Adaptive changes in redox response and decreased platelet aggregation in lead-exposed workers.

Chronic lead exposure can generate pro-oxidative and pro-inflammatory conditions in the blood, related to high platelet activation and aggregation, altering cell functions. We studied ADP-stimulated aggregation and the oxidant/antioxidant system of platelets from chronically lead-exposed workers and non-exposed workers. Platelet aggregation was low in lead-exposed workers (62 vs. 97%), who had normal platelet counts and showed no clinical manifestations of hemostatic failure. ADP-activated platelets from lead-exposed workers failed to increase superoxide release (3.3 vs. 6.6 µmol/g protein), had low NADPH concentration (60 vs. 92 nmol/mg protein), high concentration of hydrogen peroxide (224 vs. 129 nmol/mg protein) and high plasma PGE2 concentration (287 vs. 79 pg/mL). Altogether, those conditions, on the one hand, could account for the low platelet aggregation and, on the other, indicate an adaptive mechanism for the oxidative status of platelets and anti-aggregating molecules to prevent thrombotic problems in the pro-oxidant and pro-inflammatory environment of chronic lead exposure.

Citicoline Modifies the Expression of Specific miRNAs Related to Cardioprotection in Patients with ST-Segment Elevation Myocardial Infarction Subjected to Coronary Angioplasty.

Extracellular vesicles are recognized as signaling mediators between cells both in physiological and pathological communication. In this work, we explored the potential effect of citicoline to modify relevant proteins or miRNAs for cardioprotection in the smallest population of such microvesicles; i.e., in exosomes from patients diagnosed with ST-segment elevation myocardial infarction (STEMI) undergoing coronary angioplasty. The plasma-exosome-enriched fraction from these patients was characterized. Their cellular origin was assessed by flow cytometry and Western blot, whereas miRNA expression was evaluated by real-time polymerase chain reaction (qRT-PCR). The content of caveolin-1, caveolin-3, and hnRNPA2B1, which play a relevant role in selective transport of miRNAs into microvesicles, along with the effect on cell viability of the exosomes obtained from citicoline-treated and untreated groups were also analyzed. Our results showed that hypoxic stress increases exosome release into the circulation. Moreover, we found that CD146+ increased in exosomes from citicoline-treated patients, while CD142+ decreased in these patients compared to the placebo group. No changes were detected in the protein levels of caveolin-1, caveolin-3, and hnRNPA2B1. Citicoline administration modified the expression of miR233-3p, miR92, and miR21-5p in exosomes. Cell viability decreased in the presence of exosomes from infarcted patients, while incubation of H9c2 cells with exosomes from patients reperfused with citicoline did not affect cell viability. In conclusion, citicoline administration modifies the expression of specific miRNAs related to cardioprotection in exosomes.

Monosubstituted Coumarins Inhibit Epinephrine-induced Platelet Aggregation.

AIM: The aim of this study was to evaluate the in vitro effect of coumarin and 15 monosubstituted derivatives on the inhibition of human platelet aggregation induced by various proaggregatory agonists, particularly by epinephrine. BACKGROUND: The emergence of residual platelet reactivity during the use of conventional antiplatelet agents (acetylsalicylic acid and clopidogrel) is one of the main causes of double therapy´s therapeutic failure. Platelet adrenoceptors participate in residual platelet reactivity. Therefore, it is necessary to develop new antiplatelet agents that inhibit epinephrine-induced platelet aggregation as a new therapeutic strategy. Information on the antiplatelet activity of coumarins in inhibiting epinephrine-induced aggregation is limited. OBJECTIVE: The objective of this study was to establish the structure-activity relationship (SAR) of coumarin derivatives with hydroxy, methoxy, and acetoxy groups in different positions of the coumarin nucleus to identify the most active molecules. Moreover, this study aimed to use in silico studies to suggest potential drug targets to which the molecules bind to produce antiplatelet effects. METHODS: The platelet aggregation was performed using a Lumi-aggregometer; the inhibitory activity of 16 compounds were evaluated by inducing the aggregation of human platelets (250 × 103/µl) with epinephrine (10 µM), collagen (2 µg/ml) or ADP (10 µM). The aggregation of control platelets was considered 100% of the response for each pro-aggregatory agonist. RESULTS: Eleven molecules inhibited epinephrine-induced aggregation, with 3-acetoxycoumarin and 7-methoxycoumarin being the most active. Only coumarin inhibited collagen-induced platelet aggregation, but no molecule showed activity when using ADP as an inducer. CONCLUSIONS: In silico studies suggest that most active molecules might have antagonistic interactions in the α2 and ß2 adrenoceptors. The antiplatelet actions of these coumarins have the potential to reduce residual platelet reactivity and thus contribute to the development of future treatments for patients who do not respond adequately to conventional agents.

Tyrame [N-(3-hydroxy-1:3:5(10)-estratrien-17ß-yl)-4-hydroxy-phenethylamine], antithrombotic aminoestrogen that decreases microvesicle formation.

BACKGROUND: Estrogens that are used as contraceptives or in replacement therapy are associated with an increase in the risk for developing thrombosis, mainly during the first year of treatment and in women with associated risk factors. OBJECTIVE: To synthesize, characterize and identify the anticoagulant, antiplatelet aggregation and microvesicle-reducing effect of the new aminoestrogen Tyrame. MATERIAL AND METHODS: CD1 strain mice were used, which had Tyrame (0, 1 and 2 mg/100 g) subcutaneously administered. At 24 h, a blood sample was obtained to determine whole-blood clotting time, microvesicles concentration and inhibitory effect on platelet aggregation. RESULTS: Blood clotting time increased up to 1.5 times in comparison with the control. Platelet aggregation inhibition had different magnitude depending on the agonist agent employed, and was complete with collagen. Both effects had a dose-dependent relationship. The microvesicles decreased up to six times with respect to the control. CONCLUSIONS: Tyrame reduces platelet aggregation and microvesicle formation, which emphasizes its potential therapeutic utility as an estrogen free of thrombotic effects.

ANTECEDENTES: Los estrógenos empleados como anticonceptivos o en la terapia de sustitución se asocian a un incremento en el riesgo de desarrollar trombosis, principalmente durante el primer año de tratamiento y en mujeres con factores de riesgo asociados. OBJETIVO: Sintetizar, caracterizar e identificar el efecto anticoagulante, antiagregante plaquetario y reductor de las microvesículas del nuevo aminoestrógeno tyrame. MATERIAL Y MÉTODOS: Se emplearon ratones cepa CD1 a los que se les administró por vía subcutánea tyrame (0, 1 y 2 mg/100 g). A las 24 h se tomó una muestra de sangre para determinar el tiempo de coagulación en sangre total, la concentración de microvesículas y el efecto inhibidor de la agregación plaquetaria. RESULTADOS: El tiempo de coagulación en sangre se incrementó hasta 1.5 veces con respecto al control. La inhibición de la agregación plaquetaria tuvo diferente magnitud dependiendo del agente agonista, siendo completa con colágena. Ambos efectos siguieron una relación dependiente de la dosis. Las microvesículas disminuyeron hasta seis veces con respecto al control. CONCLUSIONES: el tyrame disminuye la agregación plaquetaria y la formación de microvesículas, lo que acentúa su posible utilidad terapéutica como un estrógeno sin efectos trombóticos.

Fibrinolytic Activity of Circulating Microvesicles Is Associated with Progression of Breast Cancer.

The fibrinolytic system plays an important role in breast cancer, favoring progression through extracellular-matrix degradation, angiogenesis, apoptosis and cellular proliferation. The expression of urokinase-type plasminogen activator (uPA) in breast cancer tissue is widely recognized as an unfavorable prognostic factor. However, fibrinolytic activity associated with uPA cannot be reliably measured in the blood because of the rapid inhibition of uPA by plasminogen activator inhibitor-1 (PAI-1). By contrast, circulating microvesicles (Mvs) in peripheral blood protect bound enzymes from inhibition. Mvs are extracellular vesicles, released from various types of cells, and their size fluctuates between 100 and 1,000 nm. Mvs carry DNA, RNA, miRNA, and proteins, thereby serving as a source of horizontal communication between cells. We investigated whether fibrinolytic activity on circulating Mvs reflects breast cancer progression. The study population consisted of 13 patients with breast cancer and 13 healthy women. The cancer patients included 4 patients in remission, 3 patients with locally advanced cancer, and 6 with metastatic disease. Mvs were isolated from peripheral blood, quantified by a protein concentration method, and their fibrinolytic potential was measured by their capacity to generate plasmin. Although the quantity of Mvs found in patients with cancer and healthy individuals was similar, plasmin generated on Mvs was twice the amount in patients with metastasis than in healthy women (P < 0.05), underlying the value of this distinctive parameter. The data suggest that in breast cancer patients, higher fibrinolytic activity of circulating Mvs could be related to progression and metastasis of breast cancer.

New Copper Compounds with Antiplatelet Aggregation Activity.

BACKGROUND: Ischemic heart disease, cerebrovascular accident, and venous thromboembolism have the presence of a thrombotic event in common and represent the most common causes of death within the population. OBJECTIVE: Since Schiff base copper(II) complexes are able to interact with polyphosphates (PolyP), a procoagulant and potentially prothrombotic platelet agent, we investigated the antiplatelet aggregating properties of two novel tridentate Schiff base ligands and their corresponding copper( II) complexes. METHODS: The Schiff base ligands (L1) and (L2), as well as their corresponding copper(II) complexes (C1) and (C2), were synthesized and characterized by chemical analysis, X-ray diffraction, mass spectrometry, and UV-Visible, IR and far IR spectroscopy. In addition, EPR studies were carried out for (C1) and (C2), while (L1) and (L2) were further analyzed by 1H and 13C NMR. Tests for antiplatelet aggregation activities of all of the four compounds were conducted. RESULTS: X-ray diffraction studies show that (L1) and (L2) exist in the enol-imine tautomeric form with a strong intramolecular hydrogen bond. NMR studies show that both ligands are found as enol-imine tautomers in CDCl3 solution. In the solid state, the geometry around the copper(II) ion in both (C1) and (C2) is square planar. EPR spectra suggest that the geometry of the complexes is similar to that observed in the solid state by X-ray crystallography. Compound (C2) exhibited the strongest antiplatelet aggregation activity. CONCLUSION: Schiff base copper(II) complexes, which are attracting increasing interest, could represent a new approach to treat thrombosis by blocking the activity of PolyP with a potential anticoagulant activity and, most importantly, demonstrating no adverse bleeding events.

Association between Stable Coronary Artery Disease and In Vivo Thrombin Generation.

Background. Thrombin has been implicated as a key molecule in atherosclerotic progression. Clinical evidence shows that thrombin generation is enhanced in atherosclerosis, but its role as a risk factor for coronary atherosclerotic burden has not been proven in coronary artery disease (CAD) stable patients. Objectives. To evaluate the association between TAT levels and homocysteine levels and the presence of coronary artery disease diagnosed by coronary angiography in patients with stable CAD. Methods and Results. We included 95 stable patients admitted to the Haemodynamics Department, including 63 patients with significant CAD and 32 patients without. We measured the thrombin-antithrombin complex (TAT) and homocysteine concentrations in all the patients. The CAD patients exhibited higher concentrations of TAT (40.76 µg/L versus 20.81 µg/L, p = 0.002) and homocysteine (11.36 µmol/L versus 8.81 µmol/L, p < 0.01) compared to the patients without significant CAD. Specifically, in patients with CAD+ the level of TAT level was associated with the severity of CAD being 36.17 ± 24.48 µg/L in the patients with bivascular obstruction and 42.77 ± 31.81 µg/L in trivascular coronary obstruction, p = 0.002. Conclusions. The level of in vivo thrombin generation, quantified as TAT complexes, is associated with the presence and severity of CAD assessed by coronary angiography in stable CAD patients.

Isolation and chemical identification of lipid derivatives from avocado (Persea americana) pulp with antiplatelet and antithrombotic activities.

Platelets play a pivotal role in physiological hemostasis. However, in coronary arteries damaged by atherosclerosis, enhanced platelet aggregation, with subsequent thrombus formation, is a precipitating factor in acute ischemic events. Avocado pulp (Persea americana) is a good source of bioactive compounds, and its inclusion in the diet as a source of fatty acid has been related to reduced platelet aggregability. Nevertheless, constituents of avocado pulp with antiplatelet activity remain unknown. The present study aims to characterize the chemical nature of avocado constituents with inhibitory effects on platelet aggregation. Centrifugal partition chromatography (CPC) was used as a fractionation and purification tool, guided by an in vitro adenosine diphosphate (ADP), arachidonic acid or collagen-platelet aggregation assay. Antiplatelet activity was initially linked to seven acetogenins that were further purified, and their dose-dependent effects in the presence of various agonists were contrasted. This process led to the identification of Persenone-C (3) as the most potent antiplatelet acetogenin (IC50=3.4 mM) among the evaluated compounds. In vivo evaluations with Persenone A (4) demonstrated potential protective effects against arterial thrombosis (25 mg kg⁻¹ of body weight), as coagulation times increased (2-fold with respect to the vehicle) and thrombus formation was attenuated (71% versus vehicle). From these results, avocado may be referred to as a functional food containing acetogenin compounds that inhibit platelet aggregation with a potential preventive effect on thrombus formation, such as those that occur in ischaemic diseases.

The structures and inhibitory effects of Buame [N-(3-hydroxy-1,3,5(10)-estratrien-17ß-yl)-butylamine] and Diebud [N,N'-bis-(3-hydroxy-1,3,5(10)-estratrien-17ß-yl)-1,4-butanediamine] on platelet aggregation.

Compounds with estrogenic effects that also inhibit platelet aggregation might be useful in reducing thrombotic events associated with estrogenic therapy. In this study, two aminoestrogens, Buame [N-(3-hydroxy-1,3,5(10)-estratrien-17ß-yl)-butylamine] and Diebud [N,N'-bis-(3-hydroxy-1,3,5(10)-estratrien-17ß-yl)-1,4-butanediamine], were synthesized and characterized using common analytical methods and spectrophotometric analyses. The location and orientation of these molecules on the estrogenic receptor α (ERα) were also evaluated. Platelet inhibitory effects were elucidated ADP-induced platelet aggregation and ADP- and collagen-induced ATP release. Molecular docking demonstrated that Buame can reach and bind to the ERα in the ligand binding domain (LBD) similar to 17ß-estradiol (co-crystallized ligand). On the other hand, Diebud binds only to the surface of ERα due to its high molecular volume compared to 17ß-estradiol and Buame.

The effect of vanadium on platelet function.

Vanadium pentoxide (V(2)O(5)) inhalation effect on platelet function in mice was explored, as well as the in vitro effect on human platelets. Mouse blood samples were collected and processed for aggregometry and flow cytometry to assess the presence of P-selectin and monocyte-platelet conjugates. Simultaneously, human platelets were processed for aggregometry(.) The mouse results showed platelet aggregation inhibition in platelet-rich-plasma (PRP) at four-week exposure time, and normality returned at eight weeks of exposure, remaining unchanged after the exposure was discontinued after four weeks. This platelet aggregation inhibition effect was reinforced with the in vitro assay. In addition, P-selectin preserved their values during the exposure, until the exposure was discontinued during four weeks, when this activation marker increased. We conclude that vanadium affects platelet function, but further studies are required to evaluate its effect on other components of the hemostatic system.