Báo cáo khoa học: A 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 ppt

A 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 of TM 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- VR1 tPA as compared to thrombin, demonstrating the importance of the VR1 loop in the interaction of thrombin 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 of TM confirms a mechanism that involves complete steric blocking of the thrombin/PAI-1 interaction. Also, it accurately decribes the biphasic inhibition profile resulting from the substantial reduction of the extremely fast rate of reversible Michaelis complex formation, which is essential for efficient inhibition of thrombin by PAI-1. Vitronectin (VN) is shown to partially relieve TM inhibitory action only by vastly increasing the initial rate of interaction 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 of TM. Collectively, these results show that VR1 is a subsite of exosite 1 on thrombin’s surface, which regulates exclusive binding of either PAI-1 or TM. This competition will be physiologically significant in con- trolling the mitogenic activity of thrombin 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 of thrombin 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 of protein C. The catalytic activity of thrombin can be inhibited by a number of serine 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 (k i )of10 3 M )1 Æs )1 ,which can be increased up to 250-fold by the cofactors vitronectin (VN) and heparin [3,4]. The VR1- or 37-loop of thrombin has been implicated in a number of intriguing interactions. First, substitution of the VR1-loop of thrombin by that of t-PA, yielding thrombin- VR1 tPA , increases the bimolecular rate constant of inhibi- tion by PAI-1 at least 1000-fold to 10 6 M )1 Æs )1 [5]. Recently, we reported that this alteration results from an increased rate of a unimolecular catalytic step [6]. It has been unambiguously evidenced that VR1 is essential for the interaction of both 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) of TM 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 of PAI-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 of PAI-1 but altered a catalytic step of the 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 of Biochemistry, Room K1-161, Meibergdreef 15, 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 of t-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 of inhibition; 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) Eur. J. Biochem. 270, 1942–1951 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03552.x [11–13]. However, previous work from our laboratory has demonstrated that thrombin inhibition by PAI-1/VN com- plexes is impaired in the presence of TM [4]. These findings have later been studied in more detail, though the mecha- nism of TM 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 of the human and rabbit aorta [14]. Also, due to the colocalization of thrombin, 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 of vascular disease [16,17]. Together, the presence of PAI-1, VN and TM in the vessel wall, including the unique property of thrombin to inactivate PAI-1, suggests a novel role of TM in controlling the behavior of vascular cells. Here, we report that binding of TM and PAI-1 to thrombin is mutually exclusive, both in the presence and absence of VN. Furthermore, the data presented here is in agreement with a mechanism in which the rate of thrombin inhibition by PAI-1 is dependent on the rate of dissociation of thrombin from TM, explaining the observed biphasic inhibition profiles. These findings are in marked contrast to the binding of all other thrombin-binding components, and comprise yet another level of specificity 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 of Maastricht, the Netherlands). Construction, expression, and activation of recombinant 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 Eppendorf tubes or in wells of a microtiter plate (Nunc Maxisorp; Gibco-BRL, Gaithersburg, MD, USA) that had been pretreated for 1 h at 37 °Cwith1% (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 n M thrombin with 1.5 l M PAI-1 at 37 °C in HBSO buffer (20 m M Hepes, pH 7.4, 150 m M NaCl, and 0.5 mgÆmL )1 ovalbumin). At specific time intervals, aliquots of 5 lL were withdrawn and the reaction was quenched by diluting 45-fold in HBSO buffer containing 0.65 m M of S2238 chromogenic substrate. Residual thrombin amidolytic activity in these aliquots was measured at 37 °C by continuously recording the absorb- ance at 405 nm in a Titertek Twinreader (Flow Laborat- ories, Irvine, UK). Plots of residual activity (relative to thrombin activity in the absence of PAI-1) vs. time were constructed and analyzed as described [6]. The effect of increasing concentrations of solulin on the inhibition of thrombin and thrombin-VR1 tPA by PAI-1 was determined. To that end, a solution of 15 n M human a-thrombin was prewarmed in NaCl/P i /Tween buffer [NaCl/P i with 0.01% (v/v) Tween 80] for 5 min at 37 °C, in the presence of increasing concentrations of solulin (0–800 n M in NaCl/P i / Tween buffer) or rl-TM (0–100 n M in 20 m M Tris buffer, pH 7.4, with 100 m M NaCl). Subsequently, the inhibition reaction was started by the addition of PAI-1 to a final concentration of 1.5 l M . At various time intervals, the residual thrombin amidolytic activity was determined as described above. Alternatively, the inhibition of 2 n M thrombin-VR1 tPA by 10 n M PAI-1 was determined in the absence or presence of 800 n M solulin. Therefore, 13 lL aliquots were quenched by diluting ninefold in HBSO buffer, containing 0.9 m M of S2238 chromogenic substrate. Surface plasmon resonance (SPR) binding studies Reversible binding of various 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 25.0 °C. All recorded sensorgrams were corrected for refractive index variations, using an empty flow cell. Thrombin-S195A and thrombin- S195A-VR1 tPA were immobilized on a sensor chip as described for thrombin [18]. Thrombin was immobilized at 40 ngÆlL )1 in 10 m M sodium acetate buffer (pH 6.0), rl-TM was immobilized at 15 ngÆlL )1 in 10 m M sodium formate buffer (pH 3.6), and vitronectin was immobilized at 120 ngÆlL )1 in 10 m M 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 m M Hepes, pH 7.4, 150 m M NaCl, 2 m M CaCl 2 , 0.005% (v/v) P20] was used ata20-lLÆmin )1 flow rate. Binding of thrombin and thrombin-VR1 tPA to immobi- lized rl-TM was monitored by applying either thrombin (0.5–20 n M ), or thrombin-VR1 tPA (10–100 n M )inHBS buffer, at 20 lLÆmin )1 . Association and dissociation rate constants were determined from the SPR sensor grams by global nonlinear regression using the BIAEVALUATION soft- ware (BIAcore AB). Binding of various analytes to rl-TM/ thrombin complexes was studied as follows: 10 lLof 200 n M human recombinant a-thrombin in HBS buffer was Ó FEBS 2003 Thrombomodulin sterically blocks the thrombin/PAI-1 interaction (Eur. J. Biochem. 270) 1943 injected 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 of thrombin and bound analyte from TM was continuously monitored in HBS buffer. Direct binding to rl-TM of 200 n M PAI-1, 200 n M latent PAI-1, 300 n M VN, or 200 n M active or latent PAI-1 preincubated with 75–300 n M VN was studied by injecting 60 lL of the respective proteins in HBS buffer. Latent PAI-1 was obtained by incubating 200 n M PAI-1 at 37 °Cforat least 20 h. Direct binding of rl-TM and solulin to VN was studied by injecting 40 lL200n M rl-TM, or 1 l M solulin, in the absence or presence of heparin (0–1000 UÆmL )1 ). Alternat- ively, previous to the TM injections, 40 lL 500 n M PAI-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 of numerical integration of the rate equa- tions derived from the mechanism shown below has been described elsewhere, including the rate constants of the suicide-substrate mechanism (k 1 to k 3 )thatwereused[6]. Briefly, at various combinations of k on and k off 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 k on and k off , 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 of thrombin inhibition by PAI-1 is significantly reduced in the presence of TM [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 half times (t 1/2 ) of PAI-1 inhibition. (A) Residual activity was monitored during the inhibition of 15 n M human thrombin by 1.5 l M PAI-1, in the absence (d)orpresenceof30n M (s), 50 n M (j), 100 n M (h), 400 n M (m)or800n M (n) TM (solulin). (B) Residual thrombin activity was monitored as in (A), but in the presence of 0 (d), 10 (s), 20 (j), 50 (h)or100(m) nM rl-TM. (C) Residual activity decrease was monitored during the inhibition of 2 n M thrombin-VR1 tPA by 15 n M PAI-1 in the absence (d) or presence of 100 n M rl-TM (s). (D) Thrombin activity decrease that was observed during the inhibition of 15 n M thrombin by 100 n M PAI-1/VN complexes, in the absence (d) or presence of 100 n M rl-TM (s). 1944 R. J. Dekker et al. (Eur. J. Biochem. 270) Ó FEBS 2003 Furthermore, we attempted to elucidate the mechanism of interference by TM on thrombin inhibition by PAI-1. The rate of thrombin inhibition by 1.5 l M PAI-1 was measured in the presence of increasing concentrations (0–800 n M )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 interactions between thrombin and TM are considered. At the highest concentration of TM (800 n M solulin), thrombin is inhibited by PAI-1 with a half-time of the reaction (t 1/2 )thatis12.5- fold longer than in the absence of TM, i.e. 6810 and 545 s, respectively (Fig. 1A). Because of the lower affinity of solulin for thrombin (due to the lack of the chondroitin sulphates) [20], high concentrations of the 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 n M rl-TM on the rate of inhibition of human plasma thrombin by PAI-1 was more substantial than that of the highest concentration of solulin (800 n M ) (Fig. 1B). Next, the effect of TM on the inhibition of the substitution variant thrombin-VR1 tPA by PAI-1 was studied. In marked contrast to thrombin, only a 1.7-fold inhibitory effect of 100 n M rl-TM is observed on the inhibition of 2 n M thrombin-VR1 tPA by 15 n M PAI-1, measured as difference of the half-time (t 1/2 )ofthereaction (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 of inhibition of thrombin by PAI-1 alone (k i  10 3 M )1 Æs )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 of magnitude higher compared to PAI-1 alone (k i  10 5 M )1 Æs )1 ). Therefore, the inhibitory effect of TM on the inhibition of 15 n M thrombin by preincubated PAI-1/VN complexes (100 n M PAI and 150 n M VN) was determined (Fig. 1D). The presence of 100 n M rl-TM in this reaction decreased the inhibition rate by 14-fold (t 1/2 ¼ 940 and 67 s, respectively). Still, even in the presence of rl-TM, VN accelerates the rate of thrombin inhibition by PAI-1 36-fold [Fig. 1B (m)vs.1D(s)]. TM binding to thrombin-VR1 tPA is substantially reduced The minor effect of TM on the rate of thrombin-VR1 tPA inhibition by PAI-1 suggests that the binding between thrombin and TM, involving the VR1 loop of thrombin, is affected in the substitution variant. Indeed, the rate of protein C activation by thrombin-VR1 tPA ,whichiscom- parable to that of thrombin, was not affected by TM, whereas TM substantially increased the rate of protein C activation by thrombin, as expected (data not shown). The affinity of TM for thrombin-VR1 tPA was determined using Surface Plasmon Resonance (SPR, data not shown). Binding of thrombin-VR1 tPA to immobilized rl-TM was significantly reduced (K d ¼ 121 ± 23 n M ) compared to thrombin (K d  0.5 n M ) [2,11]. Thus, the minor effect of TM on the thrombin-VR1 tPA –PAI-1 interaction appears to be the result of the decreased ability of TM to bind thrombin-VR1 tPA . 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 of the inhibition of thrombin by PAI-1 can be described by the so-called Ôsuicide-substrateÕ mechanism as previously elaborated by our group [6,21]. In this mecha- nism, each productive encounter of serpin and protease can either lead to formation of the enzyme/inhibitor complex or can result in cleavage of the inhibitor and release of active enzyme. A decreased overall inhibition rate can thus be the result of a shift of the rate constants of the 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 of increased cleavage indicating that TM does not alter the product distribution of the 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 of PAI-1 to immobilized thrombin/TM complexes was studied in real-time using SPR. The high rate constant of initial thrombin/PAI-1 complex formation (k 1  10 6 m )1 Æs )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 of the products of the reaction of 600 n M thrombin with 3.5 l M PAI-1 in the presence of 0 (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 of Fig. 4. Note that after 16 h incubation, the thrombin–PAI-1 complexes formed in the presence of TM (lanes 5, 7 and 9) have mostly been degraded to lower molecular mass species by the remaining free, active thrombin as noted before [21]. Ó FEBS 2003 Thrombomodulin sterically blocks the thrombin/PAI-1 interaction (Eur. J. Biochem. 270) 1945 ternary thrombin/PAI-1/TM complexes occurs. First, thrombin/TM complexes were formed by applying a solution of 200 n M thrombin 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 of the thrombin/TM complex in the subnanomolar range [2,11]. Binding was mass transport- limited, precluding exact determination of the rate constants for this interaction under these conditions. Alternatively, an estimate of k off can be given using the half time of throm- bin/TM dissociation, i.e. t 1/2 ¼ 470 s and k off  10 )3 Æs )1 . Second, immediately following the thrombin injection, either a solution of 800 n M PAI-1, 800 n M 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 of PAI-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 of ternary 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 of PAI-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 of throm- 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 of the thrombin/ATIII complex from TM, Fig. 3. TM and PAI-1 competitively bind thrombin. Binding of various 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 lL200n 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 lL800n 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 of the following solutions was directly applied (at 0 s) to the immobilized rl-TM in the absence of thrombin, 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 of rl-TM to VN was studied by immobilizing VN on a sensor chip. Next, at 0 s 40 lL200n M rl-TM was directly injected in the absence (––) or presence of 200 (- - -), 500 (-Æ-) or 1000 (ÆÆÆ)UÆmL )1 heparin. Subsequently, dissociation was monitored under continuous buffer flow (beyond 120 s). 1946 R. J. Dekker et al. (Eur. J. Biochem. 270) Ó FEBS 2003 resulting in a net decrease in TM-associated mass on the chip surface. Moreover, these data are consistent with the previously described strong reduction of the 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 of VN (0–300 n M ) were preincubated with 200 n M PAI-1 and binding to preformed thrombin/rl-TM complexes was monitored (Fig. 3B). In contrast to PAI-1 and ATIII, binding of PAI-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 of active 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 of PAI-1 (data not shown). Therefore, binding of VN 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 of the chondroitin sulphate moiety on solulin, in conjunction with its inability to bind PAI-1/VN complexes, suggests the involvement of the chondroitin sulphate of rl-TM in the binding of PAI-1/VN. Indeed, both in the absence and presence of PAI-1, the inter- action of rl-TM with immobilized VN could be com- peted by including increasing concentrations of heparin (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 of rl-TM independent of TM-bound thrombin. Kinetic modeling using TM as a competitive inhibitor correctly predicts the inhibitory effect of TM We decided to model the kinetics of the 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 of rl-TM to thrombin displayed a sigmoidal character due to the cooperative effects of both protein/protein and protein/glycosamino- glycan interactions. Complete sterical blocking of the thrombin/PAI-1 interaction would suggest the ability of TM to completely prevent the inhibition of thrombin by PAI-1 at high TM concentrations. However, this is not observed experimentally at TM concentrations that are several fold higher than K d (rl-TM  200-fold; solulin > 10-fold). An indication that could explain this apparent discrepancy is our previous description of the high reversible association rate of thrombin 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 of free enzyme together with cleaved, inactive inhibitor (P*) with the rate constant rÆk 3 . The partition ratio (r) represents the number of catalytic turnovers per inactivation event, where 1 + r is the apparent stoichiometry. The rate of PAI-1 conversion to its latent form (P L ) 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 on and k off , respectively. Ó FEBS 2003 Thrombomodulin sterically blocks the thrombin/PAI-1 interaction (Eur. J. Biochem. 270) 1947 dissociation rate of thrombin/TM complexes. Upon disso- ciation of the 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 of TM and PAI-1 throughout the course of the reaction. Therefore, an infinite concentration of TM would completely compete PAI-1 binding to thrombin. To test this concept, numerical integration of the rate equations that describe the mechan- isminFig.4wasperformedtoobtaintheoreticalinsight into the effect of the second-order rate constants and concentrations of TM and PAI-1 on the rate of thrombin/ PAI-1 complex formation. For various combinations of k on and k off for the thrombin/TM interaction, the concentration of all reactants and intermediates was calculated through- out the course of the reaction. The model fits the experi- mental data best when k on ¼ 3 · 10 4 M )1 Æs )1 and k off ¼ 1 · 10 )3 Æs )1 (data not shown). The K d for solulin ( 30 n M ) 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 of the reaction, is dependent on the starting concentration of free thrombin molecules (which itself is dependent on the total TM concentration) (Fig. 5B). Consequently, the maximal concentration of this reversible intermediate determines the rate of formation of the first irreversible intermediate TP¢ and thus establishes the overall rate of thrombin inhibition, i.e. the rate at which TP disappears in time at steady state after the rapid initial increase. The pronounced biphasic character of the inhibi- tion profiles in Fig. 1A–C is explained by the presteady state and steady state phases of the 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 of thrombin activity that is observed during the first minutes. The second phase in the inhibition profiles describes the steady state phase of the reaction where thrombin is slowly released by TM and inhibited by PAI-1. The reduced affinity of TM for thrombin-VR1 tPA 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 of 800 n M TM, 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 of all 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 of residual 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 of rate constants for all TM concentra- tions. The lower panel shows the residuals of the fit expressed as the difference between the experimental and calculated values. (B) Shows the calculated change in concentration of the thrombin–PAI-1 reversible Michaelis complex ([TP]), as predicted throughout the course of the reaction modeled in panel A. The maximal amount of TP 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 of the thrombin–TM complex. Symbols are identical to Fig. 1A. Lines represent theTM concentration thatwas used, i.e.0 (––),30 (– – –), 50 (- - -), 100 (-Æ-), 400 (-ÆÆ) and 800 (ÆÆÆ)n M . 1948 R. J. Dekker et al. (Eur. J. Biochem. 270) Ó FEBS 2003 effect of TM, by allowing thrombin/PAI-1/TM complex formation, did not fit the experimentally observed TM- dependent decrease of the 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 of thrombin, being in close vicinity of the compact functional epitope for TM [27], is the subsite of exosite-1 that regulates exclusive binding of either 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 of the t-PA VR1 loop residues to inhibition by PAI-1 strongly suggests binding of PAI-1 to VR1 residues in t-PA and thrombin-VR1 tPA . In contrast, the interaction of thrombin with ATIII is primarily determined by subsites located in the Western-exit of thrombin (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 of thrombin 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 of ternary complex formation between thrombin (–VR1 tPA ), PAI-1 and hirugen demonstrates that PAI-1 does not bind the part of the anion binding exosite-1 of thrombin that is utilized by hirugen as well as by TM [22,29,30]. However, as demonstrated in this study, binding of TM does sterically hinder PAI-1 binding to thrombin (–VR1 tPA ). Therefore, the binding of PAI-1 to thrombin either involves a small part of the VR1 loop that is physically blocked by TM, or the bulkiness of TM bound to exosite-1 decreases the accessibility of the 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 of TM 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 of substantial importance to the inhibition of thrombin by PAI-1 [5,6,21]. In addition, the reduced affinity of TM for thrombin-VR1 tPA is in agreement with the significant contribution of this part of the VR1 loop to the binding of TM by thrombin. Binding of the carboxy-terminal part of the reactive center loop of PAI-1 in the small cleft formed by the 60-loop and VR1 loop can be envisioned. The kinetics of the interaction of thrombin 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 (k off  10 )3 Æs )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 of TM binding to thrombin [10]. Hydrophobic residues of TM are buried in a surface hydrophobic pocket that is partly formed by VR1 and exosite-1. This would explain the significantly reduced binding of TM to thrombin-VR1 tPA as compared to thrombin, despite the fact that according to the structure the lower, highly charged rim of exosite-1 is unchanged [6]. This hydrophobic interaction, involving Phe34 in the VR1 loop, would then exclude the interaction of PAI-1 with the VR1 if thrombin is bound to TM. The kinetic model of TM/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 (k 2 in 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 of the 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. k 2 and k 3 in Fig. 4). When the amidolytic activity of thrombin is assayed during the course of the reaction, quenching of the reaction mixture leads to dissociation of the majority of initial Michaelis complexes, and thus releases active throm- bin. In the presence of a theoretical infinite concentration of TM, all thrombin would be in complex with TM at the start of the 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 of PAI-1 and TM, including their rate constants of association 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 l M )are sufficient to have most thrombin in complex with TM. At the high PAI-1 concentration (> 1 l M )thatwasused, PAI-1 will compete efficiently with TM as k 1 > k on 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 of the 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 Ó FEBS 2003 Thrombomodulin sterically blocks the thrombin/PAI-1 interaction (Eur. J. Biochem. 270) 1949 has probably no effect on the rate of thrombin inhibition by PAI-1 in the presence of TM. A slightly larger inhibitory effect of TM on the rate of thrombin 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 of any free thrombin that is at equilibrium with the thrombin/TM species by competing more efficiently for reassociating with TM. The physiologic relevance of TM 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, of which PAR1 was found to be expressed by SMC in vivo [33]. In this respect, the interplay between thrombin and PAI-1 in thevesselwallhastwofaces.First,PAI-1isabletoinhibit the mitogenic potential of thrombin. On the other hand, cleavage and inactivation of PAI-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 ÔunfavorableÕ for the thrombin/PAI-1 protease/serpin pair, especially in the presence of VN, being six inactivated (cleaved) PAI-1 molecules for each thrombin molecule that is inhibited (r ¼ 5) [21]. Probably the main physiologic consequence of this interaction is an inactivation of the 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 of vascular SMC [4,34]. In the context of the vessel wall, TM might therefore function as a regulator of PAI-1 inactivation by thrombin in the presence of the abundant matrix protein VN. The presence of TM 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 of TM 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 of PAI-1 by thrombin. The ability of PAI-1 to compete with the SMC surface-exposed integrin a v b 3 and u-PA receptor for binding VN therefore suggests a possible migratory role of TM in neointimal hyperplasia. This concept is in agreement with the expression of TM by neointimal vascular SMC that was found in vivo [14,34]. In conclusion, this study provides a mechanistic concept, elucidating a multicomponent system of proteases, 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 of vascular 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. References 1. Esmon, C.T. (1987) The regulation of natural anticoagulant pathways. Science 235, 1348–1352. 2. Esmon, C.T. (1995) Thrombomodulin as a model of molecular mechanisms that modulate protease specificity and function at the vessel surface. FASEB J. 9, 946–955. 3. Ehrlich, H.J., Klein-Gebbink, R., Keijer, J., Linders, M., Preiss- ner, K.T. & Pannekoek, H. (1990) Alteration of serpin specificity by a protein cofactor: Vitronectin endows plasminogen activator inhibitor 1 with thrombin inhibitory properties. J. Biol. Chem. 265, 13029–13035. 4. Ehrlich, H.J., Klein-Gebbink, R., Preissner, K.T., Keijer, J., Esmon, N., Mertens, N. & Pannekoek, H. (1991) Thrombin neutralizes plasminogen activator inhibitor 1 (PAI-1) that is complexed with vitronectin in the endothelial cell matrix. J. Cell. Biol. 115, 1773–1781. 5. Horrevoets, A.J.G., Tans, G., Smilde, A.E., van Zonneveld, A J. & Pannekoek, H. (1993) Thrombin-variable region 1 (VR1). Evidence for the dominant contribution of VR1 of serine proteases to their interaction with plasminogen activator inhibitor 1. J. Biol. Chem. 268, 779–782. 6. Dekker, R.J., Eichinger, A., Stoop, A.A., Bode, W., Pannekoek, H. & Horrevoets, A.J. (1999) The variable region-1 from tissue type plasminogen activator confers specificity for plasminogen activator inhibitor-1 to thrombin by facilitating catalysis: release of a kinetic block by a heterologous protein surface loop. J. Mol. Biol. 293, 613–627. 7. Madison, E.L., Goldsmith, E.J., Gerard, R.D., Gething, M J.H., Sambrook, J.F. & Bassel-Duby, R.S. (1990) Amino acid residues that affect interaction of tissue-type plasminogen activator with plasminogen activator inhibitor 1. Proc. Natl Acad. Sci. USA 87, 3530–3533. 8. Baerga-Ortiz, A., Rezaie, A.R. & Komives, E.A. (2000) Electro- static dependence of the thrombin–thrombomodulin interaction. J. Mol. Biol. 296, 651–658. 9. Mathews, I.I., Padmanabhan, K.P. & Tulinsky, A. (1994) Struc- ture of a nonadecapeptide of the fifth EGF domain of thrombo- modulin complexed with thrombin. Biochemistry 33, 13547– 13552. 10. Fuentes-Prior,P.,Iwanaga,Y.,Huber,R.,Pagila,R.,Rumennik, G.,Seto,M.,Morser,J.,Light,D.R.&Bode,W.(2000)Structural basis for the anticoagulant activity of the thrombin-thrombo- modulin complex. Nature 404, 518–525. 11. Hofsteenge, J., Taguchi, H. & Stone, S.R. (1986) Effect of thrombomodulin on the kinetics of the interaction of thrombin with substrates and inhibitors. Biochem. J. 237, 243–251. 12. Rezaie, A.R., Cooper, S.T., Church, F.C. & Esmon, C.T. (1995) Protein C inhibitor is a potent inhibitor of the thrombin-throm- bomodulin complex. J. Biol. Chem. 270, 25336–25339. 13. Bajzar, L., Morser, J. & Nesheim, M. (1996) TAFI, or plasma procarboxypeptidase B, couples the coagulation and fibrinolytic cascades through the thrombin-thrombomodulin complex. J. Biol. Chem. 271, 16603–16608. 14.Tohda,G.,Oida,K.,Okada,Y.,Kosaka,S.,Okada,E., Takahashi, S., Ishii, H. & Miyamori, I. (1998) Expression of thrombomodulin in atherosclerotic lesions and mitogenic activity of recombinant thrombomodulin in vascular smooth muscle cells. Arterioscler. Thromb. Vasc. Biol. 18, 1861–1869. 1950 R. J. Dekker et al. (Eur. J. Biochem. 270) Ó FEBS 2003 15. Rezaie, A.R. (1999) Role of exosites 1 and 2 in thrombin reaction with plasminogen activator inhibitor-1 in the absence and presence of cofactors. Biochemistry 38, 14592–14599. 16. Smith, E.B., Crosbie, L. & Carey, S. (1996) Prothrombin-related antigens in human aortic intima Semin. Thromb. Hemost. 22, 347–350. 17. Stoop, A.A., Lupu, F. & Pannekoek, H. (2000) Colocalization of thrombin, PAI-1, and vitronectin in the atherosclerotic vessel wall: a potential regulatory mechanism of thrombin activity by PAI-1/ vitronectin complexes. Arterioscler. Thromb. Vasc. Biol. 20, 1143–1149. 18. van Meijer, M., Stoop, A.A., Smilde, A., Preissner, K.T., van Zonneveld, A J. & Pannekoek, H. (1997) The composition of complexes between plasminogen activator inhibitor 1, vitronectin and either thrombin or tissue-type plasminogen activator. Thromb. Haemost. 77, 516–521. 19. Parkinson, J.F., Grinnell, B.W., Moore, R.E., Hoskins, J., Vlahos, C.J. & Bang, N.U. (1990) Stable expression of a secretable deletion mutant of recombinant human thrombomodulin in mammalian cells. J. Biol. Chem. 265, 12602–12610. 20. Clarke, J.H., Light, D.R., Blasko, E., Parkinson, J.F., Nagashima, M., McLean, K., Vilander, L., Andrews, W.H., Morser, J. & Glaser, C.B. (1993) The short loop between epidermal growth factor-like domains 4 and 5 is critical for human thrombomodulin function. J. Biol. Chem. 268, 6309–6315. 21. van Meijer, M., Smilde, A.E., Tans, G., Nesheim, M.E., Panne- koek, H. & Horrevoets, A.J. (1997) The suicide substrate reaction between plasminogen activator inhibitor 1 and thrombin is regu- lated by the cofactors vitronectin and heparin. Blood 90, 1874– 1882. 22. Bock, P.E., Olson, S.T. & Bjo ¨ rk, I. (1997) Inactivation of thrombin by antithrombin is accompanied by inactivation of regulatory exosite I. J. Biol. Chem. 272, 19837–19845. 23. Seiffert, D. & Loskutoff, D.J. (1991) Kinetic analysis of the interaction between type 1 plasminogen activator inhibitor and vitronectin and evidence that the bovine inhibitor binds to a thrombin-derived amino-terminal fragment of bovine vitronectin. Biochim. Biophys. Acta 1078, 23–30. 24. Light, D.R., Glaser, C.B., Betts, M., Blasko, E., Campbell, E., Clarke,J.H.,McCaman,M.,McLean,K.,Nagashima,M., Parkinson,J.F.,Rumennik,G.,Young,T.&Morser,J.(1999) The interaction of thrombomodulin with Ca 2+ . Eur. J. Biochem. 262, 522–533. 25. Dang, Q.D. & Di Cera, E. (1996) Residue 225 determines the Na(+)-induced allosteric regulation of catalytic activity in serine proteases. Proc. Natl Acad. Sci. USA 93, 10653–10656. 26. Pineda, A.O., Savvides, S.N., Waksman, G. & Di Cera, E. (2002) Crystal structure of the anticoagulant slow form of thrombin. J. Biol. Chem. 277, 40177–40180. 27. Pineda, A.O., Cantwell, A.M., Bush, L.A., Rose, T. & Di Cera, E. (2002) The thrombin epitope recognizing thrombomodulin is a highly cooperative hot spot in exosite I. J. Biol. Chem. 277, 32015– 32019. 28. Tsiang, M., Jain, A.K. & Gibbs, C.S. (1997) Functional require- ments for inhibition of thrombin by antithrombin III in the presence and absence of heparin. J. Biol. Chem. 272, 12024– 12029. 29. Skrzypczak-Jankun, E., Carperos, V.E., Ravichandran, K.G., Tulinsky, A., Westbrook, M. & Maraganore, J.M. (1991) Struc- ture of the hirugen and hirulog 1 complexes of a-thrombin. J. Mol. Biol. 221, 1379–1393. 30. Liu, L.W., Vu, T.K., Esmon, C.T. & Coughlin, S.R. (1991) The region of the thrombin receptor resembling hirudin binds to thrombin and alters enzyme specificity. J. Biol. Chem. 266, 16977– 16980. 31. Hall, S.W., Nagashima, M., Zhao, L., Morser, J. & Leung, L.L. (1999) Thrombin interacts with thrombomodulin, protein C, and thrombin-activatable fibrinolysis inhibitor via specific and distinct domains. J. Biol. Chem. 274, 25510–25516. 32. Stockmann, A., Hess, S., Declerck, P., Timpl, R. & Preissner, K.T. (1993) Multimeric vitronectin. Identification and characterization of conformation–dependent self association of the adhesive pro- tein. J. Biol. Chem. 268, 22874–22882. 33. Nelken, N.A., Soifer, S.J., O’Keefe, J., Vu, T.K., Charo, I.F. & Coughlin, S.R. (1992) Thrombin receptor expression in normal and atherosclerotic human arteries. J. Clin. Invest. 90, 1614–1621. 34. Kanse, S.M., Kost, C., Wilhelm, O.G., Andreasen, P.A. & Pre- issner, K.T. (1996) The urokinase receptor is a major vitronectin- binding protein on endothelial cells. Exp. Cell. Res. 224, 344–353. Ó FEBS 2003 Thrombomodulin sterically blocks the thrombin/PAI-1 interaction (Eur. J. Biochem. 270) 1951 . A steady-state competition model describes the modulating effects of thrombomodulin on thrombin inhibition by plasminogen activator inhibitor-1 in the. PAI-1 in thevesselwallhastwofaces.First,PAI-1isabletoinhibit the mitogenic potential of thrombin. On the other hand, cleavage and inactivation of PAI-1 by thrombin