High resolution crystal structures of free thrombin in the presence of K(+) reveal the molecular basis of monovalent cation selectivity and an inactive slow form

Structural biology has recently advanced our understanding of the molecular mechanisms of activation and selectivity in monovalent cation activated enzymes. Here we report a 1.9 Angstrom resolution crystal structure of free thrombin, a Na(+) selective enzyme, in the presence of KCl. There are two molecules in the asymmetric unit, one with the cation site bound to K(+) and the other with this site free. The K(+)-bound form shows key differences compared with the Na(+)-bound structure that explain the different kinetics of activation. The cation-free form, on the other hand, assumes a conformation where the monovalent cation binding site is completely disordered, the S1 pocket is inaccessible to substrate and binding to exosite I is compromised by an unprecedented >20 Angstrom shift in the position of the autolysis loop. This form, named S(*), corresponds to the inactive Na(+)-free slow form identified by early kinetic studies. A simple model of thrombin allostery that incorporates the contribution of S(*) is proposed.     

Molecular dissection of Na+ binding to thrombin

Na(+) binding near the primary specificity pocket of thrombin promotes the procoagulant, prothrombotic, and signaling functions of the enzyme. The effect is mediated allosterically by a communication between the Na(+) site and regions involved in substrate recognition. Using a panel of 78 Ala mutants of thrombin, we have mapped the allosteric core of residues that are energetically linked to Na(+) binding. These residues are Asp-189, Glu-217, Asp-222, and Tyr-225, all in close proximity to the bound Na(+). Among these residues, Asp-189 shares with Asp-221 the important function of transducing Na(+) binding into enhanced catalytic activity. None of the residues of exosite I, exosite II, or the 60-loop plays a significant role in Na(+) binding and allosteric transduction. X-ray crystal structures of the Na(+)-free (slow) and Na(+)-bound (fast) forms of thrombin, free or bound to the active site inhibitor H-d-Phe-Pro-Arg-chloromethyl-ketone, document the conformational changes induced by Na(+) binding. The slow --> fast transition results in formation of the Arg-187:Asp-222 ion pair, optimal orientation of Asp-189 and Ser-195 for substrate binding, and a significant shift of the side chain of Glu-192 linked to a rearrangement of the network of water molecules that connect the bound Na(+) to Ser-195 in the active site. The changes in the water network and the allosteric core explain the thermodynamic signatures linked to Na(+) binding and the mechanism of thrombin activation by Na(+). The role of the water network uncovered in this study establishes a new paradigm for the allosteric regulation of thrombin and other Na(+)-activated enzymes involved in blood coagulation and the immune response.     

Crystal structure of thrombin in a self-inhibited conformation

The activating effect of Na(+) on thrombin is allosteric and depends on the conformational transition from a low activity Na(+)-free (slow) form to a high activity Na(+)-bound (fast) form. The structures of these active forms have been solved. Recent structures of thrombin obtained in the absence of Na(+) have also documented inactive conformations that presumably exist in equilibrium with the active slow form. The validity of these inactive slow form structures, however, is called into question by the presence of packing interactions involving the Na(+) site and the active site regions. Here, we report a 1.87A resolution structure of thrombin in the absence of inhibitors and salts with a single molecule in the asymmetric unit and devoid of significant packing interactions in regions involved in the allosteric slow --> fast transition. The structure shows an unprecedented self-inhibited conformation where Trp-215 and Arg-221a relocate >10A to occlude the active site and the primary specificity pocket, and the guanidinium group of Arg-187 penetrates the protein core to fill the empty Na(+)-binding site. The extreme mobility of Trp-215 was investigated further with the W215P mutation. Remarkably, the mutation significantly compromises cleavage of the anticoagulant protein C but has no effect on the hydrolysis of fibrinogen and PAR1. These findings demonstrate that thrombin may assume an inactive conformation in the absence of Na(+) and that its procoagulant and anticoagulant activities are closely linked to the mobility of residue 215.     

Crystal structure of the thrombin mutant D221A/D222K: the Asp222:Arg187 ion-pair stabilizes the fast form

The thrombin mutant D221A/D222K (ARK) does not bind Na+ and has interesting functional properties intermediate between those of the slow and fast forms of wild type. We solved the X-ray crystal structure of ARK bound at exosite I with a fragment of hirudin at 2.4-A resolution. The structure shows a slight collapse of the 186 and 220 loops into the Na+ binding site due to disruption of the Asp222:Arg187 ion-pair. The backbone O atoms of Arg221a and Lys224 are shifted into conformations that eliminate optimal interaction with Na+. A paucity of solvent molecules in the Na+ binding site is also noted, by analogy to what is seen in the structure of the slow form. These findings reinforce the crucial role of the Asp222:Arg187 ion-pair in stabilizing the fast form of thrombin.     

Sodium ions as the effector of catalytic action of alpha-thrombin

A process of thrombin interaction with synthetic and natural substrates in the presence of Na+ ions has been analyzed in the survey. Molecular bases of this interaction have been presented, interrelation between the structure and function of thrombin has been noted; the nature of the unique site of its active centre which determines high thrombin affinity for the substrates and increase of its catalytic activity defined by the term of "specificity to univalent cations" have been considered in detail. Na+ ions play the role of allosteric effector in realization of two informational states of thrombin which penform, respectively, two fundamental and competing functions in the process of hemostasis. The molecular basis of the process of Na+ binding with thrombin is rather simple and depends only on the single site which importance for the enzyme function is marked by numerous investigations of a number of authors, and it is shown that Na(+)-binding site is distributed in the other zone of thrombin molecule as compared to exosites I and II, which do not take part in Na(+)-binding and allosteric transduction. Considerable attention was given to conformational conversions of a thrombin molecule caused by Na+ ions binding. It was shown that the transition slow <--> fast of the enzyme forms leads to formation of the ion pair Arg-187: Asp-222, optimal orientation of Asp-189 and Ser-195 for binding of substrates and considerable shift of the lateral chain Glu-192 determined by the disturbance of the lattice of water molecules which connects Na(+)-binding site with aminoacid Ser-195 of the active centre of the enzyme. New data have been presented which indicate that the changes in the lattice of water molecules and allosteric nucleus of Na(+)-binding site of the enzyme are the basic link of raising the affinity between the thrombin and substrate and mechanism of the enzyme activation by Na(+)-ions. The survey touches some problems of creation of allosteric inhibitors of thrombin which can take essential effect on Na(+)-binding site and favor stabilization of the anticoagulant slow-form of thrombin, and of enzyme rational mutants with selective specificity in respect of protein C which display effective and safe anticoagulant and antithrombotic effects in vivo.     

Hirudin binding reveals key determinants of thrombin allostery

Thrombin exists in two allosteric forms, slow (S) and fast (F), that recognize natural substrates and inhibitors with significantly different affinities. Because under physiologic conditions the two forms are almost equally populated, investigation of thrombin function must address the contribution from the S and F forms and the molecular origin of their differential recognition of ligands. Using a panel of 79 Ala mutants, we have mapped for the first time the epitopes of thrombin recognizing a macromolecular ligand, hirudin, in the S and F forms. Hirudin binding is a relevant model for the interaction of thrombin with fibrinogen and PAR1 and is likewise influenced by the allosteric S-->F transition. The epitopes are nearly identical and encompass two hot spots, one in exosite I and the other in the Na+ site at the opposite end of the protein. The higher affinity of the F form is due to the preferential interaction of hirudin with Lys-36, Leu-65, Thr-74, and Arg-75 in exosite I; Gly-193 in the oxyanion hole; and Asp-221 and Asp-222 in the Na+ site. Remarkably, no correlation is found between the energetic and structural involvements of thrombin residues in hirudin recognition, which invites caution in the analysis of protein-protein interactions in general.     

Expression of allosteric linkage between the sodium ion binding site and exosite I of thrombin during prothrombin activation

The specificity of thrombin for procoagulant and anticoagulant substrates is regulated allosterically by Na+. Ordered cleavage of prothrombin (ProT) at Arg320 by the prothrombinase complex generates proteolytically active, meizothrombin (MzT), followed by cleavage at Arg271 to produce thrombin and fragment 1.2. The alternative pathway of initial cleavage at Arg271 produces the inactive zymogen form, the prethrombin 2 (Pre 2).fragment 1.2 complex, which is cleaved subsequently at Arg320. Cleavage at Arg320 of ProT or prethrombin 1 (Pre 1) activates the catalytic site and the precursor form of exosite I (proexosite I). To determine the pathway of expression of Na+-(pro)exosite I linkage during ProT activation, the effects of Na+ on the affinity of fluorescein-labeled hirudin-(54-65) ([5F]Hir-(54-65)(SO-3)) for the zymogens, ProT, Pre 1, and Pre 2, and for the proteinases, MzT and MzT-desfragment 1 (MzT(-F1)) were quantitated. The zymogens showed no significant linkage between proexosite I and Na+, whereas cleavage at Arg320 caused the affinities of MzT and MzT(-F1) for [5F]Hir-(54-65)(SO-3) to be enhanced by Na+ 8- to 10-fold and 5- to 6-fold, respectively. MzT and MzT(-F1) showed kinetically different mechanisms of Na+ enhancement of chromogenic substrate hydrolysis. The results demonstrate for the first time that MzT is regulated allosterically by Na+. The results suggest that the distinctive procoagulant substrate specificity of MzT, in activating factor V and factor VIII on membranes, and the anticoagulant, membrane-modulated activation of protein C by MzT bound to thrombomodulin are regulated by Na+-induced allosteric transition. Further, the Na+ enhancement in MzT activity and exosite I affinity may function in directing the sequential ProT activation pathway by accelerating thrombin formation from the MzT fast form.     

Thrombin inhibition by serpins disrupts exosite II

Thrombin uses three principal sites, the active site, exosite I, and exosite II, for recognition of its many cofactors and substrates. It is synthesized in the zymogen form, prothrombin, and its activation at the end of the blood coagulation cascade results in the formation of the active site and exosite I and the exposure of exosite II. The physiological inhibitors of thrombin are all serpins, whose mechanism involves significant conformational change in both serpin and protease. It has been shown that the formation of the thrombin-serpin final complex disorders the active site and exosite I of thrombin, but exosite II is thought to remain functional. It has also been hypothesized that thrombin contains a receptor-binding site that is exposed upon final complex formation. The position of this cryptic site may depend on the regions of thrombin unfolded by serpin complexation. Here we investigate the conformation of thrombin in its final complex with serpins and find that in addition to exosite I, exosite II is also disordered, as reflected by a loss of affinity for the γ'-peptide of fibrinogen and for heparin and by susceptibility to limited proteolysis. This disordering of exosite II occurs for all tested natural thrombin-inhibiting serpins. Our data suggest a novel framework for understanding serpin function, especially with respect to thrombin inhibition, where serpins functionally "rezymogenize" proteases to ensure complete loss of activity and cofactor binding.     

An extensive interaction interface between thrombin and factor V is required for factor V activation

The interaction interface between human thrombin and human factor V (FV), necessary for complex formation and cleavage to generate factor Va, was investigated using a site-directed mutagenesis strategy. Fifty-three recombinant thrombins, with a total of 78 solvent-exposed basic and polar residues substituted with alanine, were used in a two-stage clotting assay with human FV. Seventeen mutants with less than 50% of wild-type (WT) thrombin FV activation were identified and mapped to anion-binding exosite I (ABE-I), anion-binding exosite II (ABE-II), the Leu(45)-Asn(57) insertion loop, and the Na(+) binding loop of thrombin. Three ABE-I mutants (R68A, R70A, and Y71A) and the ABE-II mutant R98A had less than 30% of WT activity. The thrombin Na(+) binding loop mutants, E229A and R233A, and the Leu(45)-Asn(57) insertion loop mutant, W50A, had a major effect on FV activation with 5, 15, and 29% of WT activity, respectively. The K52A mutant, which maps to the S' specificity pocket, had 29% of WT activity. SDS-polyacrylamide gel electrophoresis analysis of cleavage reactions using the thrombin ABE mutants R68A, Y71A, and R98A, the Na(+) binding loop mutant E229A, and the Leu(45)-Asn(57) insertion loop mutant W50A showed a requirement for both ABEs and the Na(+)-bound form of thrombin for efficient cleavage at the FV residue Arg(709). Several basic residues in both ABEs have moderate decreases in FV activation (40-60% of WT activity), indicating a role for the positive electrostatic fields generated by both ABEs in enhancing complex formation with complementary negative electrostatic fields generated by FV. The data show that thrombin activation of FV requires an extensive interaction interface with thrombin. Both ABE-I and ABE-II and the S' subsite are required for optimal cleavage, and the Na(+)-bound form of thrombin is important for its procoagulant activity.     

Synergistic effect of aptamers that inhibit exosites 1 and 2 on thrombin

Thrombin is a multifunctional protease that plays a key role in hemostasis, thrombosis, and inflammation. Most thrombin inhibitors currently used as antithrombotic agents target thrombin''s active site and inhibit all of its myriad of activities. Exosites 1 and 2 are distinct regions on the surface of thrombin that provide specificity to its proteolytic activity by mediating binding to substrates, receptors, and cofactors. Exosite 1 mediates binding and cleavage of fibrinogen, proteolytically activated receptors, and some coagulation factors, while exosite 2 mediates binding to heparin and to platelet receptor GPIb-IX-V. The crystal structures of two nucleic acid ligands bound to thrombin have been solved. Previously Padmanabhan and colleagues solved the structure of a DNA aptamer bound to exosite 1 and we reported the structure of an RNA aptamer bound to exosite 2 on thrombin. Based upon these structural studies we speculated that the two aptamers would not compete for binding to thrombin. We observe that simultaneously blocking both exosites with the aptamers leads to synergistic inhibition of thrombin-dependent platelet activation and procoagulant activity. This combination of exosite 1 and exosite 2 inhibitors may provide a particularly effective antithrombotic approach.     

Thrombin

Thrombin is a Na+-activated, allosteric serine protease that plays opposing functional roles in blood coagulation. Binding of Na+ is the major driving force behind the procoagulant, prothrombotic and signaling functions of the enzyme, but is dispensable for cleavage of the anticoagulant protein C. The anticoagulant function of thrombin is under the allosteric control of the cofactor thrombomodulin. Much has been learned on the mechanism of Na+ binding and recognition of natural substrates by thrombin. Recent structural advances have shed light on the remarkable molecular plasticity of this enzyme and the molecular underpinnings of thrombin allostery mediated by binding to exosite I and the Na+ site. This review summarizes our current understanding of the molecular basis of thrombin function and allosteric regulation. The basic information emerging from recent structural, mutagenesis and kinetic investigation of this important enzyme is that thrombin exists in three forms, E*, E and E:Na+, that interconvert under the influence of ligand binding to distinct domains. The transition between the Na+ -free slow from E and the Na+ -bound fast form E:Na+ involves the structure of the enzyme as a whole, and so does the interconversion between the two Na+ -free forms E* and E. E* is most likely an inactive form of thrombin, unable to interact with Na + and substrate. The complexity of thrombin function and regulation has gained this enzyme pre-eminence as the prototypic allosteric serine protease. Thrombin is now looked upon as a model system for the quantitative analysis of biologically important enzymes.     

Crystal structure of thrombin in complex with fibrinogen gamma' peptide

Elevated levels of heterodimeric gamma(A)/gamma' fibrinogen 2 have been associated with an increased incidence of coronary artery disease, whereas a lowered content of gamma' chains is associated with an increased risk of venous thrombosis. Both situations may be related to the unique features of thrombin binding to variant gamma' chains. The gamma' peptide is an anionic fragment that binds thrombin with high affinity without interfering directly with substrate binding. Here we report the crystal structure of thrombin bound to the gamma' peptide, solved at 2.4 A resolution. The complex reveals extensive interactions between thrombin and the gamma' peptide mediated by electrostatic contacts with residues of exosite II and hydrophobic interactions with a pocket in close proximity to the Na(+) binding site. In its binding mode, the gamma' peptide completely overlaps with heparin bound to exosite II. These findings are consistent with functional data and broaden our understanding of how thrombin interacts with fibrinogen at the molecular level.     

Binding of exosite ligands to human thrombin. Re-evaluation of allosteric linkage between thrombin exosites I and II.

The substrate specificity of thrombin is regulated by binding of macromolecular substrates and effectors to exosites I and II. Exosites I and II have been reported to be extremely linked allosterically, such that binding of a ligand to one exosite results in near-total loss of affinity for ligands at the alternative exosite, whereas other studies support the independence of the interactions. An array of fluorescent thrombin derivatives and fluorescein-labeled hirudin(54-65) ([5F]Hir(54-65)(SO(3)(-))) were used as probes in quantitative equilibrium binding studies to resolve whether the affinities of the exosite I-specific ligands, Hir(54-65)(SO(3)(-)) and fibrinogen, and of the exosite II-specific ligands, prothrombin fragment 2 and a monoclonal antibody, were affected by alternate exosite occupation. Hir(54-65)(SO(3)(-)) and fibrinogen bound to exosite I with dissociation constants of 16-28 nm and 5-7 microm, respectively, which were changed < or =2-fold by fragment 2 binding. Native thrombin and four thrombin derivatives labeled with different probes bound fragment 2 and the antibody with dissociation constants of 3-12 microm and 1.8 nm, respectively, unaffected by Hir(54-65)(SO(3)(-)). The results support a ternary complex binding model in which exosites I and II can be occupied simultaneously. The thrombin catalytic site senses individual and simultaneous binding of exosite I and II ligands differently, resulting in unique active site environments for each thrombin complex. The results indicate significant, ligand-specific allosteric coupling between thrombin exosites I and II and catalytic site perturbations but insignificant inter-exosite thermodynamic linkage.     

PAR-3 is a low-affinity substrate,high affinity effector of thrombin

A polypeptide corresponding to the extracellular domain of protease-activated receptor 3 (PAR-3) is hydrolyzed by thrombin slowly because of high K(M) (>100 microM). However, thrombin is found to bind two PAR-3, one without catalyzing hydrolysis or blocking the active site, while the other is hydrolyzed. In a solvent lacking Na(+), hydrolysis of a nitroanilide substrate is enhanced 1.6-fold by addition of PAR-3 polypeptide, with half-saturation at 2.5 microM. In contrast, the fibrinogen clotting activity of thrombin is inhibited completely by PAR-3, with a K(I) of 3 microM. None of the activities of thrombin are affected by addition of 50 microM PAR-4 polypeptide. Thus, PAR-3 in low concentrations binds thrombin in a configuration that blocks the anion-binding exosite but not the catalytic site, while hydrolysis of PAR-3, PAR-4, and other substrates that do not interact with exosite I persists. The allosteric effect of PAR-3 is characteristic of that of Na(+).     

Synthesis,structure, and structure-activity relationships of divalent thrombin inhibitors containing an α-keto-amide transition-state mimetic

A new class of divalent thrombin inhibitors is described that contains an α-keto-amide transition-state mimetic linking an active site binding group and a group that binds to the fibrinogen-binding exosite. The X-ray crystallographic structure of the most potent member of this new class, CVS995, shows many features in common with other divalent thrombin inhibitors and clearly defines the transition-state-like binding of the α-keto-amide group. The structure of the active site part of the inhibitor shows a network of water molecules connecting both the side-chain and backbone atoms of thrombin and the inhibitor. Direct peptide analogues of the new transition-state-containing divalent thrombin inhibitors were compared using in vitro assays of thrombin inhibition. There was no direct correlation between the binding constants of the peptides and their α-keto-amide counterparts. The most potent cv-keto-amide inhibitor, CVS995, with a K_i = 1 pM, did not correspond to the most potent divalent peptide and contained a single amino acid deletion in the exosite binding region with respect to the equivalent region of the natural thrombin inhibitor hirudin. The interaction energies of the active site, transition state, and exosite binding regions of these new divalent thrombin inhibitors are not additive.     

The reaction of thrombin with platelet-derived nexin requires a secondary recognition site in addition to the catalytic site

A protease nexin released by activated platelets forms stable complexes with alpha-thrombin. Active-site-blocked thrombin does not form the stable complex, but it inhibits formation of the stable complex by active alpha-thrombin. gamma-Thrombin, which has a damaged substrate recognition site (the anion-binding exosite), did not form the complex and did not inhibit formation of the stable complex by alpha-thrombin. Complex formation was inhibited by the C-terminal dodecapeptide of hirudin, which has been shown to bind to the anion-binding exosite. A monoclonal antibody that blocks reactions of thrombin that involve the anion-binding exosite also inhibited formation of a stable complex of alpha-thrombin and the platelet-derived protease nexin. It is concluded that the anion-binding exosite of thrombin, a site that confers a high degree of specificity for substrates with a complementary site, binds to the platelet nexin prior to reaction of the catalytic site with the serpin.     

Effects of activation peptide bond cleavage and fragment 2 interactions on the pathway of exosite I expression during activation of human prethrombin 1 to thrombin

Activation of prothrombin (Pro) by factor Xa to form thrombin occurs by proteolysis of Arg271-Thr272 and Arg320-Ile321, resulting in expression of regulatory exosites I and II. Cleavage of Pro by thrombin liberates fragment 1 and generates the zymogen analog, prethrombin 1 (Pre 1). The properties of exosite I on Pre 1 and its factor Xa activation intermediates were characterized in spectroscopic and equilibrium binding studies using the fluorescein-labeled probe, hirudin(54-65) ([5F]Hir(54-65)-(SO3-)). Prethrombin 2 (Pre 2), formed by factor Xa cleavage of Pre 1 at Arg271-Thr272, had the same affinity for hirudin(54-65) peptides as Pre 1 in the absence or presence of near-saturating fragment 2 (F2). Pre 2 and thrombin also had indistinguishable affinities for F2. By contrast, cleavage of Pre 1 at Arg320-Ile321, to form active meizothrombin des-fragment 1 MzT(-F1), showed a 11- to 20-fold increase in affinity for hirudin(54-65), indistinguishable from the 13- to 20-fold increase seen for conversion of Pre 2 to thrombin. Thus, factor Xa cleavage of Pre 1 at Arg271-Thr272 does not effect exosite I expression, whereas cleavage at Arg320-Ile321 results in concomitant activation of the catalytic site and exosite I. Furthermore, expression of exosite I on the Pre 1 activation intermediates is not modulated by F2, and exosite II is not activated conformationally. The differential expression of exosite I affinity on the Pre 1 activation intermediates and the previously demonstrated role of (pro)exosite I in factor Va-dependent substrate recognition suggest that changes in exosite I expression may regulate the rate and direction of the Pre 1 activation pathway.     

Rapid kinetics of Na+ binding to thrombin

The kinetic mechanism of Na(+) binding to thrombin was resolved by stopped-flow measurements of intrinsic fluorescence. Na(+) binds to thrombin in a two-step mechanism with a rapid phase occurring within the dead time of the spectrometer (<0.5 ms) followed by a single-exponential slow phase whose k(obs) decreases hyperbolically with increasing [Na(+)]. The rapid phase is due to Na(+) binding to the enzyme E to generate the E:Na(+) form. The slow phase is due to the interconversion between E(*) and E, where E(*) is a form that cannot bind Na(+). Temperature studies in the range from 5 to 35 degrees C show significant enthalpy, entropy, and heat capacity changes associated with both Na(+) binding and the E to E(*) transition. As a result, under conditions of physiologic temperature and salt concentrations, the E(*) form is negligibly populated (<1%) and thrombin is almost equally partitioned between the E (40%) and E:Na(+) (60%) forms. Single-site Phe mutations of all nine Trp residues of thrombin enabled assignment of the fluorescence changes induced by Na(+) binding mainly to Trp-141 and Trp-215, and to a lesser extent to Trp-148, Trp-207, and Trp-237. However, the fast phase of fluorescence increase is influenced to different extents by all Trp residues. The distribution of these residues over the entire thrombin surface demonstrates that Na(+) binding induces long-range effects on the structure of the enzyme as a whole, contrary to the conclusions drawn from recent structural studies. These findings elucidate the mechanism of Na(+) binding to thrombin and are relevant to other clotting factors and enzymes allosterically activated by monovalent cations.     

Crystal structure of anticoagulant thrombin variant E217K provides insights into thrombin allostery

Thrombin is the ultimate protease of the blood clotting cascade and plays a major role in its own regulation. The ability of thrombin to exhibit both pro- and anti-coagulant properties has spawned efforts to turn thrombin into an anticoagulant for therapeutic purposes. This quest culminated in the identification of the E217K variant through scanning and saturation mutagenesis. The antithrombotic properties of E217K thrombin are derived from its inability to convert fibrinogen to a fibrin clot while maintaining its thrombomodulin-dependent ability to activate the anticoagulant protein C pathway. Here we describe the 2.5-A crystal structure of human E217K thrombin, which displays a dramatic restructuring of the geometry of the active site. Of particular interest is the repositioning of Glu-192, which hydrogen bonds to the catalytic Ser-195 and which results in the complete occlusion of the active site and the destruction of the oxyanion hole. Substrate binding pockets are further blocked by residues previously implicated in thrombin allostery. We have concluded that the E217K mutation causes the allosteric inactivation of thrombin by destabilizing the Na(+) binding site and that the structure thus may represent the Na(+)-free, catalytically inert "slow" form.     

Murine thrombin lacks Na+ activation but retains high catalytic activity

Human thrombin utilizes Na+ as a driving force for the cleavage of substrates mediating its procoagulant, prothrombotic, and signaling functions. Murine thrombin has Asp-222 in the Na+ binding site of the human enzyme replaced by Lys. The charge reversal substitution abrogates Na+ activation, which is partially restored with the K222D mutation, and ensures high activity even in the absence of Na+. This property makes the murine enzyme more resistant to the effect of mutations that destabilize Na+ binding and shift thrombin to its anticoagulant slow form. Compared with the human enzyme, murine thrombin cleaves fibrinogen and protein C with similar k(cat)/K(m) values but activates PAR1 and PAR4 with k(cat)/K(m) values 4- and 26-fold higher, respectively. The significantly higher specificity constant toward PAR4 accounts for the dominant role of this receptor in platelet activation in the mouse. Murine thrombin can also cleave substrates carrying Phe at P1, which potentially broadens the repertoire of molecular targets available to the enzyme in vivo.