Fucosylated Chondroitin Sulfates from the Sea Cucumbers Paracaudina chilensis and Holothuria hilla: Structures and Anticoagulant Activity.

Fucosylated chondroitin sulfates (FCSs) PC and HH were isolated from the sea cucumbers Paracaudina chilensis and Holothuria hilla, respectively. The purification of the polysaccharides was carried out by anion-exchange chromatography on a DEAE-Sephacel column. The structural characterization of the polysaccharides was performed in terms of monosaccharide and sulfate content, as well as using a series of nondestructive NMR spectroscopic methods. Both polysaccharides were shown to contain a chondroitin core [→3)-ß-d-GalNAc (N-acethyl galactosamine)-(1→4)-ß-d-GlcA (glucuronic acid)-(1→]n, bearing sulfated fucosyl branches at O-3 of every GlcA residue in the chain. These fucosyl residues were different in their pattern of sulfation: PC contained Fuc2S4S and Fuc4S in a ratio of 2:1, whereas HH included Fuc2S4S, Fuc3S4S, and Fuc4S in a ratio of 1.5:1:1. Moreover, some GalNAc residues in HH were found to contain an unusual disaccharide branch Fuc4S-(1→2)-Fuc3S4S-(1→ at O-6. Sulfated GalNAc4S6S and GalNAc4S units were found in a ratio of 3:2 in PC and 2:1 in HH. Both polysaccharides demonstrated significant anticoagulant activity in a clotting time assay, which is connected with the ability of these FCSs to potentiate the inhibition of thrombin and factor Xa in the presence of anti-thrombin III (ATIII) and with the direct inhibition of thrombin in the absence of any cofactors.

Innovative Three-Step Microwave-Promoted Synthesis of N-Propargyltetrahydroquinoline and 1,2,3-Triazole Derivatives as a Potential Factor Xa (FXa) Inhibitors: Drug Design, Synthesis, and Biological Evaluation.

The coagulation cascade is the process of the conversion of soluble fibrinogen to insoluble fibrin that terminates in production of a clot. Factor Xa (FXa) is a serine protease involved in the blood coagulation cascade. Moreover, FXa plays a vital role in the enzymatic sequence which ends with the thrombus production. Thrombosis is a common causal pathology for three widespread cardiovascular syndromes: acute coronary syndrome (ACS), venous thromboembolism (VTE), and strokes. In this research a series of N-propargyltetrahydroquinoline and 1,2,3-triazole derivatives as a potential factor Xa (FXa) inhibitor were designed, synthesized, and evaluated for their FXa inhibitor activity, cytotoxicity activity and coagulation parameters. Rational design for the desired novel molecules was performed through protein-ligand complexes selection and ligand clustering. The microwave-assisted synthetic strategy of selected compounds was carried out by using Ullmann-Goldberg, N-propargylation, Mannich addition, Friedel-Crafts, and 1,3-dipolar cycloaddition type reactions under microwave irradiation. The microwave methodology proved to be an efficient way to obtain all novel compounds in high yields (73-93%). Furthermore, a thermochemical analysis, optimization and reactivity indexes such as electronic chemical potential (µ), chemical hardness (η), and electrophilicity (ω) were performed to understand the relationship between the structure and the energetic behavior of all the series. Then, in vitro analysis showed that compounds 27, 29-31, and 34 exhibited inhibitory activity against FXa and the corresponding half maximal inhibitory concentration (IC50) values were calculated. Next, a cell viability assay in HEK293 and HepG2 cell lines, and coagulation parameters (anti FXa, Prothrombin time (PT), activated Partial Thromboplastin Time (aPTT)) of the most active novel molecules were performed to determine the corresponding cytotoxicity and possible action on clotting pathways. The obtained results suggest that compounds 27 and 29 inhibited FXa targeting through coagulation factors in the intrinsic and extrinsic pathways. However, compound 34 may target coagulation FXa mainly by the extrinsic and common pathway. Interestingly, the most active compounds in relation to the inhibition activity against FXa and coagulation parameters did not show toxicity at the performed coagulation assay concentrations. Finally, docking studies confirmed the preferential binding mode of N-propargyltetrahydroquinoline and 1,2,3-triazole derivatives inside the active site of FXa.

Factor Xa inhibition and sPESI failure in intermediate-high-risk pulmonary embolism.

We report the case of a 61-year-old man who presented at the Emergency Department (ED), complaining of sudden-onset dyspnea and chest pain after a long flight from Tokyo to Houston. Considering his clinical stability and sPESI 0, enoxaparin 1 mg/kg BID was started for 24 h, and the patient was then considered for early discharge with apixaban 10 mg BID. Direct-factor Xa inhibition did not improve extensive thrombus burden and right ventricular dysfunction despite D-dimer measurement reduction. Because of the treatment failure, we considered thrombolysis. Currently, recommendations to use thrombolysis in patients under non-vitamin K antagonist oral anticoagulants (NOACs) do not exist. Hence, the one dose of apixaban was stopped, and 12 h later, we performed successful thrombolysis. A systematic review from 2007 to 2017 did not identify any cases related to NOACs failure to reduce thrombus burdens in patients with PE and persistent right ventricular dysfunction. We also did not find any evidence of cases that reported strategies for urgent thrombolysis in PE patients on NOACs. To the best of our knowledge, apixaban's failure to reduce thrombus burden, persistent right ventricular dysfunction, and a NOACs-thrombolysis bridge in patients with PE on apixaban has not been previously described. Both the bedside risk stratification and the therapeutic failures should alert clinicians in the ED to the potential limitations of low-molecular-weight heparin, NOACs therapy, and sPESI in the setting of intermediate-high-risk PE.

Novel FXa Inhibitor Identification through Integration of Ligand- and Structure-Based Approaches.

Factor Xa (FXa), a vitamin K-dependent serine protease plays a pivotal role in the coagulation cascade, one of the most interesting targets for the development of new anticoagulants. In the present work, we performed a virtual screening campaign based on ligand-based shape and electrostatic similarity search and protein-ligand docking to discover novel FXa-targeted scaffolds for further development of inhibitors. From an initial set of 260,000 compounds from the NCI Open database, 30 potential FXa inhibitors were identified and selected for in vitro biological evaluation. Compound 5 (NSC635393, 4-(3-methyl-4H-1,4-benzothiazin-2-yl)-2,4-dioxo-N-phenylbutanamide) displayed an IC50 value of 2.02 nM against human FXa. The identified compound may serve as starting point for the development of novel FXa inhibitors.

Design, synthesis, and biological activity of novel tetrahydropyrazolopyridone derivatives as FXa inhibitors with potent anticoagulant activity.

A series of novel tetrahydropyrazolopyridone derivatives containing 1,3,4-triazole, triazolylmethyl, and partially saturated heterocyclic moieties as P2 binding element was designed, synthesized, and evaluated in vitro for anticoagulant activity in human and rabbit plasma. All compounds showed moderate to significant potency, and compounds 15b, 15c, 20b, 20c, and 22b were further examined for their inhibitory activity against human FXa in vitro. While compounds 15c and 22b were tested for rat venous thrombosis in vivo. The most promising compound 15c, with an IC50 (FXa) value of 0.14µM and 98% inhibition rate, warranted further investigation as an FXa inhibitor.

Design, synthesis, and structure-activity relationship of novel and effective apixaban derivatives as FXa inhibitors containing 1,2,4-triazole/pyrrole derivatives as P2 binding element.

Four series of novel and potent FXa inhibitors possessing the 1,2,4-triazole moiety and pyrrole moiety as P2 binding element and dihydroimidazole/tetrahydropyrimidine groups as P4 binding element were designed, synthesized, and evaluated for their anticoagulant activity in human and rabbit plasma in vitro. Most compounds showed moderate to excellent activity. Compounds 14a, 16, 18c, 26c, 35a, and 35b were further examined for their inhibition activity against human FXa in vitro and rat venous thrombosis in vivo. The most promising compound 14a, with an IC50 (FXa) value of 0.15µM and 99% inhibition rate, was identified for further evaluation as an FXa inhibitor.

Evaluación de la anticoagulación con rivaroxaban, en pacientes con fibrilación auricular no valvular de reciente diagnóstico / Evaluation of oral anticoagulation with rivaroxaban, in patients with new onset non valvular atrial fibrillation

Background: Atrial fibrillation (AF) generates a hypercoagulable state with an increased thrombin generation and raised levels of thrombin-antithrombin complexes, which results in a high risk of stroke and thromboembolism. Aim: To evaluate the anticoagulant effect of rivaroxaban by anti-Xa factor activity and its correlation with thrombin-antithrombin complexes, thrombin generation and prothrombin time in patients newly diagnosed with non-valvular AF. Patients and Methods: Prospective study in patients with indication of anticoagulation. Demographic variables, cardiovascular risk factors, CHA2DS2-VASc and HAS-BLED scores were recorded. Blood samples were taken at baseline, at 3 and 24 hours after the administration of the drug and at 30 days. Rivaroxaban levels, anti-Xa activity, prothrombin time, thrombin generation and plasma levels of thrombin-antithrombin complexes were determined. Results: We studied 20 patients aged 76.3 ± 8.0 years (60% female) with a CHA2DS2-VASc score > 2 points. The anti-Xa factor activity correlated with rivaroxaban plasma levels at 3 hours (r = 0.61, p < 0.01), at 24 hours (r = 0.85, p < 0.01) and at 30 days (r = 0.99, p < 0.01), with prothrombin time at 3 hours (r = -0.86, p = 0.019) and at 30 days (r = -0.63, p = 0.02) and with a sustained decrease in thrombin generation at 30 days of follow-up (r = -0.74, p < 0.01). There was no correlation with thrombin-antithrombin complexes (r = -0.02, p = 0.83). Conclusions: Rivaroxaban consistently inhibited the mild pro-coagulant state found in newly diagnosed non-valvular AF patients through the first 24 hours and this effect was maintained at 30 days. Plasma levels of the drug correlated with anti-Xa factor activity, thrombin generation and prothrombin time

Amblyomin-X having a Kunitz-type homologous domain, is a noncompetitive inhibitor of FXa and induces anticoagulation in vitro and in vivo.

BACKGROUND: Cancer has long been associated with thrombosis and many of the standard chemotherapeutics used to treat cancer are pro-thrombotic. Thus, the identification of novel selective anticancer drugs that also have antithrombotic properties is of enormous significance. Amblyomin-X is an anticancer protein derived from the salivary glands of the Amblyomma cajennense tick. METHODS: In this work, we determined the inhibition profile of Amblyomin-X and its effect on activated partial thromboplastin time (aPTT) and prothrombin time (PT), using various approaches such as, kinetic analyses, amidolytic assays, SDS-PAGE, and mass spectrometry. RESULTS: Amblyomin-X inhibited factor Xa, prothrombinase and tenase activities. It was hydrolyzed by trypsin and plasmin. MS/MS data of tryptic hydrolysate of Amblyomin-X suggested the presence of Cys(8)-Cys(59) and Cys(19)-Cys(42) but not Cys(34)-Cys(55) disulfide bond. Instead of Cys(34)-Cys(55), two noncanonical Cys(34)-Cys(74) and Cys(55)-Cys(74) disulfide bonds were identified. Furthermore, when Amblyomin-X (1mg/kg) injected in rabbits, it prolonged aPTT and PT. CONCLUSION: Amblyomin-X is a noncompetitive inhibitor (Ki=3.9µM) of factor Xa. It is a substrate for plasmin and trypsin, but not for factor Xa and thrombin. The disulfide Cys(34)-Cys(55) bond probably scrambles with interchain seventh free cysteine residues (Cys(74)) of Amblyomin-X. The prolongation of PT and aPTT is reversible. GENERAL SIGNIFICANCE: In term of anticoagulant property, this is structural and functional characterization of Amblyomin-X. All together, these results and previous findings suggest that Amblyomin-X has a potential to become an anticancer drug with antithrombotic property.

[Evaluation of oral anticoagulation with rivaroxaban, in patients with new onset non valvular atrial fibrillation]. / Evaluación de la anticoagulación con rivaroxaban, en pacientes con fibrilación auricular no valvular de reciente diagnóstico.

BACKGROUND: Atrial fibrillation (AF) generates a hypercoagulable state with an increased thrombin generation and raised levels of thrombin-antithrombin complexes, which results in a high risk of stroke and thromboembolism. AIM: To evaluate the anticoagulant effect of rivaroxaban by anti-Xa factor activity and its correlation with thrombin-antithrombin complexes, thrombin generation and prothrombin time in patients newly diagnosed with non-valvular AF. PATIENTS AND METHODS: Prospective study in patients with indication of anticoagulation. Demographic variables, cardiovascular risk factors, CHA2DS2-VASc and HAS-BLED scores were recorded. Blood samples were taken at baseline, at 3 and 24 hours after the administration of the drug and at 30 days. Rivaroxaban levels, anti-Xa activity, prothrombin time, thrombin generation and plasma levels of thrombin-antithrombin complexes were determined. RESULTS: We studied 20 patients aged 76.3 ± 8.0 years (60% female) with a CHA2DS2-VASc score > 2 points. The anti-Xa factor activity correlated with rivaroxaban plasma levels at 3 hours (r = 0.61, p < 0.01), at 24 hours (r = 0.85, p < 0.01) and at 30 days (r = 0.99, p < 0.01), with prothrombin time at 3 hours (r = -0.86, p = 0.019) and at 30 days (r = -0.63, p = 0.02) and with a sustained decrease in thrombin generation at 30 days of follow-up (r = -0.74, p < 0.01). There was no correlation with thrombin-antithrombin complexes (r = -0.02, p = 0.83). CONCLUSIONS: Rivaroxaban consistently inhibited the mild pro-coagulant state found in newly diagnosed non-valvular AF patients through the first 24 hours and this effect was maintained at 30 days. Plasma levels of the drug correlated with anti-Xa factor activity, thrombin generation and prothrombin time.

Nuevos anticoagulantes y anestesia regional / New anticoagulants and regional anesthesia

En los últimos años se ha producido un incremento de la práctica de la anestesia regional en pacientes que reciben fármacos que afectan el sistema fisiológico de la coagulación. La posibilidad de producirse un hematoma espinal, luego de una punción neuroaxial, en este tipo de pacientes, intranquiliza al médico anestesista. Por lo tanto, es indispensable que el médico anestesista conozca los mecanismos de acción de los diferentes anticoagulantes, sus propiedades farmacológicas y farmacocinéticas para poder así definir el intervalo entre la administración de los fármacos anticoagulantes y el bloqueo neuroaxial, la retirada del catéter y el reinicio de la anticoagulación permitiendo asociar la anestesia regional y la anticoagulación de forma segura para el paciente¹. Este trabajo tiene como objetivo principal realizar una revisión de los nuevos fármacos anticoagulantes y analizar las recomendaciones existentes como para poder hacer un uso racional y seguro en nuestra práctica diaria.

New anticoagulants and regional anesthesia In recent years there has been an increase in the practice of regional anesthesia in patients receiving drugs affecting the physiological coagulation system. The possibility of a spinal hematoma occurs after neuraxial puncture in these patients, uneasy the anesthesiologist. Therefore, it is essential that the anesthesiologist know the mechanism of action of different anticoagulants, their pharmacological and pharmacokinetic properties to well define the interval between administration of anticoagulants and neuraxial blockade, catheter removal and resetting anticoagulation allowing associate regional anesthesia and anticoagulation safely for patient1. This work has as main objective to carry out a review of the new anticoagulant drugs and analyze existing recommendations as to make rational and safe use in our daily practice.

Nuevos anticoagulantes y anestesia regional / New anticoagulants and regional anesthesia

En los últimos años se ha producido un incremento de la práctica de la anestesia regional en pacientes que reciben fármacos que afectan el sistema fisiológico de la coagulación. La posibilidad de producirse un hematoma espinal, luego de una punción neuroaxial, en este tipo de pacientes, intranquiliza al médico anestesista. Por lo tanto, es indispensable que el médico anestesista conozca los mecanismos de acción de los diferentes anticoagulantes, sus propiedades farmacológicas y farmacocinéticas para poder así definir el intervalo entre la administración de los fármacos anticoagulantes y el bloqueo neuroaxial, la retirada del catéter y el reinicio de la anticoagulación permitiendo asociar la anestesia regional y la anticoagulación de forma segura para el paciente. Este trabajo tiene como objetivo principal realizar una revisión de los nuevos fármacos anticoagulantes y analizar las recomendaciones existentes como para poder hacer un uso racional y seguro en nuestra práctica diaria.