Results TM is an effective inhibitor of the thrombin interaction with PAI-1 in the absence and presence of VN Earlier studies both from our group and others have shown that the rate ofth
Trang 1A steady-state competition model describes the modulating effects
of thrombomodulin on thrombin inhibition by plasminogen activator inhibitor-1 in the absence and presence of vitronectin
Rob J Dekker, Hans Pannekoek and Anton J G Horrevoets
Department of Biochemistry, Academic Medical Center, University of Amsterdam, the Netherlands
Thrombomodulin (TM) slows down the interaction rate
between thrombin and plasminogen activator inhibitor 1
(PAI-1) We now show that the 12-fold reduced inhibition
rate in the presence ofTM does not result from an altered
distribution between PAI-1 cleavage and irreversible
com-plex formation Surface plasmon resonance (SPR) revealed
an over 200-fold reduced affinity of TM for
thrombin-VR1tPA as compared to thrombin, demonstrating the
importance ofthe VR1 loop in the interaction ofthrombin
with both TM and PAI-1 Furthermore, in contrast to ATIII,
PAI-1 was not able to bind the thrombin/TM complex
demonstrating complete competitive binding between PAI-1
and TM Kinetic modeling on the inhibitory effect ofTM
confirms a mechanism that involves complete steric blocking
ofthe thrombin/PAI-1 interaction Also, it accurately
decribes the biphasic inhibition profile resulting from the
substantial reduction ofthe extremely fast rate ofreversible
Michaelis complex formation, which is essential for efficient inhibition ofthrombin by PAI-1 Vitronectin (VN) is shown
to partially relieve TM inhibitory action only by vastly increasing the initial rate ofinteraction between free thrombin and PAI-1 In addition, SPR established that solution-phase PAI-1/VN complexes and non-native VN (extracellular matrix form) bind TM directly via the chon-droitin sulphate moiety ofTM Collectively, these results show that VR1 is a subsite ofexosite 1 on thrombin’s surface, which regulates exclusive binding ofeither PAI-1 or TM This competition will be physiologically significant in con-trolling the mitogenic activity ofthrombin during vascular disease
Keywords: serine protease; serpin; suicide substrate mecha-nism; competitive inhibitor; kinetic modeling
Classically, the serine protease thrombin is known for its
dual role in hemostasis, exhibiting coagulant as well as
anticoagulant properties Reversible binding ofthrombin to
the endothelial cell surface cofactor thrombomodulin (TM)
endows thrombin with potent anticoagulant properties [1,2]
The thrombin/TM complex is no longer able to bind and
cleave fibrinogen and various other substrates and inhibitors
but becomes a potent activator ofprotein C The catalytic
activity ofthrombin can be inhibited by a number ofserine
protease inhibitors (serpins), including antithrombin III (ATIII), heparin cofactor II, and PAI-1 Inactivation of thrombin by PAI-1, however, is a very inefficient process with a second-order rate constant (ki) of 103M )1Æs)1, which can be increased up to 250-fold by the cofactors vitronectin (VN) and heparin [3,4]
The VR1- or 37-loop ofthrombin has been implicated in
a number ofintriguing interactions First, substitution ofthe VR1-loop ofthrombin by that oft-PA, yielding thrombin-VR1tPA, increases the bimolecular rate constant ofinhibi-tion by PAI-1 at least 1000-fold to 106M )1Æs)1[5] Recently,
we reported that this alteration results from an increased rate ofa unimolecular catalytic step [6] It has been unambiguously evidenced that VR1 is essential for the interaction ofboth t-PA [7] and thrombin [5,6] with PAI-1 Second, binding kinetics and structural studies have esta-blished that the epidermal growth factor domains 4–6 (EGF4–6) ofTM bind thrombin electrostatically at exosite
1 [8], but are also involved in hydrophobic contacts with VR1-loop residues [9,10] As a result, a marked influence of
TM binding on the interaction ofPAI-1 and thrombin can
be envisioned The hirudin-derived decapeptide hirugen, however, also interacts with thrombin by utilizing exosite I [1,2,11], although it did not prevent binding ofPAI-1 but altered a catalytic step ofthe reaction between thrombin and PAI-1 [6]
TM acts as a positive effector on other thrombin interactions, e.g with ATIII, protein C inhibitor, thrombin-activatable fibrinolysis inhibitor (TAFI) and protein C
Correspondence to A J G Horrevoets, Academic Medical Center,
Department ofBiochemistry, Room K1-161, Meibergdreef15,
1105 AZ Amsterdam, the Netherlands.
Fax: + 31 20 6915519, Tel.: + 31 20 5665153,
E-mail: a.j.horrevoets@amc.uva.nl
Abbreviations: VR1, variable region-1 (also 37-loop); t-PA, tissue-type
plasminogen activator; u-PA, urokinase-type plasminogen activator;
VR1 tPA , VR1 loop oft-PA; serpin, serine protease inhibitor; PAI-1,
plasminogen activator inhibitor type 1; RCL, reactive center loop;
PPACK, Phe-Pro-Arg-chloromethylketone; VN, vitronectin;
TM, thrombomodulin; rl-TM, rabbit-lung thrombomodulin;
solulin, soluble human recombinant thrombomodulin; SPR, surface
plasmon resonance; ATIII, antithrombin III; k i , second-order rate
constant ofinhibition; r, partition ratio; k on , association rate constant;
k off , dissociation rate constant; K d , thermodynamic equilibrium
dissociation constant.
(Received 15 October 2002, revised 24 December 2002,
accepted 3 March 2003)
Trang 2[11–13] However, previous work from our laboratory has
demonstrated that thrombin inhibition by PAI-1/VN
com-plexes is impaired in the presence ofTM [4] These findings
have later been studied in more detail, though the
mecha-nism ofTM interference in this interaction has not yet been
elucidated, nor is any evidence available on the possible
physiologic role [14,15] Interestingly, using
immunohisto-chemistry, TM antigen was demonstrated on vascular
smooth muscle cells (SMC), monocytes, and macrophages
in atherosclerotic lesions ofthe human and rabbit aorta [14]
Also, due to the colocalization ofthrombin, PAI-1 and VN
in the vessel wall, increasing attention is being paid to the
mitogenic effect of thrombin, and its control by PAI-1/VN
in the pathogenesis ofvascular disease [16,17] Together, the
presence ofPAI-1, VN and TM in the vessel wall, including
the unique property ofthrombin to inactivate PAI-1,
suggests a novel role ofTM in controlling the behavior of
vascular cells
Here, we report that binding ofTM and PAI-1 to
thrombin is mutually exclusive, both in the presence and
absence ofVN Furthermore, the data presented here is in
agreement with a mechanism in which the rate ofthrombin
inhibition by PAI-1 is dependent on the rate ofdissociation
ofthrombin from TM, explaining the observed biphasic
inhibition profiles These findings are in marked contrast to
the binding ofall other thrombin-binding components, and
comprise yet another level ofspecificity switching of
thrombin that is controlled by TM
Materials and methods
Materials
The chromogenic substrate H-D-Phe-Pip-Arg-p-nitroaniline
(where Pip is l-pipecolic acid; S2238) was obtained from
Chromogenix (Mo¨lndal, Sweden) All additional chemicals
were obtained from Sigma (St Louis, MO, USA)
Poly-sorbate-20 (Surfactant P20), and all additional BIAcore
materials were obtained from BIAcore AB (Uppsala,
Sweden)
Proteins
Ovalbumin (grade V) was obtained from Sigma
Rabbit-lung TM (rl-TM) was purchased from American
Diagnos-tica Inc (Lot #970117A, Veenendaal, the Netherlands)
Recombinant soluble human TM (solulin) was a gift of
J Morser (Berlex Biosciences, Richmond, CA, USA)
Active PAI-1 was generously provided by T M Reilly
(Dupont de Nemours, Wilmington, DE, USA) Human
a-thrombin purified from plasma was a gift of G Tans
(University ofMaastricht, the Netherlands) Construction,
expression, and activation ofrecombinant prothrombin
variants were described [6] VN was a kind gift of K T
Preissner (Justus Liebig University, Giessen, Germany)
Antithrombin III was obtained from the Sanquin
Founda-tion (CLB, Amsterdam, the Netherlands)
Determination of the PAI-1 inhibition rates
To prevent protein adsorption, all experiments were
performed in Eppendorftubes or in wells ofa microtiter
plate (Nunc Maxisorp; Gibco-BRL, Gaithersburg, MD, USA) that had been pretreated for 1 h at 37C with 1% (w/v) polyethylene glycol 20 000 and subsequently washed with distilled water Prior to all experiments, PAI-1 dilutions were titrated on a calibrated t-PA standard The decrease of thrombin amidolytic activity during the inhibition by PAI-1 was determined after incubating 15 nM thrombin with 1.5 lM PAI-1 at 37C in HBSO buffer (20 mM Hepes,
pH 7.4, 150 mM NaCl, and 0.5 mgÆmL)1ovalbumin) At specific time intervals, aliquots of5 lL were withdrawn and the reaction was quenched by diluting 45-fold in HBSO buffer containing 0.65 mMofS2238 chromogenic substrate Residual thrombin amidolytic activity in these aliquots was measured at 37C by continuously recording the absorb-ance at 405 nm in a Titertek Twinreader (Flow Laborat-ories, Irvine, UK) Plots ofresidual activity (relative to thrombin activity in the absence ofPAI-1) vs time were constructed and analyzed as described [6] The effect of increasing concentrations ofsolulin on the inhibition of thrombin and thrombin-VR1tPAby PAI-1 was determined
To that end, a solution of15 nM human a-thrombin was prewarmed in NaCl/Pi/Tween buffer [NaCl/Piwith 0.01% (v/v) Tween 80] for 5 min at 37C, in the presence of increasing concentrations ofsolulin (0–800 nMin NaCl/Pi/ Tween buffer) or rl-TM (0–100 nM in 20 mMTris buffer,
pH 7.4, with 100 mM NaCl) Subsequently, the inhibition reaction was started by the addition ofPAI-1 to a final concentration of1.5 lM At various time intervals, the residual thrombin amidolytic activity was determined as described above Alternatively, the inhibition of2 nM thrombin-VR1tPAby 10 nMPAI-1 was determined in the absence or presence of800 nM solulin Therefore, 13 lL aliquots were quenched by diluting ninefold in HBSO buffer, containing 0.9 mMofS2238 chromogenic substrate Surface plasmon resonance (SPR) binding studies Reversible binding ofvarious components was studied using SPR in a BIAcore 2000 system (BIAcore AB, Uppsala, Sweden) Binding experiments were performed using CM5 Sensor Chips (BIAcore AB) at 25.0C All recorded sensorgrams were corrected for refractive index variations, using an empty flow cell Thrombin-S195A and thrombin-S195A-VR1tPA were immobilized on a sensor chip as described for thrombin [18] Thrombin was immobilized at
40 ngÆlL)1in 10 mMsodium acetate buffer (pH 6.0), rl-TM was immobilized at 15 ngÆlL)1in 10 mMsodium formate buffer (pH 3.6), and vitronectin was immobilized at
120 ngÆlL)1 in 10 mM sodium acetate buffer (pH 4.8), resulting in approximately 4000, 2000, and 17 000 immobi-lized resonance units, respectively In all SPR experiments, HBS buffer [20 mMHepes, pH 7.4, 150 mM NaCl, 2 mM CaCl2, 0.005% (v/v) P20] was used at a 20-lLÆmin)1flow rate Binding ofthrombin and thrombin-VR1tPAto immobi-lized rl-TM was monitored by applying either thrombin (0.5–20 nM), or thrombin-VR1tPA (10–100 nM) in HBS buffer, at 20 lLÆmin)1 Association and dissociation rate constants were determined from the SPR sensor grams by global nonlinear regression using theBIAEVALUATION soft-ware (BIAcore AB) Binding ofvarious analytes to rl-TM/ thrombin complexes was studied as follows: 10 lL of
200 n human recombinant a-thrombin in HBS buffer was
Trang 3injected on a sensorchip with immobilized rl-TM, directly
followed by a 40-lL injection (using the coinject option) of
the respective proteins or HBS buffer alone Hereafter,
dissociation ofthrombin and bound analyte from TM was
continuously monitored in HBS buffer
Direct binding to rl-TM of200 nMPAI-1, 200 nMlatent
PAI-1, 300 nM VN, or 200 nM active or latent PAI-1
preincubated with 75–300 nMVN was studied by injecting
60 lL ofthe respective proteins in HBS buffer Latent PAI-1
was obtained by incubating 200 nMPAI-1 at 37C f or at
least 20 h
Direct binding ofrl-TM and solulin to VN was studied by
injecting 40 lL 200 nM rl-TM, or 1 lM solulin, in the
absence or presence ofheparin (0–1000 UÆmL)1)
Alternat-ively, previous to the TM injections, 40 lL 500 nMPAI-1
solution was injected to form PAI-1/VN complexes on the
chip surface Hereafter, during the slow dissociation of
PAI-1/VN, rl-TM or solulin was injected as described above
Kinetic modeling
The procedure ofnumerical integration ofthe rate
equa-tions derived from the mechanism shown below has been
described elsewhere, including the rate constants ofthe suicide-substrate mechanism (k1 to k3) that were used [6] Briefly, at various combinations of kon and koff for the thrombin/TM interaction, the total thrombin amidolytic activity was calculated at various time intervals, i.e the sum
of actual free thrombin, thrombin/TM complex, and free thrombin resulting from completion of all thrombin/PAI-1 intermediates after quenching of the reaction The throm-bin/TM complex has a similar amidolytic activity towards S2238 as thrombin (data not shown) For all combinations
of konand koff, the calculated total thrombin activity was compared to the experimental activity decrease shown in Fig 1A
Results
TM is an effective inhibitor of the thrombin interaction with PAI-1 in the absence and presence of VN
Earlier studies both from our group and others have shown that the rate ofthrombin inhibition by PAI-1 is significantly reduced in the presence ofTM [4,15] In this study, we performed quantitative measurements of this inhibition
Fig 1 Effect of TM and VN on the rates of thrombin inhibition by PAI-1 Residual thrombin amidolytic activity was measured at various time intervals and used to calculate the halftimes (t 1/2 ) ofPAI-1 inhibition (A) Residual activity was monitored during the inhibition of15 n M human thrombin by 1.5 l M PAI-1, in the absence (d) or presence of 30 n M (s), 50 n M (j), 100 n M (h), 400 n M (m) or 800 n M (n) TM (solulin) (B) Residual thrombin activity was monitored as in (A), but in the presence of0 (d), 10 (s), 20 (j), 50 (h) or 100 (m) nM rl-TM (C) Residual activity decrease was monitored during the inhibition of2 n M thrombin-VR1tPAby 15 n M PAI-1 in the absence (d) or presence of100 n M rl-TM (s) (D) Thrombin activity decrease that was observed during the inhibition of15 n M thrombin by 100 n M PAI-1/VN complexes, in the absence (d)
or presence of100 n M rl-TM (s).
Trang 4Furthermore, we attempted to elucidate the mechanism of
interference by TM on thrombin inhibition by PAI-1 The
rate ofthrombin inhibition by 1.5 lMPAI-1 was measured
in the presence ofincreasing concentrations (0–800 nM) of
solulin Solulin lacks the transmembrane domain and does
not contain chondroitin sulphate, which might have an
additional heparin-like effect on the thrombin/PAI-1
inter-action [19] In this way, only protein–protein interinter-actions
between thrombin and TM are considered At the highest
concentration ofTM (800 nMsolulin), thrombin is inhibited
by PAI-1 with a half-time of the reaction (t1/2) that is
12.5-fold longer than in the absence of TM, i.e 6810 and 545 s,
respectively (Fig 1A) Because ofthe lower affinity of
solulin for thrombin (due to the lack of the chondroitin
sulphates) [20], high concentrations ofthe TM preparation
were necessary to obtain the observed effect Hence,
rabbit-lung TM (rl-TM), which has a higher affinity for thrombin
due to its chondroitin sulphate moiety, was also used to
study the effect on the thrombin/PAI-1 interaction The
inhibitory effect of only 100 nM rl-TM on the rate of
inhibition ofhuman plasma thrombin by PAI-1 was more
substantial than that ofthe highest concentration ofsolulin
(800 nM) (Fig 1B) Next, the effect of TM on the inhibition
ofthe substitution variant thrombin-VR1tPAby PAI-1 was
studied In marked contrast to thrombin, only a 1.7-fold
inhibitory effect of 100 nM rl-TM is observed on the
inhibition of2 nM thrombin-VR1tPA by 15 nM PAI-1,
measured as difference of the half-time (t1/2) of the reaction
(Fig 1C) Opposed to the accelerating effect of TM on
thrombin inhibition by ATIII and protein C inhibitor, these
findings indicate that TM considerably reduces the rate of
thrombin inhibition by PAI-1
The poor rate ofinhibition ofthrombin by PAI-1 alone
(ki 103
M )1Æs)1) can be substantially increased by the
cofactor VN [3] Complexed to VN, PAI-1 inhibits
throm-bin at a rate that is at least two orders ofmagnitude higher
compared to PAI-1 alone (ki 105
M )1Æs)1) Therefore, the inhibitory effect ofTM on the inhibition of15 nMthrombin
by preincubated PAI-1/VN complexes (100 nM PAI and
150 nM VN) was determined (Fig 1D) The presence of
100 nMrl-TM in this reaction decreased the inhibition rate
by 14-fold (t1/2¼ 940 and 67 s, respectively) Still, even in
the presence ofrl-TM, VN accelerates the rate ofthrombin
inhibition by PAI-1 36-fold [Fig 1B (m) vs 1D (s)]
TM binding to thrombin-VR1tPAis substantially
reduced
The minor effect of TM on the rate of thrombin-VR1tPA
inhibition by PAI-1 suggests that the binding between
thrombin and TM, involving the VR1 loop ofthrombin, is
affected in the substitution variant Indeed, the rate of
protein C activation by thrombin-VR1tPA, which is
com-parable to that of thrombin, was not affected by TM,
whereas TM substantially increased the rate ofprotein C
activation by thrombin, as expected (data not shown) The
affinity of TM for thrombin-VR1tPAwas determined using
Surface Plasmon Resonance (SPR, data not shown)
Binding ofthrombin-VR1tPA to immobilized rl-TM was
significantly reduced (Kd¼ 121 ± 23 nM) compared to
thrombin (Kd 0.5 nM) [2,11] Thus, the minor effect of
TM on the thrombin-VR1tPA–PAI-1 interaction appears to
be the result ofthe decreased ability ofTM to bind thrombin-VR1tPA Moreover, these results demonstrate that, as for PAI-1, the VR1 loop of thrombin is an essential interaction site for TM
The stoichiometry of the suicide-substrate mechanism is not influenced by TM The kinetics ofthe inhibition ofthrombin by PAI-1 can be described by the so-called suicide-substrate mechanism as previously elaborated by our group [6,21] In this mecha-nism, each productive encounter ofserpin and protease can either lead to formation of the enzyme/inhibitor complex or can result in cleavage ofthe inhibitor and release ofactive enzyme A decreased overall inhibition rate can thus be the result ofa shift ofthe rate constants ofthe branched part of mechanism, i.e increased cleavage at the expense of complex formation Therefore, the products of the reaction were analyzed by SDS/PAGE (Fig 2) We found no evidence ofincreased cleavage indicating that TM does not alter the product distribution ofthe suicide-substrate reaction between thrombin and PAI-1 These findings leave
a role for TM open in altering the initial binding step between thrombin and PAI-1 or in changing the ability of thrombin to catalyze subsequent steps that are common
to both branches in the mechanism, i.e steric hindrance
vs allosteric modulation, respectively
PAI-1 and TM compete for an overlapping binding site on thrombin
At this point, the mechanism of the inhibitory effect of TM
on the interaction between PAI-1 and thrombin remains to
be elucidated To that end, binding ofPAI-1 to immobilized thrombin/TM complexes was studied in real-time using SPR The high rate constant ofinitial thrombin/PAI-1 complex formation (k1 106m)1Æs)1) [6] would result in a significant increase in surface-bound mass if formation of
Fig 2 TM does not alter the distribution of the cleavage and substrate pathway Analysis by SDS/PAGE ofthe products ofthe reaction of
600 n M thrombin with 3.5 l M PAI-1 in the presence of0 (lanes 2–3),
430 (lanes 1, 4–5), 630 (lanes 6–7), and 885 (lanes 8–9) nM TM (sol-ulin) After 0 min (lane 1), 3 min (lanes 2, 4, 6 and 8) or 16 h (lanes 3, 5,
7 and 9) the samples were immediately quenched by adding sample buffer, subjected to 10% (w/v) SDS/PAGE and stained with Coo-massie Brilliant Blue Indicated are free thrombin (T), intact PAI-1 (P), cleaved PAI-1 (P*), SDS-stable thrombin/PAI-1 complex (T-P), and thrombomodulin (TM), as described in the legend ofFig 4 Note that after 16 h incubation, the thrombin–PAI-1 complexes formed in the presence ofTM (lanes 5, 7 and 9) have mostly been degraded to lower molecular mass species by the remaining free, active thrombin as noted before [21].
Trang 5ternary thrombin/PAI-1/TM complexes occurs First,
thrombin/TM complexes were formed by applying a
solution of200 nMthrombin on rl-TM that was
immobi-lized on a sensor chip A high affinity interaction between
thrombin and TM was observed, consistent with a
disso-ciation constant ofthe thrombin/TM complex in the
subnanomolar range [2,11] Binding was mass
transport-limited, precluding exact determination ofthe rate constants
for this interaction under these conditions Alternatively, an
estimate of koffcan be given using the halftime
ofthrom-bin/TM dissociation, i.e t1/2¼ 470 s and koff 10)3Æs)1
Second, immediately following the thrombin injection,
either a solution of800 nM PAI-1, 800 nM latent PAI-1
or buffer was applied to study the formation of ternary
TM/thrombin/PAI-1 complexes on the chip surface
How-ever, thrombin slowly and continuously dissociated from
the immobilized TM at the same rate as in the absence of
PAI-1 and no increase in surface-bound mass was observed
as a result ofPAI-1 binding to TM/thrombin complexes
(Fig 3A) Also, no significant difference in binding between
the active and latent form of PAI-1 was observed, the latter rendered unable to bind thrombin The absence ofternary complex formation implies that TM has a steric effect on the interaction between thrombin and PAI-1 An allosteric effect of TM is not consistent with these results as the formation of ternary complexes, that would be more slowly converted to stable protease/serpin complexes due to TM-induced allosteric changes in thrombin, is not observed, even at the high concentrations ofPAI-1 used
As a positive control, identical experiments were per-formed with ATIII that inhibits thrombin at a rate comparable to PAI-1, and in contrast to PAI-1 is known
to inhibit the thrombin/TM complex even slightly more efficient than thrombin alone [11] Injection of ATIII after formation of thrombin/TM complexes on the chip surface, resulted in a considerably increased dissociation ofthrom-bin from TM, depending on the ATIII concentration that was used (Fig 3A) These findings are in agreement with fast binding of ATIII to thrombin/TM followed by rapid dissociation ofthe thrombin/ATIII complex from TM,
Fig 3 TM and PAI-1 competitively bind thrombin Binding ofvarious proteins to immobilized rl-TM was studied using SPR (A–C) Plots show the increase in surface-associated mass (D Resonance Units) measured in real-time, resulting from binding to rl-TM that was immobilized on the sensor chip surface (A) At 0 s 10 lL 200 n M recombinant thrombin was injected directly followed (at 30 s) by 40 lL 800 n M active PAI-1 (––), 800 n M
latent PAI-1 (- - -), buffer (ÆÆÆ), 600 n M (-Æ-) or 6 l M (-ÆÆ) ATIII Hereafter, dissociation was continuously monitored by injecting buffer alone (beyond
150 s) (B) Again, 10 lL 200 n M rII A was injected directly followed by 40 lL 800 n M active PAI-1 (ÆÆÆ), 300 n M VN (—), 200 n M latent PAI-1 preincubated with 300 n M VN (- - -), or 200 n M active PAI-1 preincubated with 75–300 n M VN (–– labeled 75–300) (C) 60 lL ofthe following solutions was directly applied (at 0 s) to the immobilized rl-TM in the absence ofthrombin, and dissociation was monitored under continuous buffer flow (180 s and beyond): 800 n M active PAI-1 (ÆÆÆ), 300 n M VN (- - -), 200 n M latent PAI-1 preincubated with 300 n M VN (––), or 200 n M
active PAI-1 preincubated with 300 n M VN (-Æ-) (D) Alternatively, direct binding ofrl-TM to VN was studied by immobilizing VN on a sensor chip Next, at 0 s 40 lL 200 n M rl-TM was directly injected in the absence (––) or presence of200 (- - -), 500 (-Æ-) or 1000 (ÆÆÆ) UÆmL)1heparin Subsequently, dissociation was monitored under continuous buffer flow (beyond 120 s).
Trang 6resulting in a net decrease in TM-associated mass on the
chip surface Moreover, these data are consistent with
the previously described strong reduction ofthe affinity of
the thrombin/ATIII complex for TM [11,22]
The PAI-1/VN complex directly binds to TM
Recently, we have shown that the PAI-1/vitronectin
com-plex can be treated as an entity with different kinetic
properties than PAI-1 alone [21] The finding that the PAI-1/
VN complex still inhibits the thrombin/TM complex at a
36-fold higher rate than thrombin alone, suggests that a
possibly different binding mode of PAI-1/VN to thrombin
might allow the formation of quaternary TM/thrombin/
PAI-1/VN complexes Therefore, binding of PAI-1/VN to
TM/thrombin complexes was tested using SPR Increasing
concentrations ofVN (0–300 nM) were preincubated with
200 nMPAI-1 and binding to preformed thrombin/rl-TM
complexes was monitored (Fig 3B) In contrast to PAI-1
and ATIII, binding ofPAI-1/VN to the chip surface was
observed without an apparent increased dissociation of
TM-bound thrombin Interestingly, neither active PAI-1 or
VN alone, nor latent PAI-1 preincubated with VN, had a
significant effect on the thrombin–TM dissociation rate
However, direct binding ofactive PAI-1/VN complexes to
rl-TM was observed making a possible interaction between
PAI-1/VN and TM-bound thrombin unlikely (Fig 3C)
Again, the observed binding was specific for active PAI-1
when preincubated with VN, as no binding was observed
for latent PAI-1 in the presence of VN Additional binding
studies demonstrate that rl-TM (glycosylated), but not
solulin (not glycosylated) binds directly to immobilized VN,
even in the absence ofPAI-1 (data not shown) Therefore,
binding ofVN to rl-TM occurs either directly to
immobi-lized VN or to VN in solution exclusively when bound to
active PAI-1 The latter is in agreement with the inability of
latent PAI-1 to bind VN [23]
Finally, the lack ofthe chondroitin sulphate moiety on solulin, in conjunction with its inability to bind PAI-1/VN complexes, suggests the involvement ofthe chondroitin sulphate ofrl-TM in the binding ofPAI-1/VN Indeed, both in the absence and presence ofPAI-1, the inter-action ofrl-TM with immobilized VN could be com-peted by including increasing concentrations ofheparin (200–1000 UÆmL)1) in the SPR experiments (Fig 3D)
In conclusion, these results suggest a mechanism in which TM sterically blocks both PAI-1 and PAI-1/VN complexes in the association with thrombin In addition, upon binding PAI-1, VN is able to bind the chondroitin sulphate moiety ofrl-TM independent ofTM-bound thrombin
Kinetic modeling using TM as a competitive inhibitor correctly predicts the inhibitory effect of TM
We decided to model the kinetics ofthe modulating effect of
TM on the thrombin/PAI-1 reaction to supply a mecha-nistic basis for the multicomponent reactions Solulin kinetic and binding data were used throughout the initial modeling, as the equilibrium binding ofrl-TM to thrombin displayed a sigmoidal character due to the cooperative effects of both protein/protein and protein/glycosamino-glycan interactions Complete sterical blocking ofthe thrombin/PAI-1 interaction would suggest the ability of
TM to completely prevent the inhibition ofthrombin by PAI-1 at high TM concentrations However, this is not observed experimentally at TM concentrations that are several fold higher than Kd(rl-TM 200-fold; solulin > 10-fold) An indication that could explain this apparent discrepancy is our previous description ofthe high reversible association rate ofthrombin and PAI-1 [6] The reaction scheme in Fig 4 implies that at infinite TM concentration all thrombin is in complex with TM, and the inhibition of thrombin activity by PAI-1 is thus dependent on the
Fig 4 Competitive mechanism of TM inhibition of the thrombin/PAI )1 interaction The inhibitor PAI-1 (P) forms a reversible Michaelis-type complex (TP) with thrombin (T), characterized by the bimolecular association rate constant k 1 and the dissociation rate constant k)1 Subsequently,
an intermediate irreversible complex (TP¢) is formed with rate constant k 2 , that can convert with a rate constant k 3 into the SDS-stable complex (T-P), or it can react according to a substrate mechanism, resulting in the release offree enzyme together with cleaved, inactive inhibitor (P*) with the rate constant rÆk 3 The partition ratio (r) represents the number ofcatalytic turnovers per inactivation event, where 1 + r is the apparent stoichiometry The rate ofPAI-1 conversion to its latent form (PL) is described by the rate constant k L Alternatively, thrombin binds to TM forming a reversible complex (T-TM) described by the association and dissociation rate constants k and k , respectively.
Trang 7dissociation rate ofthrombin/TM complexes Upon
disso-ciation ofthe thrombin/TM complex, there will be
compe-tition between PAI-1 and TM for (re)associating with
thrombin Competition is dependent on the second-order
rate constants for the thrombin/PAI-1 and thrombin/TM
interaction, including the actual concentration ofTM and
PAI-1 throughout the course ofthe reaction Therefore, an
infinite concentration ofTM would completely compete
PAI-1 binding to thrombin To test this concept, numerical
integration ofthe rate equations that describe the
mechan-ism in Fig 4 was perf ormed to obtain theoretical insight
into the effect of the second-order rate constants and
concentrations ofTM and PAI-1 on the rate ofthrombin/
PAI-1 complex formation For various combinations of kon
and kofffor the thrombin/TM interaction, the concentration
ofall reactants and intermediates was calculated
through-out the course ofthe reaction The model fits the experi-mental data best when kon¼ 3 · 104
M )1Æs)1 and
koff¼ 1 · 10)3Æs)1 (data not shown) The Kd for solulin ( 30 nM) that was derived from these values is in good agreement with literature [24] The total thrombin amido-lytic activity that is thus predicted was compared to the experimentally observed decrease in thrombin activity (Fig 5A) The modeling adequately predicts the effect of
TM on the thrombin/PAI-1 inhibition kinetics The extremely fast formation of the initial thrombin/PAI-1 Michaelis-type complex, which is predicted during the presteady state phase ofthe reaction, is dependent on the starting concentration offree thrombin molecules (which itselfis dependent on the total TM concentration) (Fig 5B) Consequently, the maximal concentration ofthis reversible intermediate determines the rate offormation ofthe first irreversible intermediate TP¢ and thus establishes the overall rate ofthrombin inhibition, i.e the rate at which TP disappears in time at steady state after the rapid initial increase The pronounced biphasic character ofthe inhibi-tion profiles in Fig 1A–C is explained by the presteady state and steady state phases ofthe reaction that are predicted by the modeling At presteady state free thrombin is quickly captured in the reversible TP complex, which accounts for the rapid decrease ofthrombin activity that is observed during the first minutes The second phase in the inhibition profiles describes the steady state phase ofthe reaction where thrombin is slowly released by TM and inhibited by PAI-1 The reduced affinity of TM for thrombin-VR1tPA results in a higher free thrombin concentration at the start of the reaction and thus a more prominent biphasic inhibition profile with a longer initial phase (Fig 1C) According to Fig 5B the difference in the maximal concentration of TP, which is reached in the absence or presence of800 nMTM,
is approximately 12-fold This value is in agreement with the inhibitory effect of TM that is observed experimentally Finally, a similar model describing an allosteric inhibitory
Fig 5 A computer-simulated competitive model correctly predicts the effect of TM on the thrombin/PAI-1 inhibition kinetics Computer-aided numerical integration was performed, using the method of Runge-Kutta, to predict the concentration ofall reactants and intermediates described in Fig 4 during the full time course of the inhibition reac-tion The rate constants k on and k off were fitted to the experimental data from Fig 1A All other rate constants have been described in a previous study [6] (A) The time-dependent decrease ofresidual thrombin activity predicted by the model fits closely to the experi-mental data Lines represent the residual thrombin activity that was calculated using the same set ofrate constants for all TM concentra-tions The lower panel shows the residuals ofthe fit expressed as the difference between the experimental and calculated values (B) Shows the calculated change in concentration ofthe thrombin–PAI-1 reversible Michaelis complex ([TP]), as predicted throughout the course ofthe reaction modeled in panel A The maximal amount ofTP complexes that is formed during the initial phase of the reaction (< 10 s) is reduced in a TM concentration-dependent fashion This decrease is related to the free thrombin concentration at the start of the inhibition reaction that is determined by the TM concentration and the
K d ofthe thrombin–TM complex Symbols are identical to Fig 1A Lines represent the TM concentration that was used, i.e 0 (––), 30 (– – –),
50 (- - -), 100 (-Æ-), 400 (-ÆÆ) and 800 (ÆÆÆ) n
Trang 8effect of TM, by allowing thrombin/PAI-1/TM complex
formation, did not fit the experimentally observed
TM-dependent decrease ofthe thrombin/PAI-1 interaction rate
(results not shown)
Discussion
TM functions as a powerful procoagulant to anticoagulant
specificity switch upon binding to thrombin with high
affinity [1,2] The binding of Na+to thrombin constitutes a
second switch that potently modulates the coagulant vs
anticoagulant functions of thrombin [25] The allosteric
effect that results from the binding of Na+makes thrombin
a significantly more efficient procoagulant [26] However, in
both its Na+-bound and Na+-free form thrombin has a
high specificity for protein C in the presence of TM The
data presented in this study demonstrates that the VR1 loop
ofthrombin, being in close vicinity ofthe compact
functional epitope for TM [27], is the subsite of exosite-1
that regulates exclusive binding ofeither PAI-1 or TM
Previous studies have unambiguously demonstrated that the
VR1 loop is responsible for the specific interaction of PAI-1
with t-PA [7] and thrombin [5,6,21] The dominant
contri-bution ofthe t-PA VR1 loop residues to inhibition by PAI-1
strongly suggests binding ofPAI-1 to VR1 residues in t-PA
and thrombin-VR1tPA In contrast, the interaction of
thrombin with ATIII is primarily determined by subsites
located in the Western-exit ofthrombin (hydrophobic
binding pocket/N-terminal subsites), which is located
distant from the VR1 loop on the opposite side of the
active-center [9,28,29] Interestingly, PAI-1 appears to be the
only serpin that utilizes the VR1 loop, in contrast to ATIII
and protein C inhibitor (PCI) In agreement with this
concept, the thrombin/TM complex can be efficiently
inhibited by ATIII and PCI [11,12] Moreover, TM acts
as a stimulator ofthrombin inhibition by these serpins, in
line with the cofactor effect of TM on protein C activation
The exclusive function of the VR1 loop is now further
supported by the results obtained with the exosite-binding
proteins TM and hirugen (this study and [6]) The possibility
ofternary complex formation between thrombin (–VR1tPA),
PAI-1 and hirugen demonstrates that PAI-1 does not bind
the part ofthe anion binding exosite-1 ofthrombin that is
utilized by hirugen as well as by TM [22,29,30] However, as
demonstrated in this study, binding ofTM does sterically
hinder PAI-1 binding to thrombin (–VR1tPA) Therefore,
the binding ofPAI-1 to thrombin either involves a small
part ofthe VR1 loop that is physically blocked by TM, or
the bulkiness ofTM bound to exosite-1 decreases the
accessibility ofthe VR1 loop for PAI-1 [9,10,27] Previous
Ala scanning mutagenesis studies have demonstrated that
the VR1 residues Phe34, Lys36, Pro37 and Gln38 are
involved in the binding ofTM to thrombin [27,31] These
residues therefore comprise the most likely overlapping
binding site for TM and PAI-1 on thrombin, as they are also
ofsubstantial importance to the inhibition ofthrombin by
PAI-1 [5,6,21] In addition, the reduced affinity of TM for
thrombin-VR1tPA is in agreement with the significant
contribution ofthis part ofthe VR1 loop to the binding
ofTM by thrombin Binding ofthe carboxy-terminal part
ofthe reactive center loop ofPAI-1 in the small cleft formed
by the 60-loop and VR1 loop can be envisioned The
kinetics ofthe interaction ofthrombin and TM were shown
to be governed by electrostatic interactions, explaining the fast association rates [8] This does not explain, however, the high affinity binding of TM to thrombin as shown by the slow dissociation rates observed in this study (koff
10)3Æs)1; Fig 3) Structural arguments were put for-ward that a major hydrophobic interaction in this strong hydrophilic environment governs the specificity and tight-ness ofTM binding to thrombin [10] Hydrophobic residues ofTM are buried in a surface hydrophobic pocket that is partly formed by VR1 and exosite-1 This would explain the significantly reduced binding ofTM to thrombin-VR1tPAas compared to thrombin, despite the fact that according to the structure the lower, highly charged rim ofexosite-1 is unchanged [6] This hydrophobic interaction, involving Phe34 in the VR1 loop, would then exclude the interaction ofPAI-1 with the VR1 ifthrombin is bound to TM The kinetic model ofTM/PAI-1 competition for throm-bin described here has the following features Previous studies from our laboratory have shown that initial Michaelis complex formation is not the rate-limiting step
in the thrombin/PAI-1 reaction, but rather a unimolecular step in the mechanism (k2in Fig 4) These findings imply that with the high PAI-1 concentrations that were used to rapidly inhibit thrombin, a major fraction of the thrombin molecules is quickly forming a reversible Michaelis complex with PAI-1 during the initial phase ofthe reaction Subsequently, the formation of stable thrombin/PAI-1 complexes is dependent on the (rate-limiting) efficiency of successive catalytic events in the inhibition pathway (i.e k2 and k3in Fig 4) When the amidolytic activity ofthrombin
is assayed during the course ofthe reaction, quenching of the reaction mixture leads to dissociation ofthe majority of initial Michaelis complexes, and thus releases active throm-bin In the presence ofa theoretical infinite concentration of
TM, all thrombin would be in complex with TM at the start ofthe reaction When PAI-1 is added, competition between
TM and PAI-1 for thrombin will occur after dissociation of each thrombin/TM complex, which was thus far at equi-librium Consequently, the concentrations ofPAI-1 and
TM, including their rate constants ofassociation with thrombin, will determine the maximum rate at which the thrombin/TM complex will be irreversibly inhibited by PAI-1 (Fig 5) Under the experimental conditions used in this study, relatively low TM concentrations (< 1 lM) are sufficient to have most thrombin in complex with TM At the high PAI-1 concentration (> 1 lM) that was used, PAI-1 will compete efficiently with TM as k1> kon and [PAI-1] > [TM] Therefore, even though TM significantly slows down the thrombin/PAI)1 interaction, eventually thrombin will be completely inhibited by PAI-1 The inhibitory effect of TM on the thrombin/PAI-1 interaction
is partly masked in the presence ofthe cofactor VN when assayed kinetically ([15] and this study) The data presented
in this study demonstrate that only PAI-1/VN complexes and immobilized VN directly bind to TM, most likely via its glycosaminoglycan sugar moiety In contrast, free VN or PAI-1 does not bind to TM In agreement with this finding,
VN is known to expose a high-affinity heparin binding-site only after it is converted to its non-native (active) confor-mation, i.e when bound to PAI-1 or when immobilized on a surface [32] Therefore, the interaction between VN and TM
Trang 9has probably no effect on the rate of thrombin inhibition by
PAI-1 in the presence ofTM A slightly larger inhibitory
effect ofTM on the rate ofthrombin inhibition by PAI-1/
VN was observed compared to inhibition by PAI-1 alone,
i.e 14 vs 12-fold, respectively In the presence of TM,
however, thrombin is still inhibited 36-fold faster by the
PAI-1/VN complex compared to PAI-1 alone The vastly
increased association rate between thrombin and PAI-1/VN
would result in an immediate capturing ofany free
thrombin that is at equilibrium with the thrombin/TM
species by competing more efficiently for reassociating
with TM
The physiologic relevance ofTM interference in the
thrombin/PAI-1 interaction can possibly be found in the
(atherosclerotic) vessel wall, where all these proteins and
cofactors are present [17], including TM on vascular SMC
[14] Both thrombin and PAI-1 can substantially influence
migration and proliferation of vascular SMC, the latter
process via the protease-activated receptors, ofwhich
PAR1 was found to be expressed by SMC in vivo [33] In
this respect, the interplay between thrombin and PAI-1 in
the vessel wall has two f aces First, PAI-1 is able to inhibit
the mitogenic potential ofthrombin On the other hand,
cleavage and inactivation ofPAI-1 by thrombin controls
the urokinase-type plasminogen activator (u-PA)-mediated
migratory effect of PAI-1 on SMC The suicide-substrate
mechanism stoichiometry is rather unfavorable for the
thrombin/PAI-1 protease/serpin pair, especially in the
presence ofVN, being six inactivated (cleaved) PAI-1
molecules for each thrombin molecule that is inhibited
(r¼ 5) [21] Probably the main physiologic consequence of
this interaction is an inactivation ofthe PAI-1 pool in the
vascular wall by thrombin, making it no longer available
for interaction with u-PA and VN, which can explain part
of the effect of thrombin on the proliferation and
migration ofvascular SMC [4,34] In the context ofthe
vessel wall, TM might therefore function as a regulator of
PAI-1 inactivation by thrombin in the presence ofthe
abundant matrix protein VN The presence ofTM on the
surface of SMC might be important in focusing its
modulatory potential to the cell surface In this respect,
physiologic significance can be attributed to the binding of
VN in its unfolded conformation (i.e as adhered matrix
protein or in solution complexed to PAI-1) to the
chondroitin sulphate moiety ofTM as was observed in
this study Neointimal vascular SMC can thus focus TM
to sites where VN is present, e.g at the leading edge of
migration, and prevent local inactivation ofPAI-1 by
thrombin The ability ofPAI-1 to compete with the SMC
surface-exposed integrin avb3 and u-PA receptor for
binding VN therefore suggests a possible migratory role
ofTM in neointimal hyperplasia This concept is in
agreement with the expression ofTM by neointimal
vascular SMC that was found in vivo [14,34]
In conclusion, this study provides a mechanistic concept,
elucidating a multicomponent system ofproteases, serpins
and cofactors Again, TM acts as a molecular switch by
excluding an interaction between thrombin and PAI-1
thereby protecting the serpin from inactivation
Further-more, these findings propose a possible novel role for TM
expressed by vascular SMC in the pathogenesis ofvascular
disease
Acknowledgements
This work was supported by the Netherlands Heart Foundation, the Hague, by grant NHS 96.094 and the Molecular Cardiology Program grant M 93.007.
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