Fibrinogen-elongated gamma 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.     

Crystal structure of thrombin in complex with fibrinogen gamma' peptide

Elevated levels of heterodimeric gamma(A)/gamma' fibrinogen 2 have been associated with an increased incidence of coronary artery disease, whereas a lowered content of gamma' chains is associated with an increased risk of venous thrombosis. Both situations may be related to the unique features of thrombin binding to variant gamma' chains. The gamma' peptide is an anionic fragment that binds thrombin with high affinity without interfering directly with substrate binding. Here we report the crystal structure of thrombin bound to the gamma' peptide, solved at 2.4 A resolution. The complex reveals extensive interactions between thrombin and the gamma' peptide mediated by electrostatic contacts with residues of exosite II and hydrophobic interactions with a pocket in close proximity to the Na(+) binding site. In its binding mode, the gamma' peptide completely overlaps with heparin bound to exosite II. These findings are consistent with functional data and broaden our understanding of how thrombin interacts with fibrinogen at the molecular level.     

An extensive interaction interface between thrombin and factor V is required for factor V activation

The interaction interface between human thrombin and human factor V (FV), necessary for complex formation and cleavage to generate factor Va, was investigated using a site-directed mutagenesis strategy. Fifty-three recombinant thrombins, with a total of 78 solvent-exposed basic and polar residues substituted with alanine, were used in a two-stage clotting assay with human FV. Seventeen mutants with less than 50% of wild-type (WT) thrombin FV activation were identified and mapped to anion-binding exosite I (ABE-I), anion-binding exosite II (ABE-II), the Leu(45)-Asn(57) insertion loop, and the Na(+) binding loop of thrombin. Three ABE-I mutants (R68A, R70A, and Y71A) and the ABE-II mutant R98A had less than 30% of WT activity. The thrombin Na(+) binding loop mutants, E229A and R233A, and the Leu(45)-Asn(57) insertion loop mutant, W50A, had a major effect on FV activation with 5, 15, and 29% of WT activity, respectively. The K52A mutant, which maps to the S' specificity pocket, had 29% of WT activity. SDS-polyacrylamide gel electrophoresis analysis of cleavage reactions using the thrombin ABE mutants R68A, Y71A, and R98A, the Na(+) binding loop mutant E229A, and the Leu(45)-Asn(57) insertion loop mutant W50A showed a requirement for both ABEs and the Na(+)-bound form of thrombin for efficient cleavage at the FV residue Arg(709). Several basic residues in both ABEs have moderate decreases in FV activation (40-60% of WT activity), indicating a role for the positive electrostatic fields generated by both ABEs in enhancing complex formation with complementary negative electrostatic fields generated by FV. The data show that thrombin activation of FV requires an extensive interaction interface with thrombin. Both ABE-I and ABE-II and the S' subsite are required for optimal cleavage, and the Na(+)-bound form of thrombin is important for its procoagulant activity.     

Identification of a thrombin-binding region in the sixth epidermal growth factor-like repeat of human thrombomodulin

The interaction of thrombin with a 28-residue peptide corresponding to the N-terminal subdomain of the sixth EGF-like repeat of human thrombomodulin plus the junction between the fifth and the sixth EGF-like domains was characterized in solution by use of NMR spectroscopy, particularly differential resonance perturbations and transferred nuclear Overhauser effects (transferred NOEs). The EGF-like thrombomodulin fragment, or hTM422-449, is conformationally flexible in the absence of thrombin. Upon addition of thrombin, differential resonance perturbations and transferred NOEs are observed for the thrombomodulin peptide, suggesting specific and rapidly reversible binding and structuring of hTM422-449 in complex with thrombin. Residue-specific analysis of the differential line broadening, resonance shifts, and transferred NOEs identified regions of hTM422-449 responding to thrombin binding as the N-terminal residues Thr422-Ile424 and residues His438-Ile447 corresponding to the central beta-hairpin, or B-loop, of the consensus EGF-like repeat. The formation of the beta-hairpin is supported by the pattern of transferred NOEs bringing the two beta-strands together and characterizing a type I beta-turn. Docking of the thrombomodulin peptide to the anion-binding exosite I of thrombin revealed structural details capturing binding contacts identified so far as essential for the thrombin-thrombomodulin interaction. Definition of specific interactions between thrombin and a minimal fragment of the sixth EGF-like domain of human TM may lead to the discovery of new peptidomimetic molecules as modulators of blood coagulation.     

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.     

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

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

The role of gamma A/gamma ' fibrinogen in plasma factor XIII activation

Factor XIII zymogen activation is a complex series of events that involve fibrinogen acting in several different roles. This report focuses on the role of fibrinogen as a cofactor in factor XIII activation by thrombin. We demonstrate that fibrinogen has two distinct activities that lead to an increased rate of factor XIII activation. First, the thrombin proteolytic activity is increased by fibrin. The cleavage rates of both a small chromogenic substrate and the factor XIII activation peptide are increased in the presence of either the major fibrin isoform, gammaA/gammaA fibrin, or a minor variant form, gammaA/gamma' fibrin. This enhancement of thrombin activity by fibrin is independent of fibrin polymerization and requires only cleavage of the fibrinopeptides. Subsequently, gammaA/gamma' fibrinogen accelerates plasma factor XIII activation by a non-proteolytic mechanism. This increased rate of activation results in a slightly more rapid cross-linking of fibrin gammaA and gamma' chains and a significantly more rapid cross-linking of fibrin alpha chain multimers. Together, these results show that although both forms of fibrin increase the rate of activation peptide cleavage by thrombin, gammaA/gamma' fibrinogen also increases the rate of factor XIII activation in a non-proteolytic manner. A revised model of factor XIII activation is presented below.     

Thrombin hydrolysis of human osteopontin is dependent on thrombin anion-binding exosites

The cytokine osteopontin (OPN) can be hydrolyzed by thrombin exposing a cryptic alpha(4)beta(1)/alpha(9)beta(1) integrin-binding motif (SVVYGLR), thereby acting as a potent cytokine for cells bearing these activated integrins. We show that purified milk OPN is a substrate for thrombin with a k(cat)/K(m) value of 1.14 x 10(5) m(-1) s(-1). Thrombin cleavage of OPN was inhibited by unsulfated hirugen (IC(50) = 1.2 +/- 0.2 microm), unfractionated heparin (IC(50) = 56.6 +/- 8.4 microg/ml) and low molecular weight (5 kDa) heparin (IC(50) = 31.0 +/- 7.9 microg/ml), indicating the involvement of both anion-binding exosite I (ABE-I) and anion-binding exosite II (ABE-II). Using a thrombin mutant library, we mapped residues important for recognition and cleavage of OPN within ABE-I and ABE-II. A peptide (OPN-(162-197)) was designed spanning the OPN thrombin cleavage site and a hirudin-like C-terminal tail domain. Thrombin cleaved OPN-(162-197) with a specificity constant of k(cat)/K(m) = 1.64 x 10(4) m(-1) s(-1). Representative ABE-I mutants (K65A, H66A, R68A, Y71A, and R73A) showed greatly impaired cleavage, whereas the ABE-II mutants were unaffected, suggesting that ABE-I interacts principally with the hirudin-like OPN domain C-terminal and contiguous to the thrombin cleavage site. Debye-Hückel slopes for milk OPN (-4.1 +/- 1.0) and OPN-(162-197) (-2.4 +/- 0.2) suggest that electrostatic interactions play an important role in thrombin recognition and cleavage of OPN. Thus, OPN is a bona fide substrate for thrombin, and generation of thrombin-cleaved OPN with enhanced pro-inflammatory properties provides another molecular link between coagulation and inflammation.     

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

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

Localization of a fibrin polymerization site

The formation of a fibrin clot is initiated after the proteolytic cleavage of fibrinogen by thrombin. The enzyme removes fibrinopeptides A and B and generates fibrin monomer which spontaneously polymerizes. Polymerization appears to occur though the interaction of complementary binding sites on the NH2-terminal and COOH-terminal (Fragment D) regions of the molecule. A peptide has been isolated from the gamma chain remnant of fibrinogen Fragment D1 which has the ability to bind to the NH2-terminal region of fibrinogen as well as to inhibit fibrin monomer polymerization. The peptide reduces the maximum rate and extent of the polymerization of thrombin or batroxobin fibrin monomer and increases the lag time. The D1 peptide does not interact with disulfide knot, fibrinogen, or Fragment D1, but it binds to thrombin-treated disulfide knot with a Kd of 1.45 X 10(-6) M at approximately two binding sites per molecule of disulfide knot. Fibrin monomer formed either by thrombin or batroxobin binds approximately two molecules of D1 peptide per molecule of fibrin monomer, indicating that the complementary site is revealed by the loss of fibrinopeptide A. The NH2-terminal sequence (Thr-Arg-Trp) and COOH-terminal sequence (Ala-Gly-Asp-Val) of the D1 peptide were determined. Therefore the gamma 373-410 region of fibrinogen contains a polymerization site which is complementary to the thrombin-activated site on the NH2-terminal region of fibrinogen.     

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.     

Antithrombin properties of C-terminus of hirudin using synthetic unsulfated N alpha-acetyl-hirudin45-65

Unsulfated N alpha-acetyl-hirudin45-65 (MDL 27 589), which corresponds to the C-terminus of hirudin1-65, was synthesized by solid-phase methods. The synthetic peptide was able to inhibit fibrin formation and the release of fibrinopeptide A from fibrinogen by thrombin. The catalytic site of thrombin was not perturbed by the synthetic peptide as H-D-Phe-Pip-Arg-pNA hydrolysis (amidase activity) was not affected. The binding of synthetic peptide and thrombin was assessed by isolation of the complex on gel-filtration chromatography. A single binding site with a binding affinity (Ka) of approx. 1.0 X 10(5) M-1 was observed for thrombin-hirudin45-65 interaction. The data suggest that the C-terminal residues 45-65 of hirudin contain a binding domain which recognizes thrombin and yet does not bind to the catalytic site of the enzyme.     

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.     

Inhibition of thrombin by synthetic hirudin peptides

To investigate the role of different regions of hirudin in the interaction with the proteinase thrombin, segments of hirudin containing 15-51 residues were synthesized. The C-terminal segment 40-65 inhibited the fibrinogen clotting activity of thrombin but not amidolysis of tosyl-Gly-Pro-Arg-p-nitroanilide. Central peptide 15–42 was insoluble at pH 7, but peptide 15-65 inhibited fibrinogen clotting and amidolysis to an equal extent. The N-terminal loop peptide 1-15 had no inhibitory activity and did not affect the potency of peptide 15-65. These data suggest that the central region inhibits catalysis.     

嵌合水蛭肽的构建与活性分析

血管成形术或动脉粥样斑块破裂等因素所致血管壁损伤而引起的血栓形成过程中 ,血小板的激活和凝血酶的形成起着关键作用 .因此 ,抗血小板和抗凝是治疗血栓的两个重要方面 .血小板膜糖蛋白GPⅡb Ⅲa受体拮抗剂 ,如含Arg Gly Asp(RGD)序列的多肽 ,在临床上已显示了良好的抗血小板     

Interactions of human fibrinogens with factor XIII: roles of calcium and the gamma' peptide

Plasma factor XIII is the zymogen of the transglutaminase factor XIIIa. This enzyme catalyzes the formation of isopeptide cross-links between fibrin molecules in nascent blood clots that greatly increase the mechanical stability of clots and their resistance to thrombolytic enzymes. We have characterized the solution interactions of factor XIII with two variants of fibrinogen, the soluble precursor of fibrin. Both the predominant fibrinogen gamma(A)/gamma(A) and the major variant gamma(A)/gamma' form complexes with a 2 fibrinogen:1 factor XIII ratio. The absence of detectable concentrations of 1:1 complexes in equilibrium mixtures containing free factor XIII and 2:1 complexes suggests that this interaction is cooperative. Factor XIII binds fibrinogen gamma(A)/gamma' approximately 20-fold more tightly than fibrinogen gamma(A)/gamma(A), and the interaction with fibrinogen gamma(A)/gamma' (but not fibrinogen gamma(A)/gamma(A)) is accompanied by a significant release of Ca(2+). Taken together, these results suggest that the strikingly anionic gamma' C-terminal sequence contains features that are important for factor XIII binding. Consistent with this notion, a synthetic 20-residue polypeptide containing the gamma' sequence was found to associate with factor XIII in a 2:1 molar ratio and act as an efficient competitor for fibrinogen gamma(A)/gamma' binding.     

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.     

A thrombin-based peptide corresponding to the sequence of the thrombomodulin-binding site blocks the procoagulant activities of thrombin.

Thrombomodulin, a cofactor in the thrombin-catalyzed activation of protein C, blocks the procoagulant activities of thrombin such as fibrinogen clotting, Factor V activation, and platelet activation. The binding site for thrombomodulin within human thrombin has been localized at a region comprising residues Thr147-Ser158 of the B-chain of thrombin. The dodecapeptide sequence, TWTANVGKGQPS, corresponding to these residues inhibits thrombin binding to thrombomodulin with an apparent Ki = 94 microM (Suzuki, K., Nishioka, J., and Hayashi, T. (1990) J. Biol. Chem. 265, 13263-13267). We have found that the inhibitory effect of the dodecapeptide on the thrombin-thrombomodulin interaction is sequence-specific, and that residues Asn151, Lys154, and Gln156 are essential for thrombomodulin binding. The dodecapeptide was also found to directly block thrombin procoagulant activities, fibrinogen clotting (concentration for half-maximum inhibition, 385 microM). Factor V activation (concentration for half-maximum inhibition, 33 microM), and platelet activation (concentration for half-maximum inhibition, 645 microM). This peptide did not block thrombin inhibition by antithrombin III, but blocked thrombin inhibition by hirudin. These findings suggest that the binding site for thrombomodulin in thrombin is shared with the sites for fibrinogen, Factor V, platelets, and hirudin, and that, therefore, the inhibition of thrombin procoagulant activities by thrombomodulin in part results from blocking of the interaction between thrombin and the procoagulant protein substrates by thrombomodulin.     

Solution structure of a platelet receptor peptide bound to bovine alpha-thrombin.

NMR experiments were carried out to study the interaction of thrombin with a synthetic peptide, ESKATNATLDPR, derived from the newly-identified platelet receptor for thrombin [Vu, T.-K. H., Hung, D. T., Wheaton, V. I., & Coughlin, S. R. (1991) Cell 64, 1057-1068]. On the basis of the observation of the thrombin-induced line broadening and transferred NOEs, binding of the peptide was found to be located exclusively within residues LDPR of the proteolytic cleavage site LDPR/S essential for receptor activation by thrombin. Measurement of transferred NOEs and molecular modeling indicate that the side chain of the Asp(P3) residue may form a hydrogen bond with thrombin and, by doing so, it is brought near a positively-charged thrombin residue Arg(221A), thereby partially neutralizing the negative charge of an Asp residue at this site of protein substrates. The hydrophobic side chains of residues Leu(P4) and Pro(P2) reside on the same side of the peptide backbone as indicated by transferred NOEs and were found by modeling to fit into a hydrophobic cage around the thrombin active site. These results suggest that the interaction of thrombin with protein substrates such as prothrombin, protein C, protein S, the platelet receptor, and the A alpha- and B beta-chains of fibrinogen all follow the same canonical binding mode in that the substrate forms an antiparallel beta-strand with thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of fibrinogen A alpha-fusion proteins. Mutants which inhibit thrombin equivalently are not equally good substrates

We have examined the interaction of thrombin with fibrinogen A alpha chain residues 7-16. Using genetically engineered constructions, we have synthesized in Escherichia coli a fibrinogen A alpha 1-50 fusion protein and seven mutant proteins with single amino acid substitutions. These are: Asp7----Ala, Phe8----Tyr, Glu11----Ala, Gly12----Val, Gly13----Val, Gly14----Val, and Arg16----Leu. Competitive immunoassay of cell lysates showed that all the mutations but one, Arg16----Leu, altered the structure of the protein such that cross-reactivity with the A alpha-specific monoclonal antibody, Y18, was significantly reduced. The fusion proteins were purified and analyzed as thrombin inhibitors and substrates. All the fusion proteins are competitive inhibitors of the amidolytic hydrolysis of Spectrozyme TH, a thrombin-specific chromogenic substrate, with inhibition constants corresponding to that for fibrinogen. We conclude that these 7 amino acid substitutions do not alter thrombin binding to the fusion proteins. The fusion proteins were tested as substrates by monitoring thrombin-dependent peptide release. The natural sequence and three mutants, Asp7----Ala, Glu11----Ala, and Gly14----Val, are good substrates. The other mutants are either poor substrates or are not cleaved by thrombin within A alpha 1-50. These results indicate that residues between Asp7 and Arg16 are critical to efficient peptide hydrolysis, whereas residues outside this region are critical to thrombin binding.