Selective Thrombin Inhibitor Research Articles

Amide-functionalized 1,2,4-Triazol-5-amines as Covalent Inhibitors of Blood Coagulation Factor XIIa and Thrombin.

To counteract thrombosis, new safe and efficient antithrombotics are required. We herein report the design, synthesis, and biological activity of a series of amide-functionalized acylated 1,2,4-triazol-5-amines as selective inhibitors of blood coagulation factor XIIa and thrombin. The introduction of an amide moiety into the main scaffold of 3-aryl aminotriazoles added certain three-dimensional properties to synthesized compounds and allowed them to reach binding sites in FXIIa and thrombin previously unaddressed by non-functionalized 1,2,4-triazol-5-amines. Among synthesized compounds, one quinoxaline-derived aminotriazole bearing N-butylamide moiety inhibited FXIIa with the IC50 value of 28 nM, whereas the N-phenylamide-derived aminotriazole inhibited thrombin with the IC50 value of 41 nM. Performed mass-shift experiments and molecular modeling studies proved the covalent mechanism of FXIIa and thrombin inhibition by synthesized compounds. In plasma coagulation tests, developed aminotriazoles showed anticoagulant properties mainly affecting the intrinsic blood coagulation pathway, activation of which is associated with thrombosis but is negligible for hemostasis.

Discovery of novel N-acylpyrazoles as potent and selective thrombin inhibitors

Direct oral anticoagulants (DOACs), which includes thrombin and factor Xa inhibitors, have emerged as the preferred therapeutics for thrombotic disorders, penetrating a market previously dominated by warfarin and heparin. This article describes the discovery and profiling of a novel series of N-acylpyrazoles, which act as selective, covalent, reversible, non-competitive inhibitors of thrombin. We describe in vitro stability issues associated with this chemotype and, importantly, demonstrate that N-acylpyrazoles successfully act in vivo as anticoagulants in basic thrombotic animal models. Crucially, this anticoagulant nature is unaccompanied by the higher bleeding risk profile that has become an undesirable characteristic of the DTIs and factor Xa inhibitors. We propose that the N-acylpyrazole chemotype shows intriguing promise as next-generation oral anticoagulants.

(1R,3S)-THCCA-Asn: To show the discovery of selective inhibitor of thrombin by successfully combining virtual screening and biological assay

Thrombin is the most potent platelet aggregator. To discover the selective inhibitor of thrombin that is important to curing platelet aggregation-related diseases, docking experiments were performed to dock (1R,3S)-2,3,4,9-tetrahydro-β-carboline-3- carboxylic acid, [(1R,3S)-THCCA], and (1S,3S)-2,3,4,9-tetrahydro-β-carboline-3- carboxylic acid, [(1S,3S)-THCCA], into the p pocket of bovine thrombin. The ideal match supported that (1R,3S)-THCCA could be used as a potential lead compound. In this case 20 natural amino acids were theoretically introduced into the 3-carboxyl of (1R,3S)-THCCA and 20 derivatives, (1R,3S)-THCCA-amino acids, were docked into p pocket of bovine thrombin to perform virtual screening. The screening revealed that comparing to (1R,3S)-THCCA itself the DockScores of 16 derivatives were higher, and (1R,3S)-THCCA-Asn (4j) got the highest DockScore. Thus, 16 derivatives were synthesized for experimental study. The in vitro anti-platelet aggregation assay showed that at 100 μM of concentration the 16 derivatives failed to inhibit the platelet aggregation induced by both adenosine diphosphate and arachidonic acid. On the other hand, however, the IC50 value of the 16 derivatives inhibiting the platelet aggregation induced by platelet activating factor and thrombin ranged from 9.44 μM to 194.64 μM and from 0.07 μM to 9.56 μM, respectively. The in vitro anti-platelet aggregation assay suggested that the 16 derivatives selectively inhibited the platelet aggregation induced by thrombin. In particular, the IC50 of (1R,3S)-THCCA-Asn (4j) had the lowest value. On rat model at 1 nmol/kg of dosage the 16 derivatives effectively prevented thrombus formation. It is worth pointing out that even at 0.01 nmol/kg of dosage, 4j still effectively prevented thrombus formation. 4j hardly has effects on the proliferation of mammalian cells and rat tail bleeding time. In conclusion, the combination of virtual screening and biological assays successfully lead to the discovery of 4j as a promising candidate of selective inhibitor of thrombin.

DEEP VEIN THROMBOSIS – A SYSTEMATIC REVIEW

Deep vein thrombosis (DVT) is the formation of blood clots in deep veins, commonly the leg veins and the pelvic veins. DVT is a potentially fatal condition leading to increased mortality and morbidity if not diagnosed and treated promptly.Incidence is more prevalent in 60- 65 years of age because of increased pro-coagulant factors like factor VIII, factor VII, homocysteine, brinogen. With the increase in age the risk of DVT development increases exponentially.All bed ridden patients should receive sequential compression device therapy as primary DVT prophylaxis in the form of intermittent pneumatic compression device (IPCDs), graduated compression stocking (GCS), and the venous foot pump.The pharmacological agents used in the prophylaxis of DVT include low-molecular-weight heparins (LMWH), fondaparinux, Unfractionated heparin (UFH), new oral direct selective thrombin inhibitors and factor Xa inhibitors.The complications of DVT include development of Pulmonary embolism (PE) , Paradoxical emboli, Recurrent DVT, Post thrombotic syndrome. Approximately 4% individuals treated for DVT develop PE. It accounts for 10-12% mortality rate in hospitalized patients.The prevention and treatment must be upgraded continuously with evidence base strategies. This article aims at reviewing the clinical presentation, diagnostic and treatment modalities of DVT

Discovery of Novel N-Acylpyrazoles as Potent and Selective Thrombin Inhibitors

Discovery of Novel N-Acylpyrazoles as Potent and Selective Thrombin Inhibitors

Acylated 1H-1,2,4-Triazol-5-amines Targeting Human Coagulation Factor XIIa and Thrombin: Conventional and Microscale Synthesis, Anticoagulant Properties, and Mechanism of Action.

We herein report the conventional and microscale parallel synthesis of selective inhibitors of human blood coagulation factor XIIa and thrombin exhibiting a 1,2,4-triazol-5-amine scaffold. Structural variations of this scaffold allowed identifying derivative 21i, a potent 29 nM inhibitor of FXIIa, with improved selectivity over other tested serine proteases and also finding compound 21m with 27 nM inhibitory activity toward thrombin. For the first time, acylated 1,2,4-triazol-5-amines were proved to have anticoagulant properties and the ability to affect thrombin- and cancer-cell-induced platelet aggregation. Performed mass spectrometric analysis and molecular modeling allowed us to discover previously unknown interactions between the synthesized inhibitors and the active site of FXIIa, which uncovered the mechanistic details of FXIIa inhibition. Synthesized compounds represent a promising starting point for the development of novel antithrombotic drugs or chemical tools for studying the role of FXIIa and thrombin in physiological and pathological processes.

Thrombin Inhibition by Argatroban: Potential Therapeutic Benefits in COVID-19.

Thrombin is a trypsin-like serine protease with multiple physiological functions. Its role in coagulation and thrombosis is well-established. Nevertheless, thrombin also plays a major role in inflammation by activating protease-activated receptors. In addition, thrombin is also involved in angiogenesis, fibrosis, and viral infections. Considering the pathogenesis of COVID-19 pandemic, thrombin inhibitors may exert multiple potential therapeutic benefits including antithrombotic, anti-inflammatory, and antiviral activities. In this review, we describe the clinical features of COVID-19, the thrombin’s roles in various pathologies, and the potential of argatroban in COVID-19 patients. Argatroban is a synthetic, small molecule, direct, competitive, and selective inhibitor of thrombin. It is approved to parenterally prevent and/or treat heparin-induced thrombocytopenia in addition to other thrombotic conditions. Argatroban also possesses anti-inflammatory and antiviral activities and has a well-established pharmacokinetics profile. It also appears to lack a significant risk of drug–drug interactions with therapeutics currently being evaluated for COVID-19. Thus, argatroban presents a substantial promise in treating severe cases of COVID-19; however, this promise is yet to be established in randomized, controlled clinical trials.

TAK-442, a Direct Factor Xa Inhibitor, Inhibits Monocyte Chemoattractant Protein 1 Production in Endothelial Cells via Involvement of Protease-Activated Receptor 1.

Oral blood coagulation inhibitors and their receptors, such as factor Xa (FXa), thrombin, and the thrombin receptor protease-activated receptor 1 (PAR1), are entered into clinical trials for acute coronary syndrome therapy; however, the results obtained so far are different for each drug. The underlying mechanisms of the results have not been fully investigated. We studied the in vitro anti-inflammatory effects of the selective FXa inhibitor TAK-442 on human endothelial cells, with comparing those of the selective thrombin inhibitor melagatran and the PAR1 antagonist vorapaxar. In human umbilical vein endothelial cells, FXa-increased production of monocyte chemoattractant protein 1 (MCP-1), a key inflammatory mediator, was inhibited by TAK-442 but not melagatran, and was also remarkably suppressed by vorapaxar. As thrombin did, FXa increased calcium mobilization in PAR1-overexpressed Chinese hamster ovary cells, which was selectively inhibited by TAK-442 and vorapaxar. We therefore confirmed the inhibitory effect of TAK-442 in endothelial MCP-1 production and the PAR1 intervention in the response. Our results suggest that TAK-442 may have anti-inflammatory potential in addition to its anti-thrombotic effects.

Antithrombotic properties of JJ1, a potent and novel thrombin inhibitor

The development of new anticoagulants is an important goal for the improvement of thrombosis treatment. Recent studies have suggested the importance of thrombin inhibitors in the modulation of thromboembolic disorders. The aim of this study was to discover a new small-molecule thrombin inhibitor. In this study, the compound JJ1, which has a novel scaffold, was selected by structure-based docking simulation to determine its potential inhibitory activity against thrombin. JJ1 was shown to inhibit the catalytic activity of human α-thrombin with a Ki of 0.019 μM by direct binding to the active site and with at least 10,000-fold selectivity relative to that reported for the inhibition of other biologically important serine proteases. JJ1 prolonged clotting times (activated partial thromboplastin time and prothrombin time) and inhibited the activity and production of thrombin. Furthermore, it inhibited thrombin-catalyzed fibrin polymerization and platelet aggregation. Similar to its in vitro antithrombotic activities, JJ1 showed enhanced antithrombotic effects in an in vivo pulmonary embolism and arterial thrombosis model. It also exhibited anticoagulant effects in mice. Collectively, these results demonstrated that JJ1 was a potent, direct, and selective thrombin inhibitor that may be useful in the management of various thrombotic disorders.

A versatile assay to determine bacterial and host factors contributing to opsonophagocytotic killing in hirudin-anticoagulated whole blood

Most bacteria entering the bloodstream will be eliminated through complement activation on the bacterial surface and opsonophagocytosis. However, when these protective innate immune systems do not work optimally, or when bacteria are equipped with immune evasion mechanisms that prevent killing, this can lead to serious infections such as bacteremia and meningitis, which is associated with high morbidity and mortality. In order to study the complement evasion mechanisms of bacteria and the capacity of human blood to opsonize and kill bacteria, we developed a versatile whole blood killing assay wherein both phagocyte function and complement activity can easily be monitored and modulated. In this assay we use a selective thrombin inhibitor hirudin to fully preserve complement activity of whole blood. This assay allows controlled analysis of the requirements for active complement by replacing or heat-inactivating plasma, phagocyte function and bacterial immune evasion mechanisms that contribute to survival in human blood.

PULMONARY EMBOLISM: SOME ISSUES OF EPIDEMIOLOGY AND TREATMENT IN CANCER PATIENTS

The risk of pulmonary embolism (PE) in cancer patients is 4–7-fold, compared to other patient categories. PE is the second most frequent cause of death in the first year after cancer diagnosis. PE is diagnosed in 7.5% of patients with malignant brain tumors, in 1 to 25% of those with gastrointestinal tumors, in 4.5 to 17.5% of those with breast cancer and in 4 to 10% of lung cancer patients. The risk of PE is higher with surgical interventions and chemotherapy, as well as in metastatic tumors. In 13% of cases, PE may be the first symptom of cancer manifestation. For prevention and treatment of PE low molecular weight heparin (LMWH) and warfarin are used. The risk of recurrent PE is 2-fold lower with LMWH. The frequency of bleeding with LMWH and warfarin treatment is from 14 to 19%. Placement of a cava filter is indicated only if anticoagulation is inefficient. New oral anticoagulants, which act as selective thrombin or Factor Xa inhibitors, are not used in cancer patients. Thus, diagnostics and treatment of PE is a very urgent problem in oncology that requires new approaches to be looked for.

The Coagulant Factor Xa Induces Protease-Activated Receptor-1 and Annexin A2–Dependent Airway Smooth Muscle Cytokine Production and Cell Proliferation

During asthma exacerbation, plasma circulating coagulant factor X (FX) enters the inflamed airways and is activated (FXa). FXa may have an important role in asthma, being involved in thrombin activation and an agonist of protease-activated receptor-1 (PAR-1). Extracellular annexin A2 and integrins are also implicated in PAR-1 signaling. In this study, the potential role of PAR-1 in mediating the effects of FXa on human airway smooth muscle (ASM) cell cytokine production and proliferation was investigated. FXa (5-50 nM), but not FX, stimulated increases in ASM IL-6 production and cell number after 24- and 48-hour incubation, respectively (P < 0.05; n = 5). FXa (15 nM) also stimulated increases in the levels of mRNA for cytokines (IL-6), cell cycle-related protein (cyclin D1), and proremodeling proteins (FGF-2, PDGF-B, CTGF, SM22, and PAI-1) after 3-hour incubation (P < 0.05; n = 4). The actions of FXa were insensitive to inhibition by hirudin (1 U/ml), a selective thrombin inhibitor, but were attenuated by SCH79797 (100 nM), a PAR-1 antagonist, or Cpd 22 (1 μM), an inhibitor of integrin-linked kinase. The selective targeting of PAR-1, annexin A2, or β1-integrin by small interfering RNA and/or by functional blocking antibodies also attenuated FXa-evoked responses. In contrast, the targeting of annexin A2 did not inhibit thrombin-stimulated ASM function. In airway biopsies of patients with asthma, FXa and annexin A2 were detected in the ASM bundle by immunohistochemistry. These findings establish FXa as a potentially important asthma mediator, stimulating ASM function through actions requiring PAR-1 and annexin A2 and involving integrin coactivation.

Outcomes of Argatroban Treatment in Patients With Atherothrombotic Stroke: Observational Nationwide Study in Japan.

Argatroban, a selective thrombin inhibitor, is recommended for the use in patients with atherothrombotic stroke by the Japanese Guidelines for the Management of Patients with Acute Ischemic Stroke. We performed a nationwide Japanese study to investigate whether argatroban improved early stroke outcomes in patients with acute atherothrombotic stroke. This retrospective observational study, using the Diagnosis Procedure Combination database in Japan, included patients who were hospitalized from July 1, 2010, to March 31, 2012, with a diagnosis of atherothrombotic stroke within 1 day of stroke onset. Patients were divided into 2 groups: those receiving argatroban on admission (argatroban group), and those who did not receive argatroban during hospitalization (control group). To balance the baseline characteristics and concomitant treatments during hospitalization between the 2 groups, one-to-one propensity-score matching analyses were performed. The main outcomes were the modified Rankin Scale score at discharge and the occurrence of hemorrhagic complications during hospitalization. An ordinal logistic regression analysis evaluated the association between argatroban use and modified Rankin Scale at discharge. After propensity-score matching, 2289 pairs of patients were analyzed. There were no significant differences in modified Rankin Scale at discharge between the argatroban and the control groups (adjusted odds ratio, 1.01; 95% confidence interval, 0.88-1.16). The occurrence of hemorrhagic complications did not differ significantly between the argatroban and the control groups (3.5% versus 3.8%; P=0.58). The present study suggested that argatroban was safe, but had no added benefit in early outcomes after acute atherothrombotic stroke.

Dabigatran Effect on Left Ventricular Thrombus in a Patient with Acute Ischemic Stroke.

Dabigatran Effect on Left Ventricular Thrombus in a Patient with Acute Ischemic Stroke.

Identification and Mechanistic Analysis of a Novel Tick-Derived Inhibitor of Thrombin.

A group of peptides from the salivary gland of the tick Hyalomma marginatum rufipes, a vector of Crimean Congo hemorrhagic fever show weak similarity to the madanins, a group of thrombin-inhibitory peptides from a second tick species, Haemaphysalis longicornis. We have evaluated the anti-serine protease activity of one of these H. marginatum peptides that has been given the name hyalomin-1. Hyalomin-1 was found to be a selective inhibitor of thrombin, blocking coagulation of plasma and inhibiting S2238 hydrolysis in a competitive manner with an inhibition constant (Ki) of 12 nM at an ionic strength of 150 mM. It also blocks the thrombin-mediated activation of coagulation factor XI, thrombin-mediated platelet aggregation, and the activation of coagulation factor V by thrombin. Hyalomin-1 is cleaved at a canonical thrombin cleavage site but the cleaved products do not inhibit coagulation. However, the C-terminal cleavage product showed non-competitive inhibition of S2238 hydrolysis. A peptide combining the N-terminal parts of the molecule with the cleavage region did not interact strongly with thrombin, but a 24-residue fragment containing the cleavage region and the C-terminal fragment inhibited the enzyme in a competitive manner and also inhibited coagulation of plasma. These results suggest that the peptide acts by binding to the active site as well as exosite I or the autolysis loop of thrombin. Injection of 2.5 mg/kg of hyalomin-1 increased arterial occlusion time in a mouse model of thrombosis, suggesting this peptide could be a candidate for clinical use as an antithrombotic.

Prediction of thrombin and factor xa inhibitory activity with associative neural networks.

Quantitative structure-activity relationship studies on a series of selective inhibitors of thrombin and factor Xa were performed by using Associative Neural Network. To overcome the problem of overfitting due to descriptor selection, 5-fold cross-validation with variable selection in each step of the analysis was performed. The predictive ability of the models was tested through leave-one-out cross-validation, giving a Q2=0.74-0.87 for regression models. Predictions for the external evaluation sets obtained accuracies in the range of 0.71-0.82 for regressions. The proposed models can be potential tools for finding new drug candidates.

Pharmacokinetics, pharmacodynamics and food effect of LB30870, a novel direct thrombin inhibitor, after single oral doses in healthy men

1. The safety, tolerability, pharmacokinetics, pharmacodynamics, and food effect of LB30870, a new selective thrombin inhibitor, were studied in 16 healthy men.2. A double-blind, placebo-controlled single ascending dose study was done at oral doses of 5, 15, 30, 60, 120, and 240 mg under fasting conditions. An open, randomized, balanced cross-over food effect study was done at 60 mg dose. Plasma and urinary concentrations were measured up to 48 h post-dose. Coagulation and thrombin activity markers were measured at selected time points.3. Cmax of LB30870 was at 1.3–3.0 h post-dose with a mean apparent terminal half-life (t1/2) of 2.8–4.1 h. AUC after doses above 15 mg appeared greater than dose-proportional. In fed state, AUC showed 80% reduction relative to fasting condition.4. At doses 60 and 120 mg, peak activated partial thromboplastin time (aPTT) increased by 1.5- and 2-fold, respectively, from baseline. The aPTT and international normalized ratio (INR) were concentration-dependent, with less within-individual variability than ecarin clotting time (ECT), prothrombin time (PT), or thrombin time (TT).5. Single oral doses of LB30870 up to 240 mg were well tolerated. The food effect must be overcome if LB30870 is to be used as an oral anti-coagulant.

L’argatroban, nouvel antithrombotique pour la thrombopénie induite par l’héparine en chirurgie cardiaque de l’adulte : utilisation en chirurgie cardiaque et en réanimation

ObjectivesAlthough heparin-induced thrombocytopemia (HIT) is uncommon, its thromboembolic complications are potentially life-threatening. The low-molecular weight heparins are less responsible of HIT than unfractionated heparin (UFH) but this latter is still indicated in some circumstances such as cardiac surgery. Argatroban, a selective thrombin inhibitor, recently available, has been indicated in HIT treatment. This review presents the main pharmacological characteristics, its indications and uses in the context of cardiac surgery and in intensive care medicine. MethodsReview of the literature in Medline database over the past 15 years using the following keywords: argatroban, cardiac surgery, circulatory assistance, cardiopulmonary bypass. ResultsDespite its short-acting pharmacokinetic, argatroban cannot be recommended during cardiopulmonary bypass. On the contrary, argatroban is indicated in many circumstances in postoperative period of various cardiac surgeries (on-pump, off-pump, circulatory assistance). Nevertheless, after cardiac surgery, doses have to be adapted according to coagulation laboratory testing (ACT), particularly in patients presenting acute organ failure (kidney injury, heart failure, liver failure). This compound has no antagonist and is excluded during severe hepatic failure. The continuous intravenous administration is a drawback. ConclusionArgatroban is a new direct competitive thrombin inhibitor well evaluated as treatment of HIT after cardiac surgery. In HIT management, argatroban is an interesting alternative to lepirudin that is not anymore available and danaparoid because of supply disturbances.

Origin of serpin-mediated regulation of coagulation and blood pressure.

Vertebrates evolved an endothelium-lined hemostatic system and a pump-driven pressurized circulation with a finely-balanced coagulation cascade and elaborate blood pressure control over the past 500 million years. Genome analyses have identified principal components of the ancestral coagulation system, however, how this complex trait was originally regulated is largely unknown. Likewise, little is known about the roots of blood pressure control in vertebrates. Here we studied three members of the serpin superfamily that interfere with procoagulant activity and blood pressure of lampreys, a group of basal vertebrates. Angiotensinogen from these jawless fish was found to fulfill a dual role by operating as a highly selective thrombin inhibitor that is activated by heparin-related glycosaminoglycans, and concurrently by serving as source of effector peptides that activate type 1 angiotensin receptors. Lampreys, uniquely among vertebrates, thus use angiotensinogen for interference with both coagulation and osmo- and pressure regulation. Heparin cofactor II from lampreys, in contrast to its paralogue angiotensinogen, is preferentially activated by dermatan sulfate, suggesting that these two serpins affect different facets of thrombin’s multiple roles. Lampreys also express a lineage-specific serpin with anti-factor Xa activity, which demonstrates that another important procoagulant enzyme is under inhibitory control. Comparative genomics suggests that orthologues of these three serpins were key components of the ancestral hemostatic system. It appears that, early in vertebrate evolution, coagulation and osmo- and pressure regulation crosstalked through antiproteolytically active angiotensinogen, a feature that was lost during vertebrate radiation, though in gnathostomes interplay between these traits is effective.

Thrombin Has Biphasic Effects on the Nitric Oxide-cGMP Pathway in Endothelial Cells and Contributes to Experimental Pulmonary Hypertension

BackgroundA potential role for coagulation factors in pulmonary arterial hypertension has been recently described, but the mechanism of action is currently not known. Here, we investigated the interactions between thrombin and the nitric oxide-cGMP pathway in pulmonary endothelial cells and experimental pulmonary hypertension.Principal FindingsChronic treatment with the selective thrombin inhibitor melagatran (0.9 mg/kg daily via implanted minipumps) reduced right ventricular hypertrophy in the rat monocrotaline model of experimental pulmonary hypertension. In vitro, thrombin was found to have biphasic effects on key regulators of the nitric oxide-cGMP pathway in endothelial cells (HUVECs). Acute thrombin stimulation led to increased expression of the cGMP-elevating factors endothelial nitric oxide synthase (eNOS) and soluble guanylate cyclase (sGC) subunits, leading to increased cGMP levels. By contrast, prolonged exposition of pulmonary endothelial cells to thrombin revealed a characteristic pattern of differential expression of the key regulators of the nitric oxide-cGMP pathway, in which specifically the factors contributing to cGMP elevation (eNOS and sGC) were reduced and the cGMP-hydrolyzing PDE5 was elevated (qPCR and Western blot). In line with the differential expression of key regulators of the nitric oxide-cGMP pathway, a reduction of cGMP by prolonged thrombin stimulation was found. The effects of prolonged thrombin exposure were confirmed in endothelial cells of pulmonary origin (HPAECs and HPMECs). Similar effects could be induced by activation of protease-activated receptor-1 (PAR-1).ConclusionThese findings suggest a link between thrombin generation and cGMP depletion in lung endothelial cells through negative regulation of the nitric oxide-cGMP pathway, possibly mediated via PAR-1, which could be of relevance in pulmonary arterial hypertension.