(Received for publication, November 8, 1995; and in revised form, January 23, 1996)
From the
Thrombin is a multifunctional protein that has both proteinase
and growth factor-like activities. Its regulation is largely mediated
by interaction with a host of inhibitors including antithrombin III
(ATIII), heparin cofactor II (HCII), -macroglobulin
(
-M), protease nexin I, and plasminogen activator
inhibitor-1 (PAI-1). ATIII, HCII, and
-M are all
abundant in blood and can inactivate blood-borne thrombin leading to
rapid hepatic clearance of the thrombin-inhibitor complex. PAI-1 alone,
a poor solution phase inhibitor of thrombin, can efficiently inhibit
thrombin in the presence of native vitronectin (VN). In this study,
active thrombin was found to be efficiently endocytosed and degraded by
cultured pre-type II pneumocyte cells, and both processes could be
blocked by polyclonal antibodies to PAI-1. When the relative efficiency
of cellular endocytosis of thrombin in complex with a number of
inhibitors was examined,
I-thrombin-PAI-1 complexes were
most efficiently cleared compared to
I-thrombin in
complex with the serpins ATIII, HCII,
-proteinase
inhibitor, or D-phenylalanyl-L-prolyl-L-arginine
chloromethyl ketone. Low density lipoprotein receptor-related proteins
1 (LRP) and 2 (gp330/megalin) mediate the endocytosis of
thrombin-PAI-1, since antagonists of receptor function such as LRP-1
and LRP-2 antibodies and the 39-kDa receptor-associated protein blocked
I-thrombin-PAI-1 endocytosis and degradation. The
LRP-mediated clearance of exogenously added
I-thrombin by
cultured cells was found to be enhanced 5-fold by inclusion of
wild-type PAI-1 but by only 2-fold when a mutant form of PAI-1 that is
unable to bind VN was included. This wild-type PAI-1 enhancement of
I-thrombin clearance was found to occur only in the
presence of native VN and not with its conformationally altered form.
The results highlight a novel mechanism for cellular clearance of
thrombin involving native VN promoting the interaction of thrombin and
PAI-1 and the subsequent endocytosis of the complex by LRP-1 or LRP-2.
This pathway is potentially important for the regulation of the potent
biological activities of thrombin, particularly at sites of vascular
injury.
The enzymatic activity of thrombin is responsible for the conversion of soluble fibrinogen to insoluble fibrin, activation of blood coagulation proteins V, VIII, and XIII(1, 2) , and activation of platelets via limited hydrolysis of the thrombin receptor(3) . Thrombin also negatively controls blood coagulation through activation of protein C, which inactivates factors Va and VIIIa. In addition, thrombin has mitogenic effects on a variety of cells including fibroblasts, endothelial cells, and macrophages (4, 5, 6, 7) and chemotactic effects on neutrophils, fibroblasts, and monocytic cells(8, 9, 10) . Considering that thrombin can also promote adhesion of polymorphonuclear cells and monocytes to endothelial cells(11, 12) , as well as increase the permeability of endothelial cell monolayers and pulmonary vessels(13) , it may be an important mediator of inflammatory and vascular wound healing events. The catalytic activity of thrombin is required for its mitogenic effects on fibroblasts (14) and endothelial cells(15) , as well as inhibition of neurite outgrowth (16) ; however, proteolytically active thrombin is not required for its chemotactic effects on neutrophils and monocytes (9, 8) or its ability to increase the permeability of endothelial cell monolayers and pulmonary vasculature(13) .
The multitude of biological actions of thrombin necessitates that
strict controls be placed on its expression, activity, and half-life.
In blood, the inhibition of proteolytic active thrombin is mediated by
interaction with a host of inhibitors including ATIII, ()HCII, and
-M. Upon interaction of blood
borne thrombin with these inhibitors, the complexes are rapidly cleared
by the liver via a receptor-mediated
process(17, 18, 19) . However, little is
known about the mechanism of inactivation and clearance of
extravascular thrombin, such as that detected on the surface of tissue
macrophage or sequestered within thrombi away from the blood flow and
the action of circulating
inhibitors(20, 21, 22) . It is likely that in
these contexts thrombin exerts its greatest effects on inflammation and
vascular wound healing. Given that plasminogen activator inhibitor-1
(PAI-1) is known to be expressed at sites of inflammation and released
from platelet granules upon activation(23, 24) , it
may under these conditions be a relevant inhibitor of thrombin. While
PAI-1 alone is a rather poor inhibitor of thrombin, complex formation
with VN has been shown to greatly augment its ability to inhibit
thrombin(25, 26) . VN is known to be present in
connective tissue extracellular matrices and released from platelets
upon their activation(24, 27) . Ehrlich et al.(28) have demonstrated that complexes of thrombin and
PAI-1 form on endothelial cell extracellular matrices and the complex
formation can be inhibited with antibodies to VN. While they speculated
that the thrombin-PAI-1 interaction might promote plasminogen activator
activity by neutralizing PAI-1, this interaction may also mediate
cellular clearance of thrombin. The latter would be similar to two
other proteinases, tissue-type plasminogen activator (tPA) and
urokinase-type plasminogen activator (uPA), whose endocytosis and
degradation via several members of the low density lipoprotein receptor
(LDLR) family are promoted after complex formation with
PAI-1(29, 30, 31, 32, 33) .
The focus of this study was to evaluate cell-mediated endocytosis as a
potential mechanism for regulating levels of extravascular thrombin and
to determine whether PAI-1, VN, and receptors of the LDLR family have
roles in the process.
Radioiodination of proteins was performed by using IODO-GEN
(Pierce). Complexes of thrombin and various inhibitors were prepared by
incubating the I-thrombin with each inhibitor at a 2:1
molar ratio for 30 min at 25 °C, followed by absorption of free
thrombin by chromatography on a column ATIII-Sepharose (2 mg of
ATIII/ml of resin). To prepare active site-inhibited thrombin,
I-thrombin (100 nM) was incubated with PPACK (5
mM) for 30 min at 25 °C in Tris-buffered saline. The
complexes were tested for thrombin activity by incubation with a
fibrinogen solution (1 mg of fibrinogen/ml in Tris-buffered saline, 5
mM CaCl
) at 25 °C for 30 min and assaying for
fibrin formation.
To evaluate the effects of wild-type and
mutant PAI-1 on the clearance (endocytosis and degradation) of
exogenously added I-thrombin, cells were grown in medium
containing 10% serum as described above. Washed monolayers were then
incubated either with wild-type PAI-1 (10 nM) or mutant PAI-1
(10 nM) for 20 min at 37 °C.
I-Thrombin (10
nM) or
I-uPA (10 nM) was added and
incubated for 4-6 h at 37 °C. Where indicated, RAP (1
µM) was incubated for 30 min prior to addition of the
ligands.
To evaluate the effects of native versus conformationally altered VN on the clearance of exogenously added
PAI-1 and active I-thrombin, cells were grown in
serum-free medium for 18 h at 37 °C on tissue culture plates coated
with 0.1% gelatin. Medium was removed and assay medium added for 1 h at
37 °C to block unoccupied binding sites with bovine serum albumin.
Cell monolayers were incubated with either native (50 nM) or
conformationally altered VN (50 nM) for 1 h at 37 °C. Cell
monolayers were then washed twice with assay medium to remove unbound
VN. Wild-type PAI-1 (10 nM) was added and incubated for 1 h at
37 °C.
I-Thrombin (10 nM) or
I-uPA (10 nM) was added and incubated for
4-6 h at 37 °C.
Figure 1:
Endocytosis and
degradation of active and active site-inhibited thrombin and effects of
PAI-1 antibodies. Pre-type II pneumocyte cell endocytosis (panel
A) and degradation (panel B) of I-thrombin-PPACK (
I-Th:PPACK) and active
I-thrombin (
I-Th) each at 20 nM.
Also shown in each panel are the effects of rabbit anti-mouse PAI-1 IgG
(0.6 mg/ml) or normal rabbit IgG (Control IgG, 0.6 mg/ml) on
the endocytosis and degradation of active
I-thrombin. The
data presented are representative of three experiments, each performed
in duplicate. Each plotted value represents the average of duplicate
determinations with the range indicated by bars.
Figure 2:
Comparison of the level of endocytosis and
degradation of I-thrombin in complex with serpins.
Pre-type II pneumocyte cells were incubated with
I-thrombin in complex with the synthetic inhibitor
phenylprolylarginylchloromethyl ketone (
I-Th:PPACK), HCII (
I-Th:HCII), ATIII (
I-Th:ATIII),
-PI (
I-Th:
PI)
each at 16 nM. Ligand endocytosis is shown in panel
A, and degradation is shown in panel B. The data
presented are representative of four experiments. Each plotted value
represents the average of duplicate determinations with the range
indicated by bars.
Figure 3:
Endocytosis and degradation of I-thrombin-PAI-1 complex are inhibited by antagonists of
LRP function.
I-Thrombin-PAI-1 complex (10 nM)
was incubated with cultured pre-type II pneumocyte cells in the
presence of RAP (1 µM), affinity-purified LRP-1 antibodies (anti-LRP-1, 150 µg/ml), affinity-purified LRP-2
antibodies (anti-LRP-2, 150 µg/ml), a mixture of the LRP-1
and LRP-2 antibodies (anti-(LRP-1+2), 300 µg/ml), or
antibody to a peptide corresponding to the cytoplasmic tail of LRP (anti-LRP-1 CD, 150 µg/ml). Specific endocytosis and
degradation was determined by co-incubation with 500-fold molar excess
of unlabeled thrombin-PAI-1. Ligand endocytosis is shown in panel
A, and degradation is shown in panel B. The data
presented are representative of two experiments. Each plotted value
represents the average of duplicate determinations with the range
indicated by bars.
Figure 4:
Binding of I-thrombin-PAI-1
complex to LRP-1 and LRP-2. The binding of
I-thrombin-PAI-1 (1 nM) to microtiter wells
coated with LRP-1 (panel A) or LRP-2 (panel B) was
measured in the presence of increasing concentrations of unlabeled
thrombin-PAI-1, thrombin, or PAI-1. The curves represent the best fit
of the data to a single class of sites. The data presented are
representative of four experiments, each performed in duplicate. Each
plotted value represents the average of duplicate determinations with
the range indicated by bars.
Figure 5:
Effect of wild-type PAI-1, or a mutant of
PAI-1 that is unable to bind VN, on the endocytosis and degradation of I-thrombin or
I-uPA. Pre-type II pneumocyte
cells were incubated with either wild-type PAI-1 (wtPAI-1, 10
nM) or a mutant form of PAI-1 (mPAI-1, 10
nM) that is unable to bind to VN. Either
I-thrombin (10 nM) or
I-uPA (10
nM) were added to the cells and incubated for 4-6 h in
the presence or absence of RAP (1 µM). The amount of
endocytosis and degradation of
I-thrombin is shown in panels A and C, respectively. The amount of
endocytosis and degradation of
I-uPA is shown in panels B and D, respectively. The data presented are
representative of four experiments, each performed in duplicate. Each
plotted value represents the average of duplicate determinations with
the range indicated by bars.
To show that the PAI-1 mutation did not
affect the ability of its complex with thrombin to bind to LRPs,
complexes of I-thrombin with either wild-type PAI-1 or
mutant PAI-1 were formed in vitro. As shown in Fig. 6,
complexes containing either form of PAI-1 were readily endocytosed (panel A) and degraded (panel B) by the pre-type II
pneumocyte cells. Both the endocytosis and degradation of the complexes
were inhibited by including RAP in the assay. These results indicate
that complexes of thrombin and either wild-type or mutant PAI-1 are
recognized equally well by LRP receptors. Therefore, when free
I-thrombin was presented to the cells as in Fig. 5, the formation of a complex with PAI-1
VN was
required for the efficient complex formation between thrombin and
PAI-1, which leads to rapid LRP-mediated endocytosis and degradation.
Figure 6:
Endocytosis and degradation of I-thrombin that has been pre-complexed to either
wild-type PAI-1 or mutant PAI-1. Pre-type II pneumocyte cells were
incubated with
I-thrombin pre-complexed with either
wild-type PAI-1 (
I-Th:wtPAI-1)
or mutant PAI-1 (
I-Th:mPAI-1)
that is unable to bind to VN.
I-Labeled complex
concentration was 10 nM. Where indicated, RAP (1
µM) was added along with the
I-labeled
complex. The amount of endocytosis and degradation of each type of
I-labeled complexes are shown in panels A and B, respectively. The data presented are representative of two
experiments. Each plotted value represents the average of duplicate
determinations with the range indicated by bars.
Figure 7:
The effect of native or conformationally
altered VN on endocytosis and degradation of I-thrombin
and
I-uPA in the presence of wtPAI-1. Pre-type II
pneumocyte cells grown in serum-free conditions were incubated with
either native VN (nVn, 50 nM) or conformationally
altered (denatured) VN (dVN, 50 nM). After washing
the cells were incubated with wild-type PAI-1 (10 nM),
followed by addition of either
I-thrombin (10
nM) or
I-uPA (10 nM). The amount of
endocytosis and degradation of
I-thrombin is shown in panels A and C, respectively. The amount of
endocytosis and degradation of
I-uPA is shown in panels B and D, respectively. The data presented are
representative of three experiments. Each plotted value represents the
average of duplicate determinations with the range indicated by bars.
In this study we have shown that: 1) exogenously administered
active thrombin is endocytosed and degraded by cultured cells; 2)
inactivation of the thrombin with PPACK or inclusion of polyclonal
antibodies to PAI-1 blocked both processes; 3) thrombin in complex with
PAI-1 was more efficiently cleared than when it was in complex with
antithrombin, HCII, or -antiproteinase; 4) RAP, the
antagonist of ligand binding to members of the LDLR family, blocked the
endocytosis and degradation of thrombin; 5) both LRP-1 and LRP-2 bound
to thrombin-PAI-1 complexes with high affinity in solid phase assays
and receptor antibodies blocked cellular clearance of the complex; 6)
wild-type PAI-1 but not a mutant form of PAI-1 that is unable to bind
VN facilitated the cellular clearance of thrombin; and 7) native but
not denatured VN augments the PAI-1-promoted clearance of thrombin.
Based on these findings, we conclude that active thrombin clearance by
pre-type II pneumocyte cells can be mediated through its complex
formation with PAI-1 and the subsequent interaction of the complex with
either LRP-1 or LRP-2. The role of native VN in this process is
critical, presumably due to the fact that it acts to augment the
formation of the thrombin-PAI-1 complex, which is otherwise an
inefficient process.
VN is known to bind both PAI-1 and
thrombin(26, 49, 50, 51, 52) .
The consequences of these binding interactions apparently leads to more
efficient interaction between PAI-1 and thrombin. It is not known
whether VN remains associated with PAI-1 and thrombin following their
interaction. VN does form a ternary complex with thrombin bound to
either ATIII HCII, proteinase nexin I, or
-PI-Pittsburgh(53, 54, 55) .
However, the complex of PA1-1 and VN dissociates following the
interaction with either uPA or tPA (56) . In our experiments we
did not evaluate whether VN was endocytosed along with thrombin-PAI-1
complex. In studies reported by Panetti and McKeown-Longo(57) ,
active thrombin but not inactivated thrombin was shown to promote the
cellular clearance of
I-native VN. Since inactive
thrombin is not able to bind serine proteinase inhibitors whereas
active thrombin can, the authors speculated that thrombin interaction
with some endogenous inhibitor facilitated native VN clearance. This is
consistent with our findings showing that active thrombin is cleared
much more efficiently than is inactivated thrombin and that PAI-1
antibodies inhibit the clearance of active thrombin. The results taken
together point to the possibility that a ternary complex of
thrombin-PAI-1 and VN may be cleared, but this remains to be
established. The fact that RAP blocks thrombin clearance to the same
extent as excess unlabeled thrombin indicates that LRP receptors are
primarily responsible for mediating the clearance process.
A major
concept to emerge from this work is that PAI-1 can serve to mediate
thrombin catabolism, but it raises the issue of when and where such a
process might occur in vivo. While PAI-1 inhibits uPA and tPA
with a second-order rate constant of 10M
s
(58) ,
by comparison the second-order rate constant for inhibition of thrombin
is
10,000-fold less(26) . The physiological relevance of
PAI-1 inhibition of thrombin therefore may not be immediately obvious,
until one considers that cofactors such as heparin and VN can
dramatically enhance the ability of PAI-1 to inhibit thrombin. For
example, in the presence of VN the second-order rate constant for the
inhibition of thrombin by PAI-1 is increased by more than 2 orders of
magnitude(26) . This effect makes PAI-1
VN a
10-20-fold better inhibitor of thrombin than ATIII in the absence
of heparin. In blood, where the concentration of ATIII is 10,000-fold
higher than PAI-1, the relevance of PAI-1 as an inhibitor of blood
borne thrombin is unlikely. However, in extravascular sites such as in
the recesses of a fibrin-containing thrombus, we speculate that it may
be a physiologically relevant inhibitor of thrombin. Fibrin is thought
to sequester thrombin, protecting it from circulating inhibitors (22, 59, 60) until lysis of the clot by
plasmin(59) . The thrombin thereby released would be available
to drive post-clotting events such as mediating mitogenesis and
chemotaxis of cells involved in clot remodeling and tissue repair.
PAI-1, either derived from platelets or synthesized by cells invading a
clot or on the boundaries of the clot, and VN, derived either from
platelets (24) or blood, could mediate inactivation of thrombin
and its clearance by LRP-expressing cells (e.g. smooth muscle
cells, macrophage, and fibroblasts). In this way the post-clotting
effects of thrombin could be negatively regulated.