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(Received for publication, July 14, 1995; and in revised form, August 29, 1995) From the
Protein C inhibitor (PCI), a plasma serine protease inhibitor,
inhibits several proteases including the anticoagulant enzyme,
activated protein C (APC), and the coagulation enzymes, thrombin and
factor Xa. Previous studies have shown that thrombin and APC are
inhibited at similar rates by PCI and that heparin accelerates PCI
inhibition of both enzymes more than 20-fold. We now demonstrate that
the thrombin-binding proteoglycan, rabbit thrombomodulin, accelerates
inhibition of thrombin by PCI
Protein C inhibitor is a heparin-binding plasma serine protease
inhibitor
(serpin)( Originally identified as an inhibitor
of activated protein C, it is now apparent that this inhibitor has a
broad specificity, inhibiting several of the blood coagulation enzymes
including thrombin and factor Xa. In fact, PCI inhibits thrombin better
than APC both in the presence and absence of
heparin(4, 10) . This paradoxical effect of inhibition
of both coagulation and anticoagulation proteins raises questions about
the physiological role of PCI. Thrombin serves a dual role in
coagulation. It clots fibrinogen, activates platelets, and feeds back
to promote coagulation by activating cofactors(11) .
Alternatively, thrombin can bind to thrombomodulin (TM), and this
complex accelerates protein C activation giving rise to the
anticoagulant serine protease, APC(12) . APC then prevents
further thrombin formation by inactivating factors Va and VIIIa, two
cofactors required for thrombin generation(12, 13) . In vivo, thrombin inhibition is usually believed to be due
primarily to antithrombin, another heparin-binding serpin. This
inhibition can be catalyzed by vascular proteoglycans or by thrombin
binding to TM(14, 15) . TM is a proteoglycan
containing a covalently associated chondroitin sulfate moiety. Only
forms of TM that contain the chondroitin sulfate enhance inhibition of
thrombin by antithrombin (16, 17, 18) . The
chondroitin sulfate moiety is not, however, required for protein C
activation, a process that appears to involve conformational changes in
the extended binding pocket of thrombin(19, 20) . The
influence of TM on thrombin inhibition by PCI has not been examined
fully. In the present study, we demonstrate that TM potently
accelerates inhibition of thrombin by PCI, a process that depends
primarily on protein-protein interactions between thrombin and TM.
These studies provide new insights into the possible physiological
functions of PCI.
where a is residual proteinase activity, t is
time, and [I] is the PCI
concentration(3, 4) . All experiments were performed
in triplicate wells, and all experiments contained control wells in
which the assay buffer had replaced PCI. In all experiments it was
ensured that less than 10% chromogenic substrate was utilized and all
inhibition assays were performed by time course analysis to obtain at
least 50% enzyme inhibition for calculation of inhibition rates. As
mentioned above, the PCI concentration in this discontinuous assay
method was 10 nM. In the presence of TM and higher
concentrations of PCI (i.e. 30 nM), the rate of
inactivation was fast, and, in 15 s, more than 90% of thrombin activity
was inhibited. To demonstrate the concentration dependence of PCI
inhibition in the presence of TM, the alternative method of continuous
inhibition assay was employed. In this method, the inhibition reaction
was carried out in the presence of a competing chromogenic substrate as
described(20, 25) . In this case, 50 µl of 1
nM human thrombin (0.5 nM final) in complex with
saturating concentrations of TM (50 nM final) or TM 4-6
(100 nM final) were added to wells of a 96-well plate that
contained 50 µl of PCI at final concentrations ranging from 3.1 to
100 nM and SPTH (0.2 mM final), and the absorbance at
405 nm was measured at 20-s time intervals immediately after thrombin
addition. S2266 (0.5 mM) with a lower affinity for thrombin
was employed to monitor thrombin inhibition by PCI in the absence of
TM. The PCI concentration had to be increased to 250 nM to
obtain reasonable rates of inhibition under these conditions. The K The apparent
pseudo-first order rate constant of inhibition was estimated by fitting
the absorbance at 405 nm versus time into the following
equation:
where t is the time of the inhibition, Ab is
the absorbance at 405 nm at time t, Ab
where [S] is the concentration of the chromogenic
substrate, SPTH or S2266, and K Thrombin inhibition by PCI was examined in the presence and
absence of saturating levels of rabbit TM containing chondroitin
sulfate and recombinant human TM fragment containing only the epidermal
growth factor-like repeats 4-6 and lacking the chondroitin
sulfate by a discontinuous assay method as shown in Fig. 1. The
rate of inactivation of the bound thrombin was increased dramatically
by both forms of TM. The acceleration of inhibition by rabbit TM was
approximately 2-3 times more effective than TM 4-6 ( Fig. 1and Table 1). These results indicate that, unlike
TM-dependent acceleration of the inhibition of thrombin with
antithrombin(16, 18) , the TM-dependent acceleration
of thrombin inhibition by PCI involves primarily protein-protein
interactions rather than glycosaminoglycan-protein interactions.
Figure 1:
Time course of thrombin inhibition by
PCI in the absence or presence of TM and TM 4-6. Thrombin (0.5
nM) was incubated with PCI (10 nM) in the absence
(
In
the presence of TM, the rate of inhibition was too fast to allow
accurate assessment of the PCI concentration dependence of thrombin
inhibition. As an alternative, the continuous assay method in the
presence of SPTH as the competing chromogenic substrate was used to
demonstrate the rate enhancement by TM and TM 4-6 with various
concentrations of PCI. As shown in Fig. 2, in the presence of
TM, inhibition was rapid, inhibitor concentration-dependent, and
complete. Fig. 3shows that the k` values are linear
with PCI concentration. Note that with 100 nM PCI, virtually
all thrombin activity was inhibited within 500 s. Similar results were
obtained with TM 4-6 (data not shown). In contrast, in the
absence of TM, the same concentration of PCI (100 nM) with
SPTH as the competing chromogenic substrate failed to inhibit thrombin
effectively (data not shown). In the absence of TM, S2266 was used as
the competing chromogenic substrate to estimate the rate constants by
the continuous assay method (Table 1).
Figure 2:
Typical progress curves for inhibition of
thrombin by PCI in the presence of TM. Thrombin (0.5 nM) in
complex with TM (50 nM) was added to reactions containing 0.2
mM SPTH and varying concentrations of PCI in TBS buffer
containing 2 mg/ml bovine serum albumin. The concentrations of PCI in
reactions were: 0 (
Figure 3:
Linear dependence of k` values versus the concentration of PCI. The pseudo-first order
association rate constants of Fig. 2are plotted versus the concentration of PCI. The slope of the straight line
represents the second order association rate constant of
inhibition.
To allow comparisons of
reaction rates, the k Comparison of the rate
constants in Table 1reveals that TM 4-6 accelerates
thrombin inhibition by PCI approximately 59-fold. Rabbit TM containing
the chondroitin sulfate is only 2-3 times more effective. Taken
together, these results indicate that the protein-protein interactions
are the most important contribution to the acceleration of thrombin
inhibition by PCI. The potential mechanisms by which the
protein-protein interactions between TM and thrombin might augment PCI
inhibition were examined by analyzing thrombin inhibition by a PCI
mutant in the presence and absence of TM 4-6. The sequence of PCI
from the P3 to the P3` residues in the reactive center is
Thr-Phe-Arg-Ser-Ala-Arg. Previous kinetic studies have illustrated that
Phe in the P2 position fits poorly into the S2 specificity pocket of
thrombin (26, 27) in contrast to Pro which is ideally
suited to fit into this pocket(28) . Mutation of Phe The observation that PCI reacts rapidly with thrombin in
complex with TM suggests that physiologically PCI functions primarily
to augment coagulation reactions and does so by inhibiting both protein
C activation and APC itself. The rate of thrombin-TM complex inhibition
by PCI is considerably more rapid than the rate of inhibition of free
thrombin by antithrombin even allowing for the higher concentrations of
antithrombin in the circulation (88 nMversus 2.3
µM). At room temperature, the calculated time to inhibit
50% of the thrombin bound to TM with plasma levels of PCI would be
3-4 s and with antithrombin would be 30 s. On thrombin-TM
complexes that contain chondroitin sulfate, antithrombin may contribute
more significantly to inhibition (calculated half-life, based on
literature values, would be 2-6 s) (16, 17) . In
cell culture and probably in vivo, the addition of chondroitin
sulfate to human thrombomodulin appears to be incomplete (29) .
With chondroitin-free TM, PCI is likely to be a major inhibitor of the
complex. In addition to cell-associated chondroitin-free forms of TM,
soluble TM generated by elastase proteolysis lacks the chondroitin
sulfate, and soluble forms of TM are found at moderately high levels in
patients with vascular diseases or inflammatory
conditions(30) . PCI may play a major role in inhibiting
thrombin bound to these different forms of TM. PCI is synthesized in
the liver, testis, prostate, and kidney(31, 32) . It
is of interest that TM has been observed on several cell types not in
contact with blood(33, 34) . Given the tissue
distribution of PCI, it is possible that PCI plays a role in inhibition
of thrombin-TM complexes in the extravascular space. Examination of
alternative sites of synthesis of this inhibitor may provide additional
insights into the physiological function of the inhibitor. At a
biochemical level, these studies draw clear distinctions between the
requirements for inhibition of thrombin bound to TM by the two serpins,
antithrombin and PCI. With PCI, occupancy of anion-binding exosite 1 by
TM 4-6 is sufficient for acceleration of inhibition by PCI with
the chondroitin contributing only a 2-3-fold additional
acceleration. In contrast, TM 4-6 or full-length TM devoid of the
chondroitin sulfate fail to accelerate thrombin inhibition by
antithrombin(16, 18) . This suggests that TM-dependent
acceleration of thrombin inhibition by PCI is largely dependent on
conformational changes in thrombin resulting from the protein-protein
interactions between thrombin and TM. The mechanisms involved in
allowing PCI to react rapidly with the thrombin-TM complex are likely
to be due to the conformational changes that occur during complex
formation in the extended binding pocket of thrombin. Comparison of TM
4-6 acceleration of thrombin inhibition by the wild type PCI (Phe
at the P2 position) with the mutant PCI with Pro at the P2 position
revealed that the TM 4-6 acceleration fell from The observation that protein-protein
interactions between thrombin and TM accelerate reaction with PCI
represents another example of significant enhancement of thrombin
reactivity with naturally occurring protein substrates and inhibitors.
The other examples are protein C activation and single-chain
urokinase-type plasminogen activator
inactivation(36, 37) . The exact mechanism of
single-chain urokinase-type plasminogen activator inactivation has not
been studied in depth, but protein C activation, like the inhibition of
thrombin by PCI, appears to involve a conformational change in the
active center of thrombin that overcomes interactions with residues
that do not interact well within the extended binding pocket of
thrombin in the absence of TM(38) . The data presented here in
combination with previous studies suggest that the S3, S2, and S3`
pockets of thrombin are all altered by interaction with TM. The
observation that reactivity of PCI with thrombin is selectively altered
by interaction with TM opens new approaches for investigating the
molecular mechanisms by which TM switches the specificity of thrombin
from a clot-promoting to a clot-inhibiting enzyme.
Volume 270,
Number 43,
Issue of October 27, 1995 pp. 25336-25339
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
140-fold (k
= 2.4 
10
in the presence of TM
compared to 1.7 10
M
s
in the absence of TM). Most of this effect is
mediated by protein-protein interactions since the active fragment of
TM composed of epidermal growth factor-like domains 4-6 (TM
4-6) accelerates inhibition by PCI
59-fold (k = 1.0 10M
s
). The mechanism by which TM alters reactivity with
PCI appears to reside in part in an alteration of the S2 specificity
pocket. Replacing Phe
with Pro at the P2 position in the
reactive loop of PCI yields a mutant that inhibits thrombin better in
the absence of TM (k
= 6.3
10
M s
), but
TM 4-6 enhances inhibition by this mutant
9-fold (k = 5.8 10M s
) indicating that TM
alleviates the inhibitory effect of the less favored Phe residue. These
results indicate that PCI is a potent inhibitor of the protein C
anticoagulant pathway at the levels of both zymogen activation and
enzyme inhibition.
)(1, 2, 3, 4) . In vivo and in plasma, a significant percentage of activated
protein C (APC) is inhibited by PCI with various studies reporting
10-50% of the APC in complex with
PCI(5, 6, 7, 8, 9) . The
other major inhibitor of APC is 
-antitrypsin. Heparin
accelerates inhibition of APC by PCI, but not by
-antitrypsin.
Proteins and Reagents
Recombinant human thrombin
was prepared as described previously(21) . The concentration of
purified recombinant thrombin was determined by active site titration
with p-nitrophenyl p`-guanidinobenzoate and agreed
with the calculated concentration based on absorbance at 280
nm(21) . Recombinant human PCI and Phe
Pro
mutant of PCI were expressed and purified from a baculovirus expression
system as described previously(3, 22) . The
concentration of recombinant PCI was calculated based on thrombin
active site titration and immunoassay as described(3) .
Recombinant human TM 4-6 fragment (23) and rabbit lung TM (24) were isolated by the cited methods. All proteins were
homogeneous by acrylamide gel electrophoresis in sodium dodecyl
sulfate. Polybrene was purchased from Sigma. Spectrozyme TH was
purchased from American Diagnostica, Greenwich, CT, and S2266 was
purchased from Kabi Pharmacia/Chromogenix, Franklin, OH.
Thrombin Inhibition with PCI
The rate of
inhibition of human thrombin was measured under pseudo-first order rate
conditions by both discontinuous and continuous assay methods. In the
discontinuous assay method, both the inhibition reaction and the
determination of residual thrombin activity were done in 96-well plates
at room temperature in 20 mM Tris-HCl (pH 7.5), 0.1 M NaCl (TBS) containing 2 mg/ml bovine serum albumin, as
described(3, 4) . Thrombin (0.5 nM) was
incubated with at least a 10-fold excess of PCI (10 nM) at
room temperature in the presence and absence of saturating
concentrations of TM (50 nM) or TM 4-6 (100
nM). For assays in the absence of TM, 0.1 mg/ml Polybrene was
also included in the buffer. Polybrene was added to ensure that trace
heparin contamination of PCI would not influence the inhibition rate.
However, the rates of PCI inhibition of thrombin in the presence or
absence of Polybrene were similar indicating that PCI preparations were
essentially free of heparin. After a period of time (30 s to 30 min
depending on the reaction rates), Spectrozyme TH (SPTH) in TBS buffer
containing 1 mg/ml Polybrene (to block the chondroitin sulfate moiety
of TM) was added to give a final concentration of 0.2 mM.
Following color development, the absorbance at 405 nm was measured with
a V
Kinetic Microplate Reader (Molecular
Devices, Menlo Park, CA). Rate constants were calculated using the
equation:
values that were determined and used in were 5.6 µM for SPTH with thrombin, 6.0
µM for the rabbit TM-thrombin complex, and 5.8 µM for the TM 4-6-thrombin complex. The K of thrombin for S2266 was 236 µM.
is
the absorbance at 405 nm at time 0, A is the
thrombin activity at time 0, and k is the
apparent pseudo-first order rate constant of inhibition. To correct for
the presence of chromogenic substrate, the pseudo-first order rate
constant of inhibition k` was given by:
is the
Michaelis-Menten constant of thrombin for SPTH or S2266. Both methods
of inhibition rate constant measurements gave similar results. The
ENZFITTER computer program (R. J. Leatherbarrow, Elsevier, Biosoft) was
used for data analysis.
) or presence of 50 nM TM (
) or 100 nM TM 4-6 (
) in TBS buffer containing 2 mg/ml bovine
serum albumin. At indicated time points, chromogenic substrate SPTH in
TBS containing 1 mg/ml Polybrene was added to a final concentration of
0.2 mM, and, after the color development, the remaining
amidolytic activity of uninhibited thrombin was determined as described
under ``Experimental Procedures.'' Solid lines were
obtained by nonlinear regression analysis of data obtained from the
average of three experiments using a first order rate
equation.
), 3.1 nM (
), 6.3 nM (
), 12.5 nM (), 25 nM (
), 50
nM (), and 100 nM (
). The pseudo-first
order association rate constant (k`) for inhibition was
determined by fitting the data to and (only
every 100-s values are plotted).
values for thrombin
inhibition by PCI in the absence and presence of TM or TM 4-6
were determined. These values were determined by both the discontinuous
and continuous assay methods from inhibition progress curves in the
presence of competing substrate as described under ``Experimental
Procedures.'' For the discontinuous assay, 17 independent
inhibition reactions were performed in the presence of rabbit TM and 12
reactions in the presence of TM 4-6. The k values determined by the discontinuous assays are given in the
top lines of each section of Table 1, and the values in the
presence of competing substrate are given in the middle line of each
section. In most cases, the values obtained by these two assay methods
agreed within a factor of 2. Therefore, to simplify presentation of the
influence of TM on the inhibition rate, the k values were averaged, and the average value was used to calculate
the fold enhancement by TM. The average value is given in the third
line of each section of Table 1. (the P2 residue) to Pro resulted in a PCI mutant that inhibited
thrombin
37-fold better (k = 6.3
10M
s
) than wild type PCI. TM 4-6 enhanced
inhibition by this mutant only 9-fold (k
=
5.8 10M
s
) versus the 59-fold enhancement for wild
type PCI suggesting that interaction of TM 4-6 with thrombin
allowed thrombin to accommodate the larger and more hydrophobic Phe
residue.
59-fold to
9-fold. We interpret these data to suggest that TM 4-6 allows
thrombin to accept bulkier and/or more hydrophobic residues at the P2
position. Based on modeling and kinetic data, Phe would not fit well
into the S2 pocket of thrombin in the absence of TM (26, 27) . TM could allow thrombin to interact with
Phe more favorably if, for instance, TM elicited conformational changes
in the large insertion loop (the 60 loop) that forms the upper portion
of the S2 specificity pocket(28) . Previous studies examining
inhibition of thrombin by bovine pancreatic trypsin inhibitor have
suggested that this loop can exist in multiple
conformations(35) .
) to Gly
prepared by the recombinant DNA
methods; PCI F353P, protein C inhibitor mutant in which Phe at position
353 is converted to Pro; SPTH, Spectrozyme TH.
We thank Gary Ferrell and Steven Carpenter for help
with cell culture, Barbara Carpenter and Bronson Sievers for isolation
of recombinant proteins (thrombin, protein C, and TM 4-6) used in
this study, and Jeff Box, Karen Deatherage, and Julie Wiseman for
assistance with preparation of the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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 S. T. Cooper, L. L. Neese, M. N. DiCuccio, D. K. Liles, M. Hoffman, and F. C. Church Vascular Localization of the Heparin-binding Serpins Antithrombin, Heparin Cofactor II, and Protein C Inhibitor Clinical and Applied Thrombosis/Hemostasis, July 1, 1996; 2(3): 185 - 191. [Abstract] [PDF]
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