Substrate-Assisted Catalysis of the PAR1 Thrombin Receptor

Platelet activation and aggregation are mediated by thrombin cleavage of the exodomain of the PAR1 receptor. The specificity of thrombin for PAR1 is enhanced by binding to a hirudin-like region (Hir) located in the receptor exodomain. Here, we examine the mechanism of thrombin-PAR1 recognition and cleavage by steady-state kinetic measurements using soluble PAR1 N-terminal exodomains. We determined that the primary role of the PAR1 Hir sequence is to reduce the kinetic barriers to formation of the docked thrombin-PAR1 complex rather than to form high affinity ground-state interactions. In addition, the exosite I-bound Hir motif facilitates the productive interaction of the PAR138LDPR/SFL44 sequence with the active site of thrombin. This locking process is the most energetically unfavorable step of the overall reaction. The subsequent irreversible steps of peptide bond cleavage are rapid and allosterically enhanced by the presence of the docked Hir sequence. Furthermore, the C-terminal exodomain product of thrombin cleavage, corresponding to the activated receptor, binds tightly to thrombin. This would suggest that an additional role of the Hir sequence in the thrombin-activated receptor is to sequester thrombin to the platelet surface and modulate cleavage of other platelet receptors such as the PAR4 thrombin receptor, which lacks a functional Hir sequence.

LDPR/SFL 44 sequence with the active site of thrombin. This locking process is the most energetically unfavorable step of the overall reaction. The subsequent irreversible steps of peptide bond cleavage are rapid and allosterically enhanced by the presence of the docked Hir sequence. Furthermore, the C-terminal exodomain product of thrombin cleavage, corresponding to the activated receptor, binds tightly to thrombin. This would suggest that an additional role of the Hir sequence in the thrombin-activated receptor is to sequester thrombin to the platelet surface and modulate cleavage of other platelet receptors such as the PAR4 thrombin receptor, which lacks a functional Hir sequence.
Cellular responses to thrombin are regulated by a novel class of seven transmembrane protease-activated receptors (PARs). 1 PAR1 plays an important role in platelet activation and blood coagulation and has been implicated in the pathological processes leading to heart disease and stroke (1). PAR1 and PAR4 mediate platelet aggregation in humans, whereas PAR3 and PAR4 are responsible for platelet aggregation in mice (2)(3)(4). PAR1 and PAR4 have markedly different kinetics of activation by thrombin that contribute to their distinct roles in signaling in human platelets (5). PAR2 is a trypsin/tryptase receptor that is not cleaved by thrombin (6,7).
Thrombin activates PAR1 by binding and cleaving the N-terminal exodomain at LDPR 41 2S 42 FL generating a new N terminus (S 42 FLLRN). The new N terminus functions as a tethered ligand, which activates PAR1 by binding to the body of the receptor (8,9). The N-terminal exodomain also contains a sequence, K 51 YEPF 55 (Hir), that resembles the C-tail of the leech anti-coagulant protein, hirudin. The PAR1 Hir sequence interacts with exosite I of thrombin as indicated by x-ray structural analysis (10). Mutagenesis studies indicate that the presence of the Hir sequence in the PAR1 exodomain confers significant enhancements in the efficiency of thrombin cleavage (11)(12)(13). In analogy to the dual interactions of hirudin and serpins with thrombin (14,15), it is possible that the Hir sequence may increase the probability that productive orientations occur at the active site by anchoring the LDPR region and by an induced fit mechanism. Thus, PAR4, which lacks a functional Hir sequence (16), is activated 20-to 70-fold slower by thrombin than PAR1 on human platelets (5). An unusual feature of thrombin-PAR interactions is that stoichiometric amounts of thrombin, rather than catalytic amounts, are required for platelet activation (5). This raises the possibility that the low turnover of thrombin could be due to tight binding and a slow off-rate from cleaved receptors. However, since fast enzymic reactions are not favored by strong ground-state interactions (17,18), it is paradoxical that thrombin activation of PAR1 on platelets is extremely rapid (5). Indeed, previous studies (19) established that the N-terminal exodomain of PAR1 (TR78) expressed in soluble form is also cleaved with high efficiency by thrombin.
In this paper, we examine the mechanism of thrombin-PAR1 recognition and cleavage by conducting steady-state kinetic measurements with soluble PAR1 N-terminal exodomains. We determined that the PAR1 Hir sequence is essential for rapid association with thrombin to form a Hir-docked complex and does not provide tight ground-state binding. The subsequent step, whereby the LDPR/SFL sequence locks into the active site of thrombin, presents the highest energy barrier to the overall reaction. Peptide bond cleavage at Arg 41 -Ser 42 is allosterically enhanced by the docked Hir sequence. Interestingly, the cleaved exodomain product, TR62, which retains the Hir sequence, binds tightly to thrombin. Thus, the Hir sequence may play an additional role in preserving thrombin association with the cleaved PAR1 receptor, consequently favoring a low turnover of thrombin and tethering the protease near adjacent naive PAR receptors on the platelet surface.

EXPERIMENTAL PROCEDURES
Materials-Pure human ␣-thrombin was obtained from Haematologic Technologies (Essex Junction, VT) (specific activity ϭ 3290 NIH units/mg). CBS 34.47 (CBS), H-D-cyclohexylglycyl-L-␣-aminobutyryl-L-arginine-p-nitroanilide, was purchased from American Bioproducts (Parsippany, NJ), and H 2 N-LDPR-pNA and N-acetyl-LDPR-pNA (pnitroanilide) were synthesized at the Kansas State University Biotech-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  Production of PAR1 Exodomain Peptides-Soluble PAR1 exodomains (TR78 and TR59) were produced in Escherichia coli as KSI-PAR1-His 6 fusions as described previously (19). Shorter PAR1 exodomain fragments TR26 (PAR1 residues Arg 36 -Glu 60 ), TR26R41Q and LBS-1 (PAR1 residues 85 PAFISEDASGYL 96 -C) were synthesized at the Tufts University School of Medicine Peptide Core Facility. TR78 was produced using the expression plasmid pET31MØTR78. This plasmid was generated by subcloning the TR78 encoding NcoI/XhoI restriction fragment from pET22-KTRMH (19) into pET31MØ, which is pET31b (Novagen, Madison, WI (20)) with NcoI replacing the AlwNI site. Expression plasmids encoding the PAR1 exodomain mutants, TR78⌬Hir (⌬ 51 KYEPF 55 ), TR78HirAla ( 51 KYEPF 55 3 AAAAK), TR78R41S, and TR78R41Q, were generated by a modification of the method of Zoller and Smith (21). The sequences encoding the exodomains were subcloned into pET22-KTRMH using the NcoI/XhoI restriction sites to produce pET22KTRMH-⌬Hir1 and pET22KTRMH-Hir1Ala. Alternatively, sequences were subcloned into pET31MØ using the NcoI/XhoI restriction sites, generating pET31TR78-R41S and pET31TR78-R41Q. The plasmid expressing the TR59 fusion protein (pET31TR59) was generated by the polymerase chain reaction using a primer containing the 3Ј-XhoI restriction site. The polymerase chain reaction product was digested with NcoI and XhoI and ligated to the NcoI/XhoI sites of pET31MØTR78. The resulting plasmids were placed into E. coli BL21(DE3)pLysS for production of the PAR1 exodomain fusion proteins. The KSI-PAR1-His 6 fusions were purified by Ni-chelate chromatography, and the PAR1 exodomains were released from KSI and His 6 by CNBr cleavage and purified by high pressure liquid chromatography as described previously (19).
Initial rates (k obs ) were obtained by analyzing base-quenched samples taken at early time points where the progress curves were linear. Quantitation of cleavage products was performed by integrating peak areas and comparing them with calibration curves of fully digested samples (19). Thrombin cleavage of TR78, TR78HirAla, and TR26 was monitored by measuring the generation of the C-terminal product (TR62, TR62HirAla, and TR20, respectively) at 278 nm. Thrombin cleavage of TR78⌬Hir and TR59 was monitored by measuring the generation of the N-terminal product (TR16) at 222 nm. TR16 was completely resolved from undigested exodomain. Initial rates were fitted to the Michaelis-Menten equation (Eq. 1) by non-linear leastsquares regression to generate the kinetic parameters k cat and K m .
Kinetics of Thrombin Cleavage of Chromogenic Substrates-Cleavage of chromogenic substrates was determined by continuously monitoring the increase in absorbance at 405 nm at 37°C in a 96-well format using a SPECTRAmax 340 microplate spectrophotometer. Cleavage assays were comprised of 5-400 M substrate (CBS, H 2 N-LDPR-pNA, or N-acetyl-LDPR-pNA) in 20 mM Tris-HCl, pH 8.3, and 150 mM NaCl (TBS) and were initiated by the addition of thrombin freshly diluted in ice-cold TBS/0.1% PEG 8000 (final thrombin concentration ϭ 208 pM). Initial rates were determined by taking the initial slopes of the progress curves using SOFTmax PRO version 2.1. Quantification of the p-nitroanilide product was determined with the extinction coefficient (⑀ 405 ϭ 8300 M Ϫ1 cm Ϫ1 ), and data were fit to the Michaelis-Menten equation. Inhibition of thrombin cleavage of CBS was performed as above, with the exception that PAR1 exodomains were added at four to five different concentrations ranging from 5 to 300 M, and assays were performed at 24°C. Inhibition data were tested against 12 inhibition models (linear, hyperbolic, and parabolic models of competitive, noncompetitive, uncompetitive, and full-noncompetitive inhibition) by non-linear leastsquares regression analysis. The model of best fit was determined by visual inspection of the slope and intercept replots, by the "goodness of fit" criterion, which is a normalized Akaike information criterion test, using EZFit 5.0 (Perrella Scientific, Inc., Amherst, NH) and by F-test using Grafit 3.0 (Erithacus Software Ltd., Staines, UK). Three patterns ESI where I is inhibitor concentration, K is is the slope inhibition constant, and K ii is the intercept inhibition constant. Effect of Solution Viscosity on PAR1 Exodomain Cleavage by Thrombin-Assays were performed as above in the presence of 0 -30% sucrose viscogen. Relative viscosity ( rel ) of the assay solution was measured relative to PBS alone for the PAR1 exodomains or TBS alone for the chromogenic substrates in quadruplicate using an Ostwald viscometer. Initial velocities were fitted to the Michaelis-Menten equation, and the kinetic parameters were analyzed as a function of relative viscosity. Because solution viscosity affects the rates of diffusion-controlled steps, such as substrate association and dissociation, one can define the parameters shown in the following one-substrate reaction: where k a is the rate constant of substrate association, k d is the rate of substrate dissociation, k f is the rate of conversion to product, E is enzyme, S is substrate, ES is the enzyme-substrate complex, and P is product. Ideally, rates of steps not controlled by diffusion such as a conformational change or a chemical reaction step will remain unaffected by relative solution viscosity. A "sticky" substrate, which has a dissociation rate comparable to or slower than the rate of reaction to product, will decrease k cat /K m with increasing relative viscosity (24). Conversely, a nonsticky substrate, which binds in rapid equilibrium prior to conversion to product, will produce a k cat /K m that is not affected by relative viscosity. Substrate stickiness was determined by plotting (k cat /K m ) o /(k cat /K m ) as a function of relative viscosity ( rel )(see Fig. 2) according to Eq. 9:   a (k cat /K m ) is the slope for the plot of (k cat /K m ) 0 /(k cat /K m ) versus relative viscosity ( rel ) according to Eq. 9. b S r is the stickiness ratio of k f /k d . c k a is the association rate constant of thrombin with substrate. d The Gibb's free energy of activation (⌬G ‡ T ) for the second-order association constant, k a , was calculated from ⌬G ‡ T ϭ RT(ln(kT/h) Ϫ ln(k a )) (17). e Lower estimate from fit of data to the Michealis-Menten equation using measured values for k 1 and k 2 . f 10 mM HEPES, pH 7.5, 150 mM NaCl, 0.1% PEG, 37°C (34). g 50 mM Tris-HCl, pH 7.8, 100 mM NaCl, 0.1% PEG 6000, 37°C (33).
where the slope, (k cat /K m ) ϭ 1/(1 ϩ k d /k f ), is equal to 1 for a sticky substrate and 0 for a nonsticky substrate, and (k cat /K m ) o is determined in the absence of viscogen. The "stickiness" ratio (S r ϭ k f /k d ) approaches zero for a nonsticky substrate, whereas it is large for sticky substrates. Association rate constants and S r were determined by linear regression analysis of the viscosity data of fits to Eq. 10.

RESULTS
The PAR1 Hirudin-like Sequence Increases the Rates of Macromolecular Association and Cleavage by Thrombin-To determine the mechanism by which the Hir sequence enhances thrombin-PAR1 interactions, we measured the effects of the Hir sequence on steady-state kinetic parameters and substrate association/dissociation rates using an array of soluble PAR1 exodomain mutants (Fig. 1). The full-length PAR1 exodomain, TR78 (Ala 26 -Thr 102 ), is an effective substrate for thrombin with a k cat of 58 s Ϫ1 and a K m of 26 M (Table I). Deletion of the 51 KYEPF 55 Hir sequence (TR78⌬Hir) or substitution with 51 AAAAK 55 (TR78HirAla) causes an 8-to 30-fold decrease in catalytic efficiency (k cat /K m ). The K m for both Hir mutants increased by 3-fold, whereas k cat decreased by 2.6-fold for TR78⌬Hir and by 10-fold for TR78HirAla.
The contribution of the Hir sequence to the binding events preceding PAR1 exodomain cleavage was obtained from the effects of viscosity on the reaction kinetics. Most strikingly, the viscosity experiments revealed that the association rate constant (k a ) for TR78⌬Hir is 73-fold slower than the k a for wildtype TR78 (Table II). We also determined the effect of the Hir sequence on the stickiness of the PAR1 exodomain to thrombin. A sticky substrate is a substrate with a slow rate of dissociation (k d ) compared with the forward rate of conversion to product (k f ) (Eq. 8). As shown in Fig. 2, the catalytic efficiency for thrombin cleavage of TR78 was not significantly affected by changes in relative viscosity as illustrated by the slope of (k cat / K m ) o /(k cat /K m ) versus rel ((k cat /K m ) ϭ 0.12) and a stickiness ratio of S r ϭ 0.12 (Table II). In contrast, a sticky substrate would display a slope of ϳ1 and a large stickiness ratio (24). Thus, the PAR1 exodomain is not a sticky substrate for thrombin and a rapid pre-equilibrium between free and bound species occurs prior to hydrolysis. Unexpectedly, TR78⌬Hir is a fully sticky substrate and its k cat /K m is affected considerably by solution viscosity 2 with (k cat /K m ) of 1.7. Stickiness, due to removal of the Hir sequence, could be attributed to a decrease in the rate of dissociation, k d , or to an increase in the rate of conversion to product, k f . Because deletion of the Hir sequence causes a 2.6-fold decrease in k f ϳ k cat , the stickiness of TR78⌬Hir likely results from a much slower rate of dissociation.
We measured the kinetics of thrombin cleavage of small peptides derived from active site-interacting P 4 -P 1 residues of PAR1 (Fig. 1). The H 2 N-LDPR-pNA and N-acetyl-LDPR-pNA peptides are surprisingly good substrates for thrombin with catalytic efficiencies similar to TR78 and 3-to 5-fold higher than those exhibited by the P 3 -P 3 Ј peptide DPR/SFL (Table I). The k cat values (120 s Ϫ1 ) for thrombin cleavage of the LDPR substrates are 2-fold larger than for TR78 and K m values (66 -113 M) are similar to TR78⌬Hir and TR78HirAla. Although these minimal LDPR substrates lack the Hir sequence, they demonstrate rapid rates of association and dissociation with thrombin and are nonsticky substrates. Association rate constants for the LDPR substrates obtained from viscosity measurements are 5-fold faster relative to TR78 and 330-to 460-fold faster relative to TR78⌬Hir (Table II). The 16-fold size difference between LDPR-pNA and TR78 predicts a 2.4-fold difference in rates of association and dissociation. Therefore, appending a large exodomain to the LDPR sequence dramatically decreases the rate of productive binding of the P 4 -P 1 residues to the active site (the locking step) unless offset by prior docking via the Hir sequence in wild-type PAR1.
Thrombin Binds Tightly to the Cleaved PAR1 Exodomain Product-The events following PAR1 cleavage by thrombin are germane to thrombin-platelet interactions, because the Hir sequence remains behind as part of the cleaved PAR1 receptor. Thus, following proteolysis and dissociation of the N-terminal PAR1 cleavage fragment TR16, thrombin may remain bound to the cleaved PAR1 and kept in proximity to adjacent naive PAR1 and PAR4 receptors on the platelet surface. To determine the affinity of thrombin for the two TR78 cleavage products, TR16 (Ala 26 -Arg 41 ) and TR62 (Ser 42 -Thr 102 ), inhibition of cleavage of a small thrombin-optimized chromogenic substrate (CBS) was measured (Table III). The N-terminal cleavage prod-2 Caution must be used when interpreting viscosity effects on reac-tion kinetics, because the large concentrations of sucrose may lead to nonspecific effects such as changes in dielectric constant and volume exclusion. However, since sucrose had no effect on the cleavage rates of wild-type TR78, H 2 N-LDPR-pNA, or N-acetyl-LDPR-pNA under identical conditions, this demonstrates that the effects of sucrose on thrombin cleavage of TR78⌬Hir are due to changes in microviscosity.

FIG. 2. Effect of relative solution viscosity on k cat /K m for thrombin cleavage of TR78 and TR78⌬Hir.
Kinetic parameters were obtained for thrombin cleavage of TR78 and TR78⌬Hir in the presence of varying concentrations of sucrose viscogen. Assays were performed as described under "Experimental Procedures." Lines represent the leastsquares fit of the data. Each data point is from k cat /K m values derived from five different substrate concentrations. uct, TR16, was unable to inhibit thrombin at concentrations as high as 290 M despite containing the LDPR sequence. In contrast, the C-terminal cleavage product, TR62, corresponding to the activated PAR1 receptor, is an excellent inhibitor of thrombin with K ii of 9.4 M. Because TR62 lacks P 4 -P 1 active site binding residues, the uncompetitive pattern of inhibition (Eq. 7) indicates that the Hir sequence of TR62 binds to exosite I of thrombin and allosterically inhibits cleavage of the CBS substrate. Indeed, TR78HirAla is only able to inhibit the active site of thrombin with a K is of 35 M in a competitive pattern (Eq. 3), and TR78⌬Hir does not inhibit cleavage of CBS at 105 M (Table III). The lower affinity of TR78⌬Hir for thrombin may be a result of repulsive forces caused by the shifted WEDEE residues. Therefore, the presence of the exosite I-binding Hir motif is required for the formation of an ESI complex and inhibition of CBS cleavage.
The C-Terminal Region of the PAR1 Exodomain Modulates Thrombin Binding to the LDPR and Hir Regions-Results from previous studies (12,25) indicate that small PAR1 exodomain fragments appear to be better substrates for thrombin than the full-length TR78 exodomain. Furthermore, removal of the Hir sequence produces surprisingly more severe effects on the cleavage efficiency of short PAR1 substrates lacking residues to the C-terminal side of the Hir sequence (TR28 -60 versus TR28 -45) as compared with the larger TR78 (Table I). These results suggest that the C-terminal half of the PAR1 exodomain modulates interactions with thrombin. Hence, we investigated the role of the C-terminal region of the exodomain (Fig.  1) on the kinetics of thrombin cleavage. We compared the catalytic efficiency of full-length PAR1 exodomain, TR78, to those of C-terminally truncated domains TR59 (Ala 26 -Leu 84 ) and TR26 (Ala 36 -Glu 60 ), a minimal PAR1 exodomain analogous to TR28 -60 (12). As shown in Fig. 3, TR59 and TR26 are cleaved ϳ2-fold faster than TR78 with 2-to 5-fold lower K m values ( Table I). The k cat /K m of TR26 cleavage is only 1.5-fold faster than the reported cleavage of TR28 -60 (12), attesting to the reproducibility of these cleavage studies using soluble exodomains. Altogether, deletion of the C-terminal portion of the PAR1 exodomain results in a 4-to 9-fold increase in k cat /K m . Because no further increase in cleavage rate was seen with TR26 as compared with TR59, we can conclude that the increase in catalytic efficiency is due to the removal of the Cterminal residues Pro 85 -Thr 102 .
To determine whether the C-terminal region was directly inhibiting thrombin, the peptide LBS-1, which contains residues Pro 85 -Leu 96 (Fig. 1), was tested for inhibition of CBS or TR78 cleavage. This region comprises a portion of the intramolecular ligand binding site as shown by mutagenesis (8,9) and NMR studies. 3 The LBS-1 peptide did not inhibit thrombin activity at 100 -300 M concentrations. Because the C-terminal region of the PAR1 exodomain does not directly inhibit thrombin cleavage, it is likely that this region confers additional structural constraints when bound to thrombin, which are lacking in the smaller exodomains TR26 and TR59.
To further delineate the linkage between the C-terminal residues and the Hir sequence, the ability of the truncated PAR1 exodomains to inhibit cleavage of the chromogenic substrate, CBS, was examined. TR78, TR59, and TR26 are all potent noncompetitive inhibitors of thrombin activity with similar inhibition constants (K is ϭ 2.3-7.6 M) due to formation of an EI complex with Hir and LDPR regions bound to thrombin (Table III). The noncompetitive patterns of inhibition (Eq. 5) reflect an additional ESI complex (K ii ϭ 7.6 -21 M) with simultaneous binding of CBS at the active site and the Hircontaining PAR1 exodomains at exosite I of thrombin. However, a point mutation (R41Q) at the P 1 site of TR26 causes a 25-fold loss in binding (K ii ) versus only 1.3-to 1.5-fold loss due to mutation of the P 1 site in the full-length exodomain (TR78R41Q, TR78R41S). Therefore, the R41Q mutation at the crucial P 1 position disrupts thrombin binding to the C-terminally deleted TR26 PAR1 fragment but does not appreciably affect binding in the context of full-length TR78. This confirms that residues located to the C-terminal side of the Hir region in the PAR1 exodomain provide hitherto unappreciated binding/ structural determinants for thrombin complexation and cleavage of PAR1.

DISCUSSION
Thrombin has many substrates and inhibitors, including fibrinogen, prothrombin, protein C, antithrombin III, ␣ 2 -macroglobulin, and factors V, VII, XI, and XIII, that circulate at concentrations ranging from 30 nM to 10 M (26). By comparison, PAR1 is a rare substrate for thrombin with only 600 -1800 receptors present on the surface of each platelet (27,28). During the time period prior to clot formation, the prothrombinase complex on the surface of platelets generates 2-15 nM thrombin in whole blood (29). Thus, to successfully compete for these low levels of thrombin generated in situ, PAR1 must increase the probability that collision between these two macromolecules leads to productive binding and cleavage on the surface of 3 S. Seeley, J. Baleja, and A. Kuliopulos, unpublished results.  3. Effect of deletion of the C terminus of PAR1 exodomain on thrombin cleavage. Initial velocity (k obs ) of thrombin cleavage was obtained for various concentrations of TR78, TR59, and TR26 as described under "Experimental Procedures." The curves represent the best fits to Eq. 1 by nonlinear leastsquares regression analysis.

FIG. 4. Energetics of thrombin interactions and cleavage of PAR1.
A two-step dock and lock mechanism is shown for thrombin cleavage of PAR1 on platelets for wild-type (WT-middle) and ⌬Hir (bottom) receptors. The individual steps of the dock and lock mechanism are illustrated as an energy diagram (top) and are aligned with the mechanisms below. The energy diagram was constructed using Gibb's free energies of activation (⌬G ‡ T ) calculated from the equation ⌬G ‡ T ϭ RT(ln(kT/h)-ln(k)) (17), using the kinetic parameters k cat /K m and k cat and the individual rates shown in Schemes 1 and 2 for TR78 and TR78⌬Hir, respectively. The rate constants of Schemes 1 and 2 were determined as follows: Association rates constants (k a ) for TR78 and TR78⌬Hir were directly determined in viscosity studies and correspond to k 1 and k 2 , respectively. The maximal cleavage rate (k cat ) of TR78 by thrombin is determined by both k 2 and k 3 , whereas it is equal to k 3 for TR78⌬Hir. As an initial estimate, we assumed that k 2 and k 3 contribute equivalently to k cat for TR78 (k 2 ϭ k 3 ϭ 116 s Ϫ1 ) and that the stickiness ratio (S r ϭ 0.12) is equal to k 2 /k Ϫ1 (k Ϫ1 ϭ 967 s Ϫ1 ). The remaining value, k Ϫ2 , for TR78 and TR78⌬Hir was determined by fitting the initial rate data (i.e. Fig. 3) to Michaelis-Menten equation where: K m ϭ (k 3 ϩ k Ϫ2 )/k 2 for TR78⌬Hir and k cat ϭ (k 2 k 3 )/(k 2 ϩ k 3 ϩ k Ϫ2 ), K m ϭ (k Ϫ1 k Ϫ2 ϩ k Ϫ1 k 3 ϩ k 2 k 3 )/(k 1 k 2 ϩ k 1 k 3 ϩ k 1 k Ϫ2 ) for TR78 (21), allowing the other parameters to float from their initial values. The thick lines represent the energetics for WT PAR1. The dotted lines represent the energetics for ⌬Hir PAR1. The concentration scale on the left depicts the energies of the initial substrate concentrations. ⌬G ‡ T values for H 2 N-LDPR-pNA (L) were determined using kinetic constants from Tables I and II. ⌬G ‡ T values for fibrinogen (F) were determined from k 1 and K d k 1 at a plasma concentration of 10 M (26, 34). Stopped-flow fluorescence studies resolved the process of association of hirulog to exosite I and the active site of thrombin into four separate steps (33). ⌬G ‡ T for hirulog (H) was determined using k 1 and k Ϫ1 from the four-step mechanism, and k 2 was approximated by the slowest of the last three steps (30 s Ϫ1 ).
platelets. Adding to the problem of low abundance, several lines of experimental evidence indicate that the PAR1 exodomain is conformationally mobile. For instance, thrombin-activated PAR1 is able to donate its tethered ligand to adjacent PAR1 receptors and activate them by an intermolecular liganding mechanism (30). Protease-susceptibility studies (19,31) and NMR structural data 3 also indicate that the PAR1 exodomain is quite flexible. Mobile PAR1 exodomains would be expected to form productive complexes more slowly with thrombin as a result of high entropic barriers due to enhanced rotational and translational degrees of freedom. Our data demonstrate that PAR1 uses the Hir motif in a "dock and lock" mechanism ( Fig. 4) to overcome the entropic and kinetic barriers leading to formation of productive thrombin-PAR1 complexes.
The dock and lock mechanism describes the two-step binding of PAR1 to thrombin. The mechanism is based in part on previous x-ray crystallographic (10) and kinetic studies (11,12), which showed that thrombin interacts with the PAR1 exodomain substrate at two distant and distinct sites. Thus, in addition to binding to the active site, TR78 is a noncompetitive inhibitor of CBS cleavage as a result of binding to exosite I of thrombin. Hirudin and a related peptide, hirulog, were shown by pre-steady-state kinetics to bind thrombin in an ordered mechanism, where the C-terminal region binds first to exosite I of thrombin followed by binding of the N-terminal region to the active site of thrombin (32,33).
Based on these observations, the interactions of the PAR1 exodomain with thrombin can be described most simply by a two-step binding model (Scheme 1). In contrast, the interaction of the Hir-deleted PAR1 exodomain (TR78⌬Hir) with thrombin is better described by a model comprised of a single binding event (Scheme 2), because this exodomain does not bind exosite I of thrombin. As shown in Scheme 1, TR78 rapidly associates with thrombin to form the Hir-docked complex. The docked exodomain is not sticky and dissociates 8-fold faster (k Ϫ1 ) back to free exodomain relative to proceeding forward (k 2 ) to the LDPR-locked complex. The rate of the locking step (k 2 ϭ 120 s Ϫ1 ), in which the LDPRSFL sequence binds to the corresponding S 4 -S 3 Ј subsites in the thrombin active site, is comparable to the rate of cleavage (k 3 ) of the Arg 41 -Ser 42 bond. Therefore, the overall rate-limiting steps (k cat ) of PAR1 exodomain proteolysis include both locking and cleavage events. In Schemes 1 and 2, the reverse rate (k Ϫ2 ϭ 0.2-0.3 s Ϫ1 ) back from the LDPRlocked complex is ϳ70to 600-fold slower than the k 2 rate of locking, which helps drive the reaction forward. Moreover, peptide bond cleavage (k 3 ) at Arg 41 -Ser 42 is allosterically enhanced by the docked Hir sequence.
The contribution of the Hir motif in reduction of these kinetic barriers is readily apparent when one examines the corresponding reaction kinetics for the TR78⌬Hir mutant (Scheme 2). Loss of the Hir motif causes a 73-fold reduction in the association rate (k 2 ) of the thrombin-TR78⌬Hir complex relative to the wild-type exodomain. The LDPR-bound complex must form without prior docking via a Hir motif. Once bound to the active site, thrombin cleaves the TR78⌬Hir mutant minus the allosteric benefit of Hir occupancy of exosite I, leading to a 5-fold drop in the rate of cleavage (k 3 ). The rate of dissociation back to free substrate and thrombin, however, is extremely slow (k Ϫ2 ϭ 0.3 s Ϫ1 ) as a reflection of the stickiness of the ⌬Hir mutant.
The energetics of thrombin association and cleavage of TR78 (WT), TR78⌬Hir (⌬Hir), and LDPR-pNA (L) are shown in Fig.  4. For comparison, the energetics of thrombin complexation with fibrinogen (F) and hirulog (H) are shown using data acquired under similar conditions (32,34). A Gibb's free energy scale for 1 nM to 1 M free substrate concentration is shown at the left of Fig. 4. This energy scale illustrates the advantage that fibrinogen has over PAR1 as a competing substrate for thrombin. Thus, the energy barrier to association and formation of a docked complex is at least 5.6 kcal/mol higher for PAR1 (ϳ1 nM) relative to fibrinogen (10 M) assuming equal partitioning of thrombin between the platelet surface and the fluid phase (29). Quite remarkably, the ⌬G ‡ T for the barrier to association of thrombin to TR78 and fibrinogen are nearly identical, suggesting similar docking mechanisms to their respective Hir-like sequences. Hirulog, on the other hand, associates 21-fold faster than TR78 and has a lower barrier to formation of the docked complex. Once docked to thrombin, WT TR78 lowers the barrier to formation of the LDPR-locked complex by starting from an elevated ground-state and by lowering the transition-state energy of locking. Deletion of the Hir sequence eliminates the first docking step and forces the ⌬Hir mutant to climb a much higher energy barrier to formation of the LDPR-bound complex. The locking step presents the highest energy barrier for both WT and ⌬Hir exodomains during the entire cleavage process. Subsequent steps of irreversible cleavage are energetically favorable and help drive the overall cleavage process forward.
Intriguingly, the cleaved exodomain product, TR62, which retains the Hir sequence, binds relatively tightly to thrombin (K ii ϭ 9.4 M) as a first indication that thrombin may remain transiently associated with cleaved PAR1 receptor on the surface of platelets. These persistent interactions might be employed to keep thrombin tethered to the platelet surface and sequestered from antithrombin molecules present in the fluid phase (␣ 2 -macroglobulin, heparin II cofactor, antithrombin III, and heparin). Too high of an affinity for the PAR1 Hir sequence might be detrimental and could seriously impair proteolysis of other thrombin substrates and further reduce thrombin turnover (35). Therefore, the macromolecular interactions of thrombin with PAR1 must strike a balance between the opposing demands of specificity and speed.