Conformational changes in thrombin when complexed by serpins

Thrombin possesses two positively charged surface domains, termed exosites, that orient substrates and inhibitors for reaction with the enzyme. Because the exosites also allosterically modulate thrombin's activity, we set out to determine whether the structure or function of the exosites changes when thrombin forms complexes with antithrombin, heparin cofactor II, or alpha(1)-antitrypsin (M358R), serpins that utilize both, one, or neither of the exosites, respectively. Using a hirudin-derived peptide to probe the integrity of exosite 1, no binding was detected when thrombin was complexed with heparin cofactor II or alpha(1)-antitrypsin (M358R), and the peptide exhibited a 55-fold lower affinity for the thrombin-antithrombin complex than for thrombin. Bound peptide or HD-1, an exosite 1-binding DNA aptamer, was displaced from thrombin by each of the three serpins. Thrombin binding to fibrin also was abrogated when the enzyme was complexed with serpins. These data reveal that, regardless of the initial mode of interaction, the function of exosite 1 is lost when thrombin is complexed by serpins. In contrast, the integrity of exosite 2 is largely retained when thrombin is complexed by serpins, because interaction with heparin or an exosite 2-directed DNA aptamer was only modestly altered. The disorganization of exosite 1 that occurs when thrombin is complexed by serpins is consistent with results of protease sensitivity studies and crystallographic analysis of a homologous enzyme-serpin complex.     

Sucrose Octasulfate Selectively Accelerates Thrombin Inactivation by Heparin Cofactor II

Inactivation of thrombin (T) by the serpins heparin cofactor II (HCII) and antithrombin (AT) is accelerated by a heparin template between the serpin and thrombin exosite II. Unlike AT, HCII also uses an allosteric interaction of its NH₂-terminal segment with exosite I. Sucrose octasulfate (SOS) accelerated thrombin inactivation by HCII but not AT by 2000-fold. SOS bound to two sites on thrombin, with dissociation constants (K_D) of 10 ± 4 μm and 400 ± 300 μm that were not kinetically resolvable, as evidenced by single hyperbolic SOS concentration dependences of the inactivation rate (k_obs). SOS bound HCII with K_D 1.45 ± 0.30 mm, and this binding was tightened in the T·SOS·HCII complex, characterized by K_complex of ∼0.20 μm. Inactivation data were incompatible with a model solely depending on HCII·SOS but fit an equilibrium linkage model employing T·SOS binding in the pathway to higher order complex formation. Hirudin-(54–65)(SO₃⁻) caused a hyperbolic decrease of the inactivation rates, suggesting partial competitive binding of hirudin-(54–65)(SO₃⁻) and HCII to exosite I. Meizothrombin(des-fragment 1), binding SOS with K_D = 1600 ± 300 μm, and thrombin were inactivated at comparable rates, and an exosite II aptamer had no effect on the inactivation, suggesting limited exosite II involvement. SOS accelerated inactivation of meizothrombin 1000-fold, reflecting the contribution of direct exosite I interaction with HCII. Thrombin generation in plasma was suppressed by SOS, both in HCII-dependent and -independent processes. The ex vivo HCII-dependent process may utilize the proposed model and suggests a potential for oversulfated disaccharides in controlling HCII-regulated thrombin generation.     

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.     

Bivalent binding to gammaA/gamma'-fibrin engages both exosites of thrombin and protects it from inhibition by the antithrombin-heparin complex

Thrombin exosite 1 binds the predominant gamma(A)/gamma(A)-fibrin form with low affinity. A subpopulation of fibrin molecules, gamma(A)/gamma'-fibrin, has an extended COOH terminus gamma'-chain that binds exosite 2 of thrombin. Bivalent binding to gamma(A)/gamma'-fibrin increases the affinity of thrombin 10-fold, as determined by surface plasmon resonance. Because of its higher affinity, thrombin dissociates 7-fold more slowly from gamma(A)/gamma'-fibrin clots than from gamma(A)/gamma(A)-fibrin clots. After 24 h of washing, however, both gamma(A)/gamma'- and gamma(A)/gamma(A)-fibrin clots generate fibrinopeptide A when incubated with fibrinogen, indicating the retention of active thrombin. Previous studies demonstrated that heparin heightens the affinity of thrombin for fibrin by simultaneously binding to fibrin and exosite 2 on thrombin to generate a ternary heparin-thrombin-fibrin complex that protects thrombin from inhibition by antithrombin and heparin cofactor II. In contrast, dermatan sulfate does not promote ternary complex formation because it does not bind to fibrin. Heparin-catalyzed rates of thrombin inhibition by antithrombin were 5-fold slower in gamma(A)/gamma'-fibrin clots than they were in gamma(A)/gamma(A)-fibrin clots. This difference reflects bivalent binding of thrombin to gamma(A)/gamma'-fibrin because (a) it is abolished by addition of a gamma'-chain-directed antibody that blocks exosite 2-mediated binding of thrombin to the gamma'-chain and (b) the dermatan sulfate-catalyzed rate of thrombin inhibition by heparin cofactor II also is lower with gamma(A)/gamma'-fibrin than with gamma(A)/gamma(A)-fibrin clots. Thus, bivalent binding of thrombin to gamma(A)/gamma'-fibrin protects thrombin from inhibition, raising the possibility that gamma(A)/gamma'-fibrin serves as a reservoir of active thrombin that renders thrombi thrombogenic.     

Monitoring complex formation in the blood-coagulation cascade using aptamer-coated SAW sensors

Specific binding of the anticoagulants heparin and antithrombin III to the blood clotting cascade factor human thrombin was recorded as a function of time with a Love-wave biosensor array consisting of five sensor elements. Two of the sensor elements were used as references. Three sensor elements were coated with RNA or DNA aptamers for specific binding of human thrombin. The affinity between the aptamers and thrombin, measured using the biosensor, was within the same range as the value of K(D) measured by filter binding experiments. Consecutive binding of the thrombin inhibitors heparin, antithrombin III or the heparin-antithrombin III complex to the immobilized thrombin molecules, and binding of a ternary complex of heparin, anithrombin III, and thrombin to aptamers was evaluated. The experiments showed attenuation of binding to thrombin due to heparin-antithrombin III complex formation. Binding of heparin activated the formation of the inhibitory complex of antithrombin III with thrombin about 2.7-fold. Binding of the DNA aptamer to exosite II appeared to inhibit heparin binding to exosite I.     

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.     

The ternary complex of antithrombin-anhydrothrombin-heparin reveals the basis of inhibitor specificity

Antithrombin, the principal physiological inhibitor of the blood coagulation proteinase thrombin, requires heparin as a cofactor. We report the crystal structure of the rate-determining encounter complex formed between antithrombin, anhydrothrombin and an optimal synthetic 16-mer oligosaccharide. The antithrombin reactive center loop projects from the serpin body and adopts a canonical conformation that makes extensive backbone and side chain contacts from P5 to P6' with thrombin's restrictive specificity pockets, including residues in the 60-loop. These contacts rationalize many earlier mutagenesis studies on thrombin specificity. The 16-mer oligosaccharide is just long enough to form the predicted bridge between the high-affinity pentasaccharide-binding site on antithrombin and the highly basic exosite 2 on thrombin, validating the design strategy for this synthetic heparin. The protein-protein and protein-oligosaccharide interactions together explain the basis for heparin activation of antithrombin as a thrombin inhibitor.     

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.     

Anticoagulant activities of a monoclonal antibody that binds to exosite II of thrombin.

A monoclonal IgG isolated from a patient with multiple myeloma has been shown to bind to exosite II of thrombin, prolong both the thrombin time and the activated partial thromboplastin time (aPTT) when added to normal plasma, and alter the kinetics of hydrolysis of synthetic peptide substrates. Although the IgG does not affect cleavage of fibrinogen by thrombin, it increases the rate of inhibition of thrombin by purified antithrombin approximately 3-fold. Experiments with plasma immunodepleted of antithrombin or heparin cofactor II confirm that prolongation of the thrombin time requires antithrombin. By contrast, prolongation of the aPTT requires neither antithrombin nor heparin cofactor II. The IgG delays clotting of plasma initiated by purified factor IXa but has much less of an effect on clotting initiated by factor Xa. In a purified system, the IgG decreases the rate of activation of factor VIII by thrombin. These studies indicate that binding of a monoclonal IgG to exosite II prolongs the thrombin time indirectly by accelerating the thrombin-antithrombin reaction and may prolong the aPTT by interfering with activation of factor VIII, thereby diminishing the catalytic activity of the factor IXa/VIIIa complex.     

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.     

Localization of the single-stranded DNA binding site in the thrombin anion-binding exosite.

Single-stranded DNA molecules containing a 15-nucleotide consensus sequence have been reported to inhibit thrombin activity. The mechanism of the inhibition was studied using a consensus 15-mer oligonucleotide and two recombinant mutant thrombins: the anion-binding exosite mutant thrombin R70E, and thrombin K154A, in which the mutation was located in a surface loop outside of the exosite. The consensus 15-mer oligonucleotide inhibited both fibrinogen-clotting and platelet-activation activities of plasma-derived thrombin, recombinant wild type thrombin, and mutant thrombin K154A in a sequence-specific and dose-dependent manner, whereas it did not inhibit either activity of mutant thrombin R70E. The 15-mer oligonucleotide also inhibited thrombomodulin-dependent protein C activation by plasma-derived thrombin. In competition equilibrium binding experiments, binding of 125I-labeled diisopropyl phosphoryl-thrombin to thrombomodulin was completely inhibited by the consensus 15-mer oligonucleotide with a Kd value of 2.68 +/- 0.16 nM. These results suggest that Arg-70 in the anion-binding exosite of thrombin is a key determinant for interaction with specific single-stranded DNA molecules, and that binding of single-stranded DNA molecules to the exosite prevents the interaction of thrombin with fibrinogen, the platelet thrombin receptor, and thrombomodulin.     

Structure-function relationships of hirulog peptide interactions with thrombin.

Using hirudin as a model, a novel class of bivalent thrombin inhibitors has been designed and characterized (Maraganore et al. (1990) Biochemistry 29, 7095-7101). These peptides, designated 'hirulogs', interact with both thrombin's catalytic center and its anion-binding exosite for fibrinogen recognition. In order to investigate structure-activity relationships in hirulog peptides, a number of peptide and peptidomimetic derivatives with alterations in catalytic-site binding and anion-binding exosite binding moieties were prepared. Replacements or modifications in the catalytic site and exosite binding moieties were achieved with the consequences of maintaining or improving antithrombin activity. In addition to showing improved affinity for thrombin, some derivatives with Ki's in the sub-nanomolar range showed increased anticoagulant activities. These findings highlight the versatility of hirulog peptides in their bivalent interactions with thrombin.     

A new blood coagulation inhibitor from the snake Bothrops jararaca plasma: isolation and characterization

A novel thrombin inhibitor, Bothrops jararaca inhibitor (BjI), has been identified and purified from B. jararaca snake blood by two anionic chromatographic steps. Purified BjI showed two polypeptide chains with molecular masses of 109 and 138 kDa, by SDS-PAGE in reducing conditions. On the other hand, in nonreducing conditions the molecular masses were 150 and 219 kDa, suggesting that the polypeptide chain 109 kDa can be a dimer form linked by disulfide bond. However, the native BjI shows a molecular mass higher than 1000 kDa by gel filtration chromatography, indicating the need of a quaternary structure formation for the blood coagulation inhibition. BjI is a specific thrombin coagulant activity inhibitor that does not affect other thrombin functions, such as: amidolytic and platelet aggregation activities. BjI is not an antithrombin-like inhibitor. Fibrinogen and heparin competition ELISA assays with BjI and thrombin showed that fibrinogen does not interfere in the BjI and thrombin binding, however, heparin interferes in BjI and thrombin interaction, suggesting that BjI binds to heparin site or other sites close to it. Our findings indicate that BjI is an exosite binding thrombin inhibitor, specific upon coagulant activity thrombin inhibitor, without any anti-platelet aggregation activity.     

Antithrombin activity of a peptide corresponding to residues 54-75 of heparin cofactor II

Heparin cofactor II (HCII) is a highly specific serine proteinase inhibitor, which complexes covalently with thrombin in a reaction catalyzed by heparin and other polyanions. The molecular basis for the thrombin specificity may be explained by the identification here of a segment of HCII including residues 54-75 that binds to thrombin. A synthetic peptide, HCII(54-75), based on this segment of HCII, Gly-Glu-Glu-Asp-Asp-Asp-Tyr-Leu-Asp-Leu-Glu- Lys-Ile-Phe-Ala-Glu-Asp-Asp-Asp-Tyr-Ile-Asp inhibited thrombin's cleavage of fibrinogen. Clotting activity of thrombin was inhibited 50% at a concentration of 28 microM. Polyacrylamide gel electrophoresis showed that HCII(54-75) inhibited thrombin's cleavage of both the A alpha and B beta polypeptides in fibrinogen. However, the peptide did not block thrombin's active site, as hydrolysis of chromogenic substrates was not inhibited. HCII(54-75) probably binds to the same site on thrombin as do carboxyl-terminal residues of hirudins, thrombin inhibitors of leeches. HCII(54-75) inhibited binding of thrombin to a synthetic peptide corresponding to residues 54-66 of hirudin PA, but the hirudin peptide was about 30-fold more potent in binding and clotting assays. Both synthetic peptides, as a result of their polyanionic character, might be expected to stimulate the reaction of HCII with thrombin. However, the hirudin-related peptide inhibited this reaction, suggesting that it blocked a site on thrombin required for interaction with HCII. HCII(54-75) had a net stimulatory effect on the thrombin-HCII reaction as a consequence of its lower affinity for thrombin and greater negative charge relative to the hirudin-related peptide. These studies suggest that residues 54-75 of HCII interact with a noncatalytic binding site on thrombin and that this interaction contributes to efficient inhibition of thrombin by HCII.     

Crystal structures of aprotinin and its complex with sucrose octasulfate reveal multiple modes of interactions with implications for heparin binding

The crystal structures of aprotinin and its complex with sucrose octasulfate (SOS), a polysulfated heparin analog, were determined at 1.7-2.6 Å resolutions. Aprotinin is monomeric in solution, which associates into a decamer at high salt concentrations. Sulfate ions serve to neutralize the basic amino acid residues of aprotinin to stabilize the decameric aprotinin. Whereas SOS interacts with heparin binding proteins at 1:1 molar ratio, SOS was surprisingly found to induce strong agglutination of aprotinins. Five molecules of aprotinin interact with one molecule of the sulfated sugar, which is stabilized by electrostatic interactions between the positively charged residues of aprotinin and sulfate groups of SOS. The multiple binding modes of SOS with five individual aprotinin molecules may represent the diverse patterns of potential heparin binding to aprotinin, reflecting the interactions of densely packed protein molecules along the heparin polymer.     

The preferred pathway of glycosaminoglycan-accelerated inactivation of thrombin by heparin cofactor II

Thrombin (T) inactivation by the serpin, heparin cofactor II (HCII), is accelerated by the glycosaminoglycans (GAGs) dermatan sulfate (DS) and heparin (H). Equilibrium binding and thrombin inactivation kinetics at pH 7.8 and ionic strength (I) 0.125 m demonstrated that DS and heparin bound much tighter to thrombin (K(T(DS)) 1-5.8 microm; K(T(H)) 0.02-0.2 microm) than to HCII (K(HCII(DS)) 236-291 microm; K(HCII(H)) 25-35 microm), favoring formation of T.GAG over HCII.GAG complexes as intermediates for T.GAG.HCII complex assembly. At [GAG] < K(HCII(GAG)) the GAG and HCII concentration dependences of the first-order inactivation rate constants (k(app)) were hyperbolic, reflecting saturation of T.GAG complex and formation of the T.GAG.HCII complex from T.GAG and free HCII, respectively. At [GAG] > K(HCII(GAG)), HCII.GAG complex formation caused a decrease in k(app). The bell-shaped logarithmic GAG dependences fit an obligatory template mechanism in which free HCII binds GAG in the T.GAG complex. DS and heparin bound fluorescently labeled meizothrombin(des-fragment 1) (MzT(-F1)) with K(MzT(-F1)(GAG)) 10 and 20 microm, respectively, demonstrating a binding site outside of exosite II. Exosite II ligands did not attenuate the DS-accelerated thrombin inactivation markedly, but DS displaced thrombin from heparin-Sepharose, suggesting that DS and heparin share a restricted binding site in or nearby exosite II, in addition to binding outside exosite II. Both T.DS and MzT(-F1).DS interactions were saturable at DS concentrations substantially below K(HCII(DS)), consistent with DS bridging T.DS and free HCII. The results suggest that GAG template action facilitates ternary complex formation and accommodates HCII binding to GAG and thrombin exosite I in the ternary complex.     

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.     

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.     

Evidence that both exosites on thrombin participate in its high affinity interaction with fibrin

Exosite 1 on thrombin mediates low affinity binding to sites on the NH2 termini of the alpha- and beta-chains of fibrin. A subpopulation of fibrin molecules (gammaA/gamma'-fibrin) has an alternate COOH terminus of the normal gamma-chain (gammaA/gammaA-fibrin) that binds thrombin with high affinity. To determine the roles of exosites 1 and 2 in the high affinity interaction of thrombin with gammaA/gamma'-fibrin, binding studies were done with thrombin variants and exosite 1- or 2-directed ligands. alpha-Thrombin bound gammaA/gamma'-fibrin via high and low affinity binding sites. A peptide analog of the COOH terminus of the gamma'-chain that binds alpha-thrombin via exosite 2 blocked the high affinity binding of alpha-thrombin to gammaA/gamma'-fibrin, suggesting that the interaction of alpha-thrombin with the gamma'-chain is exosite 2-mediated. In support of this concept, (a) gamma-thrombin, which lacks a functional exosite 1, bound to gammaA/gamma'-fibrin, but not to gammaA/gammaA-fibrin; (b) thrombin R93A/R97A/R101A, an exosite 2-defective variant, bound only to gammaA/gamma'-fibrin via low affinity sites; and (c) exosite 2-directed ligands reduced alpha-thrombin binding to gammaA/gamma'-fibrin. However, several lines of evidence indicate that exosite 1 contributes to the high affinity interaction of thrombin with gammaA/gamma'-fibrin. First, the affinity of gamma-thrombin for gammaA/gamma'-fibrin was lower than that of alpha-thrombin. Second, removal of a low affinity binding site on the beta-chain of gammaA/gamma'-fibrin reduced its affinity for alpha-thrombin. Third, exosite 1-directed ligands reduced alpha-thrombin binding to gammaA/gamma'-fibrin. Taken together, these data suggest that, although exosite 2 mediates the interaction of thrombin with the gamma'-chain of gammaA/gamma'-fibrin, simultaneous ligation of exosite 1 by low affinity binding sites is essential for the high affinity interaction of thrombin with gammaA/gamma'-fibrin.     

Structure of the antithrombin-thrombin-heparin ternary complex reveals the antithrombotic mechanism of heparin

The maintenance of normal blood flow depends completely on the inhibition of thrombin by antithrombin, a member of the serpin family. Antithrombin circulates at a high concentration, but only becomes capable of efficient thrombin inhibition on interaction with heparin or related glycosaminoglycans. The anticoagulant properties of therapeutic heparin are mediated by its interaction with antithrombin, although the structural basis for this interaction is unclear. Here we present the crystal structure at a resolution of 2.5 A of the ternary complex between antithrombin, thrombin and a heparin mimetic (SR123781). The structure reveals a template mechanism with antithrombin and thrombin bound to the same heparin chain. A notably close contact interface, comprised of extensive active site and exosite interactions, explains, in molecular detail, the basis of the antithrombotic properties of therapeutic heparin.