From the Holland Laboratory, American Red
Cross, Rockville, Maryland 20855, ¶ George Washington
University, Washington D. C. 20037, and the § Department
of Pediatrics, Nara Medical University, Kashihara City, Nara
634-8522, Japan
Received for publication, September 1, 2000, and in revised form, January 4, 2001
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ABSTRACT |
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We have demonstrated previously that
catabolism of a coagulation factor VIII (fVIII) from its complex with
von Willebrand factor (vWf) is mediated by low density lipoprotein
receptor-related protein (LRP) (Saenko, E. L., Yakhyaev, A. V., Mikhailenko, I., Strickland, D. K., and Sarafanov, A. G. (1999) J. Biol. Chem. 274, 37685-37692). In the
present study, we found that this process is facilitated by cell
surface heparan sulfate proteoglycans (HSPGs). This was demonstrated by
simultaneous blocking of LRP and HSPGs in model cells, which completely
prevented fVIII internalization and degradation from its complex with
vWf. In contrast, the selective blocking of either receptor had a
lesser effect. In vivo studies of clearance of
125I-fVIII-vWf complex in mice also demonstrated that the
simultaneous blocking of HSPGs and LRP led to a more significant
prolongation of fVIII half-life (5.5-fold) than blocking of LRP alone
(3.5-fold). The cell culture and in vivo experiments
revealed that HSPGs are also involved in another, LRP-independent
pathway of fVIII catabolism. In both pathways, HSPGs act as receptors
providing the initial binding of fVIII-vWf complex to cells. We
demonstrated that this binding occurs via the A2 domain of fVIII, since
A2, but not other portions of fVIII or isolated vWf, strongly inhibited
cell surface binding of fVIII-vWf complex, and the affinities of A2 and
fVIII-vWf complex for the cells were similar. The A2 site involved in
binding to heparin was localized to the region 558-565, based on the
ability of the corresponding synthetic peptide to inhibit A2 binding to heparin, used as a model for HSPGs.
Factor VIII (fVIII)1 is
an essential component of the intrinsic pathway of blood coagulation,
since genetic deficiency in fVIII results in a coagulation disorder
known as hemophilia A and occurs in 1 per 5000 males. In the intrinsic
pathway, activated fVIII (fVIIIa) functions as a cofactor for the
serine protease factor IXa, and their membrane-bound complex
(Xase complex) activates factor X to factor Xa (1). Factor Xa
subsequently participates in activation of prothrombin into thrombin,
the key enzyme of the coagulation cascade.
FVIII is a glycoprotein (~300 kDa, 2332 amino acid residues)
consisting of three homologous A domains, two homologous C domains, and
the unique B domain, which are arranged in the order of
A1-A2-B-A3-C1-C2 (2). Prior to its secretion to plasma, fVIII is
processed intracellularly to a series of Me2+-linked
heterodimers produced by cleavage at the B-A3 junction (3) and by a
number of additional cleavages within the B domain (2). These cleavages
generate a heavy chain (HCh) consisting of the A1 (residues 1-336), A2
(residues 373-740), and B domains (residues 741-1648), and a light
chain (LCh) composed of the domains A3 (residues 1690-2019), C1
(residues 2020-2172), and C2 (residues 2173-2332).
In circulation, most of fVIII is bound to vWf, which confers from
physiological concentrations of the proteins, which are ~1 (4) and
~50 nM (5), respectively, and a high affinity (0.2-0.5
nM) of their interaction (6, 7). Binding to vWf prevents
fVIII from premature interaction with components of Xase complex and is
also required for maintenance of the normal fVIII level in plasma (8),
since vWf deficiency in both humans (8, 9) and animals (10, 11) leads
to a secondary deficiency of fVIII.
We have recently shown that fVIII catabolism from its complex with vWf
in vitro and in vivo is mediated by low density
lipoprotein receptor-related protein (LRP) (12). LRP, a member of the
low density lipoprotein receptor family (13), is responsible for plasma
clearance of lipoprotein remnants, serine proteinases, and their
complexes with inhibitors (serpins) (13, 14). LRP is most prominent in
liver on hepatocytes, and in vasculature it is presented on the surface
of smooth muscle cells, fibroblasts, and macrophages (15). Besides
fVIII, LRP mediates the clearance of a number of other proteins
involved in blood coagulation and fibrinolysis, such as factors IXa
(16) and Xa (17, 18), plasminogen activators, and their complexes with
plasminogen activator inhibitor (19-21). A unique place among LRP
ligands belongs to 39-kDa receptor-associated protein (RAP), which
binds to LRP with a high affinity (Kd = 4 nM) and efficiently inhibits binding and endocytosis of all
known LRP ligands (22).
The sites of fVIII involved in interaction with LRP were
localized within the A2 domain residues 484-509 (12) and within the
C-terminal portion of the C2 domain (23). Since the latter region of
fVIII is likely to be blocked by vWf (23, 24), the C2 site could
contribute to the clearance of fVIII only in the absence of vWf.
This is consistent with the reported faster clearance of fVIII in
vWf-deficient patients and animals (8, 25, 26), which was shown to be
mediated by LRP (11).
The LRP-mediated endocytosis of many ligands is facilitated by cell
surface heparan sulfate proteoglycans (HSPGs), one of the components
constituting extracellular matrix. Among the LRP ligands, lipoprotein
lipase (27), apoE-containing lipoproteins (28, 29), thrombospondin
(30), thrombin-protease nexin 1 complex (31), and tissue factor pathway
inhibitor (19, 32) are HSPGs-binding proteins. HSPGs serve either as
coreceptors of LRP providing the initial binding of the ligands to the
cell surface and their subsequent presentation to LRP (14, 29), or
function as catabolic receptors themselves, acting independently of LRP
(33). All LRP ligands interacting with HSPGs are also able to bind to
heparin (34), which is structurally similar to carbohydrate portions of
HSPG molecules, and represent a useful model for studying these
interactions in a purified system.
Noteworthy, the recently reported Kd of 116 nM for fVIII interaction with LRP (12) is much higher than
the normal (~1 nM) concentration of fVIII in plasma (4).
This implies that the direct binding of plasma fVIII-vWf complex to LRP
is negligible and suggests possible involvement of other receptor(s) in
this process. In the present study, we examined participation of cell
surface HSPGs in the binding and catabolism of fVIII-vWf complex, based
on the ability of fVIII to interact with heparin (35). We demonstrated
that HSPGs are indeed responsible for the initial binding of fVIII-vWf
complex to the surface of various LRP-expressing cells and subsequent
facilitation of fVIII catabolism both in cell culture and in
vivo. We found that the binding occurs via the fVIII moiety of
fVIII-vWf complex and localized the major heparin-binding site of fVIII
within its A2 domain.
Reagents--
Chondroitin sulfate A, heparin (average molecular
weight 17-19 kDa), and biotinylated heparin were purchased from
Sigma and Celsus Laboratories Inc., respectively. Chondroitin sulfate A was biotinylated using EZ-Link Biotin-LC-Hydrazide (Pierce) as described (36). Human coagulation factors IXa, X, and Xa were purchased
from Enzyme Research Laboratories, and heparinase I was purchased from
Sigma. Active site fluorescently labeled factor IXa (Fl-FFR-fIXa) was a
generous gift of Dr. Philip Fay. Monoclonal antibody 8860 recognizing
the A2 domain of fVIII was kindly provided by Baxter/Hyland Healthcare
Inc. The rabbit polyclonal anti-LRP antibody Rab 2629, RAP, and
125I-labeled activated Proteins--
FVIII was purified from therapeutic concentrates
prepared by Method M, American Red Cross (38). HCh and LCh of fVIII
were prepared as described previously (6). The A1 and A2 subunits were
obtained from thrombin-activated fVIII using ion exchange chromatography on a Mono S column (Amersham Pharmacia Biotech) (12).
Radiolabeling of fVIII and Its A2 Subunit--
Prior to
labeling, fVIII and A2 were dialyzed into 0.2 M sodium
acetate, pH 6.8, containing 5 mM calcium nitrate. Five µg of fVIII or A2 in 30 µl of the above buffer were added to
lactoperoxidase beads (Worthington) containing 5 µl of
Na125I (100 mCi/ml, Amersham Pharmacia Biotech) and 5 µl
of 0.03% H2O2 (Mallinckrodt) and incubated for
4 min at room temperature. Unreacted Na125I was removed by
chromatography on a PD10 column (Amersham Pharmacia Biotech). The
specific radioactivities of 125I-labeled fVIII and A2 were
3-6 µCi/µg of protein. The activity of 125I-fVIII
(3650 units/mg) determined in the one-stage clotting assay (39) was
similar to that of unlabeled fVIII (3840 units/mg).
Assays for Cell-mediated Surface Binding, Internalization, and
Degradation of Ligands--
LRP-expressing mouse embryonic fibroblast
cells (MEF) and mouse embryonic fibroblast cells genetically deficient
in LRP biosynthesis (PEA 13) were obtained from Dr. Joachim Herz
(University of Texas Southwestern Medical Center, Dallas, TX) and
maintained as described (40). Cells were grown to a density of 2 × 105 cells/well as we described previously (12). Human
smooth muscle cells (SMC) and human alveolar epithelial cells (T2) were
obtained from American Tissue Culture Collection. SMC and T2 cells were grown to a density of 105 cells/well in DMEM and
Leibovitz's L-15 medium, respectively, containing 10% fetal bovine
serum (Life Technologies, Inc.). The complex of 125I-fVIII
or unlabeled fVIII with vWf was prepared by incubating the proteins at
a 1:50 molar ratio in HBS, 5 mM CaCl2 for 30 min at 25 °C. The complex formation was verified by gel filtration as described previously (12). To assess the contribution of HSPGs in
fVIII uptake, the cells were preincubated in the medium containing
heparinase-I (Sigma) at a concentration of 0.005 IU/ml for 30 min at
37 °C followed by three washes with HBS containing 0.1% bovine
serum albumin. Surface binding, internalization, and degradation assays
were conducted as described previously (41). In some experiments,
surface binding was determined after incubation at 4 °C to prevent
endocytosis (42). Surface binding of radiolabeled ligands was defined
as the amount of radioactivity released by treatment with mixture of
trypsin (50 µg/ml) and proteinase K (50 µg/ml) (Sigma) as described
(43). The radioactivity, which remained associated with the cells, was
considered as internalized (41). Degradation was defined as
radioactivity in the medium that was soluble in 10% trichloroacetic
acid. The value of degradation was corrected for noncellular
degradation by subtracting the acid-soluble radioactivity in parallel
wells lacking cells.
Factor Xa Generation Assay--
The rate of conversion of factor
X to factor Xa was measured in a purified system (39), in which fVIIIa
was substituted by its A2 subunit as described (44, 45). The A2 subunit
(200 nM) was preincubated with varying concentrations of
heparin (0-100 µg/ml) in HBS, 5 mM CaCl2,
0.01% Tween 20, and 200 µg/ml bovine serum albumin for 30 min at
room temperature. This was followed by the addition of factor IXa (5 nM) and PSPC vesicles (10 µM) and incubation
for 10 min, prior to addition of factor X (300 nM). To
determine the initial rates of factor Xa generation, the aliquots were
taken at 10, 20, 30, and 45 min, and the reaction was stopped with 0.05 M EDTA. Factor Xa generation was determined from conversion
of synthetic chromogenic substrate S-2765 (Chromogenix, Sweden)
as described (45).
Fluorescence Anisotropy Measurements--
The measurements of
interaction of the A2 subunit with Fl-FFR-fIXa were performed as
described (45). A2 was preincubated with varying concentrations of
heparin for 15 min at 25 °C in HBS, 5 mM
CaCl2. The anisotropy was measured in a 0.2-ml cell upon
addition of PSPC vesicles (50 µM) and Fl-FFR-fIXa (30 nM) in the presence or absence of factor X (400 nM). The measurements were carried out using SLM 8000C
spectrofluorometer (SLM Instrument Inc.) at the excitation wavelength
of 495 nm and emission wavelength of 524 nm. The data were recorded 5 times for each reaction and averaged.
Kinetic Measurements Using Surface Plasmon Resonance
(SPR)--
The kinetics of interaction of fVIII-vWf complex, fVIII,
its fragments, and vWf with heparin or chondroitin sulfate was measured by SPR technique using Biacore 3000 (Biacore, Sweden). Biotinylated heparin (100 µg/ml) or chondroitin sulfate (100 µg/ml) was
immobilized at the level of 300 resonance units on the surface of a
biosensor SA chip in HBS, 5 mM CaCl2, 0.05%
Tween 20. The binding of the above ligands was measured in the same
buffer at a flow rate of 10 µl/min. Dissociation was measured upon
replacement of the ligand solution for the buffer without ligand. The
chip surface was regenerated by washing with 1 M NaCl,
0.05% Tween 20. The kinetic parameters were derived from the kinetic
curves using Biacore BIA evaluation 3.1 software.
Immunofluorescence Microscopy--
Human hepatocellular
carcinoma cells HEP G2 (American Tissue Culture Collection) were grown
on coverslips to 80% confluence in DMEM containing 10% fetal bovine
serum at 37 °C, 6% CO2. Intact cells or cells treated
with heparinase as above were incubated with 10 nM of
fVIII-vWf complex in 0.5 ml of DMEM, 1% bovine serum albumin for
2 h at 4 °C in the absence or presence of RAP (1 µM). The cells were washed twice with phosphate-buffered
saline, fixed in 2% formaldehyde in phosphate-buffered saline, and
stained for fVIII, LRP, and HSPGs by triple label immunofluorescence
staining. Staining for fVIII was performed by subsequent incubation of
cells with mouse anti-fVIII monoclonal antibody 8860, biotinylated
anti-mouse antibody, and Texas Red-conjugated Avidin D (2.5 µg/ml).
Staining for LRP was performed by subsequent incubation with rabbit
polyclonal anti-LRP antibody Rab 2629, biotinylated anti-rabbit IgG,
and fluorescein Avidin DCS (2.5 µg/ml). Staining for HSPGs was
performed by subsequent incubation with mouse monoclonal anti-heparan
sulfate antibody 10E4 (Seikagaku Corp.), biotinylated anti-mouse IgG, and 7-amino-4-methylcoumarin-3-acetic acid Avidin D (5 µg/ml). The primary antibodies were added at 5 µg/ml and incubated with the
cells for 1 h at 25 °C. The secondary biotinylated antibodies and fluorescent reagents were purchased from Vector Laboratories and
used according to the supplied protocols. Avidin/biotin blocking kit
(Vector Laboratories) was applied after staining with each fluorescent
probe. The specificity of the staining was controlled using normal
mouse or rabbit immunoglobulins instead of the primary antibodies. For
microscopy, the coverslips with triple-stained cells were mounted on
slides with ProLong Antifade mounting medium (Molecular Probes, Inc.).
The images were obtained using an Eclipse E800 microscope (Nikon)
equipped with a set of selective fluorescent filter blocks and digital
SPOT RT camera (Diagnostic Instruments, Inc.). Simultaneous
visualization of fVIII, LRP, and HSPGs was performed by merging the
single-dye images using the SPOT Advanced Program Mode.
Clearance of 125I-fVIII-vWf Complex in
Mice--
Prior to the experiment, 125I-fVIII, vWf, and
RAP were dialyzed into HBS, 5 mM CaCl2 buffer.
BALB/c mice (12-14 weeks old, weight 20-24 g) were injected in the
tail vein with 100-µl solutions of either 200 µM
protamine or 150 µM RAP alone or with 100 µl of 200 µM protamine and 150 µM RAP together in the
above buffer. After a 2-min interval, 100-µl samples of
125I-fVIII-vWf complex formed from 125I-fVIII
(15 nM) and vWf (750 nM) were injected into
mice either in the absence or in the presence of 100 units (1 mg) of
heparin. In the control experiment, 125I-fVIII-vWf complex
was injected in the absence of protamine and RAP. Blood samples of
30-40 µl were withdrawn from each mouse via retroorbital puncture
into 15 µl of 0.1 M sodium citrate buffer, pH 7.4, at
selected time intervals (1, 5, 10, 15, 30, 60, 120, 240, 360, and 480 min). The radioactivity per ml of blood at each time point was
calculated from radioactivities of the samples and their volumes. The
percentage of 125I-fVIII remaining in circulation was
calculated assuming the radioactivity of aliquots taken 1 min after
injection of 125I-fVIII-vWf complex as 100% (46, 47). To
verify that no significant fVIII clearance occurred during the 1st min,
we compared the radioactivities of 1-min aliquots (n = 20) with the corresponding initial (0 min) radioactivity values. The
0-min values were calculated from the total amount of injected
radioactivity and the blood volume of each mouse determined using the
formula V = 0.09 × W0.88
(48), where W is the animal weight in grams and V
is the total blood volume in milliliters. Lack of statistically
significant differences (p = 0.34 according to
Student's t test) between these two groups of values
justified our assumption that the radioactivity in blood at 1 min after
injection of 125I-fVIII-vWf complex can be considered as
100%. The time course of each of the above conditions was examined in
four mice and averaged. The kinetics of 125I-fVIII
clearance from circulation was fitted to the previously used
double-exponential model (12) by using the Sigmaplot 3.0 computer
program (Jandel Scientific).
HSPGs Are the Primary Receptors Responsible for the Initial Binding
of fVIII-vWf Complex to LRP-expressing Cells--
We demonstrated
previously that RAP inhibited endocytosis and degradation of fVIII from
its complex with vWf by LRP-expressing cells, indicating that LRP is
involved in catabolism of fVIII (12). Therefore, we examined the role
of LRP in the initial binding of fVIII-vWf complex to the cell surface.
As seen in Fig. 1A, the
binding levels of 125I-fVIII-vWf complex were similar for
both LRP-expressing MEF cells and LRP-deficient PEA13 cells. Moreover,
RAP did not have a significant inhibitory effect on the binding to MEF
cells. These findings suggest that receptor(s) other than LRP is
responsible for the surface binding of 125I-fVIII-vWf
complex. We next examined whether these receptors could be HSPGs by
testing the effect of heparin on the surface binding. As seen in Fig.
1A, heparin significantly reduced the cell surface binding
for both LRP-expressing and LRP-deficient cells, supporting our
assumption that HSPGs are the major surface receptors responsible for
the initial binding of fVIII-vWf complex. Consistent with this
assumption, heparin also inhibited degradation of fVIII by MEF cells
(Fig. 1B). To exclude the possibility that heparin inhibited
fVIII degradation by interfering with fVIII binding to LRP, we tested
the effect of heparin on 125I-fVIII binding to immobilized
LRP in the assay described previously (12). We found that heparin did
not inhibit the binding (data not shown), suggesting the following: (i)
inhibition of fVIII degradation in LRP-expressing cells by heparin was
not related to inhibition of fVIII interaction with LRP and (ii)
heparin- and LRP-binding sites of fVIII do not overlap. Notably,
LRP-deficient PEA13 cells also degraded fVIII at the level constituting
22% of that degraded by LRP-expressing cells (Fig. 1B).
Although this level was significantly lower in comparison with MEF
cells, the ability of LRP-deficient cells to degrade fVIII implies the
existence of an alternative, LRP-independent pathway of fVIII
catabolism. Since it was inhibited by heparin (Fig. 1B),
HSPGs appear to be also required for this LRP-independent pathway.
The Major fVIII Site Involved in Binding to HSPGs Is Located within
Its A2 Domain--
Since the LRP binding site of fVIII is located
within its A2 domain (12), it is expected that LRP-expressing cells
will catabolize the isolated A2 domain as well. To examine whether catabolism of A2 and fVIII from its complex with vWf occurs in a
quantitatively similar fashion, we studied dose dependence of their
surface binding and degradation by MEF cells. As seen in Fig.
2, the corresponding curves of the
surface binding and degradation of A2 and fVIII from its complex with
vWf are similar.
We next examined whether HSPGs play a similar role in catabolism of the
A2 subunit and fVIII. The preliminary evidence of involvement of HSPGs
in A2 catabolism was derived from the inhibitory effect of heparin on
the surface binding and degradation of A2 (data not shown). To confirm
the role of HSPGs in catabolism of A2 and fVIII, we studied the effect
of heparinase, which removes carbohydrate portions of proteoglycans, on
the surface binding and degradation of these ligands. As seen in Fig.
2, this treatment reduced the surface binding and degradation of
125I-A2 and 125I-fVIII to the same level,
indicating that interaction of fVIII-vWf complex with HSPGs is likely
to occur primarily via the A2 domain. Notably, the addition of RAP to
heparinase-treated cells did not inhibit the surface binding of the
ligands (data not shown); however, it further reduced their degradation
(Fig. 2B). In the control experiment, we confirmed that the
functional activity of LRP was not impaired by heparinase treatment,
since this treatment did not have any effect on internalization of a
direct LRP ligand 125I-activated
To examine whether involvement of HSPGs in LRP-mediated
catabolism of A2 is a common feature of LRP-expressing cells, we tested the binding of A2 to human smooth muscle cells (SMC) and human alveolar
epithelial cells (T2) expressing LRP and HSPGs (15). In both cell types
heparin and heparinase significantly inhibited the surface binding,
internalization, and degradation of 125I-A2 (Fig.
3). Addition of RAP to heparinase-treated
cells had no effect on the 125I-A2 binding but led to a
further decrease of its internalization and degradation. Thus, the
effects of heparinase and RAP on A2 catabolism in MEF (Fig. 2), SMC,
and T2 (Fig. 3) are similar, indicating that LRP and HSPGs are both
involved in the A2 catabolism by different LRP-expressing cells.
To confirm that the A2 domain is fully responsible for the
binding of fVIII-vWf complex to cell surface HSPGs, we studied the
effects of increasing concentrations of fVIII fragments and vWf on the
surface binding of 125I-fVIII-vWf complex to MEF cells. As
seen in Fig. 4, at a concentration of 200 nM, A2 inhibited this binding by 84%. Notably,
pretreatment of MEF cells with heparinase resulted in a similar (87%)
reduction of 125I-fVIII-vWf binding as shown in Fig.
2A. In contrast, neither A1/A3-C1-C2 heterodimer nor vWf
were able to inhibit the binding of 125I-fVIII-vWf complex.
This indicates that fVIII but not vWf is responsible for the binding of
fVIII-vWf complex to cell surface HSPGs, and the major HSPGs-binding
site of fVIII is located within the A2 domain.
The A2 Domain and fVIII-vWf Complex Bind to the Cells with
Similar Affinities--
The presence of the major HSPGs-binding site
within A2 implies that affinities of A2 and fVIII-vWf complex for the
cell surface should be similar. To verify this, we first
determined the affinity of 125I-A2 to MEF cells in a
saturation binding experiment similar to that presented in Fig. 2 but
performed at 4 °C to exclude internalization (data not shown). The
nonspecific binding measured in the presence of 100-fold excess of
unlabeled A2 constituted 18% of the total 125I-A2 binding.
The specific binding was adequately described by a model implying
existence of a single class of binding sites (9.6 × 104 sites per cell) with Kd of 15 ± 2.8 nM. To verify that A2 and fVIII-vWf complex bind to
the same sites, we performed displacement of 125I-A2 (1 nM) by unlabeled A2 or fVIII-vWf complex. In this assay, A2
and fVIII-vWf complex were found to be equal as competitors (Fig.
5) with Ki values of
18.8 ± 2.2 and 21.4 ± 1.9 nM, respectively. The
similarity of the Ki values further supports that
the binding of fVIII-vWf complex to HSPGs is mediated by the A2 domain
of fVIII.
The Major Site within A2 and the Minor Site within LCh Are Involved
in fVIII Binding to Heparin--
To examine whether A2 is the only
site responsible for interaction of isolated fVIII with HSPGs, we
tested the direct binding of fVIII, A2, and other fVIII fragments to
heparin in SPR-based assay (Fig. 6). The
kinetic parameters for fVIII and its fragments derived from Fig. 6 are
shown in Table I. We found that fVIII, its A2 domain, and HCh (containing A2), but not A1, were able to bind
to heparin, consistent with the presence of the heparin-binding site
within the A2 subunit. The determined affinity of the A2 subunit for
heparin is 25.8 nM. Unexpectedly, LCh was also able to bind
heparin with a low affinity (Kd = 571 nM), indicating that it contains another heparin-binding
site. Consistent with this observation, the fVIII binding kinetics was
optimally fitted to a model implying existence of two heparin-binding
sites, which affinities (Kd values of 28 and 652 nM) are similar to the values determined for A2 and LCh,
respectively. The 23-fold lower affinity of the LCh site for heparin
implies that its contribution to fVIII binding to HSPGs is not
significant. In control experiments, the specificity of fVIII and A2
interaction with heparin was demonstrated using Biacore SA chip coated
with biotinylated chondroitin sulfate which, similarly to heparin, is
composed of negatively charged sulfated polysaccharides. The binding of
fVIII (Fig. 6, curve 1b) and its A2 subunit (data not shown)
constituted less than 7.3% of the corresponding binding of the ligands
to heparin.
We next compared the parameters of fVIII-vWf complex and vWf binding to
heparin (derived from Fig. 6, inset). As seen in Table I,
although the parameters of fVIII-vWf interaction with heparin are
similar to those of isolated fVIII, isolated vWf bound to heparin with
a low affinity (Kd = 8.4 µM). This
confirms that its contribution to interaction of fVIII-vWf complex with heparin is negligible. Remarkably, the affinities of fVIII-vWf complex
and A2 for heparin determined in a purified system (Table I) are close
to the affinities of fVIII-vWf complex and A2 (18.8 and 21.4 nM, respectively) for the surface of MEF cells. Altogether, these data further support our hypothesis that the major fVIII site
responsible for the binding to HSPGs is located within the A2 domain.
The A2 Domain Heparin-binding Site Includes the Residues
558-565--
Localization of the heparin-binding site within the A2
domain was initiated by the previous findings that heparin inhibits Xase activity (35, 49), and fVIIIa can be substituted by its A2 subunit
in the Xase assay (45). As seen in Fig.
7A, heparin was inhibitory in
the A2-dependent Xase assay; the effect was dose-dependent; and 90% inhibition was observed at 10 µg/ml (~600 nM).
Since it was previously shown that heparin does not inhibit interaction
of the Xase complex with its substrate factor X (49), we proposed that
heparin inhibits Xase assembly by preventing the A2 binding to factor
IXa. To examine this possibility, we tested the effect of heparin on
the A2 binding to factor IXa by fluorescent anisotropy technique. The
experiment was based on the previous observation that anisotropy of
Fl-FFR-fIXa increases moderately upon the binding of A2 (45), and this
effect becomes more pronounced in the presence of factor X (44, 45). We
found that heparin inhibited the increase of anisotropy in a
dose-dependent fashion, both in the absence or presence of
factor X (Fig. 7B). The maximal effect of heparin was
observed at its concentration
The above findings suggest that heparin blocks interaction between the
A2 subunit and factor IXa, which might be due to the overlapping of the
A2 domain binding sites for heparin and factor IXa. Since the A2 domain
regions comprising residues 484-509 and 558-565 are directly involved
in the interaction with factor IXa (45, 50), we tested the effects of
the corresponding synthetic peptides on the A2 binding to heparin. In
the SPR-based experiment, the peptide 558-565 inhibited the binding by
78% at a concentration of 800 µM (Fig.
8). In contrast, at the same
concentration, the peptide 484-509 inhibited the binding by ~25%,
and the peptide 417-428 was not inhibitory at all. This suggests that
the A2 domain region 558-565 is involved in the fVIII binding to
heparin and, possibly, to cell surface HSPGs.
Cell Surface Proteoglycans Participate in fVIII Catabolism in
Vivo--
To examine whether HSPGs contribute to fVIII clearance
in vivo, we performed clearance studies in mice in the
presence of protamine, which prevents HSPGs from interaction with their
ligands (32, 51). The data shown in Fig.
9 were fitted to the previously used
double exponential model (12), implying existence of the fast and slow
phases of fVIII clearance. This model is described by the following
Equation 1:
FVIII Is Colocalized with HSPGs on the Surface of LRP-expressing
Hepatic Cells--
We found previously that injection of
125I-fVIII-vWf complex into mice led to accumulation of
most of the radioactivity in liver (12), where LRP is present in high
abundance (15). To verify whether HSPGs are involved in the initial
fVIII binding to the liver cells, we performed direct visualization of
fVIII, HSPGs, and LRP in human hepatic cells HEP G2, expressing both
LRP and HSPGs (53). The cells were incubated with fVIII-vWf complex at
4 °C followed by triple label immunofluorescent staining for fVIII, LRP, and HSPGs and microscopy
(Fig. 10). For each preparation, the distribution of fVIII, HSPGs, or
LRP is shown in red, blue and green images,
respectively. For control cells, the individual stainings for fVIII,
HSPGs, and LRP are represented by the images a,
b, and c, respectively. FVIII was distributed on the
cell surface in a grainy pattern, typical for cell surface but not for
cytoplasmic staining. The merged image (image d)
demonstrates that fVIII colocalized predominantly with HSPGs as the
purple areas, resulting from the superimposing of
red and blue staining for fVIII and HSPGs,
respectively. Colocalization of surface-bound fVIII with LRP was
negligible, since large areas in the merged image remained
green but not yellow, as would be expected for
superimposed red and green images. Consistent with this observation,
treatment of the cells with heparinase removing glycosamine residues
from HSPGs (image f) led to a dramatic reduction of bound
fVIII (image e) and to disappearance of
purple areas on the merged image (image
h). In contrast, blocking of LRP by RAP (images
i, j, k, and l) did not appreciably alter the level of fVIII binding (image i) when compared
with the control cells (image a). In the merged
image (image l) fVIII remained colocalized with
HSPGs, consistent with a negligible role of LRP in the initial surface
binding of fVIII-vWf complex. Thus, the microscopy study confirmed that
HSPGs are the major receptors responsible for the initial binding of
fVIII-vWf complex to the surface of LRP-expressing hepatic cells.
In the present study we found that cell surface HSPGs facilitate
LRP-mediated catabolism of fVIII from its complex with vWf in cell
culture and in vivo. In LRP-expressing cells, the bulk of
the initial binding of fVIII-vWf complex occurs via HSPGs, which
cooperate with LRP receptor in the consequent internalization of the
fVIII molecule. In mice, the simultaneous blocking of HSPGs and LRP led
to a significant prolongation of the fVIII half-life compared with
fVIII half-lives when HSPGs and LRP were blocked independently.
The interaction of fVIII-vWf complex with HSPGs occurs via the A2
domain of fVIII, which is based on the following findings: (i) the cell
surface binding of A2 and fVIII-vWf complex displayed a similar dose
dependence and was inhibited by heparinase treatment to the same
extent; (ii) the A2 subunit, but not other portions of fVIII or
isolated vWf, strongly inhibited the cell surface binding; (iii) the A2
subunit and fVIII-vWf complex bound to the cell surface with similar
affinities; (iv) the A2 subunit and fVIII had similar affinities for
heparin in a purified system. Although vWf was previously shown to
interact with heparin, its contribution to the binding of fVIII-vWf
complex to the cells surface is negligible, which follows from the low
affinity of vWf for heparin determined in our experiments
(Kd = 8.4 µM) and by others
(Kd values are 2 (54) and 78 µM (55)). The heparin-binding affinity of vWf in the present study was 300-fold lower than the affinities determined for fVIII and A2 (~28
nM). The interaction of fVIII and its A2 subunit with
heparin was specific as demonstrated by a negligible binding of these
ligands to chondroitin sulfate, which represents another type of
carbohydrates associated with cell surface proteoglycans. This suggests
that fVIII-vWf complex selectively binds to cell surface heparan
sulfate proteoglycans.
The A2 site involved in the binding to heparin was localized to the
region 558-565, based on the ability of the synthetic peptide
encompassing this region to inhibit the A2 binding to heparin. Notably,
the peptide 484-509 corresponding to the previously localized
LRP-binding site (12) did not appreciably inhibit the binding,
suggesting that the A2 sites responsible for the binding to LRP and
HSPGs are distinct.
Heparin-binding sites of proteins are commonly represented by cationic
clusters formed by Arg and Lys residues, which interact with anionic
groups (sulfate and carboxyl groups) of glycosaminoglycan chains of the
proteoglycans (56). According to the previously published
three-dimensional model of the A2 domain (57), there are
Lys556, Lys562, Lys570, and
Arg571 within the 558-565 region and in the close
proximity to it, which are exposed on the A2 surface. We also found
another, low affinity heparin-binding site within LCh of fVIII, which
precise localization remains to be performed.
Cooperation of HSPGs with LRP in catabolism of fVIII is similar to
their role in catabolism of most heparin-binding LRP ligands (19, 27,
31, 41, 42, 58). The proposed role of HSPGs is to concentrate ligands
on the cell surface and to facilitate their subsequent internalization
by presenting them to LRP (14, 31). This role of HSPGs is consistent
with our data, since the affinity of fVIII-vWf complex for HSPGs and
heparin (Kd = 15-30 nM) is higher than
that for LRP (Kd = 116 nM (12)).
Internalization and degradation of fVIII by LRP-expressing MEF, SMC,
and T2 cells could be effectively inhibited by RAP, which confirms the
involvement of LRP in fVIII catabolism previously reported by us (12).
In LRP-deficient PEA13 cells the level of fVIII degradation was similar
to that determined in the previous study (12) and significantly lower
(22%) in comparison with LRP-expressing MEF cells. We found that this
less effective, LRP-independent degradation by PEA13 cells was strongly
inhibited by heparinase treatment, suggesting existence of a different
pathway of fVIII catabolism, which involves HSPGs. These findings are
consistent with the biphasic character of fVIII clearance in
vivo, reflecting the existence of two distinct pathways of fVIII
catabolism. The inhibitory effect of protamine on the fast and slow
phases of clearance points to involvement of HSPGs in both pathways of
fVIII catabolism in vivo. Since only the fast phase of fVIII
clearance was RAP-sensitive, we propose that in this phase fVIII bound
to cell surface HSPGs undergoes LRP-mediated endocytosis, whereas in
the slow phase, also facilitated by HSPGs, fVIII follows
LRP-independent pathway. Unlike in cell culture, the simultaneous
blocking of HSPGs and LRP by protamine and RAP, respectively, did not
completely block the fVIII clearance in mice. This can be explained by
either incomplete inhibition of these receptors due to clearance of RAP and protamine or by the existence of another mechanism, which does not
involve HSPGs and LRP. It cannot be completely excluded, however, that
the inhibitory effect of protamine on fVIII clearance could be due to
prevention of fVIII interactions with negatively charged cell
surface-associated molecules other than HSPGs.
The proposed role of HSPGs is depicted in Fig.
11, which indicates that catabolism of
fVIII from its complex with vWf occurs via initial binding of the
complex to HSPGs, followed by both LRP-mediated and LRP-independent
endocytosis and degradation of fVIII. The model implies that vWf
dissociates prior to fVIII internalization, based on our previous
finding that vWf does not follow fVIII in the endocytic pathway (12).
Our demonstration that catabolism of the A2 subunit is equivalent to
that of fVIII from its complex with vWf suggests that dissociation of
the complex is not a rate-limiting step of the catabolic process. The
previous finding that the isolated fVIII is catabolized more
efficiently than from its complex with vWf (12) can be explained by the
presence of the second LRP-binding site within the C2 domain of LCh
(23), which is likely to be blocked by interaction with vWf. The
overlapping of the C2 domain LRP- and vWf-binding sites (24) was
suggested from the inhibitory effect of monoclonal anti-C2 domain
antibody ESH4 on fVIII binding to both LRP (23) and
vWf.2 The proposed
contribution of the LRP-binding site located within the C2 domain to
catabolism of fVIII is consistent with a significantly faster clearance
of fVIII in vWf-deficient patients (8).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-macroglobulin were
kindly provided by Dr. Dudley Strickland. Phosphatidylserine (PS) and
phosphatidylcholine (PC) were purchased from Sigma. Phospholipid
vesicles containing 25% PS and 75% PC were prepared as described
previously (37). The fVIII peptides 432-456, 484-509, and 558-565
were synthesized using a 9050 Milligen synthesizer (Millipore) by the
Fmoc ((9-fluorenyl)methoxycarbonyl) method and pentafluoro-ester
activation chemistry and were purified by reverse phase high pressure
liquid chromatography using a C18 column (Waters) in a
gradient of 0-70% acetonitrile in 0.1% trifluoroacetic acid. The
2.2-3.5 mM solutions of peptides were dialyzed
versus 20 mM HEPES, pH 7.4, 0.15 M
NaCl (HBS) using membrane with 1-kDa cut-off (Pierce).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (20K):
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Fig. 1.
Effect of RAP and heparin on the surface
binding and degradation of 125I-fVIII-vWf by MEF and PEA13
cells. 125I-fVIII-vWf complex (1 nM) was
added to wells containing 2 × 105 of LRP-expressing
MEF cells (solid bars) or LRP-deficient PEA 13 cells
(gray bars) in the absence or presence of heparin (100 µg/ml) or RAP (1 µM) and incubated for 6 h at
37 °C. The surface binding of 125I-fVIII (A)
and its degradation (B) were subsequently determined as
described under "Experimental Procedures." Each data point
represents the mean value and S.D. of duplicate determinations.
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Fig. 2.
Comparison of the surface binding and
degradation of 125I-A2 and 125I-fVIII from its
complex with vWf by MEF cells. Wells containing 2 × 105 of LRP-expressing MEF cells were preincubated without
(closed symbols) or with (open symbols)
heparinase as described under "Experimental Procedures." This was
followed by the addition of increasing concentrations of
125I-fVIII-vWf complex ( ,
) or 125I-A2
(
,
), incubation for 6 h at 37 °C, and determination of
the surface binding (A) and degradation (B). In
additional experiments shown in B, 125I-fVIII
(
) and 125I-A2 (
) were incubated with
heparinase-treated MEF cells in the presence of RAP (1 µM). Each data point represents the mean value and S.D.
of duplicate determinations.
2-macroglobulin (40), data not shown. Altogether, the
above experiments demonstrated that the A2 subunit behaves equivalently
to fVIII-vWf complex in the catabolic process.
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Fig. 3.
Effect of RAP, heparin, and heparinase on the
surface binding, internalization, and degradation of
125I-A2 subunit of fVIII by LRP-expressing SMC and T2
cells. Wells containing 105 SMC (gray bars)
or T2 cells (hatched bars) were preincubated without or with
heparinase as described under "Experimental Procedures." This was
followed by the addition of 125I-A2 (1 nM) in
the absence or presence of RAP (1 µM) or heparin (100 µg/ml) and determination of the surface binding (A and
D), internalization (B and E), and
degradation (C and F) of 125I-A2
after 6 h of incubation at 37 °C. Each data point represents
the mean value and S.D. of duplicate determinations.
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Fig. 4.
Effect of fVIII fragments and vWf on the
surface binding of fVIII-vWf complex to MEF cells. One
nM of 125I-fVIII-vWf complex was added to wells
containing 2 × 105 of LRP-expressing MEF cells in the
presence of varying concentrations of A2 ( ), A1/A3-C1-C2 (
), or
vWf (
) and incubated for 2 h at 4 °C, followed by
determination of surface-bound radioactivity, as described under
"Experimental Procedures." Each data point represents the mean
value and S.D. of duplicate determinations.
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Fig. 5.
Determination of affinities of A2 and
fVIII-vWf complex for binding to MEF cells. MEF cells were
incubated for 2 h at 4 °C with 125I-A2 (1 nM) in the presence of increasing concentrations of
unlabeled A2 ( ) or fVIII-vWf complex (
) formed from varying
concentrations of fVIII (4-128 nM) and the fixed
concentration of vWf (1000 nM) as described under
"Experimental Procedures." This was followed by determination of
125I-A2 binding to the cells. Each data point represents
the mean value and S.D. of quadruplicate determinations. The
solid lines show the best fit of the data to a model
describing homologous or heterologous ligand displacement from a single
class of binding sites using the LIGAND program. The dashed
line is shown for the control experiment in which vWf (
) was
used as a competitor.
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Fig. 6.
Interaction of fVIII-vWf complex, fVIII, its
fragments, and vWf with heparin in SPR-based assay. Heparin was
immobilized to a biosensor chip at a level of 300 resonance units
(RU) as described under "Experimental Procedures." The
binding of 500 nM of either fVIII (curve 1a),
its HCh (curve 2), LCh (curve 3), A2 (curve
4), or A1 (curve 5) was measured for 5 min at a flow
rate of 10 µl/min. In the control experiment (dotted curve
1b), fVIII binding to chondroitin sulfate, immobilized on the chip
at the same level as heparin, was tested. Dissociation kinetics was
measured upon replacement of the ligand solution for the buffer without
a ligand. Inset shows interaction of 1000 nM vWf
(curve 6) and 500 nM fVIII-vWf complex formed
from 500 nM fVIII and 1000 nM vWf (curves
7) with heparin-coated chip.
Kinetic parameters of the binding of fVIII-vWf complex, fVIII, its
fragments and vWf to heparin
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Fig. 7.
Inhibition of the A2-dependent
factor Xa generation and interaction between A2 and factor IXa by
heparin. A, effect of heparin on the generation of
factor Xa. The mixtures containing factor IXa (5 nM), PSPC
vesicles (10 µM), A2 subunit (200 nM), and
the indicated concentrations of heparin were incubated for 10 min, and
the reactions were started by the addition of factor X (300 nM). The initial rates of factor Xa generation ( ) were
determined as described under "Experimental Procedures."
B, effect of heparin on interaction between A2 subunit and
factor IXa. The A2 subunit (300 nM) was preincubated with
indicated concentrations of heparin for 15 min. The anisotropy was
measured upon the addition of PSPC vesicles (50 µM) and
Fl-FFR-fIXa (30 nM) in the presence (
) or absence (
)
of factor X (400 nM). In control experiments, the A2
subunit was omitted from the reaction mixtures with (
) or without
(
) factor X. Each point represents the mean value ± S.D. of
five measurements.
30 µg/ml, which is similar to the
concentration completely suppressing the factor Xase assay (Fig.
7A). In the control experiment performed in the absence of
A2, heparin did not affect the anisotropy of Fl-FFR-fIXa in either
absence or presence of factor X.
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Fig. 8.
Effect of synthetic peptides on the A2
binding to heparin. A, effect of the synthetic peptide
558-565 on A2 binding to heparin measured by SPR technique. Heparin
was immobilized on the chip surface as described under "Experimental
Procedures." The binding of the A2 subunit (200 nM) was
measured in the absence (curve 1) or presence of increasing
concentrations of the peptide (25, 50, 100, 200, 400, and 800 µM, curves 2-7, respectively). B,
effect of synthetic peptides 432-456 ( ), 484-509 (
), and
558-565 (
) on A2 binding to heparin. The equilibrium binding of A2
to immobilized heparin at indicated concentrations of each peptide was
determined as in A. The A2 binding in the presence of
peptides is expressed as the percentage of the A2 binding when no
peptide was added.
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Fig. 9.
Effect of protamine and RAP on clearance of
125I-fVIII-vWf in vivo. BALB/c mice
were injected with 100 µl of either 200 µM (1 mg)
protamine ( ) or 150 µM RAP (
) alone, or with 100 µl of 200 µM protamine and 150 µM RAP
together (
) 2 min prior to injection of 100-µl samples containing
125I-fVIII (15 nM) and vWf (750 nM). In the experiment (
), 100 units (1 mg) of heparin
were administered 2 min after injection of protamine and RAP. In the
control experiment (
), clearance of 125I-fVIII-vWf
complex was studied in the absence of any agent. At the indicated time
points, blood samples were taken, and their radioactivity was counted.
The percentage of the ligand remaining in circulation was calculated
considering radioactivity of 1-min aliquot as 100%.
125I-fVIII clearance was examined in four mice for each of
the above conditions. The curves show the best fit of the
experimental data to Equation 1 ("Experimental Procedures")
describing biphasic exponential clearance of fVIII. The dotted
curve represents the best fit for the experiment (
).
where C is the percentage of 125I-fVIII
remaining in plasma at a given time; k1 and
k2 are the kinetic rate constants corresponding to the fast and slow phases of fVIII clearance; and
C1 and C2 are percentages
of radioactivity removed during the fast and slow phases of clearance.
The values of k1, k2,
C1, and C2 constants were
derived for each clearance curve by fitting C versus
t to the above Equation 1. At the saturating concentration
of RAP, the rate of the fast phase of clearance was dramatically
reduced (Table II), resulting in
prolongation of the half-life of fVIII by 3.5-fold, similar to that
reported previously (12). Administration of protamine prolonged the
fVIII half-life by 1.6-fold and reduced the rates of both phases of
clearance (Table II). This indicates that HSPGs contribute to both
RAP-sensitive and RAP-independent pathways of fVIII clearance. Notably,
coinjection of RAP and protamine resulted in a greater increase of the
fVIII half-life (5.5-fold) than injection of RAP alone (3.5-fold),
suggesting that LRP (12) and HSPGs are simultaneously involved in fVIII
clearance. To confirm that the effect of protamine on fVIII clearance
in vivo was specific, we performed a control experiment in
which protamine was administered together with 100 units (1 mg) of
heparin. This amount of heparin is sufficient to neutralize 1 mg of
protamine by irreversible binding, thus preventing interaction of
protamine with HSPGs (51, 52). Administration of heparin abolished the
effect of protamine both in the presence of RAP (Fig. 9) and in its
absence (data not shown), supporting our assumption that the effect of
protamine is due to prevention of fVIII-vWf binding to HSPGs. Thus, the above data suggest that HSPGs participate in fVIII clearance in vivo and are involved in LRP-mediated and LRP-independent
catabolic pathways.
(Eq. 1)
Effect of RAP and protamine on the parameters of fVIII clearance from
plasma of mice
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Fig. 10.
Microscopy studies of surface binding
of fVIII from its complex with vWf by HEP G2 cells. Control
untreated HEP G2 cells (upper panel, images
a-d) and the cells treated with heparinase (middle
panel, images e-h) or RAP (lower
panel, images i-l) were incubated with 10 nM of fVIII-vWf complex for 2 h at 4 °C. This was
followed by fixing the cells and triple label staining for fVIII using
Texas Red (red images a, e, and i),
for HSPGs using AMCA (blue images b, f, and
g) and for LRP using FITC (green images c,
g, and k) as fluorophores, as described under
"Experimental Procedures." Each type of staining was visualized
using a selective fluorescent filter block. The merged images (d,
h, and l) were obtained by superimposing single-stained
images as described under "Experimental Procedures."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (28K):
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Fig. 11.
Molecular model of catabolism of fVIII from
its complex with vWf. Initial binding of fVIII-vWf complex occurs
mainly via interaction with HSPGs, followed by either LRP-mediated
endocytosis of fVIII occurring via clathrin-coated pits (61) or its
LRP-independent endocytosis. Since vWf does not follow fVIII in the
endocytic pathway in the cell culture experiments (12), we propose that
it dissociates from fVIII prior to entry of the complex into endosomal
compartments.
Our finding that the isolated A2 domain of fVIII can also be catabolized by HSPGs- and LRP-mediated mechanisms may reflect the existence of a specific pathway for clearance of activated fVIII. Heterotrimeric fVIIIa (A1/A2/A3-C1-C2) is an unstable molecule due to its rapid but reversible dissociation to A2 and A1/A3-C1-C2 (59, 60). Since the A2 subunit retains a weak fVIIIa-like ability to support Xase and may also reassemble with A1/A3-A3-C1, it is tempting to speculate that clearance of the isolated A2 subunit may have evolved as a mechanism preventing formation of the Xase complex at inappropriate coagulation sites.
In summary, we demonstrated that fVIII catabolism from its complex with
vWf involves the initial binding of the complex to cell surface HSPGs
due to interaction between polysaccharide portions of HSPGs and the
major heparin-binding site of fVIII localized within the A2 domain. The
fVIII molecule is subsequently catabolized via LRP-mediated and
LRP-independent pathways. Our finding that the simultaneous blocking of
LRP and HSPGs receptors dramatically prolonged the lifetime of fVIII in
a mouse model supports the physiological role of LRP and HSPGs in fVIII catabolism.
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FOOTNOTES |
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* This work was supported in part by Scientist Development Grant 9630065N (to E. L. S.) from the American Heart Association and Junior Faculty Award (to E. L. S.) from the American Society of Hematology.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: American Red
Cross, 15601 Crabbs Branch Way, Rockville, MD 20855. Tel.:
301-738-0743; Fax: 301-738-0794.
Published, JBC Papers in Press, January 12, 2001, DOI 10.1074/jbc.M008046200
2 E. L. Saenko, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: fVIII, factor VIII; fVIIIa, activated factor VIII; HCh, heavy chain of factor VIII; LCh, light chain of factor VIII; vWf, von Willebrand factor; HSPGs, heparan sulfate proteoglycans; LRP, low density lipoprotein receptor-related protein; RAP, receptor-associated protein; Fl-FFR-fIXa, fluorescently labeled factor IXa; MEF, mouse embryonic fibroblast cells expressing LRP; PEA 13, mouse embryonic fibroblast cells genetically deficient in LRP; SMC, human smooth muscle cells; T2, human alveolar epithelial cells; HEP G2, human hepatocellular carcinoma cells; SPR, surface plasmon resonance; PC, phosphatidylcholine; PS, phosphatidylserine; DMEM, Dulbecco's modified Eagle's medium; Xase, membrane-bound complex of fVIIIa and factor IXa.
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