems to be driven by a particularly efficient process, the mechanism by which this occurs has remained poorly understood. In 1999, we and others reported that FVIII binds to the multifunctional endocytic receptor LRP and that this receptor mediates the cellular uptake and subsequent degradation of FVIII in vitro.11,12 If LRP were to play a major role in the clearance of FVIII in vivo, one would expect LRP deficiency to be associated with elevated FVIII levels in the circulation. In the present study, we have addressed this question employing the cre/loxP homologous recombination system to inactivate the LRP gene in mice. This unique model of LRP deficiency allowed us to investigate the potential role of LRP in regulating plasma FVIII in vivo. We found that both constituents of the FVIII-VWF complex are elevated
in LRP-deficient mice (Figure 1; Table 1). Although the increase of
plasma VWF was statistically significant, it was minor in comparison
with FVIII. This is reflected by an upward shift in stoichiometry
within the FVIII-VWF complex in LRP-deficient mice (Figure
1C). Why VWF is slightly increased in LRP-deficient mice
remains unclear. Unlike FVIII, VWF has been reported not to be a ligand
of LRP in vitro.11,12 Therefore, we have no reason to
suppose that LRP is involved in the clearance of VWF. On the other
hand, it might be possible that the MX1-induced expression of cre
recombinase causes some VWF increase as part of an inflammatory response. In humans, elevation in plasma concentration of VWF usually
results in corresponding changes of FVIII. In mice, however, VWF
increase has been reported without concomitant rise in
FVIII.35 Similarly, we did not observe any rise in FVIII
after increasing the plasma VWF level using DDAVP or endotoxin (Figure
2), or after adenovirus-mediated Ad- The molecular mechanisms that underlie elevated plasma FVIII in LRP-deficient mice can be due to increased FVIII biosynthesis, impaired FVIII clearance, or a combination thereof. As LRP is known to play a role in endocytosis of its ligands, we studied the elimination of FVIII from the circulation in both LRP-deficient and control mice. Indeed, hepatic LRP contributes to FVIII clearance and, in particular, to the initial 120 minutes of FVIII decay (Figure 3). However, the question may arise whether or not this effect is sufficient to fully explain FVIII elevation in LRP deficiency. In this context, we cannot exclude a role for LRP in the biosynthesis of FVIII. Our current understanding of the mechanism of FVIII biosynthesis in mice is limited by the lack of a positive correlation between FVIII mRNA and FVIII protein levels.39,40 Because FVIII mRNA and LRP mRNA have been identified in the same cell type,3,4,16,41 we cannot exclude the possibility that LRP plays an as-yet-unidentified role along the intracellular secretory pathway of FVIII. This would be consistent with the recent observation that the LDL receptor within the endoplasmic reticulum mediates the presecretory degradation of its ligand apolipoprotein B.42,43 As plasma FVIII is elevated in LRP-deficient mice, one would expect
that, in normal mice, plasma FVIII should accumulate following the
administration of the LRP-antagonist RAP. As RAP is rapidly cleared
from the circulation,44 bolus injections are impractical for maintaining RAP in the circulation for an extended period of time. Therefore, we employed adenoviral gene transfer to evoke a
sustained level of RAP in the circulation. Indeed, we found that RAP
overexpression resulted in a marked increase of plasma FVIII in normal
mice (Table 2). It is important to note that, while the control
Ad- It has been generally accepted that elevated FVIII in plasma is a risk factor for venous thrombosis.48 In this report, we show that disruption of the LRP gene results in the accumulation of FVIII in plasma (Figure 1). Moreover, the mechanism of plasma FVIII regulation may not be limited to LRP, as overexpression of RAP further increases plasma FVIII in LRP-deficient mice (Table 2). These observations open the possibility that LRP or another RAP-sensitive receptor is associated with an increased risk for developing thrombosis. The mechanisms by which FVIII levels are elevated in plasma are poorly understood. So far, attempts to associate elevated plasma FVIII with mutations in the FVIII gene have remained unsuccessful. To date, numerous variations that are associated with familial hypercholesterolemia have been found in the gene encoding the LDL receptor.49 The molecular consequences of these genetic variations include loss of LDL receptor function, resulting in the accumulation of its ligand in plasma.49-51 Whether or not such genetic variations that are associated with increased plasma FVIII or thrombosis exist within the LRP gene or other LDL receptor family member genes is an intriguing question that deserves further study.
We thank Dr J. A. van Mourik for critically reading the manuscript and Dr B. Teusink for advice on pharmacokinetics.
Submitted July 15, 2002; accepted December 30, 2002.
Prepublished online as Blood First Edition Paper, January 9, 2003; DOI 10.1182/blood-2002-07-2081.
Supported by grants from the Royal Netherlands Academy of Art and Sciences (B.J.M.v.V.) and the Landsteiner Foundation for Blood Transfusion Research (K.M. and B.J.M.v.V.).
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.
Reprints: Koen Mertens, Department of Plasma Proteins, Sanquin Research at CLB, Plesmanlaan 125, 1066 CX Amsterdam, the Netherlands; e-mail: k.mertens{at}sanquin.nl.
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© 2003 by The American Society of Hematology.
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  S. Lacroix-Desmazes, A.-M. Navarrete, S. Andre, J. Bayry, S. V. Kaveri, and S. Dasgupta Dynamics of factor VIII interactions determine its immunologic fate in hemophilia A Blood, July 15, 2008; 112(2): 240 - 249. [Abstract] [Full Text] [PDF]
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S. Dasgupta, Y. Repesse, J. Bayry, A.-M. Navarrete, B. Wootla, S. Delignat, T. Irinopoulou, C. Kamate, J.-M. Saint-Remy, M. Jacquemin, et al. VWF protects FVIII from endocytosis by dendritic cells and subsequent presentation to immune effectors Blood, January 15, 2007; 109(2): 610 - 612. [Abstract] [Full Text] [PDF]
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S. M. S. Espirito Santo, N. M. M. Pires, L. S. M. Boesten, G. Gerritsen, N. Bovenschen, K. W. van Dijk, J. W. Jukema, H. M. G. Princen, A. Bensadoun, W.-P. Li, et al. Hepatic low-density lipoprotein receptor-related protein deficiency in mice increases atherosclerosis independent of plasma cholesterol Blood, May 15, 2004; 103(10): 3777 - 3782. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
)
(n = 29) and control LRPflox/flox mice (
) (n = 18)
were injected 3 times intraperitoneally with 250 µg pI:pC at 2-day
intervals. At 10 days after the final pI:pC injection, blood
was drawn and plasma was analyzed for FVIII and VWF, as
described in "Materials and methods." (C) Replotting of individual
FVIII levels (A) against the corresponding VWF levels (B).
) and VWF (
). Values represent geometric
means and 68% confidence intervals. *P < .05,
significantly different from plasma VWF levels before endotoxin
administration, with the use of the Mann-Whitney U
test.
