Thrombin activation of factor XI on activated platelets requires the interaction of factor XI and platelet glycoprotein Ib alpha 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.     

Role of thrombin exosites in inhibition by heparin cofactor II.

We determined the role of specific thrombin "exosites" in the mechanism of inhibition by the plasma serine proteinase inhibitors heparin cofactor II (HC) and antithrombin (AT) in the absence and presence of a glycosaminoglycan by comparing the inhibition of alpha-thrombin to epsilon- and gamma T-thrombin (produced by partial proteolysis of alpha-thrombin by elastase and trypsin, respectively). All of the thrombin derivatives were inhibited in a similar manner by AT, either in the absence or presence of heparin, which confirmed the integrity of both heparin binding abilities and serpin reactivities of epsilon- and gamma T-thrombin compared to alpha-thrombin. Antithrombin activities of HC in the absence of a glycosaminoglycan with alpha-, epsilon, and gamma T-thrombin were similar with rate constants of 3.5, 2.4, and 1.2 x 10(4) M-1 min-1, respectively. Interestingly, in the presence of glycosaminoglycans the maximal inhibition rate constants by HC with heparin and dermatan sulfate, respectively, were as follows: 30.0 x 10(7) and 60.5 x 10(7) for alpha-thrombin, 14.6 x 10(7) and 24.3 x 10(7) for epsilon-thrombin, and 0.017 x 10(7) and 0.034 x 10(7) M-1 min-1 for gamma T-thrombin. A hirudin carboxyl-terminal peptide, which binds to anion-binding exosite-I of alpha-thrombin, dramatically reduced alpha-thrombin inhibition by HC in the presence of heparin but not in its absence. We analyzed our results in relation to the recently determined x-ray structure of D-Phe-Pro-Arg-chloromethyl ketone-alpha-thrombin (Bode, W., Mayr, I., Baumann, U., Huber, R., Stone, S. R., and Hofsteenge, J. (1989) EMBO J. 8, 3467-3475). Our results suggest that the beta-loop region of anion-binding exosite-I in alpha-thrombin, which is not present in gamma T-thrombin, is essential for the rapid inhibition reaction by HC in the presence of a glycosaminoglycan. Therefore, alpha-thrombin and its derivatives would be recognized and inhibited differently by HC and AT in the presence of a glycosaminoglycan.     

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.     

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.     

The role of thrombin exosites I and II in the activation of human coagulation factor V

Human blood coagulation Factor V (FV) is a plasma protein with little procoagulant activity. Limited proteolysis at Arg(709), Arg(1018), and Arg(1545) by thrombin or Factor Xa (FXa) results in the generation of activated FV, which serves as a cofactor of FXa in prothrombin activation. Both thrombin exosites I and II have been reported to be involved in FV activation, but the relative importance of these regions in the individual cleavages remains unclear. To investigate the role of each exosite in FV activation, we have used recombinant FV molecules with only one of the three activation cleavage sites available, in combination with exosite I- or II-specific aptamers. In addition, structural requirements for exosite interactions located in the B-domain of FV were probed using FV B-domain deletion mutants and comparison with FV activating enzymes from the venom of Russell's viper (RVV-V) and of Levant's viper (LVV-V) known to activate FV by specific cleavage at Arg(1545). Our results indicate that thrombin exosite II is not involved in cleavage at Arg(709) and that both thrombin exosites are important for recognition and cleavage at Arg(1545). Efficient thrombin-catalyzed FV activation requires both the N- and C-terminal regions of the B-domain, whereas only the latter is required by RVV-V and LVV-V. This indicates that proteolysis of FV by thrombin at Arg(709), Arg(1018), and Arg(1545) show different cleavage requirements with respect to interactions mediated by thrombin exosites and areas that surround the respective cleavage sites. In addition, interactions between exosite I of thrombin and FV are primarily responsible for the different cleavage site specificity as compared with activation by RVV-V or LVV-V.     

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.     

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.     

The role of the anion-binding site of transferrin in its interaction with the reticulocyte

Specific and tight binding of Fe(III) by transferrin does not occur unless a suitable anion is concomitantly bound. Bicarbonate, which normally occupies the anion binding site of the protein, may be replaced by an oxalate ion. The resulting ternary complex of Fe(III), transferrin and oxalate is less than 35% as effective as the bicarbonate complex in serving as an iron donor for heme synthesis by the reticulocyte. However, the binding of transferrin to the reticulocyte is not altered by the substitution of oxalate for bicarbonate. When both the oxalate and bicarbonate forms are incubated with reticulocytes, the uptake of iron from the bicarbonate complex is substantially depressed. The free oxalate ion, at the same concentration as the ternary Fe-transferrin-oxalate complex, does not alter the uptake of iron by reticulocytes from the native form of transferrin. The ternary Fe-transferrin-malonate complex is also less efficient than the bicarbonate complex as an iron donor to the reticulocyte, but the effect is less striking than that observed with the oxalate complex. The hypothesis is advanced that the mechanism of iron uptake from transferrin during the transferrin-reticulocyte interaction first entails an attack upon the anion bound to the protein, following which iron release to the heme-synthesizing apparatus of the cell takes place.     

Conformational changes induced in troponin I by interaction with troponin T and actin/tropomyosin.

Troponin I (TnI) is the inhibitory component of the striated muscle Ca2+ regulatory protein troponin (Tn). The other two components of Tn are troponin C (TnC), the Ca2+-binding component, and troponin T (TnT), the tropomyosin-binding component. We have used limited chymotryptic digestion to probe the local conformation of TnI in the free state, the binary TnC*TnI complex, the ternary TnC*. TnI*TnT (Tn) complex, and in the reconstituted Tn*tropomyosin*F-actin filament. The digestion of TnI alone or in the TnC*TnI complex produced initially two major fragments via a cleavage of the peptide bond between Phe100 and Asp101 in the so-called inhibitory region. In the ternary Tn complex cleavage occurred at a new site between Leu140 and Lys141. In the absence of Ca2+ this was followed by digestion of the 1-140 fragment at Leu122 and Met116. In the reconstituted thin filament the same fragments as in the case of the ternary complex were produced, but the rate of digestion was slower in the absence than in the presence of Ca2+. These results indicate firstly that in both free TnI and TnI complexed with TnC there is an exposed and flexible site in the inhibitory region. Secondly, TnT affects the conformation of TnI in the inhibitory region and also in the region that contains the 140-141 bond. Thirdly, the 140-141 region of TnI is likely to interact with actin in the reconstituted thin filament when Ca2+ is absent. These findings are discussed in terms of the role of TnI in the mechanism of thin filament regulation, and in light of our previous results [Y. Luo, J.-L. Wu, J. Gergely, T. Tao, Biochemistry 36 (1997) 13449-13454] on the global conformation of TnI.     

Conformational changes in actin induced by its interaction with gelsolin.

Actin cleaved by the protease from Escherichia coli A2 strain between Gly42 and Val43 (ECP-actin) is no longer polymerizable when it contains Ca2+ as a tightly bound cation, but polymerizes when Mg2+ is bound. We have investigated the interactions of gelsolin with this actin with regard to conformational changes in the actin molecule induced by the binding of gelsolin. ECP-(Ca)actin interacts with gelsolin in a manner similar to that in which it reacts with intact actin, and forms a stoichiometric 2:1 complex. Despite the nonpolymerizability of ECP-(Ca)actin, this complex can act as a nucleus for the polymerization of intact actin, thus indicating that upon interaction with gelsolin, ECP-(Ca)actin undergoes a conformational change that enables its interaction with another actin monomer. By gel filtration and fluorometry it was shown that the binding of at least one of the ECP-cleaved actins to gelsolin is considerably weaker than of intact actin, suggesting that conformational changes in subdomain 2 of actin monomer may directly or allosterically affect actin-gelsolin interactions. On the other hand, interaction with gelsolin changes the conformation of actin within the DNase I-binding loop, as indicated by inhibition of limited proteolysis of actin by ECP and subtilisin. Cross-linking experiments with gelsolin-nucleated actin filaments using N,N-phenylene-bismaleimide (which cross-links adjacent actin monomers between Cys374 and Lys191) reveal that gelsolin causes a significant increase in the yield of the 115-kDa cross-linking product, confirming the evidence that gelsolin stabilizes or changes the conformation of the C-terminal region of the actin molecule, and these changes are propagated from the capped end along the filament. These results allow us to conclude that nucleation of actin polymerization by gelsolin is promoted by conformational changes within subdomain 2 and at the C-terminus of the actin monomer.     

Dual role of Lys206-Lys296 interaction in human transferrin N-lobe: iron-release trigger and anion-binding site.

The unique structural feature of the dilysine (Lys206-Lys296) pair in the transferrin N-lobe (hTF/2N) has been postulated to serve a special function in the release of iron from the protein. These two lysines, which are located in opposite domains, hydrogen bond to each other in the iron-containing hTF/2N at neutral pH but are far apart in the apo-form of the protein. It has been proposed that charge repulsion resulting from the protonation of the dilysines at lower pH may be the trigger to open the cleft and facilitate iron release. The fact that the dilysine pair is positively charged and resides in a location close to the metal-binding center has also led to the suggestion that the dilysine pair is an anion-binding site for chelators. The present report provides comprehensive evidence to confirm that the dilysine pair plays this dual role in modulating release of iron. When either of the lysines is mutated to glutamate or glutamine or when both are mutated to glutamate, release of iron is much slower compared to the wild-type protein. This is due to the fact that the driving force for cleft opening is absent in the mutants or is converted to a lock-like interaction (in the case of the K206E and K296E mutants). Direct titration of the apo-proteins with anions as well as anion-dependent iron release studies show that the dilysine pair is part of an active anion-binding site which exists with the Lys296-Tyr188 interaction as a core. At this site, Lys296 serves as the primary anion-binding residue and Tyr188 is the main reporter for electronic spectral change, with smaller contributions from Lys206, Tyr85, and Tyr95. In iron-loaded hTF/2N, anion binding becomes invisible as monitored by UV-vis difference spectra since the spectral reporters Tyr188 and Tyr95 are bound to iron. Our data strongly support the hypothesis that the apo-hTF/2N exists in equilibrium between the open and closed conformations, because only in the closed form is Lys296 in direct contact with Tyr188. The current findings bring together observations, ideas, and experimental data from a large number of previous studies and shed further light on the detailed mechanism of iron release from the transferrin N-lobe. In iron-containing hTF/2N, Lys296 may still function as a target to introduce an anion (or a chelator) near to the iron-binding center. When the pH is lowered, the protonation of carbonate (synergistic anion for metal binding) and then the dilysine pair form the driving force to loosen the cleft, exposing iron; the nearby anion (or chelator) then binds to the iron and releases it from the protein.     

Evidence for a single catalytic site of honeybee alpha-glucosidase I by chemical modification with diethylpyrocarbonate.

Honeybee alpha-glucosidase I was inactivated with diethylpyrocarbonate (DEPC). The inactivation followed pseudo-first-order kinetics. The rate of the loss of activity was decreased by the addition of a substrate, maltose. Since there was no spectral change in the tyrosine absorption region, it was recognized that DEPC did not react with this residue. The alpha-glucosidase had one free sulfhydryl group, which was not involved in the catalytic reaction, and was not modified by DEPC. On the other hand, the specific reaction of DEPC with a histidyl residue was spectrophotometrically confirmed by an increase in absorption near 240 nm, and the activity of the inactivated enzyme was restored by hydroxylamine. The modification rate of one histidyl residue by DEPC was almost equal to the rate of the activity loss. These results indicate that there is one histidyl residue at or near the catalytic site, and that honeybee alpha-glucosidase I has a single active site.     

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

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

Discovery of a new class of catalytic topoisomerase II inhibitors targeting the ATP-binding site by structure based design. Part I

Topoisomerase II is a validated target in oncology. Among the different ways of blocking the function of this enzyme, inhibiting its ATPase activity has been relatively less investigated. In an effort to identify topoisomerase II inhibitors of a novel type, exerting their action by this mechanism, we have designed a purine inhibitor scaffold targeting the ATP-binding site of the enzyme. Searching the Novartis compound collection for molecules containing this purine motif has allowed the identification of two micromolar hits providing access to a new class of catalytic topoisomerase II inhibitors.     

Heparin and calcium ions dramatically enhance antithrombin reactivity with factor IXa by generating new interaction exosites

Blood coagulation factor IXa has been presumed to be regulated by the serpin, antithrombin, and its polysaccharide activator, heparin, but it has not been clear whether factor IXa is inhibited by the serpin with a specificity comparable to that for thrombin and factor Xa or what determinants govern this specificity. Here we show that antithrombin is essentially unreactive with factor IXa in the absence of heparin (k(ass) approximately 10 M(-1) s(-1)) but undergoes a remarkable approximately 1 million-fold enhancement in reactivity with this proteinase to the physiologically relevant range (k(ass) approximately 10(7) M(-1) s(-1)) when activated by heparin in the presence of physiologic levels of calcium. This rate enhancement is shown to derive from three sources: (i) allosteric activation of antithrombin by a sequence-specific heparin pentasaccharide (300-500-fold), (ii) allosteric activation of factor IXa by calcium ions (4-8-fold), and (iii) heparin bridging of antithrombin and factor IXa augmented by calcium ions (130-1000-fold depending on heparin chain length). Mutagenesis of P6-P3' reactive loop residues of antithrombin further reveals that the reactivity of the unactivated inhibitor is principally determined by the P1 Arg residue, whereas exosites outside the loop which are present on the activated serpin and on heparin are responsible for heparin enhancement of this reactivity. These results together with our previous findings demonstrate that exosites are responsible for the unusual specificity of antithrombin and heparin for three clotting proteases with quite distinct substrate specificities.