(Received for publication, September 6, 1995; and in revised form, December 12, 1995)
From the
The inhibition of proteinase activity by members of the serine
proteinase inhibitor (serpin) family is a critical regulatory mechanism
for a variety of biological processes. Once formed, the serpin enzyme
complexes (SECs) are removed from the circulation by a hepatic
receptor. The present study suggests that this receptor is very likely
the low density lipoprotein receptor-related protein (LRP), a prominent
liver receptor. In vitro binding studies revealed that
antithrombin III (ATIII)thrombin, heparin cofactor II
(HCII)
thrombin, and
-antitrypsin
(
AT)
trypsin bound to purified LRP, and their
binding was inhibited by the 39-kDa receptor-associated protein (RAP),
an antagonist of LRP-ligand binding activity. In contrast, native or
modified forms of the inhibitors were unable to bind to LRP. Mouse
embryonic fibroblasts, which express LRP, mediate the cellular
internalization leading to degradation of these SECs, while mouse
fibroblasts genetically deficient in LRP showed no capacity to
internalize and degrade these complexes. SECs were also degraded by
HepG2 cells, and this process was inhibited by LRP antibodies, RAP, and
chloroquine. The cellular-mediated uptake and degradation was specific
for SECs; native or modified forms of the inhibitors were not
internalized and degraded. Finally, in vivo clearance studies
in rats demonstrated that RAP inhibited the clearance of
ATIII
I-thrombin complexes from the circulation.
Together, these results indicate that LRP functions as a liver receptor
responsible for the plasma clearance of SECs.
Serine proteinase inhibitors (serpins) are a supergene family
comprised of more than 40 members (Carrell and Travis, 1985). Most
serpins function to inhibit serine proteinases through the formation of
a stable serpin-enzyme complex (SEC) ()(Laskowski and Kato,
1980, Travis and Salvesen, 1983); however, some members such as
angiotensinogen and ovalbumin are unable to inhibit proteinases
(Doolittle, 1983; Hunt and Dayhoff, 1980). The inhibitory serpins are
found in three distinct structural conformations; native (active
inhibitor), cleaved (an inactive inhibitor generated after cleavage of
its reactive center loop), or complexed with a proteinase. The
inhibitory action of serpins affect a broad range of biological
processes including coagulation, fibrinolysis, inflammation, connective
tissue turnover, complement activation, and phagocytosis (for reviews,
see Travis and Salvesen(1983), Bjork and Danielsson(1986), and Carrell
and Travis(1985)).
-Antitrypsin
(
AT, also referred to as
-proteinase
inhibitor) is the prototypic member of the serpin family and primarily
acts to inhibit neutrophil elastase and proteinase 3 (PR3) (Travis and
Salvesen, 1982; Carrell and Travis, 1985; Rao et al., 1991).
Neutrophil elastase and PR3 degrade connective tissue matrix proteins,
and both enzymes can induce emphysema in animal models (Janoff et
al., 1977; Senior et al., 1977; Kao et al.,
1988).
AT is present in high concentration in the
plasma (>25 µM) and protects the connective tissue-rich
lung against excessive proteolysis through its inhibitory action on
neutrophil elastase and PR3. The protection of lung tissue by
AT is evidenced by studies that have genetically
linked decreased functional levels of
AT with the
early onset of pulmonary emphysema (Laurell and Eriksson, 1963; Travis
and Salvesen, 1983; Carrell, 1986).
Antithrombin III (ATIII), a
serpin with extensive sequence similarity to AT, plays
a major role in hemostasis by acting as the principle physiological
inhibitor of thrombin and coagulation Factor X
(for reviews
see Bjork and Danielsson(1986), Bjork et al.(1989), and Olson
and Bjork(1992)). This inhibitor is present in high concentration in
the plasma (5 µM), and its importance is underscored by
the observation that reduced functional levels of ATIII are associated
with increased risks of thromboembolic disorders (Edeburg, 1965; Lane et al., 1991).
Heparin cofactor II (HCII) is a 65,000-dalton inhibitor of thrombin, cathepsin G, and chymotrypsin (Tollefsen et al., 1982; Church and Hoffman, 1994; Church et al., 1995) and is present in the human plasma at a concentration of 1.4 µM. Like ATIII, its ability to react with target proteinases is greatly accelerated in the presence of heparin. Individuals exhibiting low levels of HCII but having normal levels of ATIII display thrombotic tendencies (Tran et al., 1985; Sie et al., 1985), suggesting that HCII, like ATIII, is an important inhibitor of thrombin in vivo.
An important
feature of serpin metabolism was originally identified when Lollar et al.(1980) showed that I-thrombin in complex
with ATIII was rapidly cleared from the circulation of rabbits by the
liver. Additional studies by several groups (Bauer et al.,
1982; Fuchs et al., 1982; Pratt et al., 1988) have
demonstrated that the liver mediates rapid clearance of complexes of
ATIII
thrombin, HCII
thrombin,
AT
trypsin, and
AT
elastase
from the circulation. Further, these complexes could compete for one
another's clearance, indicating that the plasma elimination of
these SECs was occurring via a common receptor pathway. Mast et
al.(1991) showed that the kinetics of plasma elimination of the
native and cleaved inhibitors were 10-50 times slower than that
of the complexed inhibitors, suggesting that the liver receptor might
preferentially clear the SEC as opposed to the free serpin.
The low density lipoprotein receptor (LDLR) and low density lipoprotein receptor-related protein (LRP) are two prominent hepatic receptors that are members of the LDLR superfamily. This receptor family also includes the very low density lipoprotein receptor and glycoprotein 330 (gp330). LRP is expressed in many tissues and cell types (Moestrup et al., 1992) and is present in high abundance in the liver. The requirement for LRP expression during development was demonstrated when targeted disruption of the LRP gene resulted in termination of the null embryos at day 13.5 during development (Herz et al., 1992). LRP interacts with numerous ligands and is thought to play key roles in both lipoprotein metabolism and proteinase regulation (for reviews, see Krieger and Herz(1994), Strickland et al.(1994, 1995), and Moestrup(1994)).
In addition to mediating the catabolism of
-macroglobulin (
M)-proteinase
complexes (Ashcom et al., 1990), LRP also binds to several
SECs, including elastase
AT (Poller et
al., 1995), urinary-type plasminogen activator (uPA)
PAI-1
(Herz et al., 1992; Nykjaer et al., 1992), and tissue
plasminogen activator
PAI-1 (Orth et al., 1992). However,
the receptor responsible for the hepatic catabolism of a number of
other predominant SECs remained obscure. Studies were therefore
initiated to determine if LRP could interact with those SECs. In this
report we demonstrate that LRP binds directly to complexes of
ATIII
thrombin, HCII
thrombin, and
AT
trypsin, can mediate their cellular uptake and
degradation, and appears responsible for their in vivo clearance from the circulation.
Figure 1:
Binding of LRP to ATIIIthrombin,
HCII
thrombin, and
AT
trypsin. In panel
A, increasing concentrations of LRP (0.6-150 nM)
were incubated for 18 h at 4 °C with wells coated with native ATIII
(
), cleaved ATIII (
), ATIII
thrombin (
), or BSA
(
). Panel B, native HCII (
), cleaved HCII
(
), HCII
thrombin (
), and BSA (
). Panel
C, native
AT (
), cleaved
AT
(
),
AT
trypsin (
), and BSA
(
). Following incubation, wells were washed and bound LRP
detected with the LRP monoclonal antibody
8G1.
Since RAP is known to inhibit the
binding of ligands to LRP (Herz et al., 1991), binding assays
were performed to test the effect of RAP on the binding of LRP to
ATIIIthrombin, HCII
thrombin, and
AT
trypsin. Fig. 2depicts an ELISA in
which LRP was incubated with serpin-proteinase-coated wells in the
presence of increasing concentrations of RAP. These data demonstrate
that RAP is capable of competing for the binding of these SECs to LRP.
Figure 2:
RAP
inhibits the binding of LRP to complexes of ATIIIthrombin,
HCII
thrombin, and
AT
trypsin. Wells coated
with ATIII
thrombin (
), HCII
thrombin (
),
AT
trypsin (
), or BSA (
) were
incubated with 20 nM LRP in the presence of increasing
concentrations of RAP (0.2-450 nM) for 18 h at 4 °C.
Following incubation, microtiter wells were washed and the bound LRP
was detected with the LRP monoclonal antibody
8G1.
Figure 3:
SDS-polyacrylamide gel electrophoresis
analysis of serpin complexes. Thrombin and trypsin were labeled with I-iodine and complexed to inhibitors as described under
``Experimental Procedures.'' Proteins were electrophoresed on
8-16% polyacrylamide gradient gels (Novex, San Diego, CA) under
non-reducing conditions, wrapped in plastic, and exposed to x-ray film
for 30 min. The migration position of the molecular mass standards
indicated were determined using Novex prestained rainbow standard
proteins. Lane 1,
I-trypsin
AT; lane 2,
I-trypsin; lane 3,
I-thrombin
HCII; lane 4,
I-thrombin
ATIII; lane 5,
I-thrombin.
Figure 4:
LRP-deficient fibroblasts do not
internalize and degrade complexes of I-thrombin
ATIII,
I-thrombin
HCII, and
I-trypsin
AT. Wells containing 2
10
MEF (LRP-expressing) or PEA13 (LRP-deficient)
cells were incubated with
I-enzyme-serpin complexes (10
nM) for selected time intervals at 37 °C in the presence
and absence of RAP (1 µM). At the indicated times, the
amount of radioligand internalized by the cell and the levels of
degraded radioligand secreted into the cell medium were determined as
described under ``Experimental Procedures.'' Panels show internalization and degradation of
ATIII
I-thrombin (A and B),
HCII
I-thrombin (C and D), and
AT
I-trypsin (E and F), respectively. The data shown are representative of three
experiments, each performed in duplicate.
Figure 5:
LRP antibodies inhibit I-thrombin
ATIII,
I-thrombin
HCII, and
I-trypsin
AT degradation by HepG2
cells. Wells containing 2
10
HepG2 cells were
incubated for 18 h at 37 °C with
I-labeled complexes
(10 nM) in the presence of affinity-purified anti-LRP IgG (100
µg/ml), control IgG against the cytoplasmic domain of LRP (100
µg/ml), chloroquine (0.1 mM), or RAP (1 µM). Panels show degradation data of
I-ATIII
thrombin (A),
I-HCII
thrombin (B), and
I-
AT
trypsin (C). Plotted
values represent means of duplicate values.
Figure 6:
LRP internalizes the proteinase
complexed form of I-ATIII,
I-HCII, and
I-
AT but not the native or modified
forms of the serpins. Mouse fibroblasts (2
10
cells/well) expressing LRP (MEF) or lacking LRP (PEA13) were
incubated for selected times at 37 °C with
I-labeled
ATIII, HCII, or
AT (5-10 nM, native,
modified, or enzyme-complexed) in the presence and absence of RAP (1
µM). At indicated intervals, the amount of radioligand
internalized by the cells was determined as detailed under
``Experimental Procedures.'' Panels show the
internalization of
I-ATIII by MEF (A) and PEA13 (B) cells,
I-HCII by MEF (C) and PEA13 (D) cells, and
I-
AT by MEF (E) and PEA13 (F) cells. Plotted values are means of
duplicate values and are representative of duplicate
experiments.
Figure 7:
Comparison of uPAPAI-1,
M*, ATIII
thrombin, HCII
thrombin, and
AT
trypsin degradation by mouse fibroblasts.
Mouse fibroblasts expressing LRP (2
10
) were
incubated with
I-labeled ligand in the absence of
competitor or in the presence of 1 µM RAP. Following
incubation, the amount of degraded (trichloroacetic-soluble)
I-ligand secreted into the medium was measured.
Degradation measured in the presence of 1 µM RAP was
subtracted from the amount of degradation measured in the absence of
competitor. Panel A,
I-labeled ligands (1
nM) were incubated for 8 h at 37 °C. The data shown are an
average of duplicate determinations ± standard error. Panel
B,
I-ATIII
thrombin (
),
I-HCII
thrombin (
), and
I-
AT
trypsin (
), at
0.6-150 nM, were incubated for 18 h at 37 °C. Panel C,
I-uPA
PAI-1 (
; 0.6-15
nM) was incubated for 18 h at 37
°C.
Figure 8:
RAP
inhibits the clearance of I-
M* (A) and ATIII
I-thrombin from the plasma of
rats (B). A, a bolus of 500 µl of
I-
M (10 nM) in the absence
(
) or presence (
) of RAP (32 µM) was
injected into the tail vein of rats. At the indicated times, blood (200
µl) was collected into 10 µl of 0.5 M EDTA and an
aliquot (50 µl) was counted for its
I content. B, a bolus of 500 µl of
ATIII
I-thrombin (100 nM) in the absence
(
) or presence (
) of RAP (110 µM) was
injected into the tail vein of rats. At the indicated times, blood (200
µl) was collected into 10 µl of 0.5 M EDTA and an
aliquot (50 µl) was counted for its
I content. The
clearance of each preparation was examined in two rats, and the data
plotted represent the average value ±
S.E.
The existence of a hepatic receptor that is responsible for
binding and removing SECs from the plasma was demonstrated in early
studies (Pizzo and Gonias, 1984; Shifman and Pizzo, 1982; Imber and
Pizzo, 1981; Fuchs et al., 1982; Pratt et al., 1988)
in which labeled SECs (ATIIIthrombin, HCII
thrombin,
AT
trypsin, and
AT
elastase) were shown to be rapidly cleared by
the liver (half-lives = 3, 10, 15, and 20 min, respectively).
These complexes apparently all bound to the same receptor, since they
were able to cross-compete with one another (Pratt et al.,
1988). However, clearance of these molecules was not competed with high
concentrations of
M
proteinase complexes (Fuchs et al., 1982). The clearance mechanism appears specific for
the enzyme-complexed form of these serpins, since studies by Mast et al.(1991) demonstrated that the clearance of native and
cleaved forms of ATIII, HCII, and
AT occurred at a
considerably slower rate (half-lives > 1 h) than the
proteinase-complexed serpins.
The data presented in the current
study suggest that LRP is the hepatic receptor responsible for the
clearance of these complexes. This conclusion is supported by several
independent lines of evidence. First of all, we demonstrated that
purified LRP can directly bind to several SECs (ATIIIthrombin,
HCII
thrombin, and
AT
trypsin) coated on
microtiter wells, and that the binding is competed by RAP. Second, SECs
are internalized and degraded in mouse fibroblasts that express LRP,
but not in mouse fibroblasts genetically deficient in LRP. Third, we
demonstrated that LRP antagonists, RAP and LRP antibodies, blocked the
cellular uptake and degradation of
I-labeled
ATIII
thrombin, HCII
thrombin, and
AT
trypsin. Fourth, our studies have shown that
the interaction of SECs with LRP has properties expected of the hepatic
clearance mechanism; LRP does not recognize the native and cleaved
forms of the inhibitors but readily internalizes the proteinase
complexed form of the inhibitors. Taken together, these data suggest
that the liver receptor responsible for the plasma clearance of
ATIII
thrombin, HCII
thrombin, and
AT
trypsin is LRP.
To demonstrate this, in
vivo clearance studies were performed in rats. The ability of RAP
to inhibit hepatic clearance of ATIII-I-thrombin
indicates that a RAP-sensitive hepatic receptor is responsible for this
process. While RAP is known to bind to all members of the LDLR
superfamily, only two of these receptors, LDLR and LRP, are known to be
expressed in high levels in the liver. Our studies indicate that the
LDLR does not play a significant role in the catabolism of SECs, since
PEA13 fibroblasts, which express LDLR and are normal in their ability
to catabolize LDL (Willnow and Herz, 1994; Kounnas et al.,
1995c), are unable to mediate the cellular catabolism of SECs. Thus,
the in vivo clearance studies suggest that LRP is playing a
prominent role in the hepatic clearance of SECs.
Perlmutter et
al. (1990a, 1990b) described a SEC receptor on monocytes and HepG2
cells which internalized complexes of AT
trypsin
and
AT
elastase, and it is highly likely that
this receptor is LRP. In the present investigation, we found that the
cellular uptake and degradation of ATIII
thrombin, HCII
thrombin, and
AT
trypsin by Hep G2 cells is
inhibited by anti-LRP IgG. In addition to a receptor that mediates the
endocytosis of SECs, studies by several investigators suggest that
binding of SECs to the cell surface may result in signal transduction
events. Perlmutter et al.(1988) demonstrated that binding of
elastase
AT complexes to monocytes or macrophages
resulted in increased gene expression of
AT, while
Banda et al.(1988) showed that cleaved
AT
stimulated neutrophil chemotaxis. Hoffman et al.(1989)
reported that HCII could be cleaved by proteinases to generate products
that stimulated neutrophil chemotaxis, suggesting that cleaved serpins
release biologically active molecules. While the mechanism of signal
transduction by these complexes is unclear, it seems likely that a
distinct signaling receptor, different from LRP, may exist and would
account for these effects.
The existence of a clearance mechanism
for SECs is likely important since such complexes are known to
dissociate with time, releasing active enzyme along with the cleaved
inhibitor (Travis and Salvesen, 1983; Carrell and Boswell, 1986). ATIII
is a primary inhibitor of thrombin and FX and plays a major
role in maintaining normal hemostasis (Bjork et al., 1989;
Olson and Bjork, 1992). Disruption of the normal balance of thrombin
and inhibitor activity results in either thrombosis (Egeberg, 1965;
Lane et al., 1991) or bleeding disorders (Owen et
al., 1983); therefore, it is clear that maintaining an appropriate
amount of thrombin activity is critical for normal hemostasis. The
principle action of ATIII may be to localize clotting by inhibiting
thrombin that has escaped into the circulation, and the LRP-mediated
clearance of ATIII
thrombin complexes may serve to help maintain
normal hemostasis.
The imbalance of AT/proteinase
activity has been associated with various pathological conditions such
as pulmonary emphysema (Laurell and Eriksson, 1963; Travis and
Salvesen, 1983; Carrell, 1986).
AT has been implicated
as a primary modulator of elastase and proteinase 3 activity during
inflammatory processes. In acute inflammation, infiltrating neutrophils
and macrophages, as well as damaged cells, release excessive amounts of
proteinases resulting in tissue damage. This damage can be minimized by
rapid inhibition of proteinase activity by inhibitors present in body
fluids. Elastase and PR3 are released by neutrophils during
inflammation and participate in the degradation of extracellular matrix
components. Once these enzymes are inhibited by
AT,
the complexes formed are known to be cleared by macrophages and
fibroblasts in a receptor-mediated process. LRP is found in high
concentration on fibroblasts and macrophages (Moestrup et al.,
1992) and has been shown to bind and mediate the uptake of at least two
AT
proteinase complexes (Poller et al.,
1995; this study) and therefore is very likely to represent the
receptor responsible for the endocytosis of
AT
proteinase complexes formed during the
inflammatory response.
Comparative experiments demonstrated that
several higher affinity LRP ligands such as uPAPAI-1 and
M* were internalized and subsequently degraded to a
greater extent than complexes of ATIII
thrombin,
HCII
thrombin, and
AT
trypsin. This is
likely related to the weaker affinity of these latter ligands for LRP.
Interestingly, the affinity for the interaction of ligands with LRP
varies considerably: uPA
PAI-1 (<1 nM; Nyjkaer et
al.(1994)),
M
methylamine (10 nM;
Ashcom et al.(1990)), LpL (18 nM; Chappell et
al.(1992)), thrombospondin (3-20 nM; Mikhailenko et al.(1995)), pro-uPA (45 nM; Kounnas et
al.(1993)), PAI-1 (55 nM; Nykjaer et al.(1994)),
hepatic lipase (52 nM; Kounnas et al. (1995a)), apoE
(54 nM; Kounnas et al. (1995b)), amyloid precursor
protein (80 nM; Kounnas et al. (1995c)), and
approximately 80-120 nM for the complexes of
ATIII
thrombin, HCII
thrombin, and
AT
trypsin. The relatively lower affinity of LRP
for the SECs (ATIII
thrombin, HCII
thrombin, and
AT
trypsin) as compared to other ligands may be
compensated for during conditions of thrombosis or inflammation where
the plasma or interstitial fluid levels of complexes are greatly
elevated. Other factors may also augment LRP-mediated uptake of the
SECs. For example cell-surface proteoglycans, which have been shown to
facilitate the uptake of a number of LRP ligands including lipoprotein
lipase (Chappell et al., 1993), hepatic lipase (Kounnas et
al., 1995), and thrombospondin (Mikhailenko et al.,
1995), may act to concentrate SECs on the surface of LRP-expressing
cells and thereby increase their local concentration.
In summary,
the current investigation demonstrates that LRP can bind and endocytose
complexes of ATIIIthrombin, HCII
thrombin, and
AT
trypsin, but not the native or the cleaved
inhibitors. In vivo clearance studies confirm that LRP
functions to remove SECs from the plasma. These findings expand the
role of LRP as a SEC receptor.