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PubMed1994

Design and synthesis of potent and highly selective thrombin inhibitors.

Thrombin, a serine protease, plays a central role in the initiation and propagation of thrombotic events. An extensive search for new thrombin inhibitors was performed, using an unconventional approach. Screening of small basic molecules for binding in the recognition pocket of thrombin led to the discovery of (aminoiminomethyl)piperidine (amidinopiperidine) as a weak, but intrinsically selective, thrombin inhibitor. Elaboration of this molecule provided compounds which inhibit thrombin with Ki's in the range of 20-50 nM and with selectivities of 1000-4000 against trypsin. These inhibitor compounds show a new and unexpected binding mode to thrombin. Modification of the central building block and then of one of the hydrophobic substituents led to the discovery of a new family of thrombin inhibitors which has reverted to the former binding mode to thrombin. This last class of compounds shows inhibitory activities in the picomolar range, low toxicity, and a short plasma half life which favors its use for an intravenous application. From this series of thrombin inhibitors, 19f(Ro 46-6240) was selected for clinical development as an antithrombotic agent for intravenous administration.

Amino Acids↗

PubMed1996

Structure of a bovine thrombin-hirudin51-65 complex determined by a combination of molecular replacement and graphics. Incorporation of known structural information in molecular replacement.

Crystals of the bovine thrombin-hirudins(51-65) complex have space group P6(1)22 with cell constants a = 116.4, and c = 200.6 A and two thrombin molecules in the asymmetric unit. Only one thrombin molecule could be located by generalized molecular replacement; the second was fit visually as a rigid body to an improved electron-density difference map. The structure was refined to R = 0.192 with two B values per residue (main chain and side chain) at 3.2 A. The polar interactions of the peptides with the exosite of thrombin show differences consistent with the known flexibility in the interactions of the C-terminal peptide of hirudin with thrombin. The hirudin peptide in complex 2 has a higher temperature factor as compared with peptide 1 which may be correlated partly with a larger number of short-range electrostatic interactions between peptide 1 and thrombin and partly with the fact that thrombin 2 is epsilon-thrombin which is cleaved at Thr149A near the peptide binding site. Later, using this structure as a test case, it was shown that the position for the second thrombin could also be determined by a novel modification of the molecular-replacement method in which the contribution of the known molecule is subtracted from the structure factors. This approach is facile and applicable to any crystal containing two or more macromolecules in the asymmetric unit in which some but not all of the molecules can be determined by molecular replacement.

Journal Article↗

PubMedDate not supplied

Peptidomimetic thrombin inhibitors.

The central position of thrombin in the coagulation cascade has made it a popular target for discovery of novel antithrombotic agents. Starting with hirudin, a natural peptide isolated from the medicinal leech, its shorter synthetic analogue hirulog, and argatroban,the first therapeutically used synthetic small-molecule thrombin active site inhibitor, hundreds of direct thrombin inhibitors have been discovered over the last 20 years. Most of them are peptidomimetic compounds,based on the amino acid sequence of fibrinogen which binds into the thrombin active site. Since elucidation of the crystal structure of human thrombin in 1989, the structure-based design of low-molecular-weight peptidomimetic thrombin inhibitors has been greatly aided by the use of x-ray crystallographic analysis of thrombin-inhibitor complexes. The ultimate goal of most research programmes and drug optimization strategies is to develop an orally bioavailable, small-molecule,direct thrombin inhibitor that would be suitable for once or twice daily dosing. An overview of the most advanced peptidomimetic direct thrombin inhibitors bivalirudin, argatroban, ximelagatran and dabigatran is presented.

Anticoagulants↗

PubMed1995

Activated clotting time as an appropriate test to compare heparin and direct thrombin inhibitors such as hirudin or Ro 46-6240 in experimental arterial thrombosis.

BACKGROUND: Specific thrombin inhibitors are considered to be more potent antithrombotics than heparin. However, the relation between the systemic anticoagulation generated by thrombin inhibitors and their antithrombotic effect is not well defined. In a guinea pig carotid thrombosis model, the activated clotting time (ACT), the activated partial thromboplastin time (aPTT), and thrombin-generation tests were evaluated for their ability to predict the arterial antithrombotic effect of direct thrombin inhibitors such as hirudin and Ro 46-6240 compared with heparin. METHODS AND RESULTS: Thrombosis of the carotid artery was induced by subendothelial damage in guinea pigs, and the subsequent cyclic flow variations were monitored. The effects of pretreatment with intravenous heparin, hirudin, and Ro 46-6240 were tested. After doubling the baseline aPTT, 1 IU.kg-1.min-1 heparin was inactive, whereas either hirudin or Ro 46-6240 (30 micrograms.kg-1.min-1) prevented thrombus formation by 80%. Heparin (10 IU.kg-1.min-1) induced the same antithrombotic effect but with indefinite aPTT prolongation. However, for similar prolongation of the ACT, the three compounds had equivalent antithrombotic effects. Thrombin generation was predictive of the antithrombotic effect of the thrombin inhibitors but not of heparin. CONCLUSIONS: The arterial antithrombotic effect of direct thrombin inhibitors, when compared with those of heparin, should be evaluated by the ACT and not the aPTT or thrombin-generation assays. For a "therapeutic" aPTT prolongation, thrombin inhibitors induce higher systemic anticoagulation than does heparin and thus might unduly have higher bleeding liability.

Animals↗

PubMed1993

Thrombin receptor agonist peptide induction of mitogenesis in CCL39 cells.

The role of the thrombin receptor tethered ligand hypothesis in mediating the mitogenic responses of cells to thrombin was explored. We have found that small (5-14 amino acid) peptides corresponding to the proposed amino terminus of thrombin activated human and hamster thrombin receptors are mitogenic for the Chinese hamster fibroblast cell line, CCL39. Hirudin and hirugen block the mitogenic effects of thrombin but not the activity of the agonist peptides. Pertussis toxin treated cells do not respond to either alpha-thrombin or the agonist peptides. The data support the idea that the thrombin receptor on CCL39 cells, which is homologous to the thrombin receptor on human platelets, is capable of transmitting mitogenic signals by a mechanism consistent with the tethered ligand hypothesis.

Amino Acid Sequence↗

PubMed2002

Dissection of protease-activated receptor-1-dependent and -independent responses to thrombin in skeletal myoblasts.

Thrombin exerts a number of effects on skeletal myoblasts in vitro. It stimulates proliferation and intracellular calcium mobilization and inhibits differentiation and apoptosis induced by serum deprivation in these cells. Many cellular responses to thrombin are mediated by protease-activated receptor-1 (PAR-1). Expression of PAR-1 is present in mononuclear myoblasts in vitro, but repressed when fusion occurs to form myotubes. In the current study, we used PAR-1-null mice to determine which of thrombin's effects on myoblasts are mediated by PAR-1. Thrombin inhibited fusion almost as effectively in cultures prepared from the muscle of PAR-1-null myoblasts as in cultures prepared from wild-type mice. Apoptosis was inhibited as effectively in PAR-1-null myoblasts as in wild-type myoblasts. These effects in PAR-1-null myoblasts were mediated by a secreted inhibitor of apoptosis and fusion, as demonstrated previously for normal rat myoblasts. Thrombin failed to induce an intracellular calcium response in PAR-1-null myoblast cultures, although these cells were able to mobilize intracellular calcium in response to activation of other receptors. PAR-1-null myoblasts also failed to proliferate in response to thrombin. These results demonstrate that thrombin's effects on myoblast apoptosis and fusion are not mediated by PAR-1 and that PAR-1 is the only thrombin receptor capable of inducing proliferation and calcium mobilization in neonatal mouse myoblasts.

Animals↗

PubMed2000

The two-receptor system PAR-1/PAR-4 mediates alpha-thrombin-induced [Ca(2+)](i) mobilization in human astrocytoma cells.

The proteinase-activated receptor 1 (PAR-1) was characterized as a functional receptor for thrombin in cells from different brain tumor entities. Whether PAR-1 alone accounts for thrombin-induced effects in human cancer cells, or whether other PAR contribute is unknown. We established primary cultures from two neurosurgically removed human astrocytomas and investigated intracellular signaling roles of PAR-1 and PAR-4 by estimating the effect of alpha-thrombin and PAR-activating peptides on [Ca(2+)](i) mobilization in single astrocytoma cells. alpha-Thrombin or the PAR-1-activating peptide SFLLRN induced a transient calcium mobilization. This suggests the involvement of PAR-1 in alpha-thrombin-induced calcium signaling in human astrocytoma cells. In addition, a second, PAR-4-dependent, mechanism exists. This was deduced from the findings that a further calcium signal could be observed in human astrocytoma cells stimulated with alpha-thrombin after SFLLRN and the PAR-4-activating peptide GYPGQV also induced a calcium response. In addition, the observation that trypsin, known to activate both PAR-2 and PAR-4, but not the specifically PAR-2-activating peptide SLIGRL induced calcium signaling is a further indication of functional PAR-4-type thrombin receptors in human astrocytoma cells. This is the first report demonstrating a signaling role for a dual thrombin receptor system in human tumor cells.

Apoptosis Regulatory Proteins↗

PubMed1990

Expression of functional thrombin receptors in xenopus oocytes injected with human endothelial cell mRNA.

Human endothelial cell thrombin receptors were functionally expressed in Xenopus laevis oocytes by injection of RNA extracted from human umbilical vein endothelial cells. Oocytes injected with endothelial cell RNA responded to thrombin with a Ca2(+)-dependent depolarizing current whose size depended on the amount of RNA injected. In oocytes expressing thrombin receptors, thrombin caused homologous but not heterologous desensitization. Both the catalytic and anion-binding exosites of thrombin were necessary to elicit depolarizing currents. Thus, Xenopus laevis oocytes injected with mRNA from human endothelial cells express Ca2(+)-dependent thrombin receptors which share many common features with thrombin receptors on intact endothelial cells. Xenopus oocytes may, therefore, be used as a screening system in the expression cloning of the endothelial cell thrombin receptor.

Animals↗

PubMed1991

A thrombin receptor in resident rat peritoneal macrophages.

Resident rat peritoneal macrophages possess 6 x 10(2) high-affinity binding sites per cell for bovine thrombin with a Kd of 11 pM, and 7.5 x 10(4) low-affinity sites with a Kd of 5.8 nM. These binding sites are highly specific for thrombin. Half-maximal binding of 125I-labeled bovine thrombin is achieved after 1 min at 37 degrees C, and after 12 min at 4 degrees C. The reversibly bound fraction of the ligand dissociates according to a biexponential time course with the rate constants 0.27 and 0.06 min-1 at 4 degrees C. Part of the tracer remains cell-associated even after prolonged incubation, but all cell-associated radio-activity migrates as intact thrombin upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The bound thrombin is minimally endocytosed as judged by the resistance to pH 3 treatment, and the receptor does not mediate a quantitatively important degradation of the ligand. The binding is not dependent on the catalytic site of thrombin, since irreversibly inactivated thrombin also binds to the receptor. 125I-labeled thrombin covalently cross-linked to its receptor migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a Mr 160,000, corresponding to an approximate receptor size of Mr 120,000.

Amino Acid Sequence↗

PubMed1984

Photoaffinity labeling of platelet thrombin-binding proteins.

The interaction of thrombin and platelets was studied with a heterobifunctional photoactivable crosslinking agent. Radiolabeled thrombin that was modified with ethyl-N-5-azido-2-nitrobenzoylaminoacetimidate formed two types of complex with platelet proteins: platelet-associated complexes and supernatant complexes. The platelet-associated complexes formed within 20 s. Autoradiography after electrophoresis with sodium dodecyl sulfate indicated that these complexes had apparent masses of 210, 185, 155 and 125 kDa. Formation of the complexes was blocked by hirudin; this is consistent with crosslinking that was a direct consequence of the binding of thrombin to a specific receptor, since hirudin blocks thrombin-induced platelet activation and the saturable binding of thrombin to platelets. The labeled supernatant complex had an apparent mass of about 490 kDa. It also formed in the supernatant solution of platelets after activation with a divalent cation ionophore, suggesting a complex of thrombin with a secreted protein. The supernatant complex did not involve fibrinogen or alpha 2-macroglobulin, but a similar complex was formed with partially purified secreted glycoprotein G (thrombin-sensitive protein, thrombospondin). Formation of the complex was blocked by hirudin. A similar complex was formed after prolonged (1 h) incubation without photoactivation. It is concluded that thrombin forms high-affinity, hirudin-sensitive complexes with secreted glycoprotein G, as well as with platelet surface proteins.

Blood Platelets↗

PubMed2005

2-(2-Br-phenyl)-8-methoxy-benzoxazinone (HPW-RX2), a direct thrombin inhibitor with a suppressive effect on thromboxane formation in platelets.

2-(2-Br-phenyl)-8-methoxy-benzoxazinone (HPW-RX2), a newly synthetic benzoxazinone derivative, has previously been shown to inhibit rabbit platelet aggregation caused by thrombin and arachidonic acid. In the present study, the mechanism for the antiplatelet effect of HPW-RX2 was further investigated. In human platelets, HPW-RX2 concentration-dependently inhibited platelet aggregation, ATP release, P-selectin expression, and intracellular calcium mobilization caused by thrombin. In contrast, HPW-RX2 had no significant effect on either SFLLRN- or GYPGKF-induced platelet aggregation, indicating that HPW-RX2 did not interfere with platelet thrombin receptors. Moreover, HPW-RX2 inhibited the amidolytic activity of thrombin and prolonged the fibrinogen clotting time. These results suggest that the inhibitory effect of HPW-RX2 on thrombin-induced platelet aggregation is via direct inhibition of thrombin proteolytic activity. Besides the inhibition on thrombin, HPW-RX2 also prevented platelet aggregation, ATP release, and increase in [Ca2+]i caused by arachidonic acid and low concentration collagen. In a parallel manner, both arachidonic acid-induced thromboxane B2 and prostaglandin D2 formations were decreased in platelets treated with HPW-RX2. This indicates that HPW-RX2 is able to inhibit the arachidonic acid cascade at the cyclooxygenase level. This is the first report of a benzoxazinone derivative possessing both thrombin and cyclooxygenase inhibitory properties. The dual effect of HPW-RX2 might provide extra therapeutic benefits for treatment of arterial thrombosis.

Adenosine Triphosphate↗

PubMed2005

Thrombin increases cardiomyocyte acute cell death after ischemia and reperfusion.

Thrombin exerts multiple actions on cardiomyocytes leading to increased intracellular Na+ and Ca2+ concentrations, and to activation of a Ca2+-independent PLA2, and has been proposed to favor the genesis of arrhythmias and ischemic injury in acute coronary syndromes. However, the influence of thrombin on cardiomyocyte cell death during ischemia-reperfusion has not been studied. A beneficial influence of low thrombin concentrations has been described in other cell types. HL-1 cardiomyocytes were subjected to simulated ischemia (SI) and reperfusion (SR) and cell death was assessed by means of LDH release to the incubation media. Thrombin dose-dependently increased cell death in normoxic cells, in cells subjected to SI, and in cells subjected to SR (by 20+/-8%, 95+/-32% and 35+/-9%, respectively, at 100 U/ml). The effects of thrombin were associated to increased cytosolic Ca2+ overload, mimicked by 100 microM PAR-1 agonist peptide SFLLRNPNDKYEPF, and reversed by the direct thrombin inhibitor lepirudin (IC50=1.3+/-0.2 microg/ml). The presence of thrombin during simulated ischemia-reperfusion increases cardiomyocyte cell death by a mechanism that involves activation of PAR-1 receptors and can be prevented by the direct thrombin inhibitor lepirudin.

Animals↗

PubMed2003

Aberrant expression and activation of the thrombin receptor protease-activated receptor-1 induces cell proliferation and motility in human colon cancer cells.

The traditional view on the role of serine proteases in tumor biology has changed with the recent discovery of a family of protease-activated receptors (PARs). In this study we explored the expression and functional role of the thrombin receptor PAR-1 in human colon cancer cells. Reverse transcriptase-polymerase chain reaction analysis showed that PAR-1 mRNAs are present in 11 of 14 human colon cancer cell lines tested but not in normal human colonic epithelial cells. This is in line with the immunolocalization of PAR-1 in human colon tumors and its absence in normal human colonic mucosa. The functional significance of the aberrant expression of PAR-1 in colon cancer cells was then investigated. We found that 1) a prompt increase in intracellular calcium concentration was observed on thrombin (10 nmol/L) or PAR-1 agonist AP1 (100 micro mol/L) challenge of HT29 cells; 2) HT29 quiescent cells treated with thrombin (0.01 to 20 nmol/L) or AP1 (1 to 300 micro mol/L) exhibited dramatic mitogenic responses (3.5-fold increase in cell number). Proliferative effects of thrombin or AP1 were also observed in other colon cancer cell lines expressing PAR-1. This effect was reversed by the MEK inhibitor PD98059 in consonance with the ability of thrombin or AP1 to induce phosphorylation of p42/p44 extracellular-regulated protein kinases. 3) PAR-1 activation by thrombin or AP1 led to a two-fold increase in cell motility of wounded HT29-D4. Our results demonstrate for the first time the aberrant expression of the functional thrombin receptor PAR-1 in colon cancers and its important involvement in cell proliferation and motility. Thrombin should now be considered as a growth factor for human colon cancer.

Calcium↗

PubMed1996

Direct evidence for two distinct G proteins coupling with thrombin receptors in human neuroblastoma SH-EP cells.

Thrombin receptor-G protein coupling was investigated in the human epithelial neuroblastoma cell line, SH-EP. In these cells, both alpha-thrombin and thrombin receptor peptides, SFLLRNP (one-letter amino-acid code), which are newly exposed following cleavage by alpha-thrombin, stimulated GTPase activity about 2-fold over basal activity. Pertussis toxin treatment only partially attenuated alpha-thrombin- and SFLLRNP-stimulated GTPase activity by 50%, whereas antibody raised against synthetic heptapeptide SFLLRNP blocked alpha-thrombin-stimulated phosphoinositide hydrolysis more than 80%. Immunoprecipitation studies using this antibody showed that both Gi2, a subtype of guanine nucleotide-binding regulatory proteins (G proteins) mediating inhibition of adenylyl cyclase, and Gq/G11, a G protein mediating stimulation of phospholipase C, were activated by alpha-thrombin. These data suggest that in these cells the thrombin receptor activates pertussis toxin-sensitive and pertussis toxin-insensitive G proteins simultaneously and directly couples to Gi2 and Gq/G11, which mediate different signaling pathways.

Antibodies↗

PubMed2000

Proteolysis and phosphorylation-mediated regulation of thrombin receptor activity in in situ endothelial cells.

The regulatory mechanism of thrombin receptor responsiveness in in situ endothelial cells was investigated by evaluating elevations of cytosolic Ca(2+) concentration ([Ca(2+)](i)) in fura-2-loaded porcine aortic valvular strips. Once stimulated with thrombin, endothelial cells did not respond to the second thrombin stimulation within 90 min. However, applying thrombin receptor activating peptide (TRAP7) at 15 min after the thrombin stimulation caused [Ca(2+)](i) elevation, which was smaller than that seen without preceding stimulation. After 90 min, response to TRAP7 recovered to the control level. When stimulated with TRAP7, the subsequent responses to thrombin and TRAP7 were attenuated at 15 min, and fully recovered after 90 min. Staurosporine partially prevented the TRAP7-induced desensitization. The recovery of responsiveness was inhibited completely by calyculin-A and partially by okadaic acid. Proteolysis and phosphorylation thus play an important role in thrombin receptor desensitization in in situ endothelial cells. Both cleaved and uncleaved receptors were desensitized through phosphorylation in part by staurosporine-sensitive kinase, and restored the responsiveness through dephosphorylation by type 1 phosphatase. The mechanism of regulation of thrombin receptor activity in in situ endothelial cells differed from those reported in cultured endothelial cells. We suggest that the cell-specific regulatory mechanism may be altered by culture conditions.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed1998

Thrombin-induced DNA synthesis of cultured human dental pulp cells is dependent on its proteolytic activity and modulated by prostaglandin E2.

To clarify the roles of alpha-thrombin and prostaglandin E2 (PGE2) in the healing and inflammatory processes of dental pulp, their effects on the DNA synthesis of human pulp cells were investigated by measurement of [3H]thymidine incorporation. At a concentration range of 1 to 25 units/ml, alpha-thrombin stimulated DNA synthesis of the pulp cells by 1.5 to 2.6-fold. On the contrary, PGE2 (> 0.05 microgram/ml) suppressed DNA synthesis by 24 to 39%. Using reverse transcriptase-polymerase chain reaction, thrombin receptor mRNA expression was identified in the pulp cells. Furthermore, alpha-thrombin-induced DNA synthesis could be inhibited by antithrombin III (2 units/ml) with heparin (2 units/ml) or D-Phe-Pro-ArgCH2Cl (50 micrograms/ml). PGE2 (0.1 to 0.5 microgram/ml) also inhibited the thrombin-induced DNA synthesis by 39 to 64%. These results imply that pulp cells express the thrombin receptor that is activated by the serine protease activity of thrombin. Interactions of thrombin and PGE2 are important in modulating the inflammatory and healing processes of the pulp.

Cells, Cultured↗

PubMed2002

Direct thrombin inhibitors in acute coronary syndromes: principal results of a meta-analysis based on individual patients' data.

BACKGROUND: To obtain more reliable and precise estimates of the effect of direct thrombin inhibitors in the management of acute coronary syndromes, including patients undergoing percutaneous coronary intervention, we undertook a meta-analysis based on individual patients' data from randomised trials comparing a direct thrombin inhibitor (hirudin, bivalirudin, argatroban, efegatran, or inogatran) with heparin. METHODS: We included trials that involved at least 200 patients. The primary efficacy outcome was death or myocardial infarction, and the primary safety outcome was major bleeding. Data from individual trials were combined by use of a modified Mantel-Haenszel method. FINDINGS: In 11 randomised trials, 35,970 patients were assigned up to 7 days' treatment with a direct thrombin inhibitor or heparin and followed up for at least 30 days. Compared with heparin, direct thrombin inhibitors were associated with a lower risk of death or myocardial infarction at the end of treatment (4.3% vs 5.1%; odds ratio 0.85 [95% CI 0.77-0.94]; p=0.001) and at 30 days (7.4% vs 8.2%; 0.91 [0.84-0.99]; p=0.02). This was due primarily to a reduction in myocardial infarctions (2.8% vs 3.5%; 0.80 [0.71-0.90]; p<0.001) with no apparent effect on deaths (1.9% vs 2.0%; 0.97 [0.83-1.13]; p=0.69). Subgroup analyses suggested a benefit of direct thrombin inhibitors on death or myocardial infarction in trials of both acute coronary syndromes and percutaneous coronary interventions. A reduction in death or myocardial infarction was seen with hirudin and bivalirudin but not with univalent agents. Compared with heparin, there was an increased risk of major bleeding with hirudin, but a reduction with bivalirudin. There was no excess in intracranial haemorrhage with direct thrombin inhibitors. INTERPRETATION: Direct thrombin inhibitors are superior to heparin for the prevention of death or myocardial infarction in patients with acute coronary syndromes. This information should prompt further clinical development of direct thrombin inhibitors for the management of arterial thrombosis.

Angina, Unstable↗

PubMed1999

Thrombin mutants with altered enzymatic activity have an impaired mitogenic effect on mouse fibroblasts and are inefficient modulators of stellation of rat cortical astrocytes.

We produced recombinant human thrombin mutants to investigate the correlation between the thrombin enzyme and mitogenic activity. Single amino acid substitutions were introduced in the catalytic triad (H43N, D99N, S205A, S205T), in the oxy-anion binding site (G203A) and in the anion binding exosite-1 region (R73E). Proteins were produced as prethrombin-2 mutants secreted in the culture medium of DXB11-derived cell lines. All mutants were activated by ecarin to the corresponding thrombin mutants; the enzymatic activity was assayed on a chromogenic substrate and on the procoagulant substrate fibrinogen. Mutations S205A and G203A completely abolished the enzyme activity. Mutations H43N, D99N and S205T dramatically impaired the enzyme activity toward both substrates. The R73E mutation dissociated the amidolytic activity and the clotting activity of the protein. The ability of thrombin mutants to induce proliferation was investigated in NIH3T3 mouse fibroblasts and rat cortical astrocytes. The ability of the thrombin mutants to revert astrocyte stellation was also studied. The mitogenic activity and the effect on the astrocyte stellation of the thrombin mutants correlated with their enzymatic activity. Furthermore the receptor occupancy by the inactive S205A mutant prevented the thrombin effects providing strong evidence that a proteolytically activated receptor is involved in cellular responses to thrombin.

3T3 Cells↗