RESUMEN

The study aimed to evaluate the antithrombotic action of Acrocomia aculeata pulp oil (AAPO) in natura, in an in vitro experimental model. AAPO was obtained by solvent extraction, and its chemical characterization was performed by gas chromatography coupled to a mass spectrometer (GC-MS). In vitro toxicity was evaluated with the Trypan Blue exclusion test and in vivo by the Galleria mellonella model. ADP/epinephrine-induced platelet aggregation after treatment with AAPO (50, 100, 200, 400, and 800 µg/mL) was evaluated by turbidimetry, and coagulation was determined by prothrombin activity time (PT) and activated partial thromboplastin time (aPTT). Platelet activation was measured by expression of P-selectin on the platelet surface by flow cytometry and intraplatelet content of reactive oxygen species (ROS) by fluorimetry. The results showed that AAPO has as major components such as oleic acid, palmitic acid, lauric acid, caprylic acid, and squalene. AAPO showed no toxicity in vitro or in vivo. Platelet aggregation decreased against agonists using treatment with different concentrations of AAPO. Oil did not interfere in PT and aPTT. Moreover, it expressively decreased ROS-induced platelet activation and P-selectin expression. Therefore, AAPO showed antiplatelet action since it decreased platelet activation verified by the decrease in P-selectin expression as well as in ROS production.

Asunto(s)

Fibrinolíticos , Selectina-P , Aceites de Plantas , Agregación Plaquetaria , Especies Reactivas de Oxígeno , Animales , Agregación Plaquetaria/efectos de los fármacos , Selectina-P/metabolismo , Humanos , Aceites de Plantas/farmacología , Aceites de Plantas/química , Especies Reactivas de Oxígeno/metabolismo , Fibrinolíticos/farmacología , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Coagulación Sanguínea/efectos de los fármacos , Activación Plaquetaria/efectos de los fármacos

RESUMEN

Metastasis is the leading cause of cancer-related deaths. Despite this relevance, there is no specific therapy targeting metastasis. The interaction of the tumor cell with platelets, forming microemboli is crucial for successful hematogenous dissemination. Heparin disrupts it by a P-selectin-mediated event. However, its clinical use for this purpose is hindered by the requirement of high doses, leading to anticoagulant-related side effects. In this study, we obtained a low-anticoagulant heparin through the fractionation of a pharmaceutical bovine heparin. This derivative was referred to as LA-hep and we investigated its efficacy in inhibiting metastases and explored its capacity of suppressing the interaction between tumor cells and platelets. Our data revealed that LA-hep is as efficient as porcine unfractionated heparin in attenuating lung metastases from melanoma and colon adenocarcinoma cells in an assay with a single intravenous administration. It also prevents platelet arrest shortly after cell injection in wild-type mice and suppresses melanoma-platelets interaction in vitro. Moreover, LA-hep blocks P-selectin's direct binding to tumor cells and platelet aggregation, providing further evidence for the role of P-selectin as a molecular target. Even in P-selectin-depleted mice which developed a reduced number of metastatic foci, both porcine heparin and LA-hep further inhibited metastasis burden. This suggests evidence of an additional mechanism of antimetastatic action. Therefore, our results indicate a dissociation between the heparin anticoagulant and antimetastatic effects. Considering the simple and highly reproducible methodology used to purify LA-hep along with the data presented here, LA-hep emerges as a promising drug for future use in preventing metastasis in cancer patients.

Asunto(s)

Adenocarcinoma , Neoplasias del Colon , Melanoma , Humanos , Animales , Bovinos , Ratones , Heparina/farmacología , Anticoagulantes/farmacología , Selectina-P/metabolismo , Melanoma/patología , Adenocarcinoma/patología , Neoplasias del Colon/patología , Plaquetas/metabolismo , Preparaciones Farmacéuticas/metabolismo , Metástasis de la Neoplasia/patología

RESUMEN

Identifying individuals and factors associated with severe cases of COVID-19 is crucial as the pandemic continues to spread globally. Effective biomarkers for predicting severe cases are essential for optimizing clinical management, therapy, and preventing unfavorable outcomes. This exploratory observational study aimed to investigate the expression of dysregulated immune response genes (ARG1, NOS2, ITGA4, and SELPLG) in total leukocytes, plasmatic levels of P-selectin and PSGL-1, and their clinical associations in patients with mild and severe COVID-19. Data from 117 confirmed COVID-19 patients (severe = 58, mild = 59) were collected upon admission. Gene expression was measured using RT-qPCR, and plasma protein levels assessed with ELISA assay. The severe COVID-19 patient group had a higher median age of 62.0 (p = 0.0001), a higher proportion of black individuals (86.2%, p < 0.0001), and more males (65.5%, p = 0.007). The neutrophil-lymphocyte ratio (NLR) and platelet-lymphocyte ratio (PLR) were significantly higher in the severe COVID-19 patient group (p < 0.0001), indicating ongoing systemic inflammation. Severe COVID-19 patients also exhibited increased expression of ARG1 (p < 0.05) and SELPLG (p < 0.0001) genes, as well as higher concentrations of soluble P-selectin (p < 0.005) and PSGL-1 (p < 0.05) proteins. Multivariate analysis revealed that NLR, PLR, the expression of SELPLG and sPSGL-1 were independent predictors of COVID-19 severity. In conclusion, this study suggests that biomarkers of endothelial dysfunction and dysregulated leukocyte responses are associated with COVID-19 severity, serving as promising predictive tools for optimizing clinical management and patient monitoring.

Asunto(s)

COVID-19 , Enfermedades Vasculares , Masculino , Humanos , Selectina-P/metabolismo , COVID-19/metabolismo , Recuento de Plaquetas , Biomarcadores , Neutrófilos/metabolismo , Inmunidad , Estudios Retrospectivos

RESUMEN

BACKGROUND: Oxidative stress derived from severe systemic inflammation promotes conversion from high-density lipoprotein HDL to oxidized HDL (oxHDL), which interacts with vascular endothelial cells (ECs). OxHDL acquires procoagulant features playing a role in modulating coagulation, which has been linked with organ failure in ICU patients. However, whether oxHDL elicits a ECs-mediated procoagulant phenotype generating organ failure and death, and the underlying molecular mechanism is not known. Therefore, we studied whether oxHDL-treated rats and high-oxHDL ICU patients exhibit a procoagulant phenotype and its association with kidney injury and mortality and the endothelial underlying molecular mechanism. METHODS: Human ECs, oxHDL-treated rats and ICU patients were subjected to several cellular and molecular studies, coagulation analyses, kidney injury assessment and mortality determination. RESULTS: OxHDL-treated ECs showed a procoagulant protein expression reprograming characterized by increased E-/P-selectin and vWF mRNA expression through specific signaling pathways. OxHDL-treated rats exhibited a procoagulant phenotype and modified E-/P-selectin, vWF, TF and t-PA mRNA expression correlating with plasma TF, t-PA and D-dimer. Also, showed increased death events and the relative risk of death, and increased creatinine, urea, BUN/creatinine ratio, KIM-1, NGAL, ß2M, and decreased eGFR, all concordant with kidney injury, correlated with plasma TF, t-PA and D-dimer. ICU patients showed correlation between plasma oxHDL and increased creatinine, cystatin, BUN, BUN/creatinine ratio, KIM-1, NGAL, ß2M, and decreased GFR. Notably, ICU high-oxHDL patients showed decreased survival. Interestingly, altered coagulation factors TF, t-PA and D-dimer correlated with both increased oxHDL levels and kidney injury markers, indicating a connection between these factors. CONCLUSION: Increased circulating oxHDL generates an endothelial-dependent procoagulant phenotype that associates with acute kidney injury and increased risk of death.

Asunto(s)

Lesión Renal Aguda , Lipoproteínas HDL , Humanos , Ratas , Animales , Lipoproteínas HDL/metabolismo , Selectina-P/metabolismo , Células Endoteliales/metabolismo , Creatinina , Lipocalina 2 , Factor de von Willebrand/metabolismo , Fenotipo , ARN Mensajero

RESUMEN

Aristotelia chilensis (Mol.) Stuntz, also known as maqui, is a plant native to Chile without chemical characterization and quantification of the bioactive compounds present in it. HPLC-UV and HPLC-MS/MS studies have shown the presence, at different concentrations, of phenolic and anthocyanin compounds in fruit and leave extracts of the domesticated maqui clones Luna Nueva, Morena, and Perla Negra. The extracts from leaves and unripe fruits of Luna Nueva and Morena clones significantly inhibit platelet aggregation induced by several agonists; the extracts inhibit platelet granule secretion by decreasing the exposure of P-selectin and CD63 at the platelet membrane. Reactive oxygen species formation in platelets is lower in the presence of maqui extracts. Statistical Pearson analysis supports the levels of phenolic and anthocyanin compounds being responsible for the antiaggregant maqui effects. This work is the first evidence of antiplatelet activity from Aristotelia chilensis giving added value to the use of leaves and unripe fruits from this species.

Asunto(s)

Antocianinas/farmacología , Elaeocarpaceae/química , Inhibidores de Agregación Plaquetaria/farmacología , Polifenoles/farmacología , Antocianinas/química , Antocianinas/aislamiento & purificación , Chile , Cromatografía Líquida de Alta Presión , Domesticación , Frutas/química , Humanos , Selectina-P/metabolismo , Extractos Vegetales/química , Extractos Vegetales/farmacología , Hojas de la Planta/química , Inhibidores de Agregación Plaquetaria/química , Inhibidores de Agregación Plaquetaria/aislamiento & purificación , Polifenoles/química , Polifenoles/aislamiento & purificación , Espectrometría de Masas en Tándem , Tetraspanina 30/metabolismo

RESUMEN

Platelet activation plays a key role in cardiovascular diseases. The generation of mitochondrial reactive oxygen species (ROS) has been described as a critical step required for platelet activation. For this reason, it is necessary to find new molecules with antiplatelet activity and identify their mechanisms of action. Mitoquinone (MitoQ) is a mitochondria-targeted antioxidant that reduces mitochondrial overproduction of ROS. In this work, the antiplatelet effect of MitoQ through platelet adhesion and spreading, secretion, and aggregation was evaluated. Thus MitoQ, in a non-toxic effect, decreased platelet adhesion and spreading on collagen surface, and expression of P-selectin and CD63, and inhibited platelet aggregation induced by collagen, convulxin, thrombin receptor activator peptide-6 (TRAP-6), and phorbol 12-myristate 13-acetate (PMA). As an antiplatelet mechanism, we showed that MitoQ produced mitochondrial depolarization and decreased ATP secretion. Additionally, in platelets stimulated with antimycin A and collagen MitoQ significantly decreased ROS production. Our findings showed, for the first time, an antiplatelet effect of MitoQ that is probably associated with its mitochondrial antioxidant effect.

Asunto(s)

Antioxidantes/farmacología , Plaquetas/metabolismo , Compuestos Organofosforados/farmacología , Especies Reactivas de Oxígeno/metabolismo , Ubiquinona/análogos & derivados , Adenosina Trifosfato/metabolismo , Animales , Plaquetas/efectos de los fármacos , Células Cultivadas , Colágeno/metabolismo , Humanos , Ratones , Mitocondrias/metabolismo , Oligopéptidos/farmacología , Selectina-P/metabolismo , Ésteres del Forbol/farmacología , Activación Plaquetaria/efectos de los fármacos , Tetraspanina 30/metabolismo , Ubiquinona/farmacología

RESUMEN

Platelets drive endothelial cell activation in many diseases. However, if this occurs in Plasmodium vivax malaria is unclear. As platelets have been reported to be activated and to play a role in inflammatory response during malaria, we hypothesized that this would correlate with endothelial alterations during acute illness. We performed platelet flow cytometry of PAC-1 and P-selectin. We measured platelet markers (CXCL4, CD40L, P-selectin, Thrombopoietin, IL-11) and endothelial activation markers (ICAM-1, von Willebrand Factor and E-selectin) in plasma with a multiplex-based assay. The values of each mediator were used to generate heatmaps, K-means clustering and Principal Component analysis. In addition, we determined pair-wise Pearson's correlation coefficients to generate correlation networks. Platelet counts were reduced, and mean platelet volume increased in malaria patients. The activation of circulating platelets in flow cytometry did not differ between patients and controls. CD40L levels (Median [IQ]: 517 [406-651] vs. 1029 [732-1267] pg/mL, P = 0.0001) were significantly higher in patients, while P-selectin and CXCL4 showed a nonsignificant trend towards higher levels in patients. The network correlation approach demonstrated the correlation between markers of platelet and endothelial activation, and the heatmaps revealed a distinct pattern of activation in two subsets of P. vivax patients when compared to controls. Although absolute platelet activation was not strong in uncomplicated vivax malaria, markers of platelet activity and production were correlated with higher endothelial cell activation, especially in a specific subset of patients.

Asunto(s)

Plaquetas/citología , Malaria Vivax/sangre , Adulto , Plaquetas/metabolismo , Ligando de CD40/genética , Ligando de CD40/metabolismo , Selectina E/genética , Selectina E/metabolismo , Células Endoteliales/metabolismo , Femenino , Humanos , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Interleucina-11/genética , Interleucina-11/metabolismo , Malaria Vivax/genética , Malaria Vivax/metabolismo , Masculino , Selectina-P/genética , Selectina-P/metabolismo , Activación Plaquetaria , Recuento de Plaquetas , Adulto Joven

RESUMEN

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emergent pathogen responsible for the coronavirus disease 2019 (COVID-19). Since its emergence, the novel coronavirus has rapidly achieved pandemic proportions causing remarkably increased morbidity and mortality around the world. A hypercoagulability state has been reported as a major pathologic event in COVID-19, and thromboembolic complications listed among life-threatening complications of the disease. Platelets are chief effector cells of hemostasis and pathological thrombosis. However, the participation of platelets in the pathogenesis of COVID-19 remains elusive. This report demonstrates that increased platelet activation and platelet-monocyte aggregate formation are observed in severe COVID-19 patients, but not in patients presenting mild COVID-19 syndrome. In addition, exposure to plasma from severe COVID-19 patients increased the activation of control platelets ex vivo. In our cohort of COVID-19 patients admitted to the intensive care unit, platelet-monocyte interaction was strongly associated with tissue factor (TF) expression by the monocytes. Platelet activation and monocyte TF expression were associated with markers of coagulation exacerbation as fibrinogen and D-dimers, and were increased in patients requiring invasive mechanical ventilation or patients who evolved with in-hospital mortality. Finally, platelets from severe COVID-19 patients were able to induce TF expression ex vivo in monocytes from healthy volunteers, a phenomenon that was inhibited by platelet P-selectin neutralization or integrin αIIb/ß3 blocking with the aggregation inhibitor abciximab. Altogether, these data shed light on new pathological mechanisms involving platelet activation and platelet-dependent monocyte TF expression, which were associated with COVID-19 severity and mortality.

Asunto(s)

Betacoronavirus/inmunología , Trastornos de la Coagulación Sanguínea/patología , Plaquetas/patología , Infecciones por Coronavirus/complicaciones , Monocitos/patología , Neumonía Viral/complicaciones , Tromboplastina/metabolismo , Adulto , Biomarcadores/metabolismo , Trastornos de la Coagulación Sanguínea/inmunología , Trastornos de la Coagulación Sanguínea/metabolismo , Trastornos de la Coagulación Sanguínea/virología , Plaquetas/metabolismo , Plaquetas/virología , COVID-19 , Estudios de Casos y Controles , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/metabolismo , Infecciones por Coronavirus/virología , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Monocitos/metabolismo , Monocitos/virología , Selectina-P/metabolismo , Pandemias , Activación Plaquetaria , Neumonía Viral/inmunología , Neumonía Viral/metabolismo , Neumonía Viral/virología , Pronóstico , Estudios Prospectivos , SARS-CoV-2 , Tasa de Supervivencia

RESUMEN

Sepsis is a clinical syndrome characterized by the presence of circulating microbial endotoxins and oxidative stress. Endotoxin and oxidative stress activate endothelial cells via a convergent signaling pathway (TLR4/MyD88/PI3 K/PLCÉ£/NF-B) that stimulates both the transcription of SELP gene (which encodes for human P-selectin) and the release of P-selectin from Weibel-Palade bodies (WPBs). However, time course pattern of P-selectin surface aggregation has not been established in endothelial cells under 24 h of endotoxic or oxidative stress. Our study shows that P-selectin has at least two waves of aggregation at the cell surface: one 10 min and the other 12 h after endotoxic or oxidative stress. The first wave depends exclusively on WPB delivery to the cell membrane, while the second depends on P-selectin translation machinery, ER-Golgi sorting, and WPB surface delivery. Understanding adhesion molecule dynamics in endothelial cells could provide further molecular insights to develop diagnostic or therapeutic tools to aid in the management of sepsis.

Asunto(s)

Células Endoteliales/metabolismo , Endotoxinas/toxicidad , Estrés Oxidativo , Selectina-P/metabolismo , Agregado de Proteínas , Línea Celular , Células Endoteliales/citología , Humanos

RESUMEN

Heparin or highly sulfated heparan sulfate (HS) has been described in different invertebrates. In ascidians (Chordata-Tunicata), these glycosaminoglycans occur in intracellular granules of oocyte accessory cells and circulating basophil-like cells, resembling mammalian mast cells and basophils, respectively. HS is also a component of the basement membrane of different ascidian organs. We have analyzed an HS isolated from the internal organs of the ascidian Phallusia nigra, using solution 1H/13C NMR spectroscopy, which allowed us to identify and quantify the monosaccharides found in this glycosaminoglycan. A variety of α-glucosamine units with distinct degrees of sulfation and N-acetylation were revealed. The hexuronic acid units occur both as α-iduronic acid and ß-glucuronic acid, with variable sulfation at the 2-position. A peculiar structural aspect of the tunicate HS is the high content of 2-sulfated ß-glucuronic acid, which accounts for one-third of the total hexuronic acid units. Another distinct aspect of this HS is the occurrence of high content of N-acetylated α-glucosamine units bearing a sulfate group at position 6. The unique ascidian HS is a potent inhibitor of the binding of human colon adenocarcinoma cells to immobilized P-selectin, being 11-fold more potent than mammalian heparin, but almost ineffective as an anticoagulant. Thus, the components of the HS structure required to inhibit coagulation and binding of tumor cells to P-selectin are distinct. Our results also suggest that the regulation of the pathway involved in the biosynthesis of glycosaminoglycans suffered variations during the evolution of chordates.

Asunto(s)

Adenocarcinoma/metabolismo , Anticoagulantes/metabolismo , Neoplasias del Colon/metabolismo , Glucuronatos/metabolismo , Heparitina Sulfato/química , Heparitina Sulfato/metabolismo , Selectina-P/metabolismo , Urocordados/metabolismo , Animales , Anticoagulantes/química , Línea Celular Tumoral , Colon/metabolismo , Ácido Glucurónico/metabolismo , Glicosaminoglicanos/metabolismo , Heparina/metabolismo , Humanos

RESUMEN

Chronic inflammation, as a consequence of the persistent infection with Trypanosoma cruzi, leads to continuous activation of the immune system in patients with chronic Chagas disease. We have previously shown that increased sera levels of soluble P-selectin are associated with the severity of the cardiomyopathy distinctive of chronic Chagas disease. In this study, we explored the expression of biomarkers of platelet and endothelial activation, tissue remodeling, and mediators of the coagulation cascade in patients at different clinical stages of chronic Chagas heart disease. The frequencies of activated platelets, measured by the expression of CD41a and CD62P were decreased in patients with chronic Chagas disease compared with those in uninfected subjects, with an inverse association with disease severity. Platelet activation in response to adenosine diphosphate was also decreased in T. cruzi-infected subjects. A major proportion of T. cruzi infected subjects showed increased serum levels of fibrinogen. Patients with severe cardiac dysfunction showed increased levels of endothelin-1 and normal values of procollagen I. In conclusion, chronic infection with T. cruzi induced hemostatic alterations, even in those patients who do not yet present cardiac symptoms.

Asunto(s)

Plaquetas/patología , Enfermedad de Chagas/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Plaquetas/metabolismo , Plaquetas/parasitología , Cardiomiopatía Chagásica/metabolismo , Cardiomiopatía Chagásica/parasitología , Cardiomiopatía Chagásica/patología , Enfermedad de Chagas/metabolismo , Enfermedad de Chagas/parasitología , Enfermedad Crónica , Endotelina-1/metabolismo , Femenino , Fibrinógeno/metabolismo , Humanos , Inflamación/metabolismo , Inflamación/patología , Masculino , Persona de Mediana Edad , Selectina-P/metabolismo , Procolágeno/metabolismo , Trypanosoma cruzi/patogenicidad , Adulto Joven

RESUMEN

BACKGROUND: Brain death (BD) induces hemodynamic instability with microcirculatory hypoperfusion, leading to increased organ inflammation and dysfunction. This study investigated the effects of 7.5% hypertonic saline solution (HSS) on mesenteric microcirculatory dysfunction and inflammation in a rat model of BD. METHODS: Male Wistar rats were anesthetized and mechanically ventilated. BD was induced by rapidly inflating an intracranial balloon catheter. The rats were randomly divided into: SH, sham-operated rats subjected to trepanation; NS, rats treated with NaCl 0.9%, 4 mL/kg immediately after BD; T1, rats treated with HSS (NaCl 7.5%, 4 mL/kg) immediately or 60 min after BD, T60. All groups were analyzed 180 min after the start of the experiment. RESULTS: Rats in BD groups presented with a similar hypertensive peak, followed by hypotension. Proportion of perfused small vessels was decreased in the NS group (46%) compared with the SH group (74%, P = 0.0039). HSS restored the proportion of perfused vessels (T1 = 71%, P = 0.0018). The anti-endothelial nitric oxide synthase (eNOS) protein expression significantly increased in rats given HSS (T1, and T60, P = 0.0002). Similar results were observed regarding endothelin-1 (P < 0.0001). Increased numbers of rolling (P = 0.0015) and migrated (P = 0.0063) leukocytes were observed in the NS group compared with the SH group. Rats given HSS demonstrated an overall reduction in leukocyte-endothelial interactions. The ICAM-1 levels increased in the NS group compared with the SH group, and decreased in the HSS-treated groups (P = 0.0002). CONCLUSIONS: HSS may improve the density of mesenteric perfused small vessels due to its effects on eNOS and endothelin-1 protein expression, and reduces inflammation by decreasing leukocyte adhesion and migration in a rat model of BD.

Asunto(s)

Muerte Encefálica/inmunología , Muerte Encefálica/fisiopatología , Solución Salina Hipertónica/farmacología , Solución Salina Hipertónica/uso terapéutico , Animales , Electrólitos , Endotelina-1/metabolismo , Hemodinámica/efectos de los fármacos , Inmunohistoquímica , Inflamación/tratamiento farmacológico , Inflamación/fisiopatología , Molécula 1 de Adhesión Intercelular/metabolismo , Masculino , Microcirculación/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo III/metabolismo , Selectina-P/metabolismo , Ratas , Ratas Wistar

RESUMEN

The main component of Alzheimer's disease (AD) is the amyloid-beta peptide (Aß), the brain of these patients is characterized by deposits in the parenchyma and cerebral blood vessels known as cerebral amyloid angiopathy (CAA). On the other hand, the platelets are the major source of the Aß peptide in circulation and once secreted can activate the platelets and endothelial cells producing the secretion of several inflammatory mediators that finally end up unchaining the CAA and later AD. In the present study we demonstrate that cAMP/PKA pathway plays key roles in the regulation of calpain activation and secretion of Aß in human platelets. We confirmed that inhibition of platelet functionality occurred when platelets were incubated with forskolin (molecule that rapidly increased cAMP levels). In this sense we found that platelets pre-incubated with forskolin (20 µM) present a complete inhibition of calpain activity and this effect is reversed using an inhibitor of protein kinase A. Consequentially, when platelets were inhibited by forskolin a reduction in the processing of the APP with the consequent decrease in the Aß peptide secretion was observed. Therefore our study provides novel insight in relation to the mechanism of processing and release of the Aß peptide from human platelets.

Asunto(s)

Péptidos beta-Amiloides/metabolismo , Plaquetas/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Adulto , Enfermedad de Alzheimer/sangre , Precursor de Proteína beta-Amiloide/metabolismo , Plaquetas/efectos de los fármacos , Calpaína/antagonistas & inhibidores , Calpaína/metabolismo , Fármacos Cardiovasculares/farmacología , Células Cultivadas , Colforsina/farmacología , Simulación por Computador , Humanos , Modelos Moleculares , Selectina-P/metabolismo , Agregación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/fisiología , Transducción de Señal/efectos de los fármacos , Adulto Joven

RESUMEN

Platelets are considered as significant players in innate and adaptive immune responses. The adhesion molecules they express, including P-selectin, CD40L, and CD42b, facilitate interactions with many cellular effectors. Upon interacting with a pathogen, platelets rapidly express and enhance their adhesion molecules, and secrete cytokines and chemokines. A similar phenomenon occurs after exposure of platelets to thrombin, an agonist extensively used for in vitro activation of these cells. It was recently reported that the dengue virus not only interacts with platelets but possibly infects them, which triggers an increased expression of adhesion molecule P-selectin as well as secretion of IL-1ß. In the present study, surface molecules of platelets like CD40L, CD42b, CD62P, and MHC class I were evaluated at 4 h of interaction with dengue virus serotype 2 (DENV-2), finding that DENV-2 induced a sharp rise in the membrane expression of all these molecules. At 2 and 4 h of DENV-2 stimulation of platelets, a significantly greater secretion of soluble CD40L (sCD40L) was found (versus basal levels) as well as cytokines such as GM-CSF, IL-6, IL-8, IL-10, and TNF-α. Compared to basal, DENV-2 elicited more than two-fold increase in these cytokines. Compared to the thrombin-induced response, the level generated by DENV-2 was much higher for GM-CSF, IL-6, and TNF-α. All these events induced by DENV end up in conspicuous morphological changes observed in platelets by confocal microscopy and transmission electron microscopy, very different from those elicited by thrombin in a more physiological scenery.

Asunto(s)

Plaquetas/metabolismo , Ligando de CD40/metabolismo , Membrana Celular/metabolismo , Virus del Dengue/fisiología , Dengue/sangre , Dengue/virología , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Plaquetas/inmunología , Ligando de CD40/sangre , Estudios de Casos y Controles , Moléculas de Adhesión Celular/metabolismo , Citocinas/metabolismo , Citosol/metabolismo , Dengue/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Selectina-P/metabolismo , Adhesividad Plaquetaria , Agregación Plaquetaria

RESUMEN

OBJECTIVE: In surgical aortic repair or cardiac surgery with aorta occlusion, the occurrence of mesenteric ischemia and bowel injury has been associated with higher short-term mortality. The vascular protection of estrogens has been investigated and is mainly mediated by increasing the availability of nitric oxide (NO). Therefore, this study investigated the role of 17ß-estradiol on visceral ischemia-reperfusion (I/R) injury after descending aorta occlusion in male rats. METHODS: Mesenteric ischemia was induced in male Wistar rats by placing a 2F Fogarty arterial embolectomy catheter (Edwards Lifesciences, Irvine, Calif) in the descending aorta, which remained occluded for 15 minutes, followed by reperfusion for up to 2 hours. Rats were divided into four groups: (1) rats that underwent surgical manipulation only (sham, n = 22); (2) rats that underwent I/R injury (n = 22); (3) rats treated with intravenous 17ß-estradiol (280 µg/kg) 30 minutes before I/R (n = 22); (4) or at the beginning of reperfusion (n = 22). Intestinal histopathologic changes were evaluated by histomorphometry. Mesenteric microcirculatory alterations were assessed by laser Doppler flowmetry and intravital microscopy technique. Protein expression of intercellular adhesion molecule-1, P-selectin, endothelial NO synthase (eNOS), and endothelin-1 was evaluated by immunohistochemistry; in addition, eNOS and endothelin-1 gene expressions were quantified by real-time polymerase chain reaction. Serum cytokines were measured by enzyme-linked immunosorbent assay. RESULTS: Relative to the sham group, the I/R group exhibited a highly pronounced loss of intestine mucosal thickness, a reduction in mesenteric blood flow (P = .0203), increased migrated leukocytes (P < .05), and high mortality rate (35%). Treatment with 17ß-estradiol before aorta occlusion preserved intestine mucosal thickness (P = .0437) and mesenteric blood flow (P = .0251), reduced the number of migrated leukocytes (P < .05), and prevented any fatal occurrence. Furthermore, 17ß-estradiol downregulated the expression of intercellular adhesion molecule-1 (P = .0001) and P-selectin (P < .0001) on the endothelium and increased the protein expression of eNOS (P < .0001). The gene expressions of eNOS and endothelin-1 did not differ between the groups. CONCLUSIONS: The prophylactic treatment with 17ß-estradiol showed better overall repercussions and was able to prevent any fatal occurrence, increase eNOS expression, thus preserving mesenteric perfusion and intestinal integrity, and reduce inflammation.

Asunto(s)

Aorta/fisiopatología , Oclusión con Balón/efectos adversos , Estradiol/farmacología , Íleon/irrigación sanguínea , Íleon/efectos de los fármacos , Isquemia Mesentérica/prevención & control , Daño por Reperfusión/prevención & control , Circulación Esplácnica/efectos de los fármacos , Animales , Modelos Animales de Enfermedad , Endotelina-1/metabolismo , Íleon/metabolismo , Íleon/patología , Molécula 1 de Adhesión Intercelular/metabolismo , Masculino , Isquemia Mesentérica/etiología , Isquemia Mesentérica/metabolismo , Isquemia Mesentérica/fisiopatología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Selectina-P/metabolismo , Ratas Wistar , Daño por Reperfusión/etiología , Daño por Reperfusión/metabolismo , Daño por Reperfusión/fisiopatología , Transducción de Señal/efectos de los fármacos , Factores de Tiempo

RESUMEN

Selected adenosine A2A receptor agonists (PSB-15826, PSB-12404, and PSB-16301) have been evaluated as new antiplatelet agents. In addition, radioligand-binding studies and receptor-docking experiments were performed in order to explain their differential biological effects on a molecular level. Among the tested adenosine derivatives, PSB-15826 was the most potent compound to inhibit platelet aggregation (EC50 0.32 ± 0.05 µmol/L) and platelet P-selectin cell-surface localization (EC50 0.062 ± 0.2 µmol/L), and to increase intraplatelets cAMP levels (EC50 0.24 ± 0.01 µmol/L). The compound was more active than CGS21680 (EC50 0.97±0.07 µmol/L) and equipotent to NECA (EC50 0.31 ± 0.05 µmol/L) in platelet aggregation induced by ADP. In contrast to the results from cAMP assays, Ki values determined in radioligand-binding studies were not predictive of the A2A agonists' antiplatelet activity. Docking studies revealed the key molecular determinants of this new family of adenosine A2A receptor agonists: differences in activities are related to π-stacking interactions between the ligands and the residue His264 in the extracellular loop of the adenosine A2A receptor which may result in increased residence times. In conclusion, these results provide an improved understanding of the requirements of antiplatelet adenosine A2A receptor agonists.

Asunto(s)

Agonistas del Receptor de Adenosina A2/farmacología , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Inhibidores de Agregación Plaquetaria/farmacología , Receptor de Adenosina A2A/metabolismo , Agonistas del Receptor de Adenosina A2/química , Adulto , AMP Cíclico/química , AMP Cíclico/metabolismo , Femenino , Expresión Génica , Humanos , Ligandos , Masculino , Modelos Moleculares , Conformación Molecular , Selectina-P/metabolismo , Activación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/química , Unión Proteica , Transporte de Proteínas , Receptor de Adenosina A2A/química , Receptor de Adenosina A2A/genética , Adulto Joven

RESUMEN

Curcumin has protective effects against toxic agents and shows preventive properties for various diseases. Particulate material with an aerodynamic diameter of ≤10 µm (PM10) and titanium dioxide nanoparticles (TiO2-NPs) induce endothelial dysfunction and activation. We explored whether curcumin is able to attenuate different events related to endothelial activation. This includes adhesion, expression of adhesion molecules and oxidative stress induced by PM10 and TiO2-NPs. Human umbilical vein endothelial cells (HUVEC) were treated with 1, 10 and 100 µM curcumin for 1 h and then exposed to PM10 at 3 µg/cm2 or TiO2-NPs at 10 µg/cm2. Cell adhesion was evaluated by co-culture with U937 human myelomonocytic cells. Adhesion molecules expression was measured by flow cytometry after 3 or 24 h of exposure. Oxidative stress was determined by 2,7-dichlorodihydrofluorescein (H2DCF) oxidation. PM10 and TiO2-NPs induced the adhesion of U937 cells and the expression of E- and P-selectins, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and platelet-endothelial cell adhesion molecule-1 (PECAM-1). The expression of E- and P-selectins matched the adhesion of monocytes to HUVEC after 3 h. In HUVEC treated with 1 or 10 µM curcumin, the expression of adhesion molecules and monocytes adhesion was significantly diminished. Curcumin also partially reduced the H2DCF oxidation induced by PM10 and TiO2-NPs. Our results suggest an anti-inflammatory and antioxidant role by curcumin attenuating the activation caused on endothelial cells by exposure to particles. Therefore, curcumin could be useful in the treatment of diseases where an inflammatory process and endothelial activation are involved.

Asunto(s)

Antioxidantes/farmacología , Curcumina/farmacología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Nanopartículas/toxicidad , Material Particulado/antagonistas & inhibidores , Biomarcadores/metabolismo , Adhesión Celular/efectos de los fármacos , Ciudades , Técnicas de Cocultivo , Selectina E/genética , Selectina E/metabolismo , Fluoresceínas/química , Expresión Génica , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , México , Estrés Oxidativo/efectos de los fármacos , Selectina-P/genética , Selectina-P/metabolismo , Material Particulado/farmacología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Titanio/farmacología , Células U937 , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/metabolismo

RESUMEN

Platelets have a major role in clotting activation and contribute to the innate immune response during systemic infections. Human platelets contain tissue factor (TF) and express functional Toll-like receptor 4 (TLR4). However, the role of TLR4 in triggering the procoagulant properties of platelets, upon challenge with bacteria, is yet unknown. Our hypothesis is that E. coli O111-TLR4 interaction activates platelets and elicits their procoagulant activity. We demonstrated that the strain, but not ultrapure LPS, increased surface P-selectin expression, platelet dependent TF procoagulant activity (TF-PCA) and prompted a faster thrombin generation (TG). Blockade of TLR4 resulted in decreased platelet activation, TF-PCA and TG, revealing the participation of this immune receptor on the procoagulant response of platelets. Our results provide a novel mechanism by which individuals with bacterial infections would have an increased incidence of blood clots. Furthermore, the identification of platelet TF and TLR4 as regulators of the effect of E. coli O111 might represent a novel therapeutic target to reduce the devastating consequences of the hemostatic disorder during sepsis.

Asunto(s)

Coagulación Sanguínea , Plaquetas/metabolismo , Plaquetas/microbiología , Escherichia coli/metabolismo , Tromboplastina/metabolismo , Receptor Toll-Like 4/metabolismo , Adulto , Anciano , Anticuerpos Monoclonales/farmacología , Coagulación Sanguínea/efectos de los fármacos , Plaquetas/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Humanos , Lipoproteínas/farmacología , Persona de Mediana Edad , Selectina-P/metabolismo , Activación Plaquetaria/efectos de los fármacos , Plasma Rico en Plaquetas/metabolismo , Trombina/metabolismo , Adulto Joven

RESUMEN

BACKGROUND: ß-d-Glucans are polysaccharides found in the cell walls of yeasts, such as Saccharomyces cerevisiae, and they have been studied because of their beneficial effects on health, mainly in terms of immunomodulation. However, information on the action of these polymers on vascular and platelet function is still scarce. This study evaluate the effect of (1→3) (1→6) ß-d-glucan (ßG-Sc) and its carboxymethylated derivative (CM-G) on vascular and platelet function in rats. METHODS: The animals received daily oral treatments with ßG-Sc (20mg/kg) and CM-G (20mg/kg) for eight days. Next, cytokine quantification, vascular reactivity and adenosine diphosphate (ADP)- and collagen-induced platelet aggregation studies were performed. In vitro platelet aggregation and P-selectin exposition assays were conducted using 100 and 300µg/mL CM-G. RESULTS: The CM-G-treated group had less IL-8 than did the control. In reactivity experiments, CM-G and ßG-Sc treatments did not change the contractile response of the vessel induced by PHE. Moreover, only CM-G improved the vasorelaxation response to Nitroprusside (SPN, a nitric oxide donor). The in vitro aggregation studies showed that at the highest concentration (300µg/mL), CM-G inhibited the agonist-induced platelet aggregation with an effect similar to that of acetylsalicylic acid and without affecting P-selectin exposition. The treatments with ßG-Sc or CM-G inhibited the platelet aggregation stimulated by ADP, but only ßG-Sc treatment was effective in affect the collagen-stimulated aggregation. CONCLUSIONS: These findings suggest that CM-G modulate positively the vascular function, mainly in responses NO-dependent. CM-G and ßG-Sc have an anti-aggregation effect, being CM-G more selective to ADP-induced platelet aggregation.

Asunto(s)

Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Saccharomyces cerevisiae/química , beta-Glucanos/farmacología , Adenosina Difosfato/metabolismo , Animales , Aspirina/farmacología , Plaquetas/efectos de los fármacos , Colágeno/metabolismo , Óxido Nítrico/metabolismo , Donantes de Óxido Nítrico/farmacología , Nitroprusiato/farmacología , Selectina-P/metabolismo , Inhibidores de Agregación Plaquetaria/administración & dosificación , Inhibidores de Agregación Plaquetaria/aislamiento & purificación , Ratas , Ratas Wistar , beta-Glucanos/administración & dosificación , beta-Glucanos/aislamiento & purificación

RESUMEN

Endosulfan (ENDO) is a widely used organochlorine (OC) pesticide and persistent organo-pollutant. Epidemiological studies have shown that high levels of OC exposure were related to colorectal cancer (CRC) incidence. The objectives of the present study were to evaluate histological changes in the colon, as well as in in situ expression of ß-catenin and P-selectin, and serum levels of select pro-inflammatory cytokines in mice administered ENDO; there is a relationship between increased serum IL-6 and P-selectin levels in CRC patients and aberrant ß-catenin signaling is important in initiation/maintenance of most CRCs. Mice were exposed to ENDO (at dose < LD50) orally once a week for up to 24 weeks, and monitored (inclusive) for a total of 42 weeks. The experiment was comprised of three groups, one that did not receive ENDO (olive oil vehicle), one administered 2 mg ENDO/kg/week and a positive control (for induction of CRC) given a weekly 20 mg 1,2-dimethylhydrazine (DMH)/kg injection. The results indicated that oral administration of ENDO provoked moderate inflammation starting at six weeks, and severe colonic inflammation with an appearance of dysplastic formations (aberrant crypts) in mice treated with ENDO (or DMH) for 12 weeks or longer. Serum IL-6 levels significantly increased starting at six weeks and rose to a peak of 15-fold higher than in controls at 42 weeks; TNFα levels likewise significantly increased, with a later peak (≈four-fold higher than controls) at 30-42 weeks. Immunohistochemical analysis of the colon also showed that expression of ß-catenin and P-selectin increased with length of exposure to ENDO. Taken together, the results indicate that continued repeated oral exposure to ENDO induces increased expression of ß-catenin and P-selectin, inflammation in the colon, and, ultimately, local tissue dysplasia.

Asunto(s)

Colitis/inmunología , Colon/inmunología , Neoplasias Colorrectales/epidemiología , Endosulfano/administración & dosificación , Inflamación/inmunología , 1,2-Dimetilhidrazina/administración & dosificación , Administración Oral , Animales , Neoplasias Colorrectales/inmunología , Endosulfano/inmunología , Femenino , Humanos , Mediadores de Inflamación/metabolismo , Interleucina-6/metabolismo , Ratones , Ratones Endogámicos C57BL , Selectina-P/metabolismo , Plaguicidas/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , beta Catenina/genética , beta Catenina/metabolismo