Regulation of the catalytic function of coagulation factor VIIa by a conformational linkage of surface residue Glu 154 to the active site

Coagulation factor VIIa is an allosterically regulated trypsin-like serine protease that initiates the coagulation pathways upon complex formation with its cellular receptor and cofactor tissue factor (TF). The analysis of a conformation-sensitive monoclonal antibody directed to the macromolecular substrate exosite in the VIIa protease domain demonstrated a conformational link from this exosite to the catalytic cleft that is independent of cofactor-induced allosteric changes. In this study, we identify Glu 154 as a critical surface-exposed exosite residue side chain that undergoes conformational changes upon active site inhibitor binding. The Glu 154 side chain is important for hydrolysis of scissile bond mimicking peptidyl p-nitroanilide substrates, and for inhibition of VIIa's amidolytic function upon antibody binding. This exosite residue is not linked to the catalytic cleft residue Lys 192 which plays an important role in thrombin's allosteric coupling to exosite I. Allosteric linkages between VIIa's active site and the cofactor binding site or between the cofactor binding site and the macromolecular substrate exosite were not influenced by mutation of Glu 154. Glu 154 thus only influences the linkage of the macromolecular substrate binding exosite to the catalytic center. These data provide novel evidence that allosteric regulation of VIIa's catalytic function involves discrete and independent conformational linkages and that allosteric transitions in the VIIa protease domain are not globally coupled.     

Platelet glycoprotein Ib alpha binds to thrombin anion-binding exosite II inducing allosteric changes in the activity of thrombin

The glycoprotein (GP) Ib-IX complex is a platelet surface receptor that binds thrombin as one of its ligands, although the biological significance of thrombin interaction remains unclear. In this study we have used several approaches to investigate the GPIb alpha-thrombin interaction in more detail and to study its effect on the thrombin-induced elaboration of fibrin. We found that both glycocalicin and the amino-terminal fragment of GPIb alpha reduced the release of fibrinopeptide A from fibrinogen by about 50% by a noncompetitive allosteric mechanism. Similarly, GPIb alpha caused in thrombin an allosteric reduction in the rate of turnover of the small peptide substrate d-Phe-Pro-Arg-pNA. The K(d) for the glycocalicin-thrombin interaction was 1 microm at physiological ionic strength but was highly salt-dependent, decreasing to 0.19 microm at 100 mm NaCl (Gamma(salt) = -4.2). The salt dependence was characteristic of other thrombin ligands that bind to exosite II of this enzyme, and we confirmed this as the GPIb alpha-binding site on thrombin by using thrombin mutants and by competition binding studies. R68E or R70E mutations in exosite I of thrombin had little effect on its interaction with GPIb alpha. Both the allosteric inhibition of fibrinogen turnover caused by GPIb alpha binding to these mutants, and the K(d) values for their interactions with GPIb alpha were similar to those of wild-type thrombin. In contrast, R89E and K248E mutations in exosite II of thrombin markedly increased the K(d) values for the interactions of these thrombin mutants with GPIb alpha by 10- and 25-fold, respectively. Finally, we demonstrated that low molecular weight heparin (which binds to thrombin exosite II) but not hirugen (residues 54-65 of hirudin, which binds to exosite I of thrombin) inhibited thrombin binding to GPIb alpha. These data demonstrate that GPIb alpha binds to thrombin exosite II and in so doing causes a conformational change in the active site of thrombin by an allosteric mechanism that alters the accessibility of both its natural substrate, fibrinogen, and the small peptidyl substrate d-Phe-Pro-Arg-pNA.     

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.     

Allosteric changes in solvent accessibility observed in thrombin upon active site occupation

The solvent accessibility of thrombin in its substrate-free and substrate-bound forms has been compared by amide hydrogen/deuterium (H/(2)H) exchange. The optimized inhibitor peptide dPhe-Pro-Arg chloromethyl ketone (PPACK) was used to simulate the substrate-bound form of thrombin. These studies were motivated by the lack of observed changes in the active site of thrombin in the crystal structure of the thrombin-thrombomodulin complex. This result appeared to contradict amide exchange studies on the thrombin-thrombomodulin complex that suggested subtle changes occur in the active site loops upon thrombomodulin binding. Our results show that two active site loops, residues 214-222 and residues 126-132, undergo decreases in solvent accessibility due to steric contacts with PPACK substrate. However, we also observe two regions outside the active site undergoing solvent protection upon substrate binding. The first region corresponds to anion binding exosite 1, and the second is a beta-strand-containing loop which runs through the core of the molecule and contains Trp141 which makes critical contacts with anion binding exosite 1. These results indicate two pathways of allosteric change that connect the active site to the distal anion binding exosite 1.     

Allosteric changes in thrombin's activity produced by peptides corresponding to segments of natural inhibitors and substrates.

Acidic synthetic peptides corresponding to segments of several nonhomologous proteins (hirudin, residues 54-65; heparin cofactor II, residues 54-75; and fibrinogen, residues 410-427 of the gamma B-chain) inhibit thrombin's cleavage of fibrinogen without blocking the enzyme's active site. Here, we examined effects of these peptides on thrombin's cleavage of protein C and small peptides. Activation of protein C by thrombin in the absence of calcium was inhibited by all of the peptides. Maximal inhibition was 60%, and no greater inhibition was produced by higher peptide concentrations. This differed from progressive inhibition of protein C activation by increasing peptide concentrations in the presence of thrombomodulin and calcium. Potencies of the peptides were in the order hirudin-(54-65) greater than heparin cofactor II-(54-75) greater than gamma B-chain-(410-427). Sulfation of the tyrosine residue in hirudin-(54-65) increased its potency about 10-fold, similar to changes in anticlotting activity. The peptides were activators rather than inhibitors of the cleavage of small chromogenic substrates. In the presence of the peptides, the affinity of thrombin for the substrates S-2366 (pyro-Glu-Pro-Arg-4-nitroanilide), Chromozyme TH (tosyl-Gly-Pro-Arg-4-nitroanilide), and S-2251 (D-Val-Leu-Lys-4-nitroanilide) increased 1.5-2-fold with little change in the Vmax of substrate cleavage. Potencies of peptides in these allosteric effects on thrombin was in the same order as for their other effects. The similar actions of these nonhomologous peptides, which are believed to bind to thrombin's anion-binding exosite, suggest that binding of any peptide to this site exerts the same allosteric effect on thrombin's active site. Interactions of these peptides with thrombin may serve as models for regulation of thrombin's interactions with natural substrates and inhibitors.     

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.     

Role of exosites 1 and 2 in thrombin reaction with plasminogen activator inhibitor-1 in the absence and presence of cofactors.

The cofactors heparin, vitronectin (VN), and thrombomodulin (TM) modulate the reactivity of alpha-thrombin with plasminogen activator inhibitor (PAI-1). While heparin and VN accelerate the reaction by approximately 2 orders of magnitude, TM protects alpha-thrombin from rapid inactivation by PAI-1 in the presence of VN. To understand how these cofactors function, we studied the kinetics of PAI-1 inactivation of alpha-thrombin, the exosite 1 variant gamma-thrombin, the exosite 2 mutant R93,97,101A thrombin, and recombinant meizothrombin in both the absence and presence of these cofactors. Heparin and VN accelerated the second-order association rate constant [k(2) = (7.9 +/- 0.5) x 10(2) M(-)(1) s(-)(1)] of alpha-thrombin with PAI-1 approximately 200- and approximately 240-fold, respectively. The k(2) value for gamma-thrombin [(7.9 +/- 0.7) x 10(1) M(-)(1) s(-)(1)] was impaired 10-fold, but was enhanced by heparin and VN approximately 280- and approximately 75-fold, respectively. Similar to inactivation of gamma-thrombin, PAI-1 inactivation of alpha-thrombin in complex with the epidermal growth factor-like domains 4-6 of TM (TM4-6) was impaired approximately 10-fold. The exosite 2 mutant R93,97,101A thrombin, which was previously shown not to bind heparin, and meizothrombin, in which exosite 2 is masked, reacted with PAI-1 at similar rates in both the absence and presence of heparin [k(2) = (1.3-1.5) x 10(3) M(-)(1) s(-)(1) for R93,97,101A thrombin and k(2) = (3.6-5.1) x 10(2) M(-)(1) s(-)(1) for meizothrombin]. Unlike heparin, however, VN enhanced the k(2) of R93,97,101A thrombin and meizothrombin inactivation approximately 80- and approximately 30-fold, respectively. Continuous kinetic analysis as well as competition kinetic studies in the presence of S195A thrombin suggested that the accelerating effect of VN or heparin occurs primarily by lowering the dissociation constant (K(d)) for formation of a noncovalent, Michaelis-type complex. Analysis of these results suggest that (1) heparin binds to exosite 2 of alpha-thrombin to accelerate the reaction by a template mechanism, (2) VN accelerates PAI-1 inactivation of alpha-thrombin by lowering the K(d) for initial complex formation by an unknown mechanism that does not require binding to either exosite 1 or exosite 2 of alpha-thrombin, (3) alpha-thrombin may have a binding site for PAI-1 within or near exosite 1, and (4) TM occupancy of exosite 1 partially accounts for the protection of thrombin from rapid inactivation by PAI-1 in the presence of vitronectin.     

Long Range Communication between Exosites 1 and 2 Modulates Thrombin Function

Although exosites 1 and 2 regulate thrombin activity by binding substrates and cofactors and by allosterically modulating the active site, it is unclear whether there is direct allosteric linkage between the two exosites. To begin to address this, we first titrated a thrombin variant fluorescently labeled at exosite 1 with exosite 2 ligands, HD22 (a DNA aptamer), γ′-peptide (an analog of the COOH terminus of the γ′-chain of fibrinogen) or heparin. Concentration-dependent and saturable changes in fluorescence were elicited, supporting inter-exosite linkage. To explore the functional consequences of this phenomenon, we evaluated the capacity of exosite 2 ligands to inhibit thrombin binding to γ_A/γ_A-fibrin, an interaction mediated solely by exosite 1. When γ_A/γ_A-fibrinogen was clotted with thrombin in the presence of HD22, γ′-peptide, or prothrombin fragment 2 there was a dose-dependent and saturable decrease in thrombin binding to the resultant fibrin clots. Furthermore, HD22 reduced the affinity of thrombin for γ_A/γ_A-fibrin 6-fold and accelerated the dissociation of thrombin from preformed γ_A/γ_A-fibrin clots. Similar responses were obtained when surface plasmon resonance was used to monitor the interaction of thrombin with γ_A/γ_A-fibrinogen or fibrin. There is bidirectional communication between the exosites, because exosite 1 ligands, HD1 (a DNA aptamer) or hirudin-(54–65) (an analog of the COOH terminus of hirudin), inhibited the exosite 2-mediated interaction of thrombin with immobilized γ′-peptide. These findings provide evidence for long range allosteric linkage between exosites 1 and 2 on thrombin, revealing further complexity to the mechanisms of thrombin regulation.As the central effector of hemostasis, thrombin is engaged in procoagulant, anticoagulant, and fibrinolytic processes. These seemingly contrasting roles are regulated, at least in part, by thrombin''s interactions with other factors in the blood and vasculature. The binding of ligands to thrombin is promoted by exosites 1 and 2, which are positively charged domains that flank the active site. These exosites facilitate the binding of substrates or cofactors and align them for optimal interaction with the active site (1).Exosite 1 is predominantly used to gain access to the active site by substrates such as fibrinogen (2), factors V (3) and VIII (4), and the protease-activated receptors (PARs)² on platelets (5). Effectors that modulate thrombin activity, including thrombomodulin (6), hirudin (7), and heparin cofactor II (8), also utilize exosite 1. Thrombomodulin alters the specificity of thrombin by hindering access of other substrates to exosite 1 (9) and by providing new binding sites for protein C and thrombin-activable fibrinolysis inhibitor, thereby promoting anticoagulant and antifibrinolytic pathways, respectively (10, 11). Fewer processes are mediated by exosite 2, which serves largely as a tether that anchors thrombin for participation in other reactions. Thus, heparin binds exosite 2 (12) and catalyzes thrombin inhibition by antithrombin and heparin cofactor II (13, 14). Exosite 2 also is used by glycoprotein 1bα on platelets to localize thrombin for activation of PARs (15–17).Although the prevailing role of the exosites is to bring substrates and cofactors into proximity with thrombin, there is evidence that the exosites also serve as allosteric regulators of thrombin activity. Crystallographic studies reveal that, when peptides derived from PAR1 or PAR3 are bound to exosite 1 on thrombin, an obstructing surface loop moves out of the active site pocket, thereby providing access to substrates (18). The binding of a thrombomodulin fragment to exosite 1 was shown to alter the environment of an active site fluorescent probe (19), which accelerates the rate of protein C and thrombin-activable fibrinolysis inhibitor activation in an allosteric fashion. In contrast, exosite 1-binding peptides from heparin cofactor II or fibrinogen decrease the rate of protein C activation (20). Additionally, the binding of ligands to exosite 1 alters the rates of chromogenic substrate hydrolysis (21, 22). Allosteric effects are not limited to exosite 1, because prothrombin fragment 2 (F2), a cleavage product of prothrombin, binds exosite 2 and decreases the rate at which thrombin converts fibrinogen to fibrin (23, 24) and is inhibited by antithrombin (25, 26). In support of the concept that these alterations are allosteric in origin, fluorescent probes bound to the active site of thrombin undergo a change in fluorescence intensity when exosite 2 is occupied (24, 27).Although there is good evidence for allosteric regulation of the active site by the exosites, it remains unclear whether there is direct allosteric connection between the exosites. Reciprocal effects between exosites 1 and 2 have been observed by some investigators (28–30), but not by others (25, 31). The aim of the current study was to use different techniques and additional ligands to resolve this controversy. First, we examined the effect of exosite 2-directed ligands on the fluorescence intensity of a thrombin variant that was labeled in exosite 1. Next, we examined the effect of these ligands on thrombin binding to fibrin. To exploit the observation that thrombin binds γ_A/γ_A-fibrinogen exclusively via exosite 1 (2, 32), leaving exosite 2 accessible, this subpopulation was isolated (32). We used intact fibrin clots and surface plasmon resonance (SPR) to examine the influence of exosite 2-directed ligands on thrombin binding to γ_A/γ_A-fibrin. In addition, diffusion studies were performed to examine the effect of exosite-directed ligands on the rate of thrombin dissociation from preformed fibrin clots. Finally, we explored whether exosite 1-directed ligands modulate the binding of thrombin to an exosite 2-directed ligand.     

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.     

Antithrombin-S195A factor Xa-heparin structure reveals the allosteric mechanism of antithrombin activation

Regulation of blood coagulation is critical for maintaining blood flow, while preventing excessive bleeding or thrombosis. One of the principal regulatory mechanisms involves heparin activation of the serpin antithrombin (AT). Inhibition of several coagulation proteases is accelerated by up to 10,000-fold by heparin, either through bridging AT and the protease or by inducing allosteric changes in the properties of AT. The anticoagulant effect of short heparin chains, including the minimal AT-specific pentasaccharide, is mediated exclusively through the allosteric activation of AT towards efficient inhibition of coagulation factors (f) IXa and Xa. Here we present the crystallographic structure of the recognition (Michaelis) complex between heparin-activated AT and S195A fXa, revealing the extensive exosite contacts that confer specificity. The heparin-induced conformational change in AT is required to allow simultaneous contacts within the active site and two distinct exosites of fXa (36-loop and the autolysis loop). This structure explains the molecular basis of protease recognition by AT, and the mechanism of action of the important therapeutic low-molecular-weight heparins.     

Exosite occupation by heparin enhances the reactivity of blood coagulation factor IXa

Blood coagulation factor IXa (fIXa) is a trypsin-like serine protease with low inherent activity that is greatly enhanced in the factor X activation complex. Molecular details of the conversion of fIXa from an inactive enzyme into a fully functional procoagulant are unclear. Recent studies have identified a heparin-binding exosite in the protease domain of fIXa. Effects of exosite occupation on fIXa activity are unclear. We used the Kunitz-type inhibitor bovine pancreatic trypsin inhibitor (BPTI) to probe fIXa reactivity in the absence and in the presence of heparin. While fIXa alone was poorly reactive with BPTI (K(i) approximately 0.7 mM), this reactivity was increased roughly 20-fold (K(i) = 37 +/- 6 microM) by heparin. This was reproducible with low-molecular-weight heparin (enoxaparin; K(i) = 70 +/- 12 microM). Surface plasmon resonance studies of the interaction between heparin and BPTI indicated an unstable interaction with very low affinity (K(d) = 172 microM). In contrast, kinetic studies revealed a high-affinity interaction between heparin and fIXa (K(d) = 128 +/- 26 nM) and showed that the enhancement of BPTI inhibition of fIXa by heparin was well described by a competitive inhibition model where heparin acts as an affecter of fIXa reactivity with inhibitor. Fluorescence studies with dansyl-EGR-fIXa supported the high-affinity interaction between heparin and fIXa and suggested an altered environment in the fIXa active-site region upon heparin binding. This modulating effect of heparin was supported by the observation of a heparin-induced increase in reactivity of fIXa toward a pentapeptide substrate. When viewed together, the results imply that specific physiological exosite interactions with heparin can induce alterations in the environment of the extended fIXa active site that can result in increased reactivity.     

Interaction of thrombin with sucrose octasulfate

The serine protease thrombin plays multiple roles in many important physiological processes, especially coagulation, where it functions as both a pro- and anticoagulant. The polyanionic glycosaminoglycan heparin modulates thrombin's activity through binding at exosite II. Sucrose octasulfate (SOS) is often used as a surrogate for heparin, but it is not known whether it is an effective heparin mimic in its interaction with thrombin. We have characterized the interaction of SOS with thrombin in solution and determined a crystal structure of their complex. SOS binds thrombin with a K(d) of ~1.4 μM, comparable to that of the much larger polymeric heparin measured under the same conditions. Nonionic (hydrogen bonding) interactions make a larger contribution to thrombin binding of SOS than to heparin. SOS binding to exosite II inhibits thrombin's catalytic activity with high potency but with low efficacy. Analytical ultracentrifugation shows that bovine and human thrombins are monomers in solution in the presence of SOS, in contrast to their complexes with heparin, which are dimers. In the X-ray crystal structure, two molecules of SOS are bound nonequivalently to exosite II portions of a thrombin dimer, in contrast to the 1:2 stoichiometry of the heparin-thrombin complex, which has a different monomer association mode in the dimer. SOS and heparin binding to exosite II of thrombin differ on both chemical and structural levels and, perhaps most significantly, in thrombin inhibition. These differences may offer paths to the design of more potent exosite II binding, allosteric small molecules as modulators of thrombin function.     

Calcium enhances heparin catalysis of the antithrombin-factor Xa reaction by promoting the assembly of an intermediate heparin-antithrombin-factor Xa bridging complex. Demonstration by rapid kinetics studies

Heparin catalyzes the inhibition of factor Xa by antithrombin mainly through an allosteric activation of the serpin inhibitor, but an alternative heparin bridging mechanism has been suggested to enhance the catalysis in the presence of physiologic calcium levels due to calcium interactions with the Gla domain exposing a heparin binding exosite in factor Xa. To provide direct evidence for this bridging mechanism, we studied the heparin-catalyzed reaction of antithrombin with factor Xa, Gla-domainless factor Xa (GDFXa), and a heparin binding exosite mutant of GDFXa in the absence and presence of calcium using rapid kinetic methods. The pseudo-first-order rate constant for factor Xa inhibition by antithrombin complexed with a long-chain approximately 70-saccharide heparin showed a saturable dependence on inhibitor concentration in the presence but not in the absence of 2.5 mM Ca(2+), indicating the formation of an intermediate heparin-serpin-proteinase encounter complex with a dissociation constant of approximately 90 nM prior to formation of the stable serpin-proteinase complex with a rate constant of approximately 20 s(-1). Similar saturation kinetics were observed for the inhibition of GDFXa by the antithrombin-heparin complex, except that Ca(2+) was not required for the effect. By contrast, no Ca(2+)-dependent saturation of the inhibition rate constant was detectable over the same range of inhibitor concentrations for reactions of either a short-chain approximately 26-saccharide high-affinity heparin-antithrombin complex with factor Xa or the long-chain heparin-antithrombin complex with the heparin binding exosite mutant, GDFXa R240A. These findings suggest that binding of full-length heparin chains to an exosite of factor Xa in the presence of Ca(2+) produces a chain-length-dependent lowering of the dissociation constant for assembly of the intermediate heparin-antithrombin-factor Xa encounter complex, resulting in a several 100-fold rate enhancement by a heparin bridging mechanism.     

Allosteric changes of thrombin catalytic site induced by interaction of bothrojaracin with anion-binding exosites I and II.

Bothrojaracin, a 27-kDa C-type lectin from Bothrops jararaca venom, is a selective and potent thrombin inhibitor (K(d) = 0.6 nM) which interacts with the two thrombin anion-binding exosites (I and II) but not with its catalytic site. In the present study, we analyzed the allosteric effects produced in the catalytic site by bothrojaracin binding to thrombin exosites. Opposite effects were observed with alpha-thrombin, which possesses both exosites I and II, and with gamma-thrombin, which lacks exosite I. On the one hand, bothrojaracin altered both kinetic parameters K(m) and k(cat) of alpha-thrombin for small synthetic substrates, resulting in an increased efficiency of alpha-thrombin catalytic activity. This effect was similar to that produced by hirugen, a peptide based on the C-terminal hirudin sequence (residues 54-65) which interacts exclusively with exosite I. On the other hand, bothrojaracin decreased the amidolytic activity of gamma-thrombin toward chromogenic substrates, although this effect was observed with higher concentrations of bothrojaracin than those used with alpha-thrombin. In agreement with these observaions, bothrojaracin produced opposite effects on the fluorescence intensity of alpha- and gamma-thrombin derivatives labeled at the active site with fluorescein-Phe-Pro-Arg-chloromethylketone. These observations support the conclusion that bothrojaracin binding to thrombin produces two different structural changes in its active site, depending on whether it interacts exclusively with exosite II, as seen with gamma-thrombin, or with exosite I (or both I and II) as observed with alpha-thrombin. The ability of bothrojaracin to evoke distinct modifications in the thrombin catalytic site environment when interacting with exosites I and II make this molecule an interesting tool for the study of allosteric changes in the thrombin molecule.     

A novel allosteric pathway of thrombin inhibition: Exosite II mediated potent inhibition of thrombin by chemo-enzymatic, sulfated dehydropolymers of 4-hydroxycinnamic acids

Thrombin and factor Xa, two important pro-coagulant proteinases, can be regulated through direct and indirect inhibition mechanisms. Recently, we designed sulfated dehydropolymers (DHPs) of 4-hydroxycinnamic acids that displayed interesting anticoagulant properties (Monien, B. H., Henry, B. L., Raghuraman, A., Hindle, M., and Desai, U. R. (2006) Bioorg. Med. Chem. 14, 7988-7998). To better understand their mechanism of action, we studied the direct inhibition of thrombin, factor Xa, factor IXa, and factor VIIa by CDSO3, FDSO3, and SDSO3, three analogs of sulfated DHPs. All three sulfated DHPs displayed a 2-3-fold preference for direct inhibition of thrombin over factor Xa, whereas this preference for inhibiting thrombin over factor IXa and factor VIIa increased to 17-300-fold, suggesting a high level of selectivity. Competitive binding studies with a thrombin-specific chromogenic substrate, a fluorescein-labeled hirudin peptide, bovine heparin, enoxaparin, and a heparin octasaccharide suggest that CDSO3 preferentially binds in or near anion-binding exosite II of thrombin. Studies of the hydrolysis of H-D-hexahydrotyrosol-Ala-Arg-p-nitroanilide indicate that CDSO3 inhibits thrombin through allosteric disruption of the catalytic apparatus, specifically through the catalytic step. Overall, designed sulfated DHPs appear to be the first molecules that bind primarily in the region defined by exosite II and allosterically induce thrombin inhibition. The molecules are radically different in structure from all the current clinically used anticoagulants and thus represent a novel class of potent dual thrombin and factor Xa inhibitors.     

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(+).     

Molecular mechanisms of antithrombin–heparin regulation of blood clotting proteinases. A paradigm for understanding proteinase regulation by serpin family protein proteinase inhibitors

Serpin family protein proteinase inhibitors regulate the activity of serine and cysteine proteinases by a novel conformational trapping mechanism that may itself be regulated by cofactors to provide a finely-tuned time and location-dependent control of proteinase activity. The serpin, antithrombin, together with its cofactors, heparin and heparan sulfate, perform a critical anticoagulant function by preventing the activation of blood clotting proteinases except when needed at the site of a vascular injury. Here, we review the detailed molecular understanding of this regulatory mechanism that has emerged from numerous X-ray crystal structures of antithrombin and its complexes with heparin and target proteinases together with mutagenesis and functional studies of heparin–antithrombin–proteinase interactions in solution. Like other serpins, antithrombin achieves specificity for its target blood clotting proteinases by presenting recognition determinants in an exposed reactive center loop as well as in exosites outside the loop. Antithrombin reactivity is repressed in the absence of its activator because of unfavorable interactions that diminish the favorable RCL and exosite interactions with proteinases. Binding of a specific heparin or heparan sulfate pentasaccharide to antithrombin induces allosteric activating changes that mitigate the unfavorable interactions and promote template bridging of the serpin and proteinase. Antithrombin has thus evolved a sophisticated means of regulating the activity of blood clotting proteinases in a time and location-dependent manner that exploits the multiple conformational states of the serpin and their differential stabilization by glycosaminoglycan cofactors.     

Heparin enhances the specificity of antithrombin for thrombin and factor Xa independent of the reactive center loop sequence. Evidence for an exosite determinant of factor Xa specificity in heparin-activated antithrombin

Heparin activates the primary serpin inhibitor of blood clotting proteinases, antithrombin, both by an allosteric conformational change mechanism that specifically enhances factor Xa inactivation and by a ternary complex bridging mechanism that promotes the inactivation of thrombin and other target proteinases. To determine whether the factor Xa specificity of allosterically activated antithrombin is encoded in the reactive center loop sequence, we attempted to switch this specificity by mutating the P6-P3' proteinase binding sequence excluding P1-P1' to a more optimal thrombin recognition sequence. Evaluation of 12 such antithrombin variants showed that the thrombin specificity of the serpin allosterically activated by a heparin pentasaccharide could be enhanced as much as 55-fold by changing P3, P2, and P2' residues to a consensus thrombin recognition sequence. However, at most 9-fold of the enhanced thrombin specificity was due to allosteric activation, the remainder being realized without activation. Moreover, thrombin specificity enhancements were attenuated to at most 5-fold with a bridging heparin activator. Surprisingly, none of the reactive center loop mutations greatly affected the factor Xa specificity of the unactivated serpin or the several hundred-fold enhancement in factor Xa specificity due to activation by pentasaccharide or bridging heparins. Together, these results suggest that the specificity of both native and heparin-activated antithrombin for thrombin and factor Xa is only weakly dependent on the P6-P3' residues flanking the primary P1-P1' recognition site in the serpin-reactive center loop and that heparin enhances serpin specificity for both enzymes through secondary interaction sites outside the P6-P3' region, which involve a bridging site on heparin in the case of thrombin and a previously unrecognized exosite on antithrombin in the case of factor Xa.     

Exosites 1 and 2 are essential for protection of fibrin-bound thrombin from heparin-catalyzed inhibition by antithrombin and heparin cofactor II

Assembly of ternary thrombin-heparin-fibrin complexes, formed when fibrin binds to exosite 1 on thrombin and fibrin-bound heparin binds to exosite 2, produces a 58- and 247-fold reduction in the heparin-catalyzed rate of thrombin inhibition by antithrombin and heparin cofactor II, respectively. The greater reduction for heparin cofactor II reflects its requirement for access to exosite 1 during the inhibitory process. Protection from inhibition by antithrombin and heparin cofactor II requires ligation of both exosites 1 and 2 because minimal protection is seen when exosite 1 variants (gamma-thrombin and thrombin Quick 1) or an exosite 2 variant (Arg93 --> Ala, Arg97 --> Ala, and Arg101 --> Ala thrombin) is substituted for thrombin. Likewise, the rate of thrombin inhibition by the heparin-independent inhibitor, alpha1-antitrypsin Met358 --> Arg, is decreased less than 2-fold in the presence of soluble fibrin and heparin. In contrast, thrombin is protected from inhibition by a covalent antithrombin-heparin complex, suggesting that access of heparin to exosite 2 of thrombin is hampered when ternary complex formation occurs. These results reveal the importance of exosites 1 and 2 of thrombin in assembly of the ternary complex and the subsequent protection of thrombin from inhibition by heparin-catalyzed inhibitors.     

Conformational Activation of Antithrombin by Heparin Involves an Altered Exosite Interaction with Protease

Heparin allosterically activates antithrombin as an inhibitor of factors Xa and IXa by enhancing the initial Michaelis complex interaction of inhibitor with protease through exosites. Here, we investigate the mechanism of this enhancement by analyzing the effects of alanine mutations of six putative antithrombin exosite residues and three complementary protease exosite residues on antithrombin reactivity with these proteases in unactivated and heparin-activated states. Mutations of antithrombin Tyr²⁵³ and His³¹⁹ exosite residues produced massive 10–200-fold losses in reactivity with factors Xa and IXa in both unactivated and heparin-activated states, indicating that these residues made critical attractive interactions with protease independent of heparin activation. By contrast, mutations of Asn²³³, Arg²³⁵, Glu²³⁷, and Glu²⁵⁵ exosite residues showed that these residues made both repulsive and attractive interactions with protease that depended on the activation state and whether the critical Tyr²⁵³/His³¹⁹ residues were mutated. Mutation of factor Xa Arg¹⁴³, Lys¹⁴⁸, and Arg¹⁵⁰ residues that interact with the exosite in the x-ray structure of the Michaelis complex confirmed the importance of all residues for heparin-activated antithrombin reactivity and Arg¹⁵⁰ for native serpin reactivity. These results demonstrate that the exosite is a key determinant of antithrombin reactivity with factors Xa and IXa in the native as well as the heparin-activated state and support a new model of allosteric activation we recently proposed in which a balance between attractive and repulsive exosite interactions in the native state is shifted to favor the attractive interactions in the activated state through core conformational changes induced by heparin binding.