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PubMed2002

Direct thrombin inhibitors.

Thrombin plays a central role in thrombosis. Consequently, most current antithrombotic treatment strategies are aimed at blocking the activity of thrombin, or preventing its generation. Although heparin has been a cornerstone of treatment, it has limitations. Thus, the anticoagulant response to heparin is unpredictable, the heparin/antithrombin complex is unable to inhibit fibrin-bound thrombin, an important trigger of thrombus growth, and heparin is neutralized by platelet factor 4. Direct thrombin inhibitors were developed to overcome these limitations. Unlike heparin, direct thrombin inhibitors produce a predictable anticoagulant response that is unaffected by platelet factor 4, and they inhibit fibrin-bound thrombin. Three parenteral direct thrombin inhibitors--hirudin, bivalirudin and argatroban--are currently licensed for use in North America, and orally available direct thrombin inhibitors are under investigation. This review summarizes the clinical trial data with direct thrombin inhibitors and provides perspective on the role of direct thrombin inhibitors in the face of other new anticoagulants currently under development.

Anticoagulants↗
PubMed1997

Cholinergic stimulation increases thrombin activity and gene expression in cultured mouse muscle.

Activity-dependent synapse reduction is a major determinant of neuromuscular innervation. Previous research has shown that nanomolar concentrations of hirudin, a specific thrombin antagonist, significantly attenuates this reduction, and protease nexin 1 (PN1), an endogenous thrombin inhibitor closely localized to the neuromuscular synapse, can inhibit synapse reduction at similar concentrations. Protease inhibitors which do not inhibit thrombin, including cystatin and aprotinin, had no effect on synapse reduction. We present a series of experiments examining whether prothrombin and/or PN1 gene expression, as well as thrombin activity, are regulated in muscle cultures by acetylcholine (ACh) receptor activation. We also studied the effect of exogenous thrombin on synapse elimination in co-cultures of muscle and cholinergic neurons. Cultured muscle cells were electrically blocked with tetrodotoxin (TTX), or co-treated with ACh in order to isolate ACh receptor activation. Electrical blockade resulted in a decrease in thrombin release to about two-thirds of control values. The application of ACh to electrically blocked muscle cultures resulted in a 2.5-fold increase in thrombin activity released into the medium and a 2-fold increase in prothrombin gene expression. In contrast, ACh treatment in the presence of TTX had no effect on PN1 gene expression compared to treatment with TTX alone. In addition, exogenous thrombin significantly increased synapse elimination in unstimulated muscle/cholinergic neuron co-cultures. These results suggest that thrombin or a thrombin-like molecule released from muscle is required for activity-dependent synapse elimination and is regulated by neuromuscular activity.

Acetylcholine↗
PubMed1998

Thrombin generation, inhibition and clinical outcomes in patients with acute myocardial infarction treated with thrombolytic therapy and heparin: results from the GUSTO-I Trial. GUSTO-I Hemostasis Substudy Group. Global Utilization of Streptokinase and TPA for Occluded Coronary Arteries.

OBJECTIVES: We sought to assess the effects of antithrombotic therapy after thrombolysis for acute myocardial infarction on markers of thrombin generation and activity and to determine the relation of these markers with clinical outcomes. BACKGROUND: Thrombin activation and generation often occur with thrombolysis for acute myocardial infarction. Antithrombotic regimens have been developed to reduce the resulting thrombotic complications. METHODS: We sampled plasma markers of thrombin generation and activity after thrombolysis in 292 patients. We assessed the relations of these markers with clinical outcomes at 30 days. RESULTS: Fibrinopeptide A (FPA), a marker of thrombin activity toward fibrinogen, was elevated at baseline (12.3 ng/ml) and increased to 18.4 ng/ml by 90 min after streptokinase and subcutaneous heparin treatment. With intravenous heparin, this increase was attenuated, but intravenous heparin did not prevent thrombin generation, as measured by prothrombin fragment 1.2 (F1.2). Heparin level, measured by anti-Xa activity, correlated with activated partial thromboplastin time (aPTT, r = 0.62 to 0.67). Thrombin activity, measured by FPA, was as closely related to aPTT as to the heparin level. Baseline levels of F1.2 were significantly related to the risk of death or reinfarction at 30 days (p = 0.008); values 12 h after enrollment also were related to 30-day mortality (p = 0.05). CONCLUSIONS: Although intravenous heparin partly suppresses the increased thrombin activity associated with thrombolysis, it does not inhibit thrombin generation. The aPTT was as good a measure of suppression of thrombin activity as the heparin level itself. Hematologic markers of thrombin generation were found to be related to the subsequent risk of thrombotic events.

Aged↗
PubMed1997

Mechanism of thrombin-induced arachidonic acid release in osteoblast-like cells.

In a previous study, we have reported that thrombin stimulates phosphatidylcholine hydrolysis by phospholipase (PL) D, but has little effect on phosphoinositide hydrolysis by PLC in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the mechanism of the thrombin-induced arachidonic acid (AA) release in MC3T3-E1 cells. Thrombin stimulated AA release dose dependently in the range between 0.1 and 1 U/ml. Quinacrine, a PLA2 inhibitor, suppressed the thrombin-induced AA release. In addition, quinacrine also suppressed the thrombin-induced prostaglandin E2 synthesis in these cells. On the other hand, propranolol, which is known to inhibit phosphatidic acid phosphohydrolase, did not affect the thrombin-induced AA release. 1(6-((17beta-3-Methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H- pyrrole-2,5-dione (U-73122), a PLC inhibitor, had no effect on the AA release by thrombin. In addition, 1,6-bis-(cyclohexyloximinocarbonylamino)-hexane (RHC-80267), a selective inhibitor of diacylglycerol lipase, had little effect on the thrombin-induced AA release. Neither propranolol, U-73122 nor RHC-80267 affect the thrombin-induced prostaglandin E2 synthesis. These results strongly suggest that thrombin induces AA release not by phosphatidylcholine hydrolysis by PLD nor phosphoinositide hydrolysis by PLC but mainly by PLA2 in osteoblast-like cells.

3T3 Cells↗
PubMed2003

Evaluation of DNA aptamers directed to thrombin as potential thrombus imaging agents.

Two DNA aptamers directed against two separate exosites on human alpha-thrombin were evaluated for thrombus-imaging potential. Aptamer ODN 1 is directed to the thrombin substrate binding site (exosite 1). Our finding that ODN 1 competes with fibrin for binding to exosite 1 on thrombin suggests that ODN 1 will not be useful for thrombus imaging. Aptamer ODN 2 is directed against the thrombin heparin binding site (exosite 2). ODN 2 bound to model thrombi that were formed either by clotting purified fibrinogen with thrombin, or by recalcifying citrated plasma. As the thrombin content of thrombi was increased the rate of ODN 2 uptake into preformed thrombi increased, whereas the rate of release of ODN 2 out of preformed thrombi decreased. This in vitro data suggested that ODN 2 might be useful for thrombus imaging because it can bind to exosite 2 on fibrin-bound thrombin. However, in a rabbit jugular vein model using thrombus supplemented with human thrombin, ODN 2 uptake was equal to the ovalbumin control, and did not reflect thrombin content. While the in vitro results with ODN 2 were consistent with thrombus imaging, the rapid clearance of ODN 2 from circulation, combined with slow mass transfer in the clot, seem to work against in vivo thrombin-dependent imaging or washout analysis.

Animals↗
PubMed1995

Electron spin resonance and fluorescence studies of the conformational environment of the thiol groups of thrombospondin: interactions with thrombin.

The free thiols of platelet thrombospondin (TSP) were modified with thiol-specific spin labels and fluorescence probes. The conformational effects of thrombin complexation with TSP were monitored by thiol-specific spin labels covalently attached to TSP and active site specific spin labels on thrombin. The results provide evidence supporting speculations that the thiols of the three polypeptide chains in TSP are not conformationally identical. Studies on the effects of Ca2+ and temperature confirm that TSP exists in multiple conformations which are under dynamic equilibrium. The ESR spectra of spin-labeled TSP are sensitive to the proteolytic effects of thrombin in the presence and absence of calcium. Phenylsulfonyl fluoride spin labels specific for the active site of thrombin are excellent indicators of thrombin: TSP complex formation in the absence of calcium. The anticoagulant thrombin inhibitor hirudin competes with TSP for the same binding locus on thrombin (which includes the requirement of an intact anion exosite). The results suggest that the species observed here is the noncovalent complex formed during the first step of the TSP--thrombin interaction, showing also that thrombin activity is not essential for complex formation. ESR and fluorescence studies of thiol-labeled TSP indicate that the sulfhydryls are not affected in the noncovalent thrombin: TSP complex, although they must be playing a major role in the second step, i.e., formation of the covalent complex, through intermolecular thiol exchange.

Animals↗
PubMed1991

Characterization of the kinetic pathway for fibrin promotion of alpha-thrombin-catalyzed activation of plasma factor XIII.

Kinetic and thermodynamic studies are presented showing that the cofactor activity of fibrin I (polymerized des-A fibrinogen) in the alpha-thrombin-catalyzed proteolysis of activation peptide (AP) from plasma factor XIII can be attributed to formation of a fibrin I-plasma factor XIII complex (Kd = 65 nM), which is processed by alpha-thrombin more efficiently (kcat/Km = 1.2 x 10(7) M-1 s-1) than free, uncomplexed plasma factor XIII (kcat/Km = 1.4 x 10(5) M-1 s-1). The increase in the specificity constant (kcat/Km) is shown to be largely due to an increase in the apparent affinity of alpha-thrombin for the complex of plasma factor XIII and fibrin I, as reflected by the 30-fold decrease in the Michaelis constant observed for fibrin I bound plasma factor XIII relative to that for uncomplexed plasma factor XIII. Analysis of the initial rates of alpha-thrombin-catalyzed hydrolysis of fibrinopeptide B (FPB) from fibrin I polymer in the presence of plasma factor XIII indicated that alpha-thrombin bound to fibrin I in the ternary complex of alpha-thrombin, plasma factor XIII, and fibrin I polymer is competent to catalyze cleavage of both FPB from fibrin I and AP from plasma factor XIII. This observation is consistent with the view that alpha-thrombin within the ternary complex is anchored to fibrin I polymer through a binding site distinct from the active site (an exosite) and that the active site is alternatively complexed with the AP moiety of plasma factor XIII or the FPB moiety of fibrin I. This conclusion is supported by the observation that a 12-residue peptide, which binds to an exosite of alpha-thrombin and blocks the interaction of alpha-thrombin with fibrinogen and fibrin, competitively inhibits alpha-thrombin-catalyzed release of both FPB and AP from the fibrin I-plasma factor XIII complex.

Binding, Competitive↗
PubMed1991

Chemical modifications and amino acid substitutions in recombinant hirudin that increase hirudin-thrombin affinity.

Recombinant hirudin (r-hirudin), unlike the naturally occurring leech protein, lacks a sulfate ester on Tyr-63 which reduces its binding affinity to thrombin by 3-10-fold. We demonstrate that nitration or iodination of Tyr-63 restores hirudin-thrombin affinity to levels similar to or exceeding that of the natural inhibitor. In contrast, nitration of Tyr-3 reduces the affinity of hirudin for thrombin. These chemical modifications results in multiple reaction products that are readily separated by reverse-phase HPLC. The mechanism of the observed changes in thrombin affinity may involve a reduction in the pK of the hydroxyl group of tyrosine due to substitution of the electrophilic iodo or nitro group on the phenyl ring, resulting in an increased negative charge at neutral pH. For Tyr-63, this effect mimics the sulfatotyrosine of natural hirudin, leading to an increased thrombin affinity at the anion-binding exosite. For Tyr-3, the increased polarity may destabilize its interaction within the apolar-binding site of thrombin. Substitution of the highly conserved Tyr-3 residue with Phe or Trp not only enables specific and quantitative chemical modification at Tyr-63 but also independently increases hirudin-thrombin affinity. Kinetic analysis of thrombin inhibition showed that enhanced binding by r-hirudin(nitro-Tyr-63) is due to an increase in the association rate between hirudin and thrombin whereas the reduced binding of r-hirudin(nitro-Tyr-3) results from a large increase in the dissociation rate. These observations indicate that specific segments within both the amino- and carboxy-terminal regions of hirudin interact with thrombin.

DNA Mutational Analysis↗
PubMed1988

Localization of the binding site on fibrin for the secondary binding site of thrombin.

Affinity chromatography of active site inhibited thrombin on immobilized fragments derived from the central (desAB-NDSK) and terminal (D1) globular domains of fibrinogen revealed that the site responsible for the binding of thrombin at its secondary fibrin binding site is located in the central domain. Chromatography of various domains of the central nodule (desAB-NDSK, fibrinogen E, and fibrin E) having nonidentical amino acid sequences showed that all of these fragments are capable of binding to PMSF-thrombin-Sepharose, suggesting that the thrombin binding site resides within the peptide regions common to all of these fragments: alpha(Gly17-Met51), beta(Val55-Met118), and gamma(Tyr1-Lys53). Competitive affinity chromatography of the same binding domains revealed that there is no detectable difference in their binding constants to PMSF-thrombin-Sepharose, indicating that the alpha(Lys52-Lys78), beta(Gly15-Lys54)/(Tyr119-Lys122), and gamma(Thr54-Met78) peptide segments do not contribute significantly to the binding of thrombin. Chromatography of the isolated chains of fibrinogen E showed that the alpha(Gly17-Lys78) peptide region itself contains a strong binding site for PMSF-thrombin-Sepharose. The location of the binding site suggests that the secondary site interaction may play an important role in determining the cleavage specificity of thrombin on fibrinogen and can affect the rate of release of the fibrinopeptides. Affinity chromatography of fragments prepared from polymerized fibrin showed that cross-linked DD (D x D) itself does not bind to thrombin, whereas the D x DE complex remained attached to the column, suggesting that the binding site on fragment E for thrombin is distinct from its binding site for D x D.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1988

Interaction of hirudin with thrombin: identification of a minimal binding domain of hirudin that inhibits clotting activity.

Hirudin, isolated from the European leech Hirudo medicinalis, is a potent inhibitor of thrombin, forming an almost irreversible thrombin-hirudin complex. Previously, we have shown that the carboxyl terminus of hirudin (residues 45-65) inhibits clotting activity and without binding to the catalytic site of thrombin. In the present study, a series of peptides corresponding to this carboxyl-terminal region of hirudin have been synthesized, and their anticoagulant activity and binding properties to thrombin were examined. Binding was assessed by their ability to displace 125I-hirudin 45-65 from Sepharose-immobilized thrombin and by isolation of peptide-thrombin complexes. We show that the carboxyl-terminal 10 amino acid residues 56-65 (Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln) are minimally required for binding to thrombin and inhibition of clotting. Phe-56 was critical for maintaining anticoagulant activity as demonstrated by the loss of activity when Phe-56 was substituted with D-Phe, Glu, or Leu. In addition, we found that the binding of the carboxyl-terminal peptide of hirudin with thrombin was associated with a significant conformational change of thrombin as judged by circular dichroism. This conformational change might be responsible for the loss of clotting activity of thrombin.

Binding Sites↗
PubMed1981

Dissociation of antithrombin III--thrombin complex. Formation of active and inactive antithrombin III.

Dissociation of the complexes formed during the reaction of thrombin with antithrombin III was studied by determining the kinetics of release of thrombin and active antithrombin III from the complexes. The rate of thrombin release increased gradually between pH 7.0 and 8.5 and then sharply increased at pH 9.0. The rate of active antithrombin III release did not change significantly over the same pH range. The results suggested that thrombin is released after reaction with antithrombin III via two pathways which are differentially affected by the pH of the solution. At pH 7.0 thrombin is released predominantly by dissociation of a non-acylated enzyme--inhibitor complex since active antithrombin III was released in relatively low amounts. The results suggested that thrombin inhibition by antithrombin III does not require the formation of an acyl bond between the active-site serine of thrombin and a carbonyl carbon of antithrombin III. The release of thrombin by acylation--deacylation appeared to be limited by the rate of enzyme acylation which was favored by more alkaline pH. This further suggests that acyl bond formation is a secondary reaction that can occur during thrombin inhibition by antithrombin III but is not necessary for thrombin inhibition.

Antithrombin III↗
PubMed2001

Crystal structure of thrombin-ecotin reveals conformational changes and extended interactions.

The protease inhibitor ecotin fails to inhibit thrombin despite its broad specificity against serine proteases. A point mutation (M84R) in ecotin results in a 1.5 nM affinity for thrombin, 10(4) times stronger than that of wild-type ecotin. The crystal structure of bovine thrombin is determined in complex with ecotin M84R mutant at 2.5 A resolution. Surface loops surrounding the active site cleft of thrombin have undergone significant structural changes to permit inhibitor binding. Particularly, the insertion loops at residues 60 and 148 in thrombin, which likely mediate the interactions with macromolecules, are displaced when the complex forms. Thrombin and ecotin M84R interact in two distinct surfaces. The loop at residue 99 and the C-terminus of thrombin contact ecotin through mixed polar and nonpolar interactions. The active site of thrombin is filled with eight consecutive amino acids of ecotin and demonstrates thrombin's preference for specific features that are compatible with the thrombin cleavage site: negatively charged-Pro-Val-X-Pro-Arg-hydrophobic-positively charged (P1 Arg is in bold letters). The preference for a Val at P4 is clearly defined. The insertion at residue 60 may further affect substrate binding by moving its adjacent loops that are part of the substrate recognition sites.

Amino Acid Sequence↗
PubMed2002

Thrombin hydrolysis of V29F and V34L mutants of factor XIII (28-41) reveals roles of the P(9) and P(4) positions in factor XIII activation.

In blood coagulation, thrombin helps to activate factor XIII by cleaving the activation peptide at the R37-G38 peptide bond. The more easily activated factor XIII V34L has been correlated with protection from myocardial infarction. V34L and V29F factor XIII mutant peptides were designed to further characterize substrate binding to thrombin. HPLC kinetic studies have been carried out on thrombin hydrolysis of FXIII activation peptide (28-41), FXIII (28-41) V34L, FXIII (28-41) V29F, and FXIII (28-41) V29F V34L. The V34L mutations lead to improvements in both K(m) and k(cat) whereas the V29F mutation primarily affects K(m). Interactions of the peptides with thrombin have been monitored by 1D proton line broadening NMR and 2D transferred NOESY studies. The results were compared with previously published X-ray crystal structures of thrombin-bound fibrinogen Aalpha (7-16), thrombin receptor PAR1 (38-60), and factor XIII (28-37). In solution, the (34)VVPR(37) and (34)LVPR(37) segments of the factor XIII activation peptide serve as the major anchor points onto thrombin. The N-terminal segments are proposed to interact transiently with the enzyme surface. Long-range NOEs from FXIII V29 or F29 toward (34)V/LVPR(37) have not been observed by NMR studies. Overall, the kinetic and NMR results suggest that the factor XIII activation peptide binds to thrombin in a manner more similar to the thrombin receptor PAR1 than to fibrinogen Aalpha. The V29 and V34 positions affect, in different ways, the ability of thrombin to effectively hydrolyze the activation peptide. Mutations at these sites may prove useful in controlling factor XIII activation.

Amino Acid Sequence↗
PubMed1996

Protein engineering thrombin for optimal specificity and potency of anticoagulant activity in vivo.

Previous alanine scanning mutagenesis of thrombin revealed that substitution of residues W50, K52, E229, and R233 (W60d, K60f, E217, and R221 in chymotrypsinogen numbering) with alanine altered the substrate specificity of thrombin to favor the anticoagulant substrate protein C. Saturation mutagenesis, in which residues W50, K52, E229, and R233 were each substituted with all 19 naturally occurring amino acids, resulted in the identification of a single mutation, E229K, that shifted the substrate specificity of thrombin by 130-fold to favor the activation of the anticoagulant substrate protein C over the procoagulant substrate fibrinogen. E229K thrombin was also less effective in activating platelets (18-fold), was resistant to inhibition by antithrombin III (33-fold and 22-fold in the presence and absence of heparin), and displayed a prolonged half-life in plasma in vitro (26-fold). Thus E229K thrombin displayed an optimal phenotype to function as a potent and specific activator of endogenous protein C and as an anticoagulant in vivo. Upon infusion in Cynomolgus monkeys E229K thrombin caused an anticoagulant effect through the activation of endogenous protein C without coincidentally stimulating fibrinogen clotting and platelet activation as observed with wild-type thrombin. In addition, E229K thrombin displayed enhanced potency in vivo relative to the prototype protein C activator E229A thrombin. This enhanced potency may be attributable to decreased clearance by antithrombin III, the principal physiological inhibitor of thrombin.

Animals↗
PubMed1997

Molecular design and characterization of an alpha-thrombin inhibitor containing a novel P1 moiety.

An inhibitor of alpha-thrombin was designed on the basis of the X-ray crystal structures of thrombin and trypsin. The design strategy employed the geometric and electrostatic differences between the specificity pockets of the two enzymes. These differences arise due to the replacement of Ser 190 in trypsin by Ala 190 in thrombin. The new inhibitor contained a tryptophan side chain instead of the arginine side chain that is present in the prototypical thrombin inhibitors. This inhibitor had a Ki value of 0.25 microM, displayed more than 400-fold specificity for thrombin over trypsin, and doubled the rat plasma APTT at a concentration of 44.9 microM. The X-ray crystal structure of the inhibitor/alpha-thrombin complex was determined. This represents the first reported three-dimensional structure of a thrombin/ inhibitor complex where the specificity pocket of the enzyme is occupied by a chemical moiety other than a guanidino or an amidino group. As was predicted by the molecular model, the tryptophan side chain docks into the specificity pocket of the enzyme. This finding is in contrast with the indole binding region of thrombin reported earlier [Berliner, L. J., & Shen, Y. Y. L. (1977) Biochemistry 16, 4622-4626]. The lower binding affinity of the new inhibitor for trypsin, compared to that for thrombin, appears to be due to (i) the extra energy required to deform the smaller specificity pocket of trypsin to accommodate the bulky indole group and (ii) the favorable electrostatic interactions of the indole group with the more hydrophobic specificity pocket of thrombin. The neutral indole group may be of pharmacological significance because the severe hypotension and respiratory distress observed following the administration of some thrombin inhibitors have been linked to the positively charged guanidino or amidino functionalities.

Alanine↗
PubMed1998

Thrombomodulin increases the rate of thrombin inhibition by BPTI.

Thrombin undergoes allosteric modulation by thrombomodulin (TM) that results in a shift in macromolecular specificity, blocking fibrinogen clotting while enhancing protein C activation. The TM enhancement of protein C activation involves both an 8-fold decrease in Km and a 200-fold increase in kcat. Although TM-mediated conformational changes in thrombin have been detected by many techniques, the nature of these changes remains obscure. Access to the active center of thrombin is relatively restricted due to the presence of a large insertion loop at residue 60 (chymotrypsin numbering) that has been implicated in modeling studies as being responsible for poor inhibition by BPTI. Thrombin and the E192Q mutant, which binds BPTI much more tightly than thrombin, are both inhibited very slowly by BPTI. TM increases the rate of thrombin or thrombin E192Q inhibition by BPTI approximately 10-fold. When analyzed as slow tight binding inhibition, the TM effect on thrombin E192Q inhibition by BPTI is primarily on the first, reversible step in the reaction. Structural studies of the thrombin E192Q-BPTI complex have previously shown that the 60 loop lies over the BPTI, a position which requires 8 A movement at the apex of the 60 loop, and that BPTI is found in the same canonical orientation as in the trypsin complex. It follows that TM enhancement of the initial interaction of thrombin results in a conformation that favors interactions with BPTI, probably involving motion of the 60 loop.

Allosteric Regulation↗
PubMed2004

Design and synthesis of novel biologically active thrombin receptor non-peptide mimetics based on the pharmacophoric cluster Phe/Arg/NH2 of the Ser42-Phe-Leu-Leu-Arg46 motif sequence: platelet aggregation and relaxant activities.

The identification of the thrombin receptor has promoted the interest for the development of new therapeutic agents capable of selectively inhibiting unwanted biological effects of thrombin on various cell types. In this study we have designed and synthesized two series of new thrombin receptor antagonists based on the thrombin receptor motif sequence S42FLLR46, one possessing two (Phe/Arg) pharmacophoric groups and the other possessing three (Phe/Arg/NH2). N-(6-Guanidohexanoyl)-N'-(phenylacetyl)piperazine (1), N-(phenylacetyl)-4-(6-guanidohexanoylamidomethyl)piperidine (2), and N-(phenylacetyl)-3-(6-guanidohexanoylamido)pyrrolidine (3) (group A) carry the two pharmacophoric side chains of Phe and Arg residues incorporated on three different templates (piperazine, 4-aminomethylpiperidine, and 3-aminopyrrolidine). Compounds with three pharmacophoric groups (group B) were built similarly to group A using the same templates with the addition of an extra methylamino group leading to (S)-N-(6-guanidohexanoyl)-N'-(2-amino-3-phenylpropionyl)piperazine (4), (S)-N-(2-amino-3-phenylpropionyl)-4-(6-guanidohexanoylamidomethyl)piperidine (5), and (S)-N-(2-amino-3-phenylpropionyl)-3-(6-guanidohexanoylamido)pyrrolidine (6). Compounds were able to inhibit thrombin-induced human platelet activation even at low concentrations. In particular, among compounds in group A, compound 3 was found to be the most powerful thrombin receptor activation inhibitor, showing an IC50 of approximately 0.11 mM on platelet aggregation assay. Among compounds in group B, compound 4 was the most powerful to inhibit thrombin-induced platelet aggregation, showing an IC50 of approximately 0.09 mM. All compounds were also found to act as agonists in the rat aorta relaxation assay. Interestingly, the order of potency of these compounds as agonists of the endothelial thrombin receptor was the inverse of the order of potency of the same compounds as antagonists of the platelet thrombin receptor. Such compounds that are causing vasodilation while simultaneously inhibiting platelet aggregation would be very useful in preventing the installation of atherosclerotic lesions and deserve further investigation as potential drugs for treating cardiovascular diseases. The above findings coupled with computational analysis molecular dynamics experiments support also our hypothesis that a cluster of phenyl, guanidino, and amino groups is responsible for thrombin receptor triggering and activation.

Amino Acid Motifs↗
PubMed2000

Structural basis for the anticoagulant activity of the thrombin-thrombomodulin complex.

The serine proteinase alpha-thrombin causes blood clotting through proteolytic cleavage of fibrinogen and protease-activated receptors and amplifies its own generation by activating the essential clotting factors V and VIII. Thrombomodulin, a transmembrane thrombin receptor with six contiguous epidermal growth factor-like domains (TME1-6), profoundly alters the substrate specificity of thrombin from pro- to anticoagulant by activating protein C. Activated protein C then deactivates the coagulation cascade by degrading activated factors V and VIII. The thrombin-thrombomodulin complex inhibits fibrinolysis by activating the procarboxypeptidase thrombin-activatable fibrinolysis inhibitor. Here we present the 2.3 A crystal structure of human alpha-thrombin bound to the smallest thrombomodulin fragment required for full protein-C co-factor activity, TME456. The Y-shaped thrombomodulin fragment binds to thrombin's anion-binding exosite-I, preventing binding of procoagulant substrates. Thrombomodulin binding does not seem to induce marked allosteric structural rearrangements at the thrombin active site. Rather, docking of a protein C model to thrombin-TME456 indicates that TME45 may bind substrates in such a manner that their zymogen-activation cleavage sites are presented optimally to the unaltered thrombin active site.

Amino Acid Sequence↗