(Received for publication, August 1, 1995)
From the
Tissue factor pathway inhibitor (TFPI) is a potent inhibitor of
the blood coagulation factor VIIa-tissue factor complex, as well as a
direct inhibitor of factor Xa. Intravenously administered TFPI is
rapidly cleared from circulation predominantly via liver. We previously
reported that the low density lipoprotein receptor-related protein
(LRP), a multifunctional endocytic receptor, mediates the uptake and
degradation of TFPI in hepatoma cells. This process is inhibited by a
39-kDa receptor-associated protein which binds to LRP and regulates its
ligand binding activity. However, a distinct, low affinity binding site
(perhaps heparin sulfate proteoglycans, HSPGs) on the endothelium and
liver is thought to be responsible for the majority of TFPI cell
surface binding. In the current study, we investigated the role of LRP
and this second binding site in the clearance of I-TFPI in vivo using competitors and inhibitors of the receptors.
Mice overexpressing the 39-kDa protein via adenoviral-mediated gene
transfer displayed diminished plasma clearance of
I-TFPI.
Blockade of cell surface HSPGs sites by incubation with the positively
charged molecule, protamine, inhibited
I-TFPI binding to
the hepatoma cells in vitro. In addition, preadministration of
protamine in vivo prolonged the plasma clearance of
I-TFPI in a dose-dependent manner. However, a dramatic
increase of the plasma half-life of
I-TFPI and virtual
elimination of
I-TFPI clearance was observed in mice
overexpressing the 39-kDa protein and administered protamine. Taken
together, our results suggest that two receptor mechanisms are involved
in the clearance of TFPI in vivo.
Tissue factor pathway inhibitor (TFPI) ()is a serine
protease inhibitor that plays a key role in regulating tissue
factor-initiated blood coagulation. Human TFPI is a trace 42-kDa plasma
glycoprotein consisting of three tandem Kunitz-type domains, followed
by a positively charged carboxyl terminus(1) . The first Kunitz
domain binds to and inhibits factor VIIa, and the second Kunitz domain
binds to and inhibits factor Xa(2) . Inhibition of tissue
factor-induced blood coagulation by TFPI has been postulated to involve
the quaternary factor Xa-TFPI-factor VIIa-tissue factor
complex(3) .
Intravenously administered I-TFPI
is cleared rapidly from the circulation with a plasma half-life of 2
min in rabbits (4) and <1 min in rats(5) . However,
the biology underlying this clearance mechanism has not been elucidated
to date. Previously, we demonstrated that the low density lipoprotein
receptor-related protein (LRP) mediates the cellular degradation of
TFPI in hepatoma cells (6) and that a 39-kDa protein, an
inhibitor of all the ligand interactions with LRP(7) , inhibits
this process. In addition, cell surface heparin sulfate proteoglycans
(HSPGs) associated with endothelial cells and liver have been proposed
to play a role in the clearance of
I-TFPI(8) .
However, the precise roles of LRP and HSPGs in the plasma clearance of
TFPI have yet to be defined.
The purpose of the present study was to elucidate the roles of LRP and HSPGs in the catabolism of TFPI both in vivo and in vitro. We took advantage of viral-mediated gene transfer to express the 39-kDa protein in liver in vivo as such an approach has allowed us to define the role of LRP in the clearance of tissue-type plasminogen activator (t-PA) in vivo(9) . The current results demonstrate a direct role for LRP as well as HSPGs in the plasma clearance of TFPI and thus suggest strategies for regulation of its catabolism.
Figure 1:
Effect of AdCMV-39-kDa on the plasma
clearance of I-TFPI. As described under ``Materials
and Methods,'' either 4
10
particles of
AdCMV-
-Gal or AdCMV-39-kDa were administered intravenously to mice
via tail vein. Five days after virus administration, mice were injected
with 34 pmol of
I-TFPI and plasma radioactivities were
determined at the indicated times. Control mice (no administration of
virus) are also shown. Clearance studies were determined for at least
three mice in each group.
To investigate the effect of LRP on I-TFPI clearance in vivo, we took advantage of
the ability of the 39-kDa protein to inhibit
I-TFPI
interaction with LRP(6) . In the current study, we used an
adenoviral vector to carry the 39-kDa protein cDNA to be expressed in
mouse liver. Overexpression of the 39-kDa protein results in plasma
accumulation of the 39-kDa protein. Previously, we demonstrated that
mice administered 4
10
particles of AdCMV-39-kDa
expressed sufficient 39-kDa protein in plasma to completely inhibit
LRP(9) , and also demonstrated that viral infection induced no
gross or microscopic morphological changes in the liver(9) .
This dose of AdCMV-39-kDa was administered intravenously to mice via
tail vein. Five days after administration, plasma clearance studies of
I-TFPI were performed. As seen in Fig. 1,
administration of AdCMV-39-kDa altered the plasma clearance of
I-TFPI: while the
phase t
was essentially unaltered compared to control mice, the
phase t
increased from
11 min to >100
min.
To confirm that the viral infection did not induce adverse
effects on the clearance of I-TFPI, studies were
performed in mice following administration of AdCMV-
-Gal. As seen
in Fig. 1, the clearance of
I-TFPI in
AdCMV-
-Gal-infected mice was essentially the same as that of the
noninfected mice.
Figure 2:
Effect of injection of recombinant 39-kDa
protein on the plasma clearance of TFPI in rats. As described under
``Materials and Methods,'' rats were injected with 0.5 mg/kg
intravenous of TFPI without () or with (
) preadministration
of recombinant 39-kDa protein given as a bolus of 50 mg/kg intravenous.
Clearance studies were determined for five animals in each group and
are displayed as mean ± S.E.
Figure 3:
Effect of heparin and protamine on the
clearance of I-TFPI in normal mice and
AdCMV-39-kDa-injected mice. 10 min after the administration of 34 pmol
of
I-TFPI, 100 units of heparin was administered 1 min
thereafter, 1 mg of protamine was administered. Plasma
I-TFPI radioactivity was determined at the indicated
times. Clearance studies were determined for at least three mice in
each group.
Figure 4:
Effect of protamine and the 39-kDa protein
on the binding of I-TFPI to MH
C
cells. A, cells were incubated for 2 h at 4 °C with
increasing concentrations of the 39-kDa protein (
) or protamine
(
). Thereafter, the cell monolayers were washed to remove unbound
ligand and directly lysed to determine cell-associated radioactivity.
Radioactivity was converted to femtomole equivalents of
I-TFPI calculated from the specific activities and is
normalized per well. Each symbol represents the average of
duplicate determinations. B, cells were incubated for 2 h at 4
°C with increasing concentrations of the 39-kDa protein in the
presence of 100 µg/ml protamine and processed as in A.
Figure 5:
Effect of preadministration of
AdCMV-39-kDa and protamine on the clearance of I-TFPI in vivo. Various doses of protamine (0.01, 0.1, and 1 mg) were
administered 1 min prior to the injection of 34 pmol of
I-TFPI to either normal mice or mice which had received 4
10
particles of AdCMV-39-kDa 5 days earlier.
Plasma
I-TFPI radioactivities were determined at the
indicated times. Clearance studies were determined for at least three
mice in each group.
The present observations demonstrate that 1) inactivation of
LRP in vivo by gene transfer of a 39-kDa protein prolongs the
plasma half-life of I-TFPI, especially the
phase,
2) in vivo degradation of exogenously administered TFPI is
inhibited about 50% by inactivation of LRP by the 39-kDa protein, 3) in vitro the 39-kDa protein inhibits the binding of
I-TFPI to hepatoma cells in a dose-dependent manner under
conditions in which
I-TFPI is unable to bind to HSPGs, 4)
blockade of HSPGs by administration of protamine in vivo prolongs the plasma half-life of
I-TFPI, and 5) a
dramatic increase of the plasma half-life of
I-TFPI is
observed in mice overexpressing the 39-kDa protein and administered
protamine, a competitor for the HSPGs-binding sites. Taken together,
these results indicate that in vivo LRP mediates the
degradation of
I-TFPI and that both LRP and HSPGs are
responsible for
I-TFPI clearance.
TFPI is a potent
inhibitor of the factor VIIa-tissue factor complex as well as a direct
inhibitior of factor Xa. In addition, in animal models TFPI is a
potential therapeutic agent in vivo for
tissue-thromboplastin-induced intravascular coagulation and prevention
of arterial reocclusion after thrombosis(20, 21) . Of
the many parameters which govern the plasma level of TFPI, its
clearance and catabolism play a central role. Elucidation of the
molecular basis thereof has recently begun. Using hepatoma cell lines,
we reported recently that LRP mediates TFPI degradation(6) .
The cellular degradation of I-TFPI was inhibited >80%
both by antibodies directed against LRP and by the LRP-associated
39-kDa protein. LRP, however, does not appear to be the major cell
surface receptor for TFPI, since
I-TFPI binding at 4
°C was not inhibited by the 39-kDa protein(6) . This major
binding species has been proposed to be HSPGs or glycosaminoglycans, as
mentioned above. Circumstantial evidence suggests that TFPI may be
bound to heparan sulfate proteoglycans on the endothelial or liver cell
surface. This hypothesis is based on the observations that (a)
TFPI binds to heparin-agarose(22) ; (b) heparin and
sulfated polysaccharides enhance the anticoagulant activity of
TFPI(19) ; and (c) after intravenous administration of
heparin, plasma levels of TFPI increase
severalfold(8, 15) .
After the intravenous
administration of I-TFPI in mice, TFPI was rapidly
cleared from the circulation (
phase t
0.3 min,
phase t
11 min).
LRP and/or HSPGs are thought to be responsible for this clearance.
Administration of 39-kDa protein either directly by injection or via
gene delivery as AdCMV-39-kDa altered the clearance of TFPI, especially
the
phase ( Fig. 1and Fig. 2). However, the effect
of 39-kDa protein on TFPI clearance is not as significant as that seen
for
I-t-PA following the 39-kDa administration via
infusion (12) or via AdCMV-39-kDa(9) . LRP is
responsible for the majority of t-PA clearance (over 50%) (10, see Fig. 1and Fig. 3), whereas for TFPI, LRP is not the major
clearance receptor. This may be because of the difference in the number
of total binding sites available to TFPI or other LRP-specific ligands.
The observation that MH
C
hepatoma cells appear
to have >10 times as many
I-TFPI-binding sites (2
10
sites/cell) as binding sites for the
LRP-specific ligands tissue-type plasminogen activator and
-macroglobulin (6) supports this hypothesis.
Previously, we have demonstrated that the 39-kDa protein inhibited I-TFPI degradation by
80% in hepatoma
cells(5, 6) . In the current study, in
AdCMV-39-kDa-injected mice, intravenously administered
I-TFPI degradation was inhibited
50%. This
observation cleary demonstrates that LRP mediates degradation of
I-TFPI in vivo as well. As expected,
I-TFPI degradation (i.e. fragment accumulation)
was inhibited more significantly in urine than in serum (Table 1). Since the sites of TFPI clearance in vivo are
the liver and kidney(4, 5) , the 39-kDa protein may
inhibit LRP activity in kidney as well as in liver. Alternatively,
kidney gp330, an LRP homolog which is also inhibited by the 39-kDa
protein(23) , may function in TFPI clearance, as well.
Previously, we reported that the 39-kDa protein did not inhibit TFPI
binding to hepatoma cells(6) . However, as seen in Fig. 4in the presence of protamine, which competes for I-TFPI binding to cell surface HSPGs, the 39-kDa protein
inhibits the binding of
I-TFPI to MH
C
cells in a dose-dependent manner. The effect of protamine on
inhibition of
I-TFPI binding to MH
C
cells is nearly saturated at 100 µg/ml of protamine. Under
these conditions, binding of
I-TFPI to
MH
C
cells is approximately 10% as that found
without protamine. These results are thus consistent with those above.
In addition and consistent with the in vitro results,
administration of protamine decreased the plasma half-life of
I-TFPI in vivo in a dose-dependent manner. In
the presence of 1 mg of protamine, a dramatic increase in the plasma
half-life of
I-TFPI (10 min) was observed in mice
overexpressing the 39-kDa protein. These results clearly demonstrate
that two independent receptor systems are involved in the clearance of
I-TFPI (one protamine-sensitive, i.e. HSPGs; the
other 39-kDa protein-sensitive, i.e. LRP). Furthermore, they
demonstrate that while the endothelial/liver cell surface HSPGs-binding
sites for TFPI are important in clearance of TFPI from the plasma, this
sequestered TFPI is readily releasable and ultimately available back in
the plasma.