| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
(Received for publication, December 26, 1995; and in revised form, February 6, 1996) From the
Coagulation factor Xa is a plasma serine protease that catalyzes
prothrombin to thrombin conversion, which, in turn, leads to the
generation of the fibrin clot. Of the several parameters that govern
the plasma level of factor Xa, control of its catabolism is of crucial
importance. However, little is known regarding the mechanisms by which
factor Xa is catabolized. In the present study we examine the cellular
basis for the uptake and degradation of factor Xa.
Factor X, a plasma glycoprotein involved in the blood
coagulation cascade, can be converted to its active serine protease
form factor Xa by both the intrinsic (factor IXa) and extrinsic (factor
VII and tissue factor) pathways (Jackson and Nemerson, 1980). Factor Xa
as the activator of prothrombin occupies a central position linking the
two blood coagulation pathways. Control of factor Xa levels by its
catabolism and by plasma protease inhibitors may therefore be pivotal
in the regulation of the coagulation process. While little is known
about factor Xa catabolism, a number of plasma serine protease
inhibitors are thought to inhibit factor Xa activity. These include
There are three sources of TFPI in
vivo: plasma TFPI, which predominantly associates with
lipoproteins and has a mean plasma concentration of 2.5 nM (Novotny et al., 1991); platelet TFPI, which is
sequestered from the circulation and represents 10% of the plasma TFPI
level (Novotny et al., 1988); and endothelial TFPI, which is
proposed to associate with endothelial glycosaminoglycans. Plasma
levels of TFPI rise severalfold following heparin infusion (Sandset et al., 1988; Novotny et al., 1991). The
physiological roles of these three pools are not well defined. Platelet
TFPI is released following thrombin stimulation and may exert an
antithrombotic effect at local sites (Novotny et al., 1988).
However, to date it has not been possible to demonstrate a direct
correlation between plasma levels of TFPI and thromboses. The fact that
patients with homozygous abetalipoproteinemia and
hypobetalipoproteinemia have very low levels of plasma TFPI and do not
suffer from thrombosis (Novotny et al., 1989) suggests that
the TFPI associated with lipoproteins may not be essential. Perhaps the
large endothelial cell surface pool of TFPI is of greater physiological
importance. In order to begin to elucidate the function of cell
surface-associated TFPI, we have investigated its role in factor Xa
catabolism. Our data show that the uptake and degradation of
Degradation
assays were generally carried out at 37 °C for 3 h in 0.5 ml of
assay buffer containing the indicated concentrations of
Figure 1:
Cell surface-bound TFPI
enhances
We first evaluated the binding of
Figure 2:
Binding of
Previously we showed that the uptake and
degradation of TFPI by hepatoma cells is blocked by anti-LRP antibody
as well as by the 39 kDa protein, an LRP-binding protein that inhibits
all known ligand interactions with LRP (Warshawsky et al.,
1994). These observations led to the conclusion that LRP is the
endocytic receptor for TFPI. To confirm this observation,
Figure 3:
Degradation of
PEA 10 and PEA 13 cells are phenotypically
different only in their expression of LRP. Thus using these two cell
lines, we next examined to what extent LRP contributed to
TFPI-dependent
Figure 4:
Figure 5:
Factor Xa stimulates the degradation of
To determine whether this stimulatory effect on
Figure 6:
Factor X or DNS-GGACK-inactivated factor
Xa has no effect on the degradation of
Figure 7:
Uptake and degradation of
It is of note that the internalized
pool of
In the current study we show that TFPI interacts with factor Xa in 1:1
stoichiometry; however, the interaction is reversible (Broze et
al., 1987, 1988). Thus one would expect an eventual transfer of
factor Xa from this complex to its major plasma inhibitors,
antithrombin III, We showed previously that cellular
degradation of Several lines of evidence
suggest that factor Xa and cell surface-bound TFPI are internalized as
a bimolecular complex. First, binding and degradation of In an
effort to identify the receptor for the TFPI-factor Xa complex, we
explored the possibility of TF as a potential candidate. TF is an
integral membrane protein capable of forming quarternary complexes with
TFPI, factor Xa, and factor VIIa (Girard et al., 1989; Gemmell et al., 1990). Adventitial fibroblasts beneath the vascular
endothelium constitutively express TF (Wilcox et al., 1989;
Drake et al., 1989; Fleck et al., 1990). On the
contrary, endothelial cells that are in intimate contact with blood,
under physiological conditions, either do or do not express very little
TF (Wilcox et al., 1989; Grake et al., 1989; Fleck et al., 1990). Assuming that the endothelium serves as a
potential site for degradation of the TFPI-factor Xa complex, one may
not predict that TF serves as this receptor due to its scarce
expression on endothelial cells. This is in fact what we have observed.
Using TF-negative fibroblasts, marked stimulation of degradation of
cell surface-bound Kazama et al.(1993) reported that in addition to
binding to cell TFPI, 30% of
Volume 271,
Number 16,
Issue of April 19, 1996 pp. 9497-9502
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
I-Factor Xa was degraded by hepatoma cells and embryonic
fibroblasts via a process which required cell surface-bound tissue
factor pathway inhibitor (TFPI), a potent inhibitor of factor Xa.
Uptake and degradation of cell surface-bound I-TFPI was
also markedly stimulated in response to factor Xa binding. The
intracellular kinetics of I-factor Xa and cell
surface-bound I-TFPI display a strikingly similar
pattern, suggesting that factor Xa and cell surface-bound TFPI are
taken up as a bimolecular complex. Using cell lines either deficient in
low density lipoprotein receptor-related protein, an endocytic receptor
that mediates the degradation of uncomplexed TFPI (Warshawsky, I.,
Broze, G. J., Jr., and Schwartz, A. L.(1994) Proc. Natl. Acad. Sci.
U. S. A. 91, 6664-6668), or deficient in tissue factor (TF),
an integral membrane protein capable of forming quarternary complexes
with factor Xa, TFPI, and factor VIIa, we demonstrated that the
receptor that mediates the uptake and degradation of factor Xa-TFPI
complex was neither low density lipoprotein receptor-related protein
nor TF. As the vascular endothelial cell surface retains a substantial
pool of TFPI (Sandset, P. M., Alildgaard, U., and Larsen, M. L.(1988) Thromb. Res. 50, 803-813; Novotny, W. F., Brown, S. G.,
Miletich, J. P., Rader, D. J., and Broze, G. J., Jr.(1991) Blood 78, 387-393), our data suggest that endothelial cell surface
TFPI may be actively involved in the clearance of factor Xa from the
circulation via mediated uptake and degradation.
-antitrypsin (Colman et al., 1982),
-macroglobulin (Jackson and Nemerson, 1980),
antithrombin III (Kurachi et al., 1976), and the recently
characterized TFPI ()(reviewed in Broze et
al.(1990)). TFPI is unique among coagulation inhibitors in having
multiple protease inhibitory domains. It contains an acidic amino
terminus followed by three tandem Kunitz-type protease inhibitory
domains and a basic carboxyl terminus (Wun et al., 1988). The
second Kunitz domain of TFPI mediates its binding to and inhibition of
factor Xa, whereas the first Kunitz domain of TFPI is required for its
inhibition of the factor VIIa/tissue factor catalytic complex (Girard et al., 1989). The carboxyl-terminal region of TFPI, including
at least a portion of the third Kunitz domain, is required for its
binding to heparin and for optimal inhibition of factor Xa
(Wesselschmidt et al., 1992, 1993). The carboxyl terminus of
TFPI is also necessary for its binding to the cell surface (Warshawsky et al., 1995).I-factor Xa by human hepatoma cells and mouse embryonic
fibroblasts require cell surface TFPI and reciprocally, the uptake and
degadation of cell surface-bound I-TFPI is enhanced in
response to factor Xa binding. Because of the similarity of the
intracellular kinetics of factor Xa and TFPI, it is likely that they
are taken up by cells as a complex. Our data further indicate that
there is a specific receptor responsible for TFPI-factor Xa degradation
and that this receptor is not the low density lipoprotein
receptor-related protein (LRP), an endocytic receptor that mediates the
uptake and degradation of uncomplexed TFPI (Warshawsky et al.,
1994), nor tissue factor, an integral membrane glycoprotein capable of
forming a quarternary complex with TFPI, factor Xa, and factor VIIa
(Girard et al., 1989; Gemmell et al., 1990). Thus,
our data suggest that in vivo endothelial cell surface TFPI
may clear activated factor Xa from the circulation by mediating its
cellular uptake and degradation.
Materials
Iodogen was purchased from Pierce.
[I]Iodide was from Amersham Corp. Pronase,
bovine serum albumin, and
dansyl-L-glutamyl-glycyl-L-arginine chloromethyl
ketone (DNS-GGACK) were obtained from Calbiochem-Novabiochem (La Jolla,
CA). Factors Xa and X were from American Diagnostica (Greenwich, CT).
Tissue culture media and plasticware were obtained from Life
Technologies, Inc. An inhibitory rabbit anti-mouse soluble TF
polyclonal antibody will be described separately. ()Proteins
Recombinant human
full-length TFPI and the 39 kDa protein were expressed in and purified
from Escherichia coli (Warshawsky et al., 1993,
1994). DNS-GGACK inactivated factor Xa was achieved by incubating
factor Xa at room temperature for 1 h in a buffer containing 20 mM Tris-HCl, pH 7.3, 100 mM NaCl, and a 100-fold molar
excess of DNS-GGACK.Protein Iodinations
Proteins (20-50 µg)
were iodinated using the Iodogen method (Bu et al., 1992).
Specific radioactivities were typically 0.5-3 
10
cpm/pmol of protein.Cell Culture
HepG2 cells were as described
(Owensby et al., 1988). PEA 10 and PEA 13 cells were obtained
from J. Herz (Willnow and Herz, 1994). TF (-/-) mouse
embryonic fibroblasts were derived from TF (-/-) chimeric
embryos. TF (-/-) chimeric embryos were collected and
sacrificed 13 days postcoitus. The embryos were eviscerated, minced,
trypsinized, and plated onto tissue culture plates. Following expansion
of the mouse embryonic fibroblasts, the cells were placed under
selection with G418 and hygromycin. After 1 week of selection,
surviving TF (-/-) fibroblasts were combined. The
fibroblasts were immortalized using a 60-day 3T3 protocol. Confirmation
of TF deficiency was assessed in a one stage clotting assay and
compared to TF activity in wild-type mouse embryonic fibroblasts and
NIH3T3 cells. Detailed methods and characterization will be described
separately. All cell lines were cultured in
Dulbecco's modified Eagle's medium (with glutamine)
supplemented with 10% fetal calf serum, penicillin at 100 units/ml, and
streptomycin at 100 µg/ml. Cells were incubated at 37 °C in
humidified air containing 5% CO.Binding and Degradation Assays
Cells were seeded
in 12-well dishes 1 day prior to assay. Cell monolayers were generally
used at 70-90% confluence. Assay buffers were Dulbecco's
modified Eagle's medium containing 3% bovine serum albumin.
Binding assays were carried out by washing cell monolayers with
prechilled assay buffer twice and binding was initiated by adding 0.5
ml of 4 °C assay buffer containing the indicated concentrations of I-labeled proteins. After incubation at 4 °C for 2 h,
overlying buffer containing unbound ligand was removed, and the cells
were washed three times with 4 °C assay buffer. The cells were then
lysed in 62.5 mM Tris-HCl, pH 6.8, 0.2% SDS, and 10% glycerol.
The radioactivity of cell lysates was determined in a 
counter
(model C5304; Packard Instruments, Meriden, CT). Nonspecfic binding was
determined in the presence of excess unlabeled ligand as specified in
the figure legends (Owensby et al., 1989).I-labeled proteins. Thereafter, the medium overlying the
cell monolayers was removed and proteins were precipitated by addition
of bovine serum albumin to 5 mg/ml and trichloroacetic acid to 10%.
Degradation of ligand was defined as the appearance of radioactive
ligand fragments in the overlying medium that were soluble in
trichloroacetic acid. Degradation of I-ligand in parallel
dishes that did not contain cells was subtracted from the total
degradation (Owensby et al., 1989).Single-cycle Endocytosis Assays
Cells were seeded
into 12-well dishes. After the cells had been washed twice with 4
°C assay buffer, 0.5 ml of assay buffer containing the indicated
concentrations of I-labeled proteins were added. After
binding for 30 min at 4 °C, cells were washed three times with 4
°C assay buffer to remove unbound ligand. Cells were then warmed
rapidly to 37 °C by adding prewarmed assay buffer and incubated at
37 °C for selected time intervals. The overlying medium was removed
at each time point, and proteins were precipitated with trichloroacetic
acid to assess the degradation. Cell monolayers were then washed three
times with 4 °C assay buffer and incubated at 4 °C for 30 min
in assay buffer containing 0.25% Pronase to remove cell suface
radioligand and also detach cells from the culture wells. The detached
cells were separated from the buffer by centrifugation and
radioactivity associated with the cell pellet (i.e. internalized radioligand) was determined.
Uptake and Degradation of Coagulation Factor Xa Are
Mediated by Cell Surface-bound TFPI
Human factor Xa has been
reported to bind to human hepatoma HepG2 cells in a specific and
saturable manner, while the zymogen factor X failed to bind to these
cells (Sakai and Kisiel, 1990). Using chemical cross-linking and
immunoprecipitation techniques, this group subsequently showed that
TFPI was the major factor Xa-binding protein on the cell surface
(Kazama et al., 1993). To investigate the role of cell
surface-bound TFPI in factor Xa degradation, HepG2 cells were
preincubated at 4 °C in the presence or absence of TFPI. Following
washing to remove the unbound ligand, the cell monolayers were
incubated with various concentrations of I-factor Xa.
Degradation of I-factor Xa was carried out at 37 °C
for 3 h, and the overlying medium containing the resultant degradation
products was assayed following trichloroacetic acid precipitation. As
shown in Fig. 1, at each concentration of I-factor
Xa added, its degradation was enhanced about 10-fold in cells which had
been preincubated with TFPI compared with those without prior TFPI
exposure. This result suggests that TFPI mediates the uptake and
degradation of factor Xa by HepG2 cells. The small amount of I-factor Xa degradation seen in cells without exogenous
TFPI addition may have resulted from either a small amount of
endogenous TFPI or more likely from fluid-phase endocytosis (Fig. 1).
I-factor Xa degradation by HepG2 cells. Cells
were incubated at 4 °C for 30 min in the presence (
) or
absence (
) of 100 nM TFPI following which unbound TFPI
was removed. The cells were then incubated at 4 °C for 30 min with
increasing concentrations of I-factor Xa, after which the
dishes were placed at 37 °C. After 3-h incubation, medium overlying
the cell monolayers was subjected to trichloroacetic acid
precipitation, and the acid-soluble radioactive material, representing
degraded ligand, was determined. Symbols represent the means of
duplicate determinations.
Degradation of Factor Xa Is Not Mediated through the LRP
Endocytic Pathway
LRP is a member of the low density lipoprotein
receptor family involved in the endocytosis of several circulating
plasma proteins and heteromeric protein complexes (reviewed in Krieger
and Herz(1994)). We have shown recently that I-TFPI
degradation by hepatoma cells is also mediated via LRP (Warshawsky et al., 1994). Because degradation of I-factor
Xa required cell surface-bound TFPI, we examined whether LRP served as
an endocytic receptor for factor Xa. To address this question directly,
LRP-negative embryonic fibroblasts (PEA 13), isolated from mice
homozygous for LRP gene-knockout (Willnow and Herz, 1994), were
utilized. Mouse embryonic fibroblasts (PEA 10), isolated from
LRP-heterozygous mice and expressing functional LRP (Willnow and Herz,
1994), were used as control.I-factor Xa to PEA 10 and PEA 13 cells as a function of
cell surface-bound TFPI. Cells were preincubated at 4 °C with
increasing concentrations (0-16 nM) of TFPI. After
washing to remove the unbound ligand, the cell monolayers were
incubated with 2 nMI-factor Xa at 4 °C for
2 h for binding assays. Fig. 2A shows that at zero
concentration of TFPI during the preincubation, the specific binding of I-factor Xa to both cell lines was minimal. As the
concentration of TFPI in preincubation buffer increased, binding of I-factor Xa to the cells increased accordingly (Fig. 2A). This result indicates that binding of factor
Xa to these cells is TFPI-dependent, supporting the observation that
TFPI is the major binding protein for factor Xa on cells (Kazama et
al., 1993). The observation that binding of I-factor
Xa to both PEA 10 and PEA 13 cells was indistinquishable (Fig. 2A) indicates that LRP does not play a role in
factor Xa binding. Fig. 2B shows that binding of I-factor Xa to PEA 10 cells was saturable at fixed levels
of prebound TFPI (similar data were obtained using PEA 13 cells, data
not shown), confirming the specific nature of the TFPI-mediated factor
Xa binding to cells.
I-factor Xa to
PEA 10 and PEA 13 cells is TFPI-dependent. A, PEA 10 and PEA
13 cells were incubated with increasing concentrations of TFPI at 4
°C for 30 min following which unbound TFPI was removed. Cells were
then incubated with 2 nMI-factor Xa at 4 °C
for 2 h to allow steady-state binding. Thereafter, the cell monolayers
were washed to remove unbound radioligand and then lysed to determine
cell-associated radioactivity. Nonspecific binding was determined in
the presence of 100 nM excess unlabeled factor Xa. Numbers shown are the specific binding derived as the difference between
total and nonspecific I-factor Xa binding. B,
PEA 10 cells were incubated at 4 °C for 30 min with 2 nM TFPI following which unbound TFPI was removed. Cells were then
incubated with increasing concentrations of I-factor Xa
at 4 °C for 2 h. The specific binding of I-factor Xa
was determined as described above. Symbols represent the means of
duplicate determinations.
I-TFPI degradation was evaluated in LRP-positive PEA 10
and LRP-negative PEA 13 cells. As shown in Fig. 3, I-TFPI degradation occurred in PEA10 cells, but not PEA
13 cells. There was an approximately 7-fold difference in I-TFPI degradation between these two cell lines (Fig. 3).
I-TFPI by PEA
10 and PEA 13 cells. Cells were incubated with increasing
concentrations (2-16 nM) of I-TFPI at 37
°C for 3 h after which the medium overlying the cell monolayers was
analyzed for I-TFPI degradation products. Each symbol
represents the mean of duplicate
determinations.
I-factor Xa degradation. Cells were
processed in an identical manner as that described in Fig. 1. As
shown in Fig. 4, upon preincubation with TFPI, identical rates
of I-factor Xa degradation were seen in both cell lines.
Parallel experiments in which cells were not preincubated with TFPI
also demonstrated identical rates between the cell lines (Fig. 4). However, as expected, the total amount of I-factor Xa degraded was much less than (approximately
10-fold) in experiments in which TFPI preincubation occurred. These
data thus demonstrate that TFPI-dependent factor Xa degradation does
not require the endocytic receptor LRP.
I-factor Xa degradation by
PEA 10 and PEA 13 cells is comparable. Treatment of cells in the
presence or absence of unlabeled TFPI and the subsequent degradation of I-factor Xa by the cells were performed as described in
the legend to Fig. 1. Symbols are the means of duplicate
determinations.
Factor Xa Stimulates the Degradation of Cell Surface
TFPI
To assess the effect of factor Xa on cell surface-bound
TFPI, degradation of cell surface I-TFPI was assayed in
the presence of factor Xa in PEA 10 and PEA 13 cells. Cells were
preincubated at 4 °C with an uniform concentration (16 nM)
of I-TFPI followed by washing to remove unbound
radioligand. The cell monolayers were subsequently incubated with
increasing concentrations (0-16 nM) of unlabeled factor
Xa at 4 °C for 30 min to allow for factor Xa binding. Thereafter
the cells were shifted to 37 °C for assessment of the I-TFPI degradation. Fig. 5shows that in the
absence of factor Xa, the amount of I-TFPI degraded was
4-fold higher in PEA 10 cells than in PEA 13 cells. With increasing
amounts of unlabeled factor Xa added to the cells, degradation of I-TFPI in both cell lines increased accordingly. This
indicates an augmentation by factor Xa on I-TFPI
degradation. Noticeably, a greater enhancement of I-TFPI
degradation was observed in LRP-negative PEA 13 cells compared to
LRP-positive PEA 10 cells (Fig. 5). Thus the difference in the
amount of I-TFPI degraded by these two cell lines, which
represents that degraded via the LRP endocytic pathway (solid
circles in Fig. 5), varied inversely with the concentration
of factor Xa added. These data therefore suggest that factor
Xa-stimulated cell surface I-TFPI degradation, similar to
TFPI-dependent I-factor Xa degradation, is not mediated
via LRP.
I-TFPI bound to both PEA 10 and PEA 13 cells. Cells were
incubated with 16 nMI-TFPI at 4 °C for 30
min and unbound radioligand removed. Cells were then incubated with
increasing concentrations of unlabeled factor Xa (0-16
nM) at 4 °C for 30 min to allow binding followed by
incubation at 37 °C for 3 h to initiate I-TFPI
degradation. Degradation of I-TFPI via LRP was derived
from the difference between the amounts degraded by PEA 10 and PEA 13
cells. Symbols represent the means of five independent
experiments.
I-TFPI degradation requires activated factor X (i.e. Xa), zymogen factor X and the DNS-GGACK-inactivated factor Xa,
both of which are unable to bind to TFPI (Broze et al., 1988),
were evaluated in similar experiments. As shown in Fig. 6,
neither factor X nor DNS-GGACK-inactivated factor Xa were able to
augment the degradation of I-TFPI in either PEA 10 or PEA
13 cells. These results clearly demonstrate that active factor Xa in
association with cell surface-bound TFPI is required for the observed
effect.
I-TFPI by PEA 10
and PEA 13 cells. The protocol for I-TFPI degradation was
identical to that described in Fig. 5, except that factor Xa was
replaced with either factor X or DNS-GGACK-inactivated factor Xa.
Symbols represent the means of duplicate
determinations.
Factor Xa-stimulated TFPI Degradation Results from
Cellular Ligand Uptake
To determine at which step(s) factor Xa
affect the cellular fate of cell surface-bound I-TFPI, a
single-cycle endocytosis analysis (Owensby et al., 1989) was
performed. This analysis was carried out with PEA 13 cells, such that
LRP does not contribute to the observed results. Fig. 7A shows that in the absence of factor Xa, there was essentially no
internalization of cell surface-bound I-TFPI, even after
60 min of incubation at 37 °C. However rapid internalization of I-TFPI was observed upon binding to factor Xa. This
indicates that factor Xa induces the uptake of cell surface-bound I-TFPI. Fig. 7B shows that the
degradation of cell surface-bound I-TFPI was also
enhanced in the presence of factor Xa, apparently as a consequence of
the increased internalization.
I-TFPI or I-factor Xa by PEA 13 cells
during a single cycle of endocytosis. A and B, cells
were incubated with 2 nMI-TFPI at 4 °C for
30 min and unbound radioligand removed. Cells were then incubated at 4
°C for 30 min in the presence () or absence () of 2
nM unlabeled factor Xa followed by incubation at 37 °C for
selected intervals. At the indicated times, the overlying medium was
removed for analysis of I-TFPI degradation (B),
while the cell monolayers were incubated with Pronase at 4 °C for
30 min to remove cell surface I-TFPI. The
Pronase-resistant radioligand associated with cells, defined as the
internalized fraction, was then determined (A). C,
cells were incubated with 2 nM TFPI at 4 °C for 30 min and
unbound ligand removed. Cells were then incubated with 2 nMI-factor Xa at 4 °C for 30 min. After washing to
remove unbound radioligand, cells were placed at 37 °C for selected
intervals for the uptake () and degradation () of I-factor Xa, determined as described above. Symbols
represent means of two independent
experiments.
I-TFPI was sustained intracellularly over a
period of at least 60 min (Fig. 7A). This feature is
distinct from that observed with ligands internalized by the
constitutive endocytic receptor LRP (Owensby et al., 1988; Bu et al., 1992; Underhill et al., 1992; Warshawsky et al., 1994), in which ligand internalization peaks at
10-15 min and declines precipitously thereafter. To determine
whether factor Xa followed similar intracellular kinetics to that of
TFPI, the fate of a cohort of I-factor Xa was examined
during single cycle endocytosis in TFPI-prebound PEA 13 cells. As shown
in Fig. 7C, similar internalization and degradation
kinetics were observed for I-factor Xa. These results,
taken together with previous observations that factor Xa-stimulated
TFPI degradation required association with factor Xa, strongly suggest
that factor Xa and TFPI are internalized as a complex.Tissue Factor Is Not the Receptor for the Factor Xa-TFPI
Complex Degradation
Data seen in Fig. 7imply that there
is a specific receptor which mediates the internalization and
degradation of factor Xa-TFPI complex. One obvious potential candidate
is tissue factor (TF). TF is an integral membrane protein capable of
forming quarternary complexes with TFPI, factor Xa, and factor VIIa
(Girard et al., 1989; Gemmell et al., 1990). To
examine this possibility we evaluated the effect of preincubation of
cells with anti-TF polyclonal antibodies. When analyzed in LRP-negative
PEA 13 cells, no significant inhibition of factor Xa-stimulated I-TFPI degradation was observed in the presence of
anti-TF antibody (data not shown). This observation suggested that TF
did not play a significant role in factor Xa-stimulated TFPI
degradation; however, alternative explanations were possible.
Therefore, in order to definitively eliminate a role for TF in this
process, we took advantage of TF-negative fibroblasts that were
generated by gene targeting and demonstrate a complete absence of TF
expression. As seen in Table 1, TF-negative cells, in
the absence of factor Xa, degraded I-TFPI at 122
fmol/10 cells. Degradation was completely abrogated by
incubation with the 39-kDa protein, indicating that uncomplexed TFPI
was degraded via LRP as has been shown (Warshawsky et al.,
1994). In the presence of factor Xa, degradation of cell surface-bound I-TFPI was further enhanced to 215 fmol/10 cells. This indicates that TF-negative cells were capable of
markedly increasing degradation of I-TFPI in response to
factor Xa addition. Furthermore, this increased degradation was not
blocked by the 39-kDa protein. Thus LRP did not contribute to the
enhancement, as observed in other cells (e.g. see above).
Taken together, these data demonstrate that TF is not the endocytic
receptor for the factor Xa-TFPI complex.
I-factor Xa
is actively endocytosed and degraded by various cell lines in a manner
that requires cell surface-bound TFPI. The uptake and degradation of
cell surface-bound I-TFPI, in response to factor Xa
binding, is also accelerated. Since the intracellular kinetics of I-factor Xa and I-TFPI are quite similar,
it is likely that they are taken up by cells as a complex. Furthermore,
our data indicate that there is a specific endocytic receptor
responsible for the TFPI-factor Xa degradation and that this receptor
is neither LRP nor TF.-proteinase inhibitor, and
macroglobulin, which form irreversible bonds with
factor Xa (Pratt and Pizzo, 1986; Colman et al., 1987). Our
data shown here indicate that rather than targeting factor Xa to other
plasma protease inhibitors, cell surface-bound TFPI directly
inactivates factor Xa by mediating its cellular degradation.
Considering the fact that the endothelial cell surface provides a
substantial reservoir of TFPI (Sandest et al., 1988; Novotny et al., 1991), it is reasonable to speculate that the vascular
endothelium, through its surface-bound TFPI, is crucial in factor Xa
catabolism in vivo.I-TFPI is inhibited by antibodies directed
against LRP and by the 39-kDa protein (Warshawsky et al.,
1994). These observations led to the conclusion that LRP is the
endocytic receptor for TFPI. In addition, recent studies with
adenoviral mediated delivery of the 39-kDa protein to mice in vivo further confirm the role of LRP as the predominant receptor
governing TFPI uptake and degradation (Narita et al., 1995).
In the present study we demonstrate that I-TFPI
degradation occurs only in LRP-positive PEA 10 cells, but not in
LRP-negative PEA 13 cells (Fig. 3). This result thus confirms
and extends our previous findings. Using these two cell lines in
degradation assays, we have observed that TFPI-mediated I-factor Xa degadation, however, is independent of the
LRP endocytic pathway (Fig. 4).I-factor Xa are mediated by cell surface-bound TFPI (Fig. 1, Fig. 2, and Fig. 4). Second, the cell
surface-bound I-TFPI, upon association with factor Xa, is
also internalized (Fig. 7). Last, the intracellular kinetics of I-TFPI and I-factor Xa are similar if not
identical (Fig. 7). The above observations also indicate that a
specific endocytic receptor is responsible for the uptake of the
complex. It is interesting to note that I-factor Xa or I-TFPI internalized by the putative receptor does not
share the internalization kinetics typical of that observed for ligands
internalized by the endocytic receptor LRP (Owensby et al.,
1988; Bu et al., 1992; Underhill et al., 1992;
Warshawsky et al., 1994). Rather than rapid declining after
10-15 min at 37 °C, I-factor Xa or I-TFPI is sustained intracellularly at plateau levels for
a significantly prolonged time. The underlying mechanism is not clear
at present. Whether this is due to an unusual intracellular pathway
traversed by the TFPI-factor Xa complex or to resistance of the complex
or its individual component proteins to proteolysis within the vacuolar
system, as is the case for PAI-1 of the t-PAPAI-1 complex
(Underhill et al., 1992), remains to be discerned.I-TFPI is manifest in response to
factor Xa binding (Table 1), similar to that seen in TF-positive
cells.I-factor Xa could be
cross-linked to protease nexin-1 on HepG2 cells. Hence they proposed
that nexin-1 is a receptor for factor Xa as well. Protease nexin-1 is
secreted by many anchorage-dependent cells, including fibroblasts,
cardiac muscle cells, and kidney epithelial cells (Eaton and Baker,
1983). However secretion by endothelial cells has not been reported.
Whether nexin-1 plays a role in factor Xa catabolism is not presently
known, nor is its physiological significance in this matter. These
issues will certainly deserve further study. As will the molecular
identity of the TFPI-factor Xa clearance receptor.
))
We thank Dr. Guojun Bu for providing the recombinant
39-kDa protein and for critical reading of this manuscript.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  E. N. Harris, J. A. Weigel, and P. H. Weigel The Human Hyaluronan Receptor for Endocytosis (HARE/Stabilin-2) Is a Systemic Clearance Receptor for Heparin J. Biol. Chem., June 20, 2008; 283(25): 17341 - 17350. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. T.B. Crawley and D. A. Lane The Haemostatic Role of Tissue Factor Pathway Inhibitor Arterioscler. Thromb. Vasc. Biol., February 1, 2008; 28(2): 233 - 242. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Butenas, T. Orfeo, M. T. Gissel, K. E. Brummel, and K. G. Mann The Significance of Circulating Factor IXa in Blood J. Biol. Chem., May 28, 2004; 279(22): 22875 - 22882. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Ho, M. Narita, G. J. Broze Jr, and A. L. Schwartz Recombinant full-length tissue factor pathway inhibitor fails to bind to the cell surface: implications for catabolism in vitro and in vivo Blood, March 15, 2000; 95(6): 1973 - 1978. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Ott, Y. Miyagi, K. Miyazaki, M. J. Heeb, B. M. Mueller, L. V. M. Rao, and W. Ruf Reversible Regulation of Tissue Factor-Induced Coagulation by Glycosyl Phosphatidylinositol-Anchored Tissue Factor Pathway Inhibitor Arterioscler. Thromb. Vasc. Biol., March 1, 2000; 20(3): 874 - 882. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. L. Saenko, A. V. Yakhyaev, I. Mikhailenko, D. K. Strickland, and A. G. Sarafanov Role of the Low Density Lipoprotein-related Protein Receptor in Mediation of Factor VIII Catabolism J. Biol. Chem., December 31, 1999; 274(53): 37685 - 37692. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Han, T. J. Girard, P. Baum, D. R. Abendschein, and G. J. Broze Jr Structural Requirements for TFPI-Mediated Inhibition of Neointimal Thickening After Balloon Injury in the Rat Arterioscler. Thromb. Vasc. Biol., October 1, 1999; 19(10): 2563 - 2567. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Hamik, H. Setiadi, G. Bu, R. P. McEver, and J. H. Morrissey Down-regulation of Monocyte Tissue Factor Mediated by Tissue Factor Pathway Inhibitor and the Low Density Lipoprotein Receptor-related Protein J. Biol. Chem., February 19, 1999; 274(8): 4962 - 4969. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Narita, A. E. Rudolph, J. P. Miletich, and A. L. Schwartz The Low-Density Lipoprotein Receptor-Related Protein (LRP) Mediates Clearance of Coagulation Factor Xa In Vivo Blood, January 15, 1998; 91(2): 555 - 560. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-B. Hansen, R. Olsen, and P. Webster Association of Tissue Factor Pathway Inhibitor With Human Umbilical Vein Endothelial Cells Blood, November 1, 1997; 90(9): 3568 - 3578. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z.-F. Huang, D. Higuchi, N. Lasky, and G. J. Broze Jr Tissue Factor Pathway Inhibitor Gene Disruption Produces Intrauterine Lethality in Mice Blood, August 1, 1997; 90(3): 944 - 951. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Ho, G. J. Broze Jr., and A. L. Schwartz Role of Heparan Sulfate Proteoglycans in the Uptake and Degradation of Tissue Factor Pathway Inhibitor-Coagulation Factor Xa Complexes J. Biol. Chem., July 4, 1997; 272(27): 16838 - 16844. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP |
