ACCELERATED PUBLICATION
Vitronectin Functions as a Cofactor for Rapid Inhibition of
Activated Protein C by Plasminogen Activator Inhibitor-1
IMPLICATIONS FOR THE MECHANISM OF PROFIBRINOLYTIC ACTION OF
ACTIVATED PROTEIN C*
Alireza R.
Rezaie
From the Edward A. Doisy Department of Biochemistry and Molecular
Biology, Saint Louis University School of Medicine, Saint
Louis, Missouri 63104
Received for publication, March 12, 2001
 |
ABSTRACT |
Activated protein C (APC) is a natural
anticoagulant in plasma that down-regulates the coagulation cascade by
degrading factors Va and VIIIa. In addition to its anticoagulant
function, APC is also known to possess a profibrinolytic property. This
property of APC has been attributed to its ability to neutralize PAI-1, thereby increasing the concentration of tissue plasminogen activator in
plasma leading to up-regulation of the fibrinolytic cascade. This
hypothesis, however, has not been well established, since the
concentration of PAI-1 in plasma is low, and its reactivity with APC is
very slow in a purified system. Here we demonstrate that vitronectin
enhances the reactivity of PAI-1 with APC ~300-fold making PAI-1 the
most efficient inhibitor of APC thus far reported (k2 = 1.8 × 105
M
1 s
1).
We further show that PAI-1 inhibition of the Glu192
Gln
mutant of APC is enhanced ~40-fold, independent of vitronectin, suggesting that vitronectin partially overcomes the inhibitory interaction of PAI-1 with Glu192. Additionally, we show
that PAI-1 inhibition of the
Lys37-Lys38-Lys39
Pro-Gln-Glu
mutant of APC is severely impaired, suggesting that, similar to tissue
plasminogen activator, the basic 39-loop of APC plays a critical
role in the reaction. Together, these results suggest that vitronectin
functions as a cofactor to promote the profibrinolytic activity of
APC.
 |
INTRODUCTION |
Activated protein C
(APC)1 is a vitamin
K-dependent serine proteinase in plasma that exhibits both
anticoagulant (2, 3) and profibrinolytic (4-8) properties. The
mechanism of anticoagulant function of APC is extensively studied. It
is established that APC inhibits thrombin generation by limited
proteolytic degradation of two essential procoagulant cofactors, Va and
VIIIa (2, 3, 9). The physiological significance of APC as an
antithrombotic factor is documented by the observation that inherited
and acquired deficiency of protein C is associated with venous
thromboembolic disease (10, 11). In contrast to its anticoagulant
function, the mechanism by which APC exerts its profibrinolytic
function is not well established. Several types of whole blood clot
lysis assays in vitro have indicated that APC functions as a
profibrinolytic factor in a plasminogen activator inhibitor-1
(PAI-1)-dependent manner (6-8). Based on these results, it
has been hypothesized that neutralization of this inhibitor by APC
increases the concentration of plasminogen activators in plasma leading
to up-regulation of the fibrinolytic cascade (6). In support of this
mechanism, incubation of active APC, but not diisopropyl
fluorophosphate-inactivated APC, with the endothelial cell culture
conditioned media or with PAI-1 partially purified from
platelets resulted in SDS-stable APC-PAI-1 complexes (7). This has
suggested that APC interacts with PAI-1 as other serine proteinases
interact with their target serine proteinase inhibitors of the serpin
superfamily (12).
Despite the evidence that APC interacts with PAI-1 by a typical serine
proteinase-serpin reaction mechanism, no kinetic studies exist in the
literature to indicate that human APC can react with PAI-1 in a
homologous system. This is probably due to the fact that prolonged
incubation of human APC with high concentrations of PAI-1 results in a
minimal decline in the activity of the proteinase. In support of this,
results of an in vitro clot lysis assay using purified
components of the APC anticoagulant pathway have disputed whether this
mechanism can contribute to the profibrinolytic property of human APC
in vivo (8). Such results have raised the possibility that
other factor(s) in blood may be involved in APC up-regulation of the
fibrinolytic cascade (8). In the current study, vitronectin, an
abundant plasma and platelet glycoprotein (13-15), has been identified
as the cofactor that dramatically improves the ability of APC to react
with PAI-1. It is shown that vitronectin enhances the reactivity of APC
with PAI-1 ~300-fold, revealing PAI-1 to be the most efficient
inhibitor of APC reported. The possible mechanism of the
APC-vitronectin-PAI-1 interaction was studied by analyzing the kinetics
of PAI-1 inactivation of two mutants of APC, in one of which the three
basic Lys37-Lys38-Lys39 residues of
APC were replaced with Pro-Gln-Glu (KKK/PQE) and in the other
Glu192 was substituted with Gln (E192Q). The reactivity of
KKK/PQE with PAI-1 was dramatically impaired, although the extent of
the cofactor effect of vitronectin was not affected. On the other hand,
the E192Q mutant of APC reacted efficiently with PAI-1 independent of
vitronectin. These results suggest that similar to plasminogen activators, the basic 39-loop of APC is critical for a productive interaction with PAI-1. In contrast, Glu192 restricts the
reactivity of APC with PAI-1 and vitronectin appears to overcome this
inhibitory interaction. The possible physiological significance of
these findings to APC regulation of coagulation and fibrinolytic
cascades is discussed.
 |
EXPERIMENTAL PROCEDURES |
Proteins and Reagents--
Methodologies for expression,
purification, and activation of the wild type and Glu192
Gln (E192Q) mutant of human protein C were described previously (16). The protein C mutant in which three basic residues,
Lys37-Lys38-Lys39, of 39-loop were
replaced with Pro-Gln-Glu (KKK/PQE, residues found at the identical
region of thrombin) was prepared by the same procedures. All
derivatives were expressed in HEK 293 cells and purified to
homogeneity on an immunoaffinity column using the
Ca2+-dependent monoclonal antibody, HPC4, as
described previously (17). Protein C (100 µg) was incubated with
thrombin (10 µg) in 0.1 M NaCl, 0.02 M
Tris-HCl, pH 7.4 (TBS buffer) containing 5 mM EDTA for
2 h at 37 °C. Activated protein C was separated from thrombin
by a fast protein liquid chromatography Mono Q column developed with
40-ml linear gradient from 0.1 to 1.0 M NaCl, 0.02 M Tris-HCl, pH 7.4. Partially and fully carboxylated APC
were eluted at ~0.3 and ~0.4 M NaCl, respectively.
Human plasma proteins APC and protein S were purchased from Hematologic
Technologies Inc. (Essex Junction, VT). Recombinant human active PAI-1
(product number PAI-A, 95 ± 5% active as titrated by
urokinase-type plasminogen activator), and plasma-derived human
native vitronectin (product number HVN) were purchased from
Molecular Innovations, Inc. (Royal Oak, MI). SDS-polyacrylamide gel
electrophoresis analysis confirmed the homogeneity of all proteins used
in this study. Phospholipid vesicles containing 80%
phosphatidylcholine and 20% phosphatidylserine (PC/PS) or 40%
phosphatidylcholine, 20% phosphatidylserine, and 40%
phosphatidylethanolamine (PC/PS/PE) were prepared as described previously (18). The chromogenic substrate Spectrozyme
PCa (SpPCa) was purchased from American Diagnostica (Greenwich, CT).
Kinetic Methods--
The PAI-1 inactivation rates of APC
derivatives were measured under pseudo-first-order rate conditions by a
discontinuous assay method as described previously (19). Briefly, 2 nM APC was incubated at room temperature with 50-2000
nM PAI-1 in 50-µl reactions in TBS buffer containing 2.5 mM Ca2+, 0.1 mg/ml bovine serum albumin and
0.1% polyethylene glycol 8000. At the end of the incubation time
(5-60 min) 50 µl of SpPCa was added to a final concentration of 0.5 mM. The remaining activity of APC was measured from the
rate of chromogenic substrate hydrolysis at 405 nm using a
Vmax Kinetics Microplate Reader (Molecular
Devices, Menlo Park, CA). The pseudo-first-order inactivation rate
constants (k') were calculated by fitting the
time-dependent change of the APC activity to a first-order
rate equation using the Enzfitter computer program (R. J. Leatherbarrow, Elsevier, Biosoft). The second-order inactivation
rate constants (k2) were calculated from the
slope of the linear plot of the k' values versus
PAI-1 concentrations as described previously (19). In all assays, less
than 10% chromogenic substrate was utilized.
For the inactivation studies in the presence of vitronectin, first, the
cofactor concentration dependence of PAI-1 inactivation of APC was
studied by incubating 2 nM APC with 50 nM PAI-1 and 0-2.5 µM vitronectin at room
temperature in 50-µl reactions in the same buffer system described
above. At the end of the incubation time (5 min), 50 µl of 1 mM SpPCa in TBS buffer was added, and the k' values at
different vitronectin concentrations were determined as described
above. Detailed kinetic studies in the presence of an optimal
concentration of vitronectin (600 nM) were then carried out
by incubating APC (2 nM) with varying concentrations of
PAI-1 (6.25-100 nM) for 2.5-20 min in the same TBS buffer
system. The k' values were calculated as described above and
plotted versus PAI-1 concentrations. The maximal inhibition
rate constant (k) and Kd values were
determined by fitting the saturable dependence of k' values
on PAI-1 concentrations to a hyperbolic equation as described
previously (20).
Determination of Inhibition Stoichiometry--
The
inhibition stoichiometry for PAI-1 inactivation of APC in the presence
of vitronectin and APC E192Q in both the absence and presence of
vitronectin were determined by titration of 20 nM APC with
increasing concentrations of PAI-1 corresponding to PAI-1/APC molar
ratios of 0-2, in the absence or presence of 600 nM
vitronectin in the same TBS buffer system as described previously (20).
The residual amidolytic activities of the wild type and mutant enzymes
were monitored for up to 12 h at room temperature by the
hydrolysis of SpPCa as described above. After completion of the
inhibition reactions, the PAI-1/APC ratios were plotted versus the residual activities of enzymes, and the
inhibition stoichiometry were determined from the
x-intercept of the linear regression fit of the
inhibition data.
 |
RESULTS AND DISCUSSION |
APC is an important anticoagulant plasma proteinase that shuts
down thrombin generation by limited proteolysis of factors Va and
VIIIa, essential cofactors of the extrinsic and intrinsic pathways of
the clotting cascade, respectively (2, 21). In addition to
down-regulation of coagulation, in vivo (4) and in
vitro (5, 6), studies have indicated that APC promotes fibrinolysis by neutralizing PAI-1. This property of APC has been hypothesized to increase the concentration of plasminogen activators in
plasma leading to up-regulation of the fibrinolytic cascade (5, 6).
Although the profibrinolytic property of APC has been relatively well
documented by several types of in vitro whole blood clot
lysis assays (6-8), there has been no study in a purified system of
the kinetics of the APC-PAI-1 interaction with human proteins. This is
due to the fact that human APC reacts with PAI-1 very slowly. As shown
in Table I, a second-order
inhibition rate constant of ~6 × 102
M
1 s
1
for PAI-1 inhibition of APC was determined. This same value was obtained if PAI-1 inhibition of APC was monitored in the presence of
220 nM protein S and 0-50 µM PC/PS or
PC/PS/PE vesicles (data not shown). These results are consistent with
other data in the literature (8). Therefore, unlike the potent
profibrinolytic effect observed for APC in vivo (4), in
whole blood clot lysis assays (6), and in endothelial cell culture
supernatants (5), kinetic data in the purified system suggest that
human APC neutralization of PAI-1 by itself may not significantly
contribute to up-regulation of the fibrinolytic cascade. Alternatively,
there may be other factor(s) in plasma or in endothelial cell culture
supernatants that enhances the ability of APC to interact with
PAI-1.
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Table I
Kinetic constants for PAI-1 inhibition of the APC derivatives in the
absence and presence of vitronectin
The second-order inhibition rate constants (k2 in
M 1 s 1) in the absence of vitronectin
were determined from the linear slope of the plot of the k'
values versus PAI-1 concentrations (50-2000 nM)
as described under "Experimental Procedures." In the presence of
vitronectin (600 nM), the maximal inhibition rate constant
(k) and Kd values were determined by
fitting the saturable dependence of k' values on PAI-1
concentrations (6-100 nM) to a hyperbolic equation as
shown in Fig. 2A and Scheme 1. The k2
values (M 1 s 1) were then calculated
from the ratio of k to the Kd values. All values are the
average of at least three independent measurements ± S.E.
|
|
PAI-1 is a serpin, which similar to other inhibitory serpins,
inactivates its target proteinases by binding to their active sites
through an exposed reactive center loop and undergoes a conformational change that traps the enzymes in inactive, stable complexes (22). Previously, it has been demonstrated that incubation of
active APC, but not diisopropyl fluorophosphate-inactivated APC, with
endothelial cell culture conditioned media (6, 7) results in
SDS-stable APC-PAI-1 complexes, suggesting that PAI-1 interacts with
APC by a similar mechanism. Unlike other inhibitory serpins, however,
PAI-1 in plasma exists in both active and latent forms (23, 24). The
active form of PAI-1 has a short half-life, unless it binds to
vitronectin (24). Vitronectin is a glycoprotein that is known to bind
PAI-1 with a high affinity to stabilize the active conformation of the
serpin (24, 25). Noting the abundance of vitronectin in plasma or
platelets (13) and in endothelial cell culture supernatants (26), it
was hypothesized that vitronectin may function as a cofactor to enhance
the reactivity of APC with PAI-1, potentially accounting for its
profibrinolytic activity observed in previous in vivo and
in vitro blood clot lysis assays.
In agreement with this hypothesis, it was found that vitronectin
dramatically accelerates the reactivity PAI-1 with APC in a
concentration-dependent manner (Fig.
1). The pseudo-first-order rate constant
(k') for PAI-1 inactivation of APC at an optimal concentration of vitronectin in large molar excess over PAI-1 was
determined at several concentrations of PAI-1 as described under
"Experimental Procedures." Based on reported Kd values for the PAI-1-vitronectin binary complex interaction, PAI-1 was
largely saturated with the cofactor protein under these conditions (27). A saturable dependence of k' on PAI-1 concentration
was observed for APC inhibition in the presence of vitronectin (Fig. 2A). Nonlinear regression
analysis of k' values according to a hyperbolic equation
yielded values for the ternary complex dissociation constant,
Kd, and the rate constant, k, for stable
complex formation as shown in Scheme 1.
The second-order inactivation rate constant
(k2), determined from the ratio of the limiting
rate constant, k, to the Kd value in
Scheme 1 was 1.8 ± 0.4 × 105
M
1 s
1,
suggesting that vitronectin enhances the rate of the APC-PAI-1 reaction
at least 300-fold (Table I). The observation that in the absence of
vitronectin, k' increases linearly with PAI-1 concentration over the same range (Fig. 2B), indicates that the
rate-enhancing effect of vitronectin is due to the promotion of
PAI-1-APC encounter complex formation. Similar results were obtained
whether the source of APC was from human plasma or from the mammalian
cell expression system. Inactivation studies in the presence of excess
hirudin further suggested that possible trace contamination of APC with thrombin does not influence the results. Unfractionated heparin maximally accelerated PAI-1 inactivation of APC only 4-fold
(k2 = 2.4 × 103
M
1 s
1)
when tested in the presence of 1 µM heparin. Inhibition
kinetic studies with activated Gla-domainless protein C suggested that the rate-accelerating effect of vitronectin requires the Gla-domain of
protein C. No effect for vitronectin was detected in PAI-1 inhibition
of activated Gla-domainless protein C (data not shown), possibly
suggesting that binding of the APC Gla domain to vitronectin was
important for the cofactor effect.

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Fig. 1.
Vitronectin concentration dependence of human
APC inactivation by human recombinant PAI-1. Recombinant human APC
(2 nM) was incubated with PAI-1 (50 nM) in the
presence of increasing concentrations of vitronectin (0-2.5
µM) in TBS buffer containing 0.1 mg/ml bovine serum
albumin, 0.1% polyethylene glycol 8000, and 2.5 mM
Ca2+. After incubation for 5 min at room temperature,
Spectrozyme PCa was added to a final concentration of 0.5 mM, and the pseudo-first-order rate constants
(k') were calculated from remaining activity of enzyme as
described under "Experimental Procedures."
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Fig. 2.
Dependence of pseudo-first-order rate
constants (k') for inactivation of APC by PAI-1 in the
absence and presence of vitronectin. The k' values for
PAI-1 inactivation of APC (2 nM) in the presence
(A) or absence (B) of vitronectin (600 nM) were determined at varying concentrations of PAI-1
(shown on x axis) and plotted versus the PAI-1
concentration. The solid lines are least squares computer
fits of data by a hyperbolic (A) or a linear equation
(B).
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|
The mechanism by which vitronectin may promote the reactivity of PAI-1
with APC was investigated by examining the kinetics of PAI-1
inactivation of two mutants of APC in both the absence and presence of
vitronectin. The reactivity of the KKK/PQE mutant of APC with PAI-1 was
markedly impaired in both the absence and presence of vitronectin,
although the extent of the cofactor function of vitronectin did not
appear to be affected with this mutant (Table I). This suggested that
the 39-loop of APC is involved in a productive interaction with the
reactive site loop of PAI-1 independent of vitronectin. The
concentration dependence of PAI-1 inactivation of the mutant in the
presence of vitronectin suggested that the Kd for
ternary complex formation was not changed, but the rate constant,
k, was impaired with this mutant (data not shown). PAI-1 has
two acidic residues at the P4' and P5' positions (28). Previously, it
has been demonstrated that the interactions of the basic residues of
the 39-loop of plasminogen activators with these acidic residues is
required for an efficient reaction with PAI-1 (29). The same appears to
be true for the reaction of the serpin with APC.
Another residue, which is known to restrict the specificity of APC with
plasma inhibitors, is Glu192 (16). Previous studies have
indicated that substitution of Glu192 with Gln, a residue
found at the identical site of trypsin and plasminogen activators,
results in a mutant (E192Q) that, unlike wild type APC, rapidly reacts
with both the serpin and Kunitz-type family of plasma protein
inhibitors, including
1-antitrypsin and tissue factor
pathway inhibitor, TFPI (30). Inhibition kinetic studies with the E192Q
mutant of APC revealed that this residue also plays a critical role in
restricting the reactivity of APC with PAI-1. As shown in Table I,
relative to wild type APC, the reactivity of PAI-1 with APC E192Q was
markedly improved by ~40-fold. Thus, similar to plasminogen
activators, vitronectin was no longer required for efficient reaction
of APC E192Q with PAI-1, although it still enhanced the rate of
inhibition ~10-fold (Table I). These results suggest that the
cofactor function of vitronectin may in large part involve overcoming
potentially inhibitory interactions occurring between the acidic
residues in the reactive site loop of PAI-1 and the extended catalytic
pocket of APC.
Vitronectin is also known to markedly accelerate the reactivity of
thrombin with PAI-1 (25, 31). The exact mechanism by which vitronectin
accelerates the reactivity of PAI-1 with thrombin is not known. Similar
to the reaction with APC (Fig. 1), previous studies of the vitronectin
concentration dependence of thrombin inactivation by PAI-1 has
indicated that the rate of reaction slightly decreases at higher
concentrations of vitronectin, possibly suggesting that vitronectin may
function by a template mechanism (31). However, other kinetic studies,
employing several mutant thrombin derivatives, have not confirmed such
a cofactor mechanism for vitronectin function (20). Thrombin also
contains a Glu at position 192. Interestingly, the reactivity of
thrombin E192Q with PAI-1 was also markedly improved independent of
vitronectin, suggesting that vitronectin may accelerate PAI-1
inhibition of both proteinases by a similar mechanism (data not shown).
In the case of thrombin, thrombomodulin occupancy of exosite 1 protects the proteinase from rapid inhibition by PAI-1 in the presence of
vitronectin (20). To determine whether complex formation of APC with
its cofactor protein S on membrane surfaces protects it from inhibition
by the vitronectin-PAI-1 complex, kinetic studies were also carried out
in the presence of human protein S (220 nM) on either PC/PS
or PC/PS/PE vesicles in the range of 0-50 µg/ml. However, protein S
did not influence the reactivity of APC with PAI-1 under either
condition (data not shown). Thrombin is also known to cleave PAI-1 in
the presence vitronectin (26). In the case of the APC-PAI-1 reaction,
however, a 1:1 stoichiometry was observed for both wild type and E192Q
APC in the presence of vitronectin (data not shown).
APC is known to circulate in plasma with a long half-life of 27 min
(32). This is thought to be due to a poor reactivity of APC with plasma
inhibitors. Protein C inhibitor and
1-antitrypsin were believed to be the best inhibitors of APC in circulation, based on
reported second-order rate constants of ~0.6-7 × 103 M
1
s
1 (33-35) and ~10
M
1 s
1
(36), respectively, and the known plasma concentrations of these
inhibitors. A poor reactivity of APC with plasma inhibitors may allow
basal levels of APC (~2 ng/ml) to circulate as an endogenous anticoagulant under normal physiological conditions (32). APC has been
demonstrated to function as a potent anticoagulant at such low
concentrations (37). However, APC has been reported to exhibit a poor
anticoagulant activity on activated platelets (38, 39). Upon
activation, platelets release very high concentrations of both PAI-1
and vitronectin (13-15). Furthermore, specific binding sites for
vitronectin on platelets following stimulation have been reported (40).
The possible physiological significance of these observations is that
the vitronectin-PAI-1 complexes on the platelet surface may inactivate
the circulating basal APC to allow factor Va to assemble into the
prothrombinase complex to generate thrombin in the initial phase of the
blood clotting cascade. This can potentially account for the poor
anticoagulant activity of APC on activated platelets. In addition, it
has been noted previously that APC resistance assays using frozen, but not fresh plasma, containing platelets are associated with frequent false-positive APC sensitivity ratios (41, 42). It has been hypothesized that components released from platelets during freezing and thawing may serve as substrates and/or inhibitors of APC
interfering with this assay (41). Results of the current study suggest
that this component is most likely vitronectin-PAI-1 complexes that are
released and remain attached to the platelet surface, leading to
inhibition of the proteinase in APC resistance assays. It appears, therefore, that vitronectin can modulate the activity of APC in both
the clotting and fibrinolytic cascades.
 |
ACKNOWLEDGEMENT |
I thank Dr. Steven Olson for his
critical reading and valuable suggestions and Dr. Akash Mathur for his
assistance with the chromatography of APC derivatives by fast protein
liquid chromatography.
 |
FOOTNOTES |
*
This work was supported by Grant R01 HL 62565 (to A. R. R.) awarded by the NHLBI of the National Institutes of Health.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: Dept. of Biochemistry
and Molecular Biology, St. Louis University School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104. Tel.: 314-577-8130; Fax:
314-577-8156; E-mail: rezaiear@slu.edu.
Published, JBC Papers in Press, March 22, 2001, DOI 10.1074/jbc.C100123200
 |
ABBREVIATIONS |
The abbreviations used are:
APC, activated protein C;
APC E192Q, APC in which Glu192 (in the
chymotrypsin numbering system of Bode et al. (1)) has been
replaced with Gln by the recombinant DNA methods;
APC KKK/PQE, APC in
which Lys37-Lys38-Lys39 in the same
numbering system has been replaced with Pro-Gln-Glu;
Gla,
-carboxyglutamic acid;
PAI-1, plasminogen activator inhibitor-1;
VN, vitronectin;
PC, phosphatidylcholine;
PS, phosphatidylserine;
PE, phosphatidylethanolamine;
SpPCa, Spectrozyme PCa.
 |
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