Anion-binding Exosite Of Thrombin Research Articles

A Novel Substrate for Assessing Thrombin Activity in Whole Blood

At sites of vessel injury, thrombin acts as the central mediator of coagulation through catalyzing fibrin clot formation and platelet activation. Currently, thrombin generation is measured using small molecule substrates. These substrates are limited in that they detect both active thrombin and thrombin bound to its inhibitor α2-macroglobulin (α2-M) and have limited utility in whole blood (Ninivaggi et al. Clin Chem 2012). In addition, these substrates contain short thrombin recognition sequences ranging from 3-4 amino acids and are unable to form additional exosite interactions with thrombin, which limit their specificity (Gallwitz et al. Plos One 2012). Furthermore, they are unfeasible for in vivo use because their hydrophobicity allows them to enter cells. However, plasma assays are limited because they ignore the hemostatic contributions of red blood cells, platelets and leukocytes and require anticoagulation and the addition of exogenous coagulation activators (Swystun & Liaw. Blood 2016 & do Amaral et al. Stem Cells Int 2016). Thus, we have developed a fluorescence resonance energy transfer (FRET)-based thrombin sensor to overcome these limitations. We cloned and expressed the construct encoding the FRET-based thrombin substrate in Escherichia coli and purified the substrate using affinity chromatography. We confirmed using Sodium dodecyl sulfate−polyacrylamide gel electrophoresis that the substrate was successfully purified and that thrombin cleavage of the purified substrate changes its FRET efficiency by monitoring its spectrum of emission. To determine whether the FRET-based thrombin substrate can form interactions with thrombin's anion binding exosites and assess its potential for in vivo use, we measured the kinetics parameters of the substrate for thrombin in the presence of exosite inhibitors and thrombin's exosite variants as well as mouse thrombin and plasmin. We then compared the resulting kinetics parameters with commercially available thrombin substrates S2238 and technothrombin. We show that unlike S2238 and technothrombin, our FRET-based thrombin sensor forms strong interactions with thrombin's anion binding exosite I and displays preference for free thrombin compared to thrombomodulin (TM) bound thrombin which implied its preference for procoagulant thrombin rather than anticoagulant TM bound thrombin that activates protein C. Furthermore, our FRET-based thrombin substrate has potential to be used in mouse models because it can be efficiently cleaved by mouse thrombin and is relatively plasmin resistant. To assess the specificity of the FRET-based thrombin substrate for thrombin, we compared the rate of hydrolysis of the substrate by thrombin and several other enzymes in circulation and compared the results to S2238 and technothrombin. Our results indicated that when compared to commercially available thrombin substrates, our FRET-based thrombin substrate is highly specific for thrombin. Since the thrombin inhibitor α2-M is present in circulation, we also compared the rate of hydrolysis of the 3 substrates for α2-M bound thrombin and free thrombin. Unlike S2238 and technothrombin, our FRET-based thrombin substrate is uncleaved by α2-M bound thrombin. The key properties of the FRET-based thrombin substrate in comparison to S22238 and technothrombin are summarized in Table 1. We then measured thrombin generation in plasma using our FRET-based thrombin substrate and showed that it is sensitive to direct oral anticoagulants in ways that are consistent with literature findings where thrombin generation was measured using technothrombin (Sivaraja et al. Plos One 2018 & Douxfils et al. Thromb Res 2012). We also confirmed that the FRET-based thrombin substrate itself is not a coagulation inhibitor since its presence did not alter thrombin generation parameters measured using technothrombin. Lastly, we confirmed that the FRET-based thrombin substrate can measure thrombin generation in whole blood since it successfully produced thrombin generation curves. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Deciphering Conformational Changes Associated with the Maturation of Thrombin Anion Binding Exosite I.

Thrombin participates in procoagulation, anticoagulation, and platelet activation. This enzyme contains anion binding exosites, ABE I and ABE II, which attract regulatory biomolecules. As prothrombin is activated to thrombin, pro-ABE I is converted into mature ABE I. Unexpectedly, certain ligands can bind to pro-ABE I specifically. Moreover, knowledge of changes in conformation and affinity that occur at the individual residue level as pro-ABE I is converted to ABE I is lacking. Such changes are transient and were not captured by crystallography. Therefore, we employed nuclear magnetic resonance (NMR) titrations to monitor development of ABE I using peptides based on protease-activated receptor 3 (PAR3). Proton line broadening NMR revealed that PAR3 (44-56) and more weakly binding PAR3G (44-56) could already interact with pro-ABE I on prothrombin. 1H-15N heteronuclear single-quantum coherence NMR titrations were then used to probe binding of individual 15N-labeled PAR3G residues (F47, E48, L52, and D54). PAR3G E48 and D54 could interact electrostatically with prothrombin and tightened upon thrombin maturation. The higher affinity for PAR3G D54 suggests the region surrounding thrombin R77a is better oriented to bind D54 than the interaction between PAR3G E48 and thrombin R75. Aromatic PAR3G F47 and aliphatic L52 both reported on significant changes in the chemical environment upon conversion of prothrombin to thrombin. The ABE I region surrounding the 30s loop was more affected than the hydrophobic pocket (F34, L65, and I82). Our NMR titrations demonstrate that PAR3 residues document structural rearrangements occurring during exosite maturation that are missed by reported X-ray crystal structures.

Hirudin and heparin enable efficient megakaryocyte differentiation of mouse bone marrow progenitors

Hematopoietic progenitors from murine fetal liver efficiently differentiate in culture into proplatelet-producing megakaryocytes and have proved valuable to study platelet biogenesis. In contrast, megakaryocyte maturation is far less efficient in cultured bone marrow progenitors, which hampers studies in adult animals. It is shown here that addition of hirudin to media containing thrombopoietin and serum yielded a proportion of proplatelet-forming megakaryocytes similar to that in fetal liver cultures (approximately 50%) with well developed extensions and increased the release of platelet particles in the media. The effect of hirudin was maximal at 100 U/ml, and was more pronounced when it was added in the early stages of differentiation. Hirugen, which targets the thrombin anion binding exosite I, and argatroban, a selective active site blocker, also promoted proplatelet formation albeit less efficiently than hirudin. Heparin, an indirect thrombin blocker, and OTR1500, a stable heparin-like synthetic glycosaminoglycan generated proplatelets at levels comparable to hirudin. Heparin with low affinity for antithrombin was equally as effective as standard heparin, which indicates antithrombin independent effects. Use of hirudin and heparin compounds should lead to improved culture conditions and facilitate studies of platelet biogenesis in adult mice.

Activation of factor XI by products of prothrombin activation

AbstractThe prothrombinase complex converts prothrombin to α-thrombin through the intermediate meizothrombin (Mz-IIa). Both α-thrombin and Mz-IIa catalyze factor (F) XI activation to FXIa, which sustains α-thrombin production through activation of FIX. The interaction with FXI is thought to involve thrombin anion binding exosite (ABE) I. α-Thrombin can undergo additional proteolysis to β-thrombin and γ-thrombin, neither of which have an intact ABE I. In a purified protein system, FXI is activated by β-thrombin or γ-thrombin, and by α-thrombin in the presence of the ABE I-blocking peptide hirugen, indicating that a fully formed ABE I is not absolutely required for FXI activation. In a FXI-dependent plasma thrombin generation assay, β-thrombin, γ-thrombin, and α-thrombins with mutations in ABE I are approximately 2-fold more potent initiators of thrombin generation than α-thrombin or Mz-IIa, possibly because fibrinogen, which binds to ABE I, competes poorly with FXI for forms of thrombin lacking ABE I. In addition, FXIa can activate factor FXII, which could contribute to thrombin generation through FXIIa-mediated FXI activation. The data indicate that forms of thrombin other than α-thrombin contribute directly to feedback activation of FXI in plasma and suggest that FXIa may provide a link between tissue factor-initiated coagulation and the proteases of the contact system.

Biophysical Investigation of GpIbα Binding to Thrombin Anion Binding Exosite II

Substrates and cofactors of the serine protease thrombin (IIa) employ two anion binding exosites (ABE-I and -II) to aid in binding. On the surface of platelets resides the GpIbalpha/beta-GpIX-GpV membrane-bound receptor complex. IIa's ABE-II is proposed to interact with an anionic portion of GpIbalpha which enhances IIa cleavage of PAR-1 and subsequent activation of platelets. In this work, one-dimensional (1D) and two-dimensional (2D) NMR, analytical ultracentrifugation (AUC), and hydrogen-deuterium exchange (HDX) coupled with MALDI-TOF MS were performed to further characterize the features of binding to IIa's ABEs. The described work builds upon investigations performed in a prior study with fibrin(ogen)'s gamma' peptide and IIa [Sabo, T. M., Farrell, D. H., and Maurer, M. C. (2006) Biochemistry 45, 7434-7445]. 1D line broadening NMR (1H and 31P) and 2D trNOESY NMR studies indicate that GpIbalpha residues D274-E285 interact extensively with the IIa surface in an extended conformation. AUC demonstrates that both GpIbalpha (269-286) and gamma' (410-427) peptides interact with IIa with a 1:1 stoichiometry. When the HDX results are compared to those for the ABE-I targeting peptide hirudin (54-65), the data imply that GpIbalpha (269-286), GpIbalpha (1-290), and gamma' (410-427) are indeed directed to ABE-II. The ABE-II binding fragments reduce HDX for sites distant from the interface, suggesting long-range conformational effects. These studies illustrate that GpIbalpha and gamma' target ABE-II with similar consequences on IIa dynamics, albeit with differing structural features.

Fibrinogen-elongated γ Chain Inhibits Thrombin-induced Platelet Response, Hindering the Interaction with Different Receptors

The expression of the elongated fibrinogen gamma chain, termed gamma', derives from alternative splicing of mRNA and causes an insertion sequence of 20 amino acids. This insertion domain interacts with the anion-binding exosite (ABE)-II of thrombin. This study investigated whether and how gamma' chain binding to ABE-II affects thrombin interaction with its platelet receptors, i.e. glycoprotein Ibalpha (GpIbalpha), protease-activated receptor (PAR) 1, and PAR4. Both synthetic gamma' peptide and fibrinogen fragment D*, containing the elongated gamma' chain, inhibited thrombin-induced platelet aggregation up to 70%, with IC(50) values of 42+/-3.5 and 0.47+/-0.03 microm, respectively. Solid-phase binding and spectrofluorimetric assays showed that both fragment D* and the synthetic gamma' peptide specifically bind to thrombin ABE-II and competitively inhibit the thrombin binding to GpIbalpha with a mean K(i) approximately 0.5 and approximately 35 microm, respectively. Both these gamma' chain-containing ligands allosterically inhibited thrombin cleavage of a synthetic PAR1 peptide, of native PAR1 molecules on intact platelets, and of the synthetic chromogenic peptide D-Phe-pipecolyl-Arg-p-nitroanilide. PAR4 cleavage was unaffected. In summary, fibrinogen gamma' chain binds with high affinity to thrombin and inhibits with combined mechanisms the platelet response to thrombin. Thus, its variations in vivo may affect the hemostatic balance in arterial circulation.

Conformational Analysis of γ‘ Peptide (410−427) Interactions with Thrombin Anion Binding Exosite II

Thrombin utilizes two anion binding exosites to supplement binding of fibrinogen to this serine protease. Approximately 7-15% of the fibrinogen gamma chain exists as the highly anionic gamma' variant (408VRPEHPAETEY(S)DSLY(S)PEDDL427). This segment has been demonstrated to target thrombin ABE-II and can accommodate sites of phosphorylation in place of sulfonation without sacrificing binding affinity. The present work employed 1D and 2D solution NMR to characterize the structural features of the bound gamma' peptide (410-427) and to evaluate the requirement of sulfonation for effective thrombin interaction. The results indicate the gamma' residues 414-427 make significant contact with the enzyme, a beta-turn exists between residues 422-425 in the presence of thrombin, and there is a large cluster of through-space interactions involving residues 418-422. Effective contact with ABE-II requires the presence of at least one phosphotyrosine residue with Y(P)422 being the more important player. Hydrogen-deuterium exchange (HDX) coupled with MALDI-TOF MS was implemented to examine the location of the gamma' peptide-thrombin interface and to screen for changes in solvent exposure at distant sites. The HDX results demonstrate that the gamma' peptide interacts with or is in close proximity to thrombin residues R93, R97, R173, and R175. The binding of the gamma' peptide also protects other regions of thrombin from deuterium exchange. Affected regions include segments of ABE-I, the autolysis loop, the edge of the active site region, and the A-chain. Finally, thrombin forms a ternary complex with the gamma' peptide and PPACK, generating an enzyme whose solvent-exposed regions are even further stabilized from HDX.

Characterization of the Interaction between Thrombin and a Pentapeptide That Interferes with Anion Binding Exosite I of the Enzyme.

We have recently characterized a pentapeptide, DYDYQ, from coagulation factor V (Beck et. al. 2004 J. Biol. Chem. 279, 3084) that inhibits both factor V activation and prothrombinase function. The pentapeptide does not interfere with the active site of thrombin but rather interferes with substrate attachment. Our aim was to characterize at the molecular level the binding site of DYDYQ on thrombin. First we used computational methods (blind and focused docking) to propose a hypothetical binding site. Blind docking of the pentapeptide (structure obtained from a 20 ns molecular dynamics simulation) on thrombin was performed using a docking grid with large spacing. This approach provided us a favorable site (−4.8 kcal/mol) that was further investigated using a smaller spaced docking grid. Hydrogen bonding was analyzed between thrombin and DYDYQ. The final free energy of binding was −9.69 kcal/mol. Amino acids Y76R77I79I82 from thrombin anion binding exosite I (ABE-I), were identified to participate in the interaction of the enzyme with DYDYQ. We next investigated the Thrombin-DYDYQ interaction following cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDC) and mass spectrometry. In these experiments purified thrombin was inhibited in the active site with a chloromethyl ketone, and treated with the DYDYQ peptide in the presence of EDC. Two bands were observed, one corresponding to thrombin cross linked to the peptide (CT) and another band corresponding to free thrombin (T). The proteins were either (i) stained with Coomassie blue for further digestion with trypsin or (ii) transferred to nitrocellulose membranes following by staining with Coomassie blue for treatment with cyanogen bromide (CNBr). Stained bands were isolated from the gel and subjected to trypsin digestion and liquid chromatography/mass spectrometry (LC-MS). Following trypsin digestion thrombin presence in both, T and CT samples was confirmed and the peptides identified in both samples covered approximately 63% of the entire thrombin sequence. The only difference observed between the sets of peptides obtained from T and CT following digestion with trypsin, was the peptide N78IEKISM*LEK87 (M* = oxidized Methionine), which was present in the T sample but was absent in the CT sample. These results suggest that the binding site of DYDYQ to thrombin is localized in the area of the above mentioned peptide protecting it from hydrolysis by trypsin. We next analyzed T and CT by LC-MS following CNBr digestion. Three important bands (peptide products from CNBr digestion) were detected in the sample containing the T, having an approximate molecular weight of ~4,500, ~7,500, and ~9,000. CNBr digestion products of CT lacked the median band (peptide mass: ~7,500). This band corresponded to the sequence L33…Y76ERN78IEKISM84 - as confirmed by ESI-ion trap mass spectrometry amino acid sequence of its first eleven amino acid residues. The difference between the two in gel CNBr digest profiles of T and CT, confirms the conclusion drawn from the MS analysis of the triptic digests which in turn was predicted by our computational analyses. Overall our data demonstrate that 1) amino acid residues Y76R77I79I82 from thrombin provide an interactive site for DYDYQ, and 2) our results from computational methods that identify protein-peptide interaction are valid and can be confirmed by mass spectrometry.

Identification of a Novel Factor V A2 Domain Tyrosine Deletion Mutation in a Patient with Factor V Deficiency and Bleeding.

The gene for coagulation Factor V (FV) is located on chromosome 1q23. FV deficiency shows an autosomal recessive mode of inheritance; heterozygotes are generally not clinically affected. The homozygous clinical phenotype occurs in approximately 1 per million individuals with variable severity of bleeding. Thus, genotype-phenotype correlations are likely to shed light on functionally important residues of FV. Here we describe a case of FV deficiency with a severe bleeding phenotype. The proband is a male infant from Argentina. His parents are unrelated. He was born healthy with no bleeding from the umbilical stump or other symptoms. He presented at eight months with a CNS hemorrhage, then suffered a second massive subdural bleed at nine months of age. Both episodes required surgical drainage and treatment with fresh frozen plasma He continues to receive prophylactic FFP infusions and has some residual neurologic impairment. The proband's FV activity ranges from 2–14%. Two siblings are unaffected. His father's FV activity is 50% and his mother's is 70%. We performed DNA sequencing spanning the entire coding region of the proband's FV gene and found two heterozygous mutations: a heterozygous single base pair deletion, del 2952T in exon 13, located in the B-domain of the FV protein, causing a frameshift mutation followed by a premature termination codon 3 amino acids downstream; and a novel 3-bp deletion in exon 10. This deletion is in-frame and results in the deletion of Y478. The del 2952T frameshift mutation was present in the father, while the del Y478 mutation was present in the mother. Y478 is in the A2 domain of FV and adjacent to another tyrosine, Y477. Evidence suggests that these tyrosine residues are important for co-factor function. Tyrosine residue sulfation has been shown to be required for full activity of the homologous co-factor, FVIII, as well as for hirudin. These sulfated tyrosines and surrounding acidic amino acids have been proposed to be important in interactions with the thrombin anion binding exosite; in the case of hirudin, sulfation of a carboxy-terminal tyrosine increases the affinity for thrombin 10-fold. The homologous tyrosines, Y718 and Y719 appear to be sulfated in FVIII. FV has been shown to be sulfated, but the precise location of the FV sulfation sites has not yet been determined. One of this patient's FV alleles is nonfunctional due to a frameshift and a premature trancation of translation. With respect to the other allele, we hypothesize that, like FVIII, one or both of FV tyrosines 477 and 478 is sulfated, and that deletion of Y478 may result in disruption of FV co-factor function. In vitro mutagenesis and expression studies to characterize the functional consequences of the del Y478 and/or del Y477 are in progress.

Thrombin Activation of Factor XI on Activated Platelets Requires the Interaction of Factor XI and Platelet Glycoprotein Ibα with Thrombin Anion-binding Exosites I and II, Respectively

Activation of factor XI (FXI) by thrombin on stimulated platelets plays a physiological role in hemostasis, providing additional thrombin generation required in cases of severe hemostatic challenge. Using a collection of 53 thrombin mutants, we identified 16 mutants with <50% of the wild-type thrombin FXI-activating activity in the presence of dextran sulfate. These mutants mapped to anion-binding exosite (ABE) I, ABE-II, the Na+-binding site, and the 50-insertion loop. Only the ABE-II mutants showed reduced binding to dextran sulfate-linked agarose. Selected thrombin mutants in ABE-I (R68A, R70A, and R73A), ABE-II (R98A, R245A, and K248A), the 50-insertion loop (W50A), and the Na+-binding site (E229A and R233A) with <10% of the wild-type activity also showed a markedly reduced ability to activate FXI in the presence of stimulated platelets. The ABE-I, 50-insertion loop, and Na+-binding site mutants had impaired binding to FXI, but normal binding to glycocalicin, the soluble form of glycoprotein Ibalpha (GPIb alpha). In contrast, the ABE-II mutants were defective in binding to glycocalicin, but displayed normal binding to FXI. Our data support a quaternary complex model of thrombin activation of FXI on stimulated platelets. Thrombin bound to one GPIb alpha molecule, via ABE-II on its posterior surface, is properly oriented for its activation of FXI bound to a neighboring GPI alpha molecule, via ABE-I on its anterior surface. GPIb alpha plays a critical role in the co-localization of thrombin and FXI and the resultant efficient activation of FXI.

Platelet Glycoprotein Ibα 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.

Inhibition of Prothrombin Activation by Bothrojaracin, a C-Type Lectin from Bothrops jararaca Venom

Bothrojaracin is a potent and specific α-thrombin inhibitor (Kd ∼ 0.6 nM) isolated from Bothrops jararaca venom. It binds to both of thrombin's anion-binding exosites (1 and 2), thus inhibiting the ability of the enzyme to act upon several natural macromolecular substrates, such as fibrinogen, platelet receptor, protein C, and factor V. Additionally, bothrojaracin interacts with prothrombin (Kd ∼ 30 nM), as previously determined by a solid-phase assay. However, there is no information concerning the effect of this interaction on prothrombin activation and whether the binding of bothrojaracin can occur in plasma. Here, we show that bothrojaracin specifically interacts with prothrombin in human plasma. It is an effective anticoagulant after activation of the intrinsic pathway of blood coagulation, and analysis of prothrombin conversion in plasma shows that bothrojaracin strongly reduces α-thrombin formation. To determine whether this effect is due exclusively to inhibition of feedback reactions involving the thrombin-induced activation of factors V and VIII, we analyzed the effect of bothrojaracin on the activation of purified prothrombin by Oxyuranus scutellatus venom. As with plasma, bothrojaracin greatly inhibited thrombin formation, suggesting a direct interference in the prothrombin activation by the enzyme found in this venom (scuterin, a prothrombin activator described as a factor Xa/factor Va-like complex). Altogether, we suggest that bothrojaracin exerts its anticoagulant effect in plasma by two distinct mechanisms: (1) it binds generated thrombin and inhibits exosite 1 dependent activities such as fibrinogen clotting and factor V activation, and (2) it interacts with prothrombin and decreases its proteolytic activation. Thus, bothrojaracin may be useful in the search for thrombin inhibitors that bind both the zymogen and the active enzyme.

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 (Kd = 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 α-thrombin, which possesses both exosites I and II, and with γ-thrombin, which lacks exosite I. On the one hand, bothrojaracin altered both kinetic parameters Km and kcat of α-thrombin for small synthetic substrates, resulting in an increased efficiency of α-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 γ-thrombin toward chromogenic substrates, although this effect was observed with higher concentrations of bothrojaracin than those used with α-thrombin. In agreement with these observaions, bothrojaracin produced opposite effects on the fluorescence intensity of α- and γ-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 γ-thrombin, or with exosite I (or both I and II) as observed with α-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.

Involvement of Thrombin Anion-binding Exosites 1 and 2 in the Activation of Factor V and Factor VIII

The role of anion-binding exosites of thrombin in the activation of factor V and factor VIII was studied using thrombin Arg93 --> Ala, Arg97 --> Ala, and Arg101 --> Ala (thrombin RA), a recombinant exosite 2 defective mutant, and a synthetic N-acetylated dodecapeptide, Ac-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-O-SO4Leu (hirugen), which competitively inhibits binding of macromolecules to exosite 1. The catalytic efficiency of the activation of factor VIII or of the first step of factor V activation by thrombin RA was approximately 10% that of wild-type thrombin. The overall rate of conversion to factor Va was not influenced by the mutation. In contrast to factor V, the slow activation of factor VIII by thrombin RA was associated with a decreased rate of cleavage at all three proteolytic sites (Arg372, Arg740, and Arg1689). Hirugen inhibited factor V and factor VIII activation. These results indicate that both anion-binding exosites of thrombin are involved in the recognition of factor V and factor VIII.

Triabin, a Highly Potent Exosite Inhibitor of Thrombin

Triabin, a new thrombin inhibitor, has been purified from the saliva of Triatoma pallidipennis, a blood-sucking triatomine bug. It forms a noncovalent complex with thrombin at a molar ratio of 1:1, inhibits thrombin-induced platelet aggregation, and prolongs thrombin clotting time and activated partial thromboplastin time. However, it only minimally suppresses the amidolytic activity of thrombin, as measured by a chromogenic peptide substrate assay. It completely blocks trypsin-catalyzed cleavage of thrombin, probably via protection of the anion-binding exosite and inhibits the effect of thrombomodulin on thrombin in a dose-dependent fashion. These results indicate that the inhibitor is directed toward the anion-binding exosite of thrombin. The protein was partially sequenced and the information used to isolate cDNA clones from a T. pallidipennis salivary gland library. Four slightly polymorphic variants coding for mature proteins of 142 amino acids preceded by a putative leader sequence were obtained. The recombinant protein expressed in the periplasmic space of Escherichia coli has a biological activity similar to that of salivary triabin, as tested in a thrombin-induced platelet aggregation assay. In addition, recombinant triabin inhibits thrombin-catalyzed hydrolysis of fibrinogen with a Ki of about 3 pM.

Structural domains of thrombin involved in the induction of mitogenesis in cultured human vascular smooth muscle cells.

Human alpha-thrombin is a known human vascular smooth muscle cell (HVSMC) mitogen. We have previously reported that gamma-thrombin, in which the anion binding exosite is disrupted, but not catalytically inactivated PPACK-thrombin is mitogenic on HVSMC. Here, the structural requirements for thrombin's mitogenic activity on HVSMC were further investigated. Total inhibition of thrombin-induced DNA synthesis was achieved by AT-III and hirudin. AT-III, added 1 h after exposure of cells to thrombin, failed to alter thrombin-induced mitogenicity. Modification of thrombin's anion binding exosite with peptides derived from the C-terminal sequence of hirudin resulted in a partial loss of thrombin's mitogenic activity. PPACK-inactivated thrombin failed to induce a significant expression of the immediate early gene, c-fos. Unlike PPACK-inactivated thrombin, alpha-thrombin and gamma-thrombin induced a significant increase in the level of the PDGF-A gene expression. A correlation between PDGF-A gene induction and thrombin-induced mitogenicity was suggested by the fact that the mitogenic forms of thrombin also stimulated PDGF-A gene expression. Together, these results indicate that the mitogenic activity of thrombin on HVSMC requires the integrity of the enzyme's active site and is altered by modifications of its anion binding exosite.

Inhibition of thrombin and SFLLR-peptide stimulation of platelet aggregation, phospholipase A 2 and Na +/H + exchange by a thrombin receptor antagonist

A thrombin receptor has been described that is activated by thrombin cleavage generating a new N-terminus. The newly exposed SFLLR-containing “tethered-ligand” then activates the receptor. In these studies, we used 3-mercapto-propionyl-Phe-Cha-Cha-Arg-Lys-Pro-Asn-Asp-Lys-amide (Mpapeptide) as a thrombin receptor antagonist. This compound was capable of preventing both thrombin- and SFLLR-peptide-induced platelet aggregation with little effect on collagen-induced platelet aggregation. It also prevented thrombin- and SFLLRNP-induced calcium mobilization with little effect on thromboxane receptor-activated platelet Ca 2+ mobilization. Platelet membrane GTPase could be activated by peptides that activated the thrombin receptor, and the thrombin receptor antagonist also prevented receptor-stimulated GTPase activity. Platelet phospholipase A 2 (PLA 2) activity (measured as the release of radiolabeled arachidonic acid) and Na +/H + exchange activation were stimulated by α-thrombin and by SFLLR-containing peptides. Activation of both processes with low concentrations of thrombin required thrombin's anion-binding exosite, as they were not activated by similar concentrations of γ-thrombin, and the α- and ζ- thrombin activation was blocked by peptides mimicking the C-terminal region of hirudin. Stimulation of PLA 2 and Na +/H + exchange by both thrombin and SFLLR-containing peptides was inhibited by the thrombin receptor antagonist Mpa-peptide. These results support the hypothesis that thrombin stimulation of PLA 2 activity and Na +/H + exchange occurs via activation of the thrombin tethered-ligand receptor. Moreover, these data are consistent with the tethered-ligand receptor mediating most actions elicited by low concentrations of α-thrombin involved in human platelet activation.

Role of the thrombin receptor in restenosis and atherosclerosis

Thrombus generation is central to thrombosis at vascular lesion sites, including post-PCTA acute reocclusion and chronic restenosis. Thrombin stimulates platelet activation, monocyte and neutrophil chemotaxis, and endothelial production of prothrombotic factors. The varied physiologic effects of thrombin are due to the widespread presence of thrombin receptors in many cell types. The receptor is uniquely activated: thrombin binds to the receptor at the thrombin anion-binding exosite, the receptor ligand (“tethered ligand”) apparently being a sequence of 6 amino acids (SFLLRN). Thus, peptides corresponding to the sequence of the tethered ligand can stimulate almost all functions of native thrombin itself. Several intracellular signaling pathways have been identified as important in the restenosis process: the G protein-related pathway, cyclic adenosine monophosphate (cAMP) mediator pathway, and tyrosine kinase activation pathway. In situ hybridization has demonstrated an increase in thrombin receptor mRNA throughout the period of neointimal and vascular lesion development. The mechanism of this increase is unknown, but may be mediated by multiple inflammatory modulators. Several strategies have been tested in animal models for inhibiting thrombin: (1) Hirudin not only prevents thrombin from cleaving fibrinogen, but also prevents thrombin receptor activation. (2) Thrombin receptor antagonist peptides block platelet aggregation effects of thrombin. (3) Mono- and polyclonal antibodies inhibit thrombin receptor activation. (4) Antisense oligonucleotides block thrombin receptor expression.

Thrombin interaction with a recombinant N-terminal extracellular domain of the thrombin receptor in an acellular system.

The cDNA of the human endothelial cell thrombin receptor has been cloned and a chimeric fusion protein consisting of glutathione-S-transferase (GST) and the portion 25-97 corresponding to the N-terminal first extracellular domain of the thrombin receptor (TRE) has been expressed in Escherichia coli. Introduction of a factor Xa cleavage site in the fusion protein allowed purification of TRE after removal from the GST carrier protein. Purified GST-TRE or TRE have been tested in solution for their ability to interact with thrombin. alpha-Thrombin cleaved the fusion protein at position Arg-41-Ser-42 of TRE in a time- and concentration-dependent manner and GST-TRE competed with the tripeptidic substrate S-2238 for hydrolysis by thrombin (Ki = 0.5 microM). gamma-Thrombin that lacks the anion-binding exosite was 100-fold less potent than alpha-thrombin at cleaving GST-TRE. TRE competed with polymerizing fibrin monomers for binding to thrombin (Ki = 7.5 microM). The cleavage of GST-TRE by alpha-thrombin was inhibited by several alpha-thrombin exosite ligands such as the C-terminal peptide of hirudin, thrombomodulin and fibrin(ogen) fragment E. In contrast, platelet glycocalicin did not inhibit GST-TRE cleavage. In conclusion, the use of purified soluble GST-TRE allowed us to derive an affinity constant for thrombin interaction with the N-terminal domain of the receptor and to confirm the location of the cleavage site at Arg41-Ser-42 of the receptor. The importance of the thrombin anion-binding exosite for thrombin receptor recognition is highlighted by the low reactivity of gamma-thrombin for GST-TRE and by competition experiments, which in addition indicate that binding sites for fibrin(ogen), thrombomodulin and GST-TRE are overlapping. In contrast, binding of thrombin to GST-TRE and glycocalicin are not mutually exclusive, indicating that glycocalicin and TRE interact with discrete subsites within the large groove that constitutes the anion-binding exosite.

An Autoantibody Directed Against Human Thrombin Anion-Binding Exosite in a Patient With Arterial Thrombosis: Effects on Platelets, Endothelial Cells, and Protein C Activation

Abstract An autoantibody, developed by a patient with severe and recurrent arterial thrombosis, was characterized to be directed against the anion- binding exosite of thrombin, and inhibited all thrombin interactions requiring this secondary binding site without interfering with the catalytic site. The effect of the antibody was studied on thrombin interactions with platelets and endothelial cells from human umbilical veins (HUVEC). The autoantibody specifically and concentration- dependently inhibited alpha-thrombin-induced platelet activation and prostacyclin (PGI2) synthesis from HUVEC. It had no effect when gamma- thrombin or the thrombin receptor activation peptide SFLLR were the inducers. The effect of the antibody on protein C activation has been studied. The antibody blocked the thrombin-thrombomodulin activation of protein C. The inhibition of the activation was maximal with a low concentration of thrombomodulin. The fact that the autoantibody inhibited concentration-dependent alpha-thrombin-induced platelet and endothelial cell functions emphasizes the crucial role of the anion- binding exosite of thrombin to activate its receptor. In regard to the pathology, the antibody inhibited two vascular processes implicated in thrombin-antithrombotic functions, PGI2 secretion, and protein C activation, which could be implicated in this arterial thrombotic disease.