(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 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
10
M
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
10
M
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.
Protein C inhibitor is a heparin-binding plasma serine protease
inhibitor
(serpin)()(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.
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 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.
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 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:
where [S] is the concentration of the chromogenic
substrate, SPTH or S2266, and K 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.
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
() 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.
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 (), 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).
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 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.
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 P2 residue) to Pro resulted in a PCI mutant that inhibited
thrombin
37-fold better (k
= 6.3
10
M
s
) than wild type PCI. TM 4-6 enhanced
inhibition by this mutant only 9-fold (k
=
5.8
10
M
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.
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 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) .
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.