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

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

Activation of heparin cofactor II by calcium spirulan

Heparin cofactor II (HCII) is a plasma serine protease inhibitor whose ability to inhibit alpha-thrombin is accelerated by a variety of sulfated polysaccharides in addition to heparin and dermatan sulfate. Previous investigations have indicated that calcium spirulan (Ca-SP), a novel sulfated polysaccharide, enhanced the rate of inhibition of alpha-thrombin by HCII. In this study, we investigated the mechanism of the activation of HCII by Ca-SP. Interestingly, in the presence of Ca-SP, an N-terminal deletion mutant of HCII (rHCII-Delta74) inhibited alpha-thrombin, as native recombinant HCII (native rHCII) did. The second-order rate constant for the inhibition of alpha-thrombin by rHCII-Delta74 was 2.0 x 10(8) M(-1) min(-1) in the presence of 50 microgram/ml Ca-SP and 10, 000-fold higher than in the absence of Ca-SP. The rates of native rHCII and rHCII-Delta74 for the inhibition of gamma-thrombin were increased only 80- and 120-fold, respectively. Our results suggested that the anion-binding exosite I of alpha-thrombin was essential for the rapid inhibition reaction by HCII in the presence of Ca-SP and that the N-terminal acidic domain of HCII was not required. Therefore, we proposed a mechanism by which HCII was activated allosterically by Ca-SP and could interact with the anion-binding exosite I of thrombin not through the N-terminal acidic domain of HCII. The Arg(103) --> Leu mutant bound to Ca-SP-Toyopearl with normal affinity and inhibited alpha-thrombin in a manner similar to native rHCII. These results indicate that Arg(103) in HCII molecule is not critical for the interaction with Ca-SP.     

The N-terminal acidic domain of heparin cofactor II mediates the inhibition of alpha-thrombin in the presence of glycosaminoglycans

Heparin cofactor II (HCII) is a glycoprotein in human plasma that inhibits thrombin and chymotrypsin. Inhibition occurs when the protease attacks the reactive site peptide bond in HCII (Leu444-Ser445) and becomes trapped as a covalent 1:1 complex. Dermatan sulfate and heparin increase the rate of inhibition of thrombin, but not of chymotrypsin, greater than 1000-fold. The N-terminal portion of HCII contains two acidic repeats (Glu56-Asp-Asp-Asp-Tyr-Leu-Asp and Glu69-Asp-Asp-Asp-Tyr-Ile-Asp) that may bind to anion-binding exosite I of thrombin to facilitate covalent complex formation. To examine the importance of the acidic domain, we have constructed a series of 5' deletions in the HCII cDNA and expressed the recombinant HCII (rHCII) in Escherichia coli. Apparent second-order rate constants (k2) for inhibition of alpha-thrombin and chymotrypsin by each variant were determined. Deletion of amino acid residues 1-74 had no effect on the rate of inhibition of alpha-thrombin or chymotrypsin in the absence of a glycosaminoglycan. Similarly, the rate of inhibition of alpha-thrombin in the presence of a glycosaminoglycan was unaffected by deletion of residues 1-52. However, deletion of residues 1-67 (first acidic repeat) or 1-74 (first and second acidic repeats) greatly decreased the rate of inhibition of alpha-thrombin in the presence of heparin, dermatan sulfate, or a dermatan sulfate hexasaccharide that comprises the minimum high-affinity binding site for HCII. Deletion of one or both of the acidic repeats increased the apparent affinity of rHCII for heparin-Sepharose, suggesting that the acidic domain may interact with the glycosaminoglycan-binding site of native rHCII. The stimulatory effect of glycosaminoglycans on native rHCII was decreased by a C-terminal hirudin peptide which binds to anion-binding exosite I of alpha-thrombin. Furthermore, the ability of native rHCII to inhibit gamma-thrombin, which lacks the binding site for hirudin, was stimulated weakly by glycosaminoglycans. These results support a model in which the stimulatory effect of glycosaminoglycans on the inhibition of alpha-thrombin is mediated, in part, by the N-terminal acidic domain of HCII.     

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.     

Multiple pathways of thrombin-induced platelet activation differentiated by desensitization and a thrombin exosite inhibitor.

Recently a thrombin receptor with a unique mechanism of activation was cloned from a megakaryocyte-like cell line (Vu et al., Cell 64:1057-1068, 1991). Thrombin cleaves a portion of this receptor creating a new N-terminus that acts as a "tethered-ligand" to activate the receptor. A thrombin receptor activating peptide (SFLLRNPNDKYEPF) homologous to the new N-terminus was shown to activate platelets. We synthesized this peptide and demonstrated that it desensitized platelets to activation by low concentrations of alpha-thrombin but not gamma-thrombin. We also synthesized a thrombin exosite inhibitor (BMS 180742) that inhibited platelet aggregation induced by low, but not high, concentrations of alpha-thrombin. In contrast, a thrombin active site inhibitor, N alpha-(2-naphthylsulfonyl-glycyl)-D,L-amidinophenylalanylpiperi dide, competitively inhibited thrombin-induced platelet aggregation. We conclude that thrombin-induced platelet activation is mediated by at least two pathways: one activated by low concentrations of alpha-thrombin and blocked by a thrombin exosite inhibitor that appears to be coupled to the "tethered-ligand" thrombin receptor, and another that is stimulated by higher concentrations of alpha-thrombin and by gamma-thrombin and does not require the thrombin exosite for activation. Both pathways are blocked by a thrombin active site inhibitor.     

Proteolytic formation of either of the two prothrombin activation intermediates results in formation of a hirugen-binding site.

Hirugen, a synthetic dodecapeptide corresponding to the carboxyl-terminal amino acids 53-64 of hirudin, binds within a deep groove in thrombin that contains a cationic region referred to as the anion-binding exosite. This region is important in many of the binary interactions of thrombin with macromolecular substrates and cofactors. Fluorescein-labeled hirugen was used to probe which steps in the prothrombin activation process generate this anion-binding exosite. Two activation cleavage sites exist in bovine prothrombin. Cleavage at Arg274-Thr275 releases the activation fragments to generate the thrombin precursor, prethrombin 2. Cleavage of prothrombin within a disulfide loop at Arg323-Ile324 leads to formation of meizothrombin with no loss of peptide material but with formation of amidolytic activity. Cleavage of the same bond in prethrombin 2 generates thrombin. Hirugen, labeled at the amino terminus with fluorescein isothiocyanate, does not bind to prothrombin but does bind to thrombin (Kd = 9.6 +/- 1.2 x 10(-8) M), prethrombin 2 (Kd = 1.3 +/- 0.1 x 10(-7) M), thrombin-fragment-2 complex (Kd = 1.1 +/- 0.2 x 10(-6) M), and meizothrombin (Kd = 1.6 +/- 0.5 x 10(-8) M). Prothrombin fragment-2 and hirugen both bind independently to thrombin. A ternary complex can form with hirugen and fragment-2 and either thrombin or prethrombin 2, suggesting that fragment-2 and hirugen bind to discrete sites. Hirugen also alters the active site conformation of thrombin as detected by modulation of synthetic substrate hydrolytic activity. These studies suggest that conformational changes, rather than alleviating steric hindrance, are responsible for the formation of the hirugen-binding site during prothrombin activation. Furthermore, this conformational change can be effected by the cleavage of either of the two bonds required for activation of prothrombin.     

The fibrinogen anion-binding exosite of thrombin is necessary for induction of rises in intracellular calcium and prostacyclin production in endothelial cells.

Thrombin stimulation of prostacyclin (PGI2) synthesis by cultured human umbilical vein endothelial cells (HUVEC) requires the active site of thrombin and involves rapid and transient rises in cytoplasmic free calcium [Ca2+]i. In this study, we investigated whether or not the anion-binding exosite for fibrinogen recognition of thrombin (which confers certain substrate specificities) is also necessary for the induction of rises in [Ca2+]i and PGI2 production. Thrombin variants which lack either the catalytic site (DIP-alpha-thrombin) or anion-binding exosite (gamma-thrombin) either alone or in combination failed to induce rises in [Ca2+]i or PGI2 production in HUVEC. To further study the role of the anion-binding exosite of thrombin in the activation of HUVEC, COOH-terminal fragments of hirudin were used. This portion of hirudin interacts with the anion-binding exosite of thrombin and inhibits thrombin-induced fibrinogen coagulation while leaving the catalytic activity of thrombin intact. A 21-amino acid COOH-terminal peptide of hirudin (N alpha-acetyldesulfato-hirudin45-65 or Hir45-65) inhibited thrombin-induced (0.5 U/ml) rises in [Ca2+]i and PGI2 production with IC50 of 0.13 and 0.71 microM, respectively. Similar results were obtained using shorter hirudin-derived peptides. Thus, the fibrinogen anion-binding exosite of thrombin is required for alpha-thrombin-induced rises in [Ca2+]i and PGI2 production in HUVEC.     

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.     

Anion-binding exosite of human alpha-thrombin and fibrin(ogen) recognition

Activation of prothrombin to alpha-thrombin generates not only the catalytic site and associated regions but also an independent site (an exosite) which binds anionic substances, such as Amberlite CG-50 resin [cross-linked poly(methylacrylic acid)]. Like human alpha-thrombin with high fibrinogen clotting activity (peak elution at I = 0.40 +/- 0.01 M, pH 7.4, approximately 23 degrees C), catalytically inactivated forms (e.g., i-Pr2P-alpha- and D-Phe-Pro-Arg-CH2-alpha-thrombins) were eluted with only slightly lower salt concentrations (I = 0.36-0.39 M), while gamma-thrombin with very low clotting activity was eluted with much lower concentrations (I = 0.29 M) and the hirudin complex of alpha-thrombin was not retained by the resin. In a similar manner, hirudin complexes of alpha-, i-Pr2P-alpha-, and gamma-thrombin were not retained by nonpolymerized fibrin-agarose resin. Moreover, the ionic strengths for the elution from the CG-50 resin of seven thrombin forms were directly correlated with those from the fibrin resin (y = 0.15 + 0.96x, r = 0.95). In other experiments, the 17 through 27 synthetic peptide of the human fibrinogen A alpha chain was not an inhibitor of alpha-thrombin, while the NH2-terminal disulfide knot (NDSK) fragment was a simple competitive inhibitor of alpha-thrombin with a Ki approximately 3 microM (0.15 M NaCl, pH 7.3, approximately 23 degrees C). These data suggest that alpha-thrombin recognizes fibrin(ogen) by a negatively charged surface, noncontiguous with the A alpha cleavage site but found within the NDSK fragment. Such interaction involving an anion-binding exosite may explain the exceptional specificity of alpha-thrombin for the A alpha cleavage in fibrinogen and alpha-thrombin incorporation into fibrin clots.     

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

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

Study on secondary structure and properties of alpha- and gamma-forms of human thrombin

Secondary structure and enzymatic properties of human a-thrombin and its gamma-form (obtaining during autolysis of the native enzyme) have been studied by differential scanning calorimetry (DSC) and circular dichroism (CD). According to DSC-data both alpha-thrombin and gamma-thrombin contained only one thermal transition peak at 58.5 and 53.3 degrees C, respectively. A comparison of these values suggested that gamma-form is less stable than initial a-thrombin. In contrast to that the thermogram of DIP-a-thrombin had two peaks (57.5 and 64.5 degrees C). CD spectra showed that conversion a- to gamma-thrombin influenced the secondary structure of the enzyme slightly. The study of the inhibitory effect of such polyanions as ATP and dextran sulfate (DS) upon thrombin-catalyzed cleavages of fibrinogen has shown that the growth of the negative charge of the polyanion molecule resulted in the increase of its inhibitory activity. The catalytically non-active DIP-alpha-thrombin, which retained the native anion-binding exosite 1, was shown to decrease the inhibitory power of the dextran sulfate. It was explained by competition of DS with the exosite 1 of both alpha- and DIP-alpha -thrombin. In contrast to that DIP-gamma-thrombin having exosite 1 destroyed neither competed nor influenced the anticoagulant capacity of dextran sulfate toward the native alpha-thrombin. In accordance with our data thrombin consists of two rather strong interacting domains. It was shown further that its anion-binding exosite 1 may play a significant role in the interaction of the enzyme with dextran sulfate.     

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.     

A fibroblast-derived urokinase-inhibitor differing from protease nexin

UK-I, a 60-kDa urokinase-inhibitor derived from human fibroblasts, inhibited 54-kDa urokinase (EC 3.4.21.31) activity dose-dependently on ordinary fibrin-agar autograms. This UK-I formed an SDS-stable approximately 75-kDa complex with radioiodinated urokinase (33 kDa) on an autoradiogram following SDS/polyacrylamide gel electrophoresis. Benzamidine hydrochloride inhibited its formation, indicating UK-I to bind at the active site of urokinase and form an inactive complex. UK-I did not form a complex with [125I]thrombin (EC 3.4.21.5). It is thus evident that UK-I is one type of urokinase-inhibitor derived from human fibroblasts with properties differing from protease nexin, another urokinase-inhibitor derived from the same source.     

Thrombin signal transduction mechanisms in rat vascular smooth muscle cells. Calcium and protein kinase C-dependent and -independent pathways.

Sustained generation of alpha-thrombin and its breakdown forms at sites of thromboses has focused attention on the roles thrombin may play in vascular responses to thrombosis and injury. We have previously shown that alpha-thrombin stimulates many growth signals in cultured rat aortic smooth muscle cells (VSMC). To characterize thrombin growth mechanisms, we studied the effects on cultured VSMC of gamma-thrombin (catalytically active with obstructed anion-binding site required for clotting activity) and D-phenylalanyl-L-prolyl-L-arginine chloromethylketone-alpha-thrombin (catalytically inactive with intact anion-binding exosite) on cultured VSMC. Either derivative alone failed to increase growth, but in combination at 130 nM each, they caused a 75 +/- 5% increase in protein synthesis, similar to that observed with alpha-thrombin. This increase in protein synthesis was related to activation of protein kinase C (PKC) and Na+/H+ exchange, because only in combination could the derivatives increase phosphorylation of a 76,000-dalton PKC substrate and alkalinize the cells. Activation of PKC was correlated with a synergistic effect of the derivatives on diacylglycerol formation at 2 min (maximum, 55 +/- 1% combined increase vs. 24 +/- 9% and 4 +/- 4% individual increases with gamma- and D-phenylalanyl-L-prolyl-L-arginine chloromethylketone-alpha-thrombin alone, respectively, p less than 0.05). The derivatives stimulated PKC without increasing inositol trisphosphate, intracellular Ca2+, or expression of the protooncogene, c-fos. Thus, thrombin stimulation of Na+/H+ exchange, diacylglycerol formation, and growth of VSMC can be distinguished from thrombin mobilization of [Ca2+]i and induction of c-fos mRNA. These data indicate the presence of more than one mechanism for thrombin-mediated signaling events in cultured VSMC. Our results also suggest that various thrombin forms retained in clots may have significant effects on VSMC growth and function.     

Affinity labeling of lysine-149 in the anion-binding exosite of human alpha-thrombin with an N alpha-(dinitrofluorobenzyl)hirudin C-terminal peptide

In order to define structural regions in thrombin that interact with hirudin, the N alpha-dinitrofluorobenzyl analogue of an undecapeptide was synthesized corresponding to residues 54-64 of hirudin [GDFEEIPEEY(O35SO3)L (DNFB-[35S]Hir54-64)]. DNFB-[35S]Hir54-64 was reacted at a 10-fold molar excess with human alpha-thrombin in phosphate-buffered saline at pH 7.4 and 23 degrees C for 18 h. Autoradiographs of the product in reducing SDS-polyacrylamide gels revealed a single 35S-labeled band of Mr approximately 32,500. The labeled product was coincident with a band on Coomassie Blue stained gels migrating slightly above an unlabeled thrombin band at Mr approximately 31,000. Incorporation of the 35S affinity reagent peptide was found markedly reduced when reaction with thrombin was performed in the presence of 5- and 20-fold molar excesses of unlabeled hirudin peptide, showing that a specific site was involved in complex formation. The human alpha-thrombin-DNFB-Hir54-64 complex was reduced, S-carboxymethylated, and treated with pepsin. Peptic fragments were separated by reverse-phase HPLC revealing two major peaks containing absorbance at 310 nm. Automated Edman degradation of the peptide fragments allowed identification of Lys-149 of human thrombin as the major site of DNFB-Hir54-64 derivatization. These data suggest that the anionic C-terminal tail of hirudin interacts with an anion-binding exosite in human thrombin removed 18-20 A from the catalytic apparatus.     

Localization of thrombomodulin-binding site within human thrombin

A binding site for thrombomodulin on human thrombin (alpha-thrombin) was elucidated by identifying an epitope for a monoclonal antibody for thrombin (MT-6) which inhibited the activation of protein C by the thrombin-thrombomodulin complex by directly inhibiting the binding of thrombin to thrombomodulin. An 8.5-kDa fragment isolated by digestion of thrombin with Staphylococcus aureus V8 protease followed by reversed-phase high performance liquid chromatography (HPLC) and a peptide isolated by reversed-phase HPLC after reduction of the 8.5-kDa fragment, which was composed of three peptides linked by disulfide-bonds, bound directly to MT-6 and thrombomodulin. The amino acid sequence of the peptide coincided with the sequence of residues Thr-147 to Asp-175 of the B-chain of thrombin. A synthetic peptide corresponding to Thr-147 to Ser-158 of the B-chain inhibited the binding of thrombin to thrombomodulin. Elastase-digested thrombin, which was cleaved between Ala-150 and Asn-151, lost its binding affinity for both MT-6 and thrombomodulin. These findings indicate that the binding site for thrombomodulin is located within the sequence between Thr-147 and Ser-158 of the B-chain.     

The interaction of thrombin with fibrinogen. A structural basis for its specificity.

The structure of the ternary complex of human alpha-thrombin with a covalently bound analogue of fibrinopeptide A and a C-terminal hirudin peptide has been determined by X-ray diffraction methods at 0.25 nm resolution. Fibrinopeptide A folds in a compact manner, bringing together hydrophobic residues that slot into the apolar binding site of human alpha-thrombin. Fibrinogen residue Phe8 occupies the aryl-binding site of thrombin, adjacent to fibrinogen residues Leu9 and Val15 in the S2 subsite. The species diversity of fibrinopeptide A is analysed with respect to its conformation and its interaction with thrombin. The non-covalently attached peptide fragment hirudin(54-65) exhibits an identical conformation to that observed in the hirudin-thrombin complex. The occupancy of the secondary fibrinogen-recognition exosite by this peptide imposes restrictions on the manner of fibrinogen binding. The surface topology of the thrombin molecule indicates positions P1'-P3', differ from those of the canonical serine-proteinase inhibitors, suggesting a mechanical model for the switching of thrombin activity from fibrinogen cleavage to protein-C activation on thrombomodulin complex formation. The multiple interactions between thrombin and fibrinogen provide an explanation for the narrow specificity of thrombin. Structural grounds can be put forward for certain congenital clotting disorders.     

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

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

Evidence that catalytically-inactivated thrombin forms non-covalently linked dimers that bridge between fibrin/fibrinogen fibers and enhance fibrin polymerization

Phe-pro-arg-chloromethyl ketone-inhibited alpha-thrombin [FPR alpha-thr] retains its fibrinogen recognition site (exosite 1), augments fibrin/fibrinogen [fibrin(ogen)] polymerization, and increases the incorporation of fibrin into clots. There are two 'low-affinity' thrombin-binding sites in each central E domain of fibrin, plus a non-substrate 'high affinity' gamma' chain thrombin-binding site on heterodimeric 'fibrin(ogen) 2' molecules (gamma(A), gamma'). 'Fibrin(ogen) 1' (gamma(A), gamma(A)) containing only low-affinity thrombin-binding sites, showed concentration-dependent FPR alpha-thr enhancement of polymerization, thus indicating that low-affinity sites are sufficient for enhancing polymerization. FPR gamma-thr, whose exosite 1 is non-functional, did not enhance polymerization of either fibrin(ogen)s 1 or 2 and DNA aptamer HD-1, which binds specifically to exosite 1, blocked FPR alpha-thr enhanced polymerization of both types of fibrin(ogen) (1>2). These results showed that exosite 1 is the critical element in thrombin that mediates enhanced fibrin polymerization. Des B beta 1-42 fibrin(ogen) 1, containing defective 'low-affinity' binding sites, was subdued in its FPR alpha-thr-mediated reactivity, whereas des B beta 1-42 fibrin(ogen) 2 (gamma(A), gamma') was more reactive. Thus, the gamma' chain thrombin-binding site contributes to enhanced FPR alpha-thr mediated polymerization and acts through a site on thrombin that is different from exosite 1, possibly exosite 2. Overall, the results suggest that during fibrin clot formation, catalytically-inactivated FPR alpha-thr molecules form non-covalently linked thrombin dimers, which serve to enhance fibrin polymerization by bridging between fibrin(ogen) molecules, mainly through their low affinity sites.