Increased Phosphatidylserine Exposure Research Articles

Assessing the impact of green and roasted coffee extracts on colorectal cancer cells in a 3D cell culture model

BackgroundColorectal cancer (CRC) is one of the most common and deadly malignant neoplasms worldwide. It is associated with multiple lifestyle risk factors such as poor diet, physical inactivity, alcohol and red meat consumption, smoking, and obesity. Consequently, studies have been conducted on the protective activity of bioactive compounds present in some foods against CRC, making it necessary to understand how diet can affect human health. In this sense, coffee, the second most consumed beverage in the world, is a major source of phytochemicals associated with a possible antitumor effect on CRC cells. PurposeThis study aimed to analyze the chemopreventive potential of green and roasted coffee extracts on HT-29 CRC cells cultured in 2D and 3D models. MethodsFlow cytometry was employed to quantify propidium iodide and DiOC6 uptake, ROS production, phosphatidylserine exposure, and cell cycle progression. In addition, cells and spheroids were analyzed by light microscopy to identify morphological changes. ResultsThe results showed that, under the employed conditions, treatment with both extracts had a concentration-dependent cytotoxic effect. Decreased cell viability was associated with mitochondrial membrane hyperpolarization, prooxidant effect, increased phosphatidylserine exposure, and changes in the G1 phase of the cell cycle. ConclusionFor the evaluated cell markers, 3D cultures required higher doses to achieve significant cytotoxic effects when compared to monolayer cultures. Since 3D cultures favor the formation of complex cell–cell interactions, this model offers a closer look at in vivo conditions for evaluating the toxicity and preclinical efficacy of potential chemopreventive agents for CRC. The experimental evidence from this study supports the 3R principles of replacement, reduction, and refinement for toxicological research in the pharmaceutical industry.

Ru(II)-based complexes containing 2-thiouracil derivatives suppress liver cancer stem cells by targeting NF-κB and Akt/mTOR signaling

Cancer stem cells (CSCs) are defined as a rare population of cancer cells related to tumor initiation and maintenance. These cells are primarily responsible for tumor growth, invasion, metastasis, recurrence, and resistance to chemotherapy. In this paper, we demonstrated the ability of Ru(II)-based complexes containing 2-thiouracil derivatives with the chemical formulas trans-[Ru(2TU)(PPh3)2(bipy)]PF6 (1) and trans-[Ru(6m2TU)(PPh3)2(bipy)]PF6 (2) (where 2TU = 2-thiouracil and 6m2TU = 6-methyl-2-thiouracil) to suppress liver CSCs by targeting NF-κB and Akt/mTOR signaling. Complexes 1 and 2 displayed potent cytotoxic effects on cancer cell lines and suppressed liver CSCs from HepG2 cells. Increased phosphatidylserine exposure, loss of mitochondrial transmembrane potential, increased PARP (Asp214) cleavage, DNA fragmentation, chromatin condensation and cytoplasmic shrinkage were detected in HepG2 cells treated with these complexes. Mechanistically, complexes 1 and 2 target NF-κB and Akt/mTOR signaling in HepG2 cells. Cell motility inhibition was also detected in HepG2 cells treated with these complexes. Complexes 1 and 2 also inhibited tumor progression in mice with HepG2 cell xenografts and exhibited tolerable systemic toxicity. Taken together, these results indicate that these complexes are new anti-HCC drug candidates that can suppress liver CSCs.

Disruption of Transmembrane Phosphatidylserine Asymmetry by HIV-1 Incorporated SERINC5 Is Not Responsible for Virus Restriction.

Host restriction factor SERINC5 (SER5) incorporates into the HIV-1 membrane and inhibits infectivity by a poorly understood mechanism. Recently, SER5 was found to exhibit scramblase-like activity leading to the externalization of phosphatidylserine (PS) on the viral surface, which has been proposed to be responsible for SER5's antiviral activity. This and other reports that document modulation of HIV-1 infectivity by viral lipid composition prompted us to investigate the role of PS in regulating SER5-mediated HIV-1 restriction. First, we show that the level of SER5 incorporation into virions correlates with an increase in PS levels in the outer leaflet of the viral membrane. We developed an assay to estimate the PS distribution across the viral membrane and found that SER5, but not SER2, which lacks antiviral activity, abrogates PS asymmetry by externalizing this lipid. Second, SER5 incorporation diminished the infectivity of pseudoviruses produced from cells lacking a flippase subunit CDC50a and, therefore, exhibited a higher baseline level of surface-accessible PS. Finally, exogenous manipulation of the viral PS levels utilizing methyl-alpha-cyclodextrin revealed a lack of correlation between external PS and virion infectivity. Taken together, our study implies that the increased PS exposure to SER5-containing virions itself is not directly linked to HIV-1 restriction.

Impact of Recombinant Factor VIII and Platelet Interaction on Platelet Functionality and Hemophilia a Treatment

Background: Platelets play an important role throughout the various stages of hemostasis. After vascular injury, platelets become activated in a pro-aggregatory state, and subsequently a subpopulation undergoes a phenotype shift to a pro-coagulant state. Pro-coagulant platelets bind to factor VIII (FVIII) and ensure efficient localization of FVIII at the site of injury. It is unclear whether modifications in recombinant FVIII (rFVIII) products impact FVIII-platelet binding and subsequent platelet signaling. Aims: To examine binding of different rFVIII products to platelets in vitro, and the impact of rFVIII on platelet phenotype and intracellular signaling. Methods: Platelet activation: Platelets isolated from healthy donors were activated with thrombin and cross-linked collagen-related peptide (CRP-XL). Platelet-FVIII binding: Activated platelets were incubated with simoctocog alfa, efmoroctocog alfa, rurioctocog alfa pegol or damoctocog alfa pegol. Binding was quantified by flow cytometry either by immunostaining with an anti-FVIII antibody conjugated to Alexa fluor (AF)647 or direct measurement of rFVIII concentrates labelled with AF647. Phenotype shift: During the shift to a pro-coagulant state, phosphatidylserine (PS) becomes exposed on the platelet membranes, with the extent of PS exposure correlating with the level of FVIII binding. To assess PS exposure, activated platelets were incubated with Annexin V-BV421, a molecular probe with high affinity for PS. Inhibition of integrin αIIbβ3 signaling: Activated platelets were incubated with the integrin αIIbβ3 inhibitor antibody 10E5. Results: Binding to pro-coagulant platelets was significantly higher with simoctocog alfa than with efmoroctocog alfa, rurioctocog alfa pegol or damoctocog alfa pegol irrespective of immunostaining method (p<0.05; Figure 1 shows detection with anti-FVIII antibody conjugated with AF647).To validate the variability in rFVIII-platelet interactions observed, we performed a dynamic platelet-binding assay with the two FVIII products exhibiting the strongest binding (simoctocog alfa and efmoroctocog alfa). The results of the dynamic assay confirmed the results of the static experiments (Figure 1). Exposure of activated platelets to simoctocog alfa and efmoroctocog alfa resulted in increased PS exposure, with a greater effect observed with simoctocog alfa, consistent with the results of the binding experiments. Immunostaining experiments revealed co-localization of simoctocog alfa and clusters of integrin αIIbβ3 in pro-aggregatory platelets. Inhibition of integrin αIIbβ3 by 10E5 decreased binding of simoctocog alfa to platelets and subsequent PS exposure in a dose-dependent manner (Figure 2). Analysis of platelet binding and phenotype shift in patients with hemophilia A are ongoing. Conclusion: Simoctocog alfa demonstrated higher binding to activated platelets in vitro compared with efmoroctocog alfa, rurioctocog alfa pegol or damoctocog alfa pegol, resulting in an increased phenotype shift of platelets from the pro-aggregatory to the pro-coagulant state. The increased binding of simoctocog alfa was associated with a phenotypic shift in platelets as evidenced by increased exposure of PS on the platelet membranes. The binding of simoctocog alfa to platelets was disrupted when integrin αIIbβ3 activation was inhibited, suggesting a role of integrin αIIbβ3 signaling following binding of FVIII to platelets. Variations in platelet binding and signaling between different rFVIIIs might impact their efficacy for the prevention of bleeds. Further experiments are ongoing to assess the impact of FVIII on the release of von Willebrand factor and thrombin generation by activated platelets, and to repeat the FVIII-binding experiments using platelets from healthy donors vs hemophilia A patients.

Crucial roles of red blood cells and platelets in whole blood thrombin generation

AbstractRed blood cells (RBCs) and platelets contribute to the coagulation capacity in bleeding and thrombotic disorders. The thrombin generation (TG) process is considered to reflect the interactions between plasma coagulation and the various blood cells. Using a new high-throughput method capturing the complete TG curve, we were able to compare TG in whole blood and autologous platelet-rich and platelet-poor plasma to redefine the blood cell contributions to the clotting process. We report a faster and initially higher generation of thrombin and shorter coagulation time in whole blood than in platelet-rich plasma upon low concentrations of coagulant triggers, including tissue factor, Russell viper venom factor X, factor Xa, factor XIa, and thrombin. The TG was accelerated with increased hematocrit and delayed after prior treatment of RBC with phosphatidylserine-blocking annexin A5. RBC treatment with ionomycin increased phosphatidylserine exposure, confirmed by flow cytometry, and increased the TG process. In reconstituted blood samples, the prior selective blockage of phosphatidylserine on RBC with annexin A5 enhanced glycoprotein VI–induced platelet procoagulant activity. For patients with anemia or erythrocytosis, cluster analysis revealed high or low whole-blood TG profiles in specific cases of anemia. The TG profiles lowered upon annexin A5 addition in the presence of RBCs and thus were determined by the extent of phosphatidylserine exposure of blood cells. Profiles for patients with polycythemia vera undergoing treatment were similar to that of control subjects. We concluded that RBC and platelets, in a phosphatidylserine-dependent way, contribute to the TG process. Determination of the whole-blood hypo- or hyper-coagulant activity may help to characterize a bleeding or thrombosis risk.

Piezo1 activation augments sickling propensity and the adhesive properties of sickle red blood cells in a calcium-dependent manner.

Haemoglobin S polymerization in the red blood cells (RBCs) of individuals with sickle cell anaemia (SCA) can cause RBC sickling and cellular alterations. Piezo1 is a mechanosensitive protein that modulates intracellular calcium (Ca2+ ) influx, and its activation has been associated with increased RBC surface membrane phosphatidylserine (PS) exposure. Hypothesizing that Piezo1 activation, and ensuing Gárdos channel activity, alter sickle RBC properties, RBCs from patients with SCA were incubated with the Piezo1 agonist, Yoda1 (0.1-10 μM). Oxygen-gradient ektacytometry and membrane potential measurement showed that Piezo1 activation significantly decreased sickle RBC deformability, augmented sickling propensity, and triggered pronounced membrane hyperpolarization, in association with Gárdos channel activation and Ca2+ influx. Yoda1 induced Ca2+ -dependent adhesion of sickle RBCs to laminin, in microfluidic assays, mediated by increased BCAM binding affinity. Furthermore, RBCs from SCA patients that were homo-/heterozygous for the rs59446030 gain-of-function Piezo1 variant demonstrated enhanced sickling under deoxygenation and increased PS exposure. Thus, Piezo1 stimulation decreases sickle RBC deformability, and increases the propensities of these cells to sickle upon deoxygenation and adhere to laminin. Results support a role of Piezo1 in some of the RBC properties that contribute to SCA vaso-occlusion, indicating that Piezo1 may represent a potential therapeutic target molecule for this disease.

Induction of Immune Responses and Phosphatidylserine Exposure by TLR9 Activation Results in a Cooperative Antitumor Effect with a Phosphatidylserine-targeting Prodrug.

Head and neck cancer is a major cancer type, with high motility rates that reduce the quality of life of patients. Herein, we investigated the effectiveness and mechanism of a combination therapy involving TLR9 activator (CpG-2722) and phosphatidylserine (PS)-targeting prodrug of SN38 (BPRDP056) in a syngeneic orthotopic head and neck cancer animal model. The results showed a cooperative antitumor effect of CpG-2722 and BPRDP056 owing to their distinct and complementary antitumor functions. CpG-2722 induced antitumor immune responses, including dendritic cell maturation, cytokine production, and immune cell accumulation in tumors, whereas BPRDP056 directly exerted cytotoxicity toward cancer cells. We also discovered a novel function and mechanism of TLR9 activation, which increased PS exposure on cancer cells, thereby attracting more BPRDP056 to the tumor site for cancer cell killing. Killed cells expose more PS in tumor for BPRDP056 targeting. Tumor antigens released from the dead cells were taken up by antigen-presenting cells, which enhanced the CpG-272-promoted T cell-mediated tumor-killing effect. These form a positive feed-forward antitumor effect between the actions of CpG-2722 and BPRDP056. Thus, the study findings suggest a novel strategy of utilizing the PS-inducing function of TLR9 agonists to develop combinational cancer treatments using PS-targeting drugs.

Effects of urban particulate matter on the quality of erythrocytes

With the acceleration of industrialisation and urbanisation, air pollution has become a serious global concern as a hazard to human health, with urban particulate matter (UPM) accounting for the largest share. UPM can rapidly pass into and persist within systemic circulation. However, few studies exist on whether UPM may have any impact on blood components. In this study, UPM standards (SRM1648a) were used to assess the influence of UPM on erythrocyte quality in terms of oxidative and metabolic damage as well as phagocytosis by macrophages in vitro and clearance in vivo. Our results showed that UPM had weak haemolytic properties. It can oxidise haemoglobin and influence the oxygen-carrying function, redox balance, and metabolism of erythrocytes. UPM increases the content of reactive oxygen species (ROS) and decreases antioxidant function according to the data of malonaldehyde (MDA), glutathione (GSH), and glucose 6 phosphate dehydrogenase (G6PDH). UPM can adhere to or be internalised by erythrocytes at higher concentrations, which can alter their morphology. Superoxide radicals produced in the co-incubation system further disrupted the structure of red blood cell membranes, thereby lowering the resistance to the hypotonic solution, as reflected by the osmotic fragility test. Moreover, UPM leads to an increase in phosphatidylserine exposure in erythrocytes and subsequent clearance by the mononuclear phagocytic system in vivo. Altogether, this study suggests that the primary function of erythrocytes may be affected by UPM, providing a warning for erythrocyte quality in severely polluted areas. For critically ill patients, transfusion of erythrocytes with lesions in morphology and function will have serious clinical consequences, suggesting that potential risks should be considered during blood donation screening. The current work expands the scope of blood safety studies.

Increased autophagy leads to decreased apoptosis during β-thalassaemic mouse and patient erythropoiesis

β-Thalassaemia results from defects in β-globin chain production, leading to ineffective erythropoiesis and subsequently to severe anaemia and other complications. Apoptosis and autophagy are the main pathways that regulate the balance between cell survival and cell death in response to diverse cellular stresses. Herein, the death of erythroid lineage cells in the bone marrow from both βIVS2-654-thalassaemic mice and β-thalassaemia/HbE patients was investigated. Phosphatidylserine (PS)-bearing basophilic erythroblasts and polychromatophilic erythroblasts were significantly increased in β-thalassaemia as compared to controls. However, the activation of caspase 8, caspase 9 and caspase 3 was minimal and not different from control in both murine and human thalassaemic erythroblasts. Interestingly, bone marrow erythroblasts from both β-thalassaemic mice and β-thalassaemia/HbE patients had significantly increased autophagy as shown by increased autophagosomes and increased co-localization between LC3 and LAMP-1. Inhibition of autophagy by chloroquine caused significantly increased erythroblast apoptosis. We have demonstrated increased autophagy which led to minimal apoptosis in β-thalassaemic erythroblasts. However, increased PS exposure occurring through other mechanisms in thalassaemic erythroblasts might cause rapid phagocytic removal by macrophages and consequently ineffective erythropoiesis in β-thalassaemia.

Effects of exhaustive exercise on coagulation state in rats

Objective: To investigate the effects of one-time exhaustive exercise on coagulation state in rats and its mechanism. Methods: Forty-eight SD rats were randomly divided into control group and exhaustive exercise group, 24 rats in each group. Rats in exhaustive exercise group were trained with treadmill training for 25～50 min at a time on non-slope treadmill and the initial speed of 5 m/min was uniformly accelerated to 25 m/min until the rats exhausted. Thrombelastography (TEG) was used to monitor the coagulation function of rats after training. The ligation model of inferior vena cava (IVC) was established to evaluate thrombosis. The phosphatidylserine (PS) exposure and Ca2+ concentration were detected by flow cytometry. The production of FXa and thrombin was detected by microplate reader. The clotting time was measured by using coagulometer. Results: Compared with the control group, the blood of rats in the exhaustive exercise group exhibited hypercoagulable state. The probability of thrombus formation, weight, length and ratio in the exhaustive exercise group were significantly higher than those in the control group (P＜0.01). The levels of PS exposure and intracellular Ca2+ concentration of red blood cells (RBCs) and platelets in the exhaustive exercise group were increased significantly (P＜0.01). The blood clotting time of RBCs and platelets was shortened (P＜0.01), and the production of FXa and thrombin was increased significantly (P＜0.01) in the exhausted exercise group, and both were inhibited by lactadherin (Lact, P＜0.01). Conclusion: The blood of exhaustive exercise rats is in a hypercoagulable state and the risk of thrombosis is increased. The increased PS exposure of RBCs and platelets caused by exhaustive exercise may be an important mechanism of thrombosis.

Extracellular Vesicles in Sickle Cell Disease: A Promising Tool

Sickle cell disease (SCD) is the most common hemoglobinopathy worldwide. It is characterized by an impairment of shear stress-mediated vasodilation, a pro-coagulant, and a pro-adhesive state orchestrated among others by the depletion of the vasodilator nitric oxide, by the increased phosphatidylserine exposure and tissue factor expression, and by the increased interactions of erythrocytes with endothelial cells that mediate the overexpression of adhesion molecules such as VCAM-1, respectively. Extracellular vesicles (EVs) have been shown to be novel actors involved in SCD pathophysiological processes. Medium-sized EVs, also called microparticles, which exhibit increased plasma levels in this pathology, were shown to induce the activation of endothelial cells, thereby increasing neutrophil adhesion, a key process potentially leading to the main complication associated with SCD, vaso-occlusive crises (VOCs). Small-sized EVs, also named exosomes, which have also been reported to be overrepresented in SCD, were shown to potentiate interactions between erythrocytes and platelets, and to trigger endothelial monolayer disruption, two processes also known to favor the occurrence of VOCs. In this review we provide an overview of the current knowledge about EVs concentration and role in SCD.

Pequi oil (Caryocar brasilense Cambess.) nanoemulsion alters cell proliferation and damages key organelles in triple-negative breast cancer cells in vitro

Pequi oil is extracted from the fruit of a Brazilian native plant (Caryocar brasiliense Camb) that contains some molecules with anticancer potential. Due to its hydrophobic property, the administration of pequi oil associated with nanoemulsion systems represents a successful strategy to improve oil bioavailability. Breast cancer is the most frequent type of cancer among women and conventional therapies used are frequently associated with several side effects. Thus, the aim of this study was to investigate the effects of pequi oil-based nanoemulsion (PeNE) on triple-negative breast cancer cells (4T1), in vitro. PeNE presented a dose- and time-dependent cytotoxic effect with lower IC50 than free pequi oil after 48 h of exposure (p < 0.001). At 180 µg/mL, PeNE demonstrated numerous cell alterations, when compared to free pequi oil, such as morphological alterations, reduction in cell proliferation and total cell number, damage to plasmatic membrane, induction of lysosomal membrane permeability and depolarization of mitochondrial membrane, alteration of intracellular ROS production and calcium level, and increase in phosphatidylserine exposure. Taken together, the results suggest an interesting induction of cell death mechanisms involving a combined action of factors that impair nucleus, mitochondria, lysosome, and ER function. In addition, more pronounced effects were observed in cells treated by PeNE at 180 µg/mL when compared to free pequi oil, thereby reinforcing the advantages of using nanometric platforms. These promising results highlight the use of PeNE as a potential complementary therapeutic approach to be employed along with conventional treatments against breast cancer in the future.

Angiotensin II-Induced Erythrocyte Senescence Contributes to Oxidative Stress.

Oxidative stress may be an important cause of erythrocyte senescence. Angiotensin II (Ang II) has recently been shown to promote vascular cell senescence. However, its effects on erythrocytes remain unclear. This study aims at investigating the role of Ang II in regulating erythrocyte lifespan through oxidative stress. Experiments were performed in C57/BL6J mice infused with Ang II (1500 ng/kg per minute) or saline for 7 days. After Ang II infusion, we found that Ang II increased erythrocyte number, hemoglobin, and red blood cell distribution width. These differences were accompanied by a decrease in glutathione (GSH) and an increase in malondialdehyde (MDA) concentration. In vitro, after 24 hours of Ang II treatment, erythrocytes showed reduced surface expression of CD47 and increased phosphatidylserine exposure. In parallel, Ang II reduced the levels of antioxidant enzymes, including Cu/ZnSOD, catalase, and peroxidase 2 (PRDX2). These effects were reversed by the addition of the antioxidant N-acetyl-L-cysteine or the Ang II type 1 (AT1) receptor blocker losartan. In addition, Ang II treatment increased pro-inflammatory oxylipin, including hydroxyeicosatetraenoic acids (HETEs) and dihydroxyoctadecenoic acids (DiHOMEs), in the erythrocyte membranes. Collectively, Ang II induced erythrocyte senescence and susceptibility to eryptosis, partially due to enhanced oxidative stress.

Influence of Anoctamin-4 and -9 on ADAM10 and ADAM17 Sheddase Function.

Ca2+-activated Cl− channels (TMEM16, also known as anoctamins) perform important functions in cell physiology, including modulation of cell proliferation and cancer growth. Many members, including TMEM16F/ANO6, additionally act as Ca2+-activated phospholipid scramblases. We recently presented evidence that ANO6-dependent surface exposure of phosphatidylserine (PS) is pivotal for the disintegrin-like metalloproteases ADAM10 and ADAM17 to exert their sheddase function. Here, we compared the influence of seven ANO family members (ANO1, 4, 5, 6, 7, 9, and 10) on ADAM sheddase activity. Similar to ANO6, overexpression of ANO4 and ANO9 led to increased release of ADAM10 and ADAM17 substrates, such as betacellulin, TGFα, and amphiregulin (AREG), upon ionophore stimulation in HEK cells. Inhibitor experiments indicated that ANO4/ANO9-mediated enhancement of TGFα-cleavage broadened the spectrum of participating metalloproteinases. Annexin V-staining demonstrated increased externalisation of PS in ANO4/ANO9-overexpressing cells. Competition experiments with the soluble PS-headgroup phosphorylserine indicated that the ANO4/ANO9 effects were due to increased PS exposure. Overexpression of ANO4 or ANO9 in human cervical cancer cells (HeLa), enhanced constitutive shedding of the growth factor AREG and increased cell proliferation. We conclude that ANO4 and ANO9, by virtue of their scramblase activity, may play a role as important regulators of ADAM-dependent cellular functions.

Characterizing the Metabolic Determinants of Thromboinflammation in Myeloproliferative Neoplasms

BACKGROUNDMyeloproliferative neoplasms (MPNs) are a group of clonal hematopoietic disorders characterized by an overproduction of terminally differentiated myeloid elements. Venous and arterial thrombotic events represent the leading causes of morbidity and mortality in patients with JAK2 positive MPNs (essential thrombocythemia [ET] and polycythemia vera [PV]) in chronic phase. Platelet hyperreactivity is a hallmark finding of MPNs but the biological mechanisms remain to be elucidated. We have previously identified that platelets from patients with PV and ET exhibit dysmorphic mitochondria, however, the detailed bioenergetic and metabolomic implications of such alterations in mitochondrial mass and morphology remain to be investigated. Here in, we aim to identify the metabolic determinants that underpin thromboinflammation in patients with JAK2 positive MPNs in chronic phase. METHODSPlatelets from sex and age-matched healthy control subjects and individuals with either JAK2 positive PV or ET were isolated and washed following standard protocols. Platelet activation by flow cytometry was determined at baseline conditions. Platelet activated fibrinogen binding site (αIIbβIII), P-selectin, and phosphatidylserine (PS) surface marker expression (as measured by mean fluorescence intensity [MFI]) was determined with PAC-1 and P-selectin antibodies and lactadherin, respectively. The bioenergetic profile of washed platelets was determined by the 24-well format Seahorse extracellular flux analyzer. Metabolomic profile was developed with the Vanquish UHPLC system coupled online to a Q Exactive mass spectrometer. Statistical analyses were performed using the unpaired 2-tailed Student t test (GraphPad Software v9.1.2). Data expressed as mean plus or minus standard error of the mean (SEM). Significance was determined at p < 0.05. RESULTSPlatelets from patients with MPNs had higher surface marker expression of PS under resting conditions. A trend of higher surface marker expression of αIIbβIII and P-selectin was seen (Figure 1). The platelet bioenergetic profile in individuals with MPN demonstrated decreased basal respiration (p = 0.0008), ATP-linked respiration (p = 0.001), and basal extracellular acidification rate (ECAR) (p = 0.09) (Figure 2). Liquid chromatography-mass spectrometry-based metabolomics revealed elevated pentose phosphate pathway metabolites (pentose phosphates and 6-Phospho-D-gluconate), and diminished adenosine diphosphate (ADP) and adenosine triphosphate (ATP) pools (Figure 3). CONCLUSIONThe findings from this study suggest that platelets from patients with JAK2 positive PV and ET have a procoagulant phenotype given the increased expression of PS, an anionic phospholipid known to facilitate the generation of thrombin. The decreased basal respiration, ATP-linked respiration, basal ECAR (a surrogate marker of glycolysis), and energy pools (ADP and ATP) in platelets from MPN patients suggest a hypometabolic phenotype. Additionally, it appears there is preferential shunting of glucose into the pentose phosphate pathway. Such metabolic switch in conjunction with the hypometabolic state of platelets in MPN, could potentially represent a compensatory mechanism of platelets against oxidative stress.The bioenergetic dysregulation of platelets in MPNs may precipitate a cascade of events that leads to the loss of plasma membrane integrity (increased PS exposure) therefore increasing its procoagulant potential. Higher amounts of platelet PS could underly the elevated incidence of thrombosis observed in patients with JAK2 positive ET and PV. Further studies are underway to identify the inciting factor(s) that lead to the platelet bioenergetic failure and elevated PS exposure seen in patients with MPNs. These investigations will provide significant mechanistic insight for identifying therapies aimed at preventing thrombotic events in patients with MPNs. [Display omitted] DisclosuresNemkov: Omix Thecnologies: Other: Co-founder. D'Alessandro: Omix Thecnologies: Other: Co-founder; Rubius Therapeutics: Consultancy; Forma Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Procoagulant Mechanisms in Patients with Myelodysplastic Syndrome

Background: MDS comprise a heterogeneous sort of disorders caused by cytogenetic changes, gene mutations, or both, responsible for altered multilineage morphological patterns that lead to a varying grade of peripheral blood cytopaenias and an increased risk of transformation to acute myeloid leukemia. Thrombocytopaenia is observed in aproximately 35% of MDS patients. Despite it is accepted that haemorrhagic events are more likely at platelet counts below 10,000/μl, in the case of MDS patients, whose platelets had a diminished ability to be activated, bleeding events are lower than expected. So, hemostasis in MDS patients is a complex issue where mechanisms additional to platelet count/function seem to be involved.Aim: This work aimed to identify procoagulant mechanisms in non-bleeders MDS patients that might protect them from bleeding.Methods: This is a prospective, observational and transversal study. Eighty four non-bleeders MDS patients (44% female, mean age of 77±25 years), were included. Sixty healthy subjects (45% female, mean age of 57±22 years) were recruited.Human peripheral blood samples were collected in 3.8% sodium citrate. Blood cell counts were performed with a Coulter Ac.T Diff cell counter (Beckman Coulter, Madrid, Spain). Platelet rich plasma (PRP) and platelet free plasma (PFP) were obtained by centrifugation.Surface exposure of phosphatidylserine (PS) and active caspase-3, -8 or -9 were assessed by flow cytometry measuring the binding of FITC-labeled Annexin V (BD Pharmingen) and with a specific kit for measuring these caspases (Millipore, Madrid, Spain).Prothrombinase complex binding to platelets was determined in quiescent washed platelets (1x108/mL) incubated with factor (F) Xa (5 nmol/L) and FVa (5nmol/L) obtained from Haemtech Inc (Cellsystems, Germany). Following fixation to cross-link platelet-bound FVa and FXa, specific or control monoclonal antibodies (0.1 μmol/L each, Haemtech Inc, Germany) were added. All samples were analized by flow cytometry.PS-associated and tissue factor (TF)-associated procoagulant activity of microparticles (MPs) was determined with the ZYMUPHEN MP-Activity kits (HYPHEN BioMed, Neuvillesur Oise, France).The identification of the MP's cellular origin was determined by flow cytometry, labeling MPs with Annexin-V-FITC and the following specific mAb conjugated with phycoerythrin: anti-CD41 mAb for platelets (Biocytex, Marseille, France); and anti-CD14 mAb for monocytes, anti-CD144 mAb for endothelial cells, anti-CD235 mAb for red cells and anti-CD45 mAb for leukocytes, all from BD Biosciences.The statistical analysis of the experimental data was using SPSS 9.0 software (SPSS Inc., Chicago, Illinois, USA).Results: Platelets from patients with MDS presented higher levels of activated caspases-3,7, -8 and -9 and this situation was accompanied by a higher PS platelet surface exposure (TABLE 1)We evaluated if increased PS exposure was functional for anchoring prothrombinase complex. Binding of FVa and of FXa on platelet membrane surface was higher in quiescent platelets from MDS patients [FVa (Mean fluorescence), control: 49.7±8.5, MDS patients: 73.9±23.2, p<0.01; FXa (Mean fluorescence), control: 46.4±17.5 , MDS patients: 77.6±12.0, p<0.05)].A significantly higher procoagulant capacity of MPs associated either to PS (control: 8.0±2.5 nanomol/L; MDS: 17.8±3.5 nanomol/L, p<0.01) or TF (control: 0.4±2.5 nanomol/L; MDS: 7.5±2.5 nanomol/L, p<0.001) were observed in the MDS group.To determine the cellular source of MPs, flow cytometry analyses were performed and a significant increase was observed in the amount of monocyte- (events, control: 94±30, MDS patients: 220±29, p<0.05)and red cell-derived MPs (events, control: 142±55, MDS patients: 252±23, p<0.01) in the samples from the patients with MDS. In patients with MDS, TF-MP-associated thrombogenic ability correlated with monocyte percentage (Spearman r=0.5019, p<0.01).Conclusion: MDS patients, even those with normal platelet count, have a reduced platelet function with enhanced apoptosis but do not bleed in many cases. This fact might be due to the increase in tissue factor-containing microparticles and in the enhanced binding of prothrombinase complex to apoptotic platelets which, in turn, augmented procoagulant capacity of these patients.Work supported by grant from FIS-FEDER PI15/01457. NB holds a Miguel Servet II (FIS-FEDER CP14/00024). [Display omitted] DisclosuresJimenez-Yuste:Roche: Consultancy; Novo Nordisk: Consultancy, Honoraria, Research Funding. Álvarez-Roman:Shire: Consultancy; Novo Nordisk: Consultancy.

Phosphatidylserine on Blood Cells and Microparticles Contributes to the Hyper-Coagulable State in Diabetic Kidney Disease

Introduction: Patients with diabetes mellitus (DM) are considered to be prothrombotic with major disturbances in hemostasis that are associated with an increased risk of venous thromboembolism, especially when microvascular complications of diabetes mellitus occur, such as diabetic kidney disease (DKD). Additionally, microthrombosis in glomerular capillaries could decrease the kidney blood flow and induce renal insufficiency. However, patients with DKD do not receive routine antithrombotic therapy. Therefore, more study is needed on the mechanisms of coagulation in DKD with the aim of identifying therapeutic targets. Recent studies have shown that phosphatidylserine (PS), a membrane constituent, plays an important role in the thrombosis. However, relatively little is known about the precise role of PS in the prothrombotic state of DKD. Our objectives were to study the effect of increased PS exposure on microparticles (MPs) and the outer membrane of MP-origin cells in DKD patients, and to evaluate its effect on procoagulant activity (PCA).Methods: DKD patients (n = 90) were divided into three groups based on urinary albumin excretion rate (UAER), defined as normoalbuminuria (<30 mg/24 h), microalbuminuria (30-299 mg/24 h), or macroalbuminuria (>300 mg/24 h), and compared to healthy controls (n = 30). Lactadherin was used to quantify PS exposure on MPs and their original cells. Healthy blood cells and human umbilical vein endothelial cells (HUVECs) were treated with 25 mM, 5 mM or 2.5 mM glucose and then PS exposure was measured. PCA of MPs and cells was evaluated by clotting time and purified coagulation complex assays. Fibrin production was determined by turbidity. Phosphatidylserine exposure and fibrin strands were observed using confocal microscopy.Results: Usingflow cytometry, we found that PS+ MPs (derived from platelets, erythrocytes, HUVECs, neutrophils, monocytes and lymphocytes) and blood cells (BCs) were significantly higher in patients than in controls. Furthermore, the number of PS+ MPs and BCs in patients with macroalbuminuria was significantly higher than in patients with normoalbuminuria. Similarly, we observed markedly elevated PS exposure on HUVECs cultured with serum from patients with macro-DKD versus serum from patients with micro-DKD or normo-DKD. In addition, circulating PS+ MPs cooperated with PS+ cells, contributing to markedly shortened coagulation time and dramatically increased FXa/thrombin generation and fibrin formation in each DKD group. Confocal microscopy images demonstrated co-localization of fibrin with PS on ECs. Moreover, blockade of exposed PS on MPs and cells with lactadherin inhibited PCA by approximately 80%. Additionally, both hyperglycemia (25 mmol/L) and hypoglycemia (2.5 mmol/L) induced increased PS exposure on cells compared to normoglycemia (5 mmol/L). Linear regression analysis showed that levels of PS+ BCs and MPs were positively correlated with uric acid and proteinuria, but negatively correlated with GFR.Conclusions: Our results suggested that PS+ MPs and blood cells play a prominent role in inducing the hypercoagulable and prothrombotic state in patients with DKD. The occurrence of abnormal glucose during the hypoglycemic therapy is associated with hypercoagulation in DKD. PS+ cells and MPs likely support thrombosis in renal vascular and result in renal dysfunction and disease progression. Our results demonstrate that PS provides binding sites for FXa and prothrombinase complexes and promotes thrombin formation. Thus, directly targeting FXa and prothrombinase complexes might decrease thrombotic and renal disease risk. The results from this study suggest that future research should focus on discovery and deployment of direct PS inhibitors.Disclosure of Interest: Jialan Shi has a patent for the use of lactadherin as a PS probe. DisclosuresNo relevant conflicts of interest to declare.

Arsenic induces autophagy-dependent apoptosis via Akt inactivation and AMPK activation signaling pathways leading to neuronal cell death

Inorganic arsenic (As3+), a well-known worldwide industrial and environmental pollutant, has been linked to neurodegenerative disorders (NDs). Autophagy plays an important role in controlling neuronal cell survival/death. However, limited information is available regarding the toxicological mechanism at the interplay between autophagy and As3+-induced neurotoxicity. The present study found that As3+ exposure induced a concomitant activation of apoptosis and autophagy in Neuro-2a cells, which was accompanied with the increase of phosphatidylserine exposure on outer membrane leaflets and apoptotic cell population, and the activation of caspase-3, -7, and PARP as well as the elevation of protein expressions of LC3-II, Atg-5, and Beclin-1, and the accumulation of autophagosome. Pretreatment of cells with autophagy inhibitor 3-MA, but not that of Z-VAD-FMK (a pan-caspase inhibitor), effectively prevented the As3+-induced autophagic and apoptotic responses, indicating that As3+-triggered autophagy was contributing to neuronal cell apoptosis. Furthermore, As3+ exposure evoked the dephosphorylation of Akt. Pretreatment with SC79, an Akt activator, could significantly attenuated As3+-induced Akt inactivation as well as autophagic and apoptotic events. Expectedly, inhibition of Akt signaling with LY294002 obviously enhanced As3+-triggered autophagy and apoptosis. Exposure to As3+ also dramatically increased the phosphorylation level of AMPKα. Pretreatment of AMPK inhibitor (Compound C) could markedly abrogate the As3+-induced phosphorylated AMPKα expression, and autophagy and apoptosis activation. Taken together, these results indicated that As3+ exerted its cytotoxicity in neuronal cells via the Akt inactivation/AMPK activation downstream-regulated autophagy-dependent apoptosis pathways, which ultimately lead to cell death. Our findings suggest that the regulation of Akt/AMPK signals may be a promising intervention to against As3+-induced neurotoxicity and NDs.

Solidagenone acts on promastigotes of L. amazonensis by inducing apoptosis-like processes on intracellular amastigotes by IL-12p70/ROS/NO pathway activation

BackgroundLeishmaniasis is a neglected tropical disease caused by protozoan parasites of the Leishmania genus. Currently, the treatment has limited effectiveness and high toxicity, is expensive, requires long-term treatment, induces significant side effects, and promotes drug resistance. Thus, new therapeutic strategies must be developed to find alternative compounds with high efficiency and low cost. Solidagenone (SOL), one of the main constituents of Solidago chilensis, has shown gastroprotective, anti-inflammatory and immunomodulatory effects. PurposeThis study assessed the in vitro effect of SOL on promastigotes and Leishmania amazonensis-infected macrophages, as well its microbicide and immunomodulatory mechanisms. MethodsSOL was isolated from the roots of S. chilensis, 98% purity, and identified by chromatographic methods, and the effect of SOL on leishmanicidal activity against promastigotes in vitro, SOL-induced cytotoxicity in THP-1, J774 cells, sheep erythrocytes, and L. amazonensis-infected J774 macrophages, and the mechanisms of death involved in this action were evaluated. ResultsIn silico predictions showed good drug-likeness potential for SOL with high oral bioavailability and intestinal absorption. SOL treatment (10–160 μM) inhibited promastigote proliferation 24, 48, and 72 h after treatment. After 24 h of treatment, SOL at the IC50 (34.5 μM) and 2 × the IC50 (69 μM) induced several morphological and ultrastructural changes in promastigotes, altered the cell cycle and cellular volume, increased phosphatidylserine exposure on the cell surface, induced the loss of plasma membrane integrity, increased the reactive oxygen species (ROS) level, induced loss of mitochondrial integrity (characterized by an apoptosis-like process), and increased the number of lipid droplets and autophagic vacuoles. Additionally, SOL induced low cytotoxicity in J774 murine macrophages (CC50 of 1587 μM), THP-1 human monocytes (CC50 of 1321 μM), and sheep erythrocytes. SOL treatment reduced the percentage of L. amazonensis-infected macrophages and the number of amastigotes per macrophage (IC50 9.5 μM), reduced TNF-α production and increased IL-12p70, ROS and nitric oxide (NO) levels. ConclusionSOL showed in vitro leishmanicidal effects against the promastigotes by apoptosis-like mechanism and amastigotes by reducing TNF-α and increasing IL-12p70, ROS, and NO levels, suggesting their potential as a candidate for use in further studies on the design of antileishmanial drugs.

New phenalenone analogues with improved activity against Leishmania species

• The five phenalenone derivatives are potential anti-leishmanial agents. • Phenalenone 8 is 9-times more selective than the reference drug miltefosine. • Derivatives decrease mitochondrial potential and increase phosphatidylserine exposure. • The five phenalenones induce apoptosis-like in Leishmania amazonensis . The in vitro activity against Leishmania spp. of five novel designed compounds, phenalenone derivatives, is described in this study. Previous works have shown that some phenalenones present leishmanicidal activity, some of which could induce programmed cell death events in L. amazonensis parasites. In this research, we focused on the determination of the programmed cell death evidence by detecting the characteristic features of the apoptosis-like process, such as phosphatidylserine exposure and mitochondrial membrane potential, among others. The results showed that the new derivatives have comparable or better activity and selectivity than the commonly prescribed anti-leishmanial drug. This result was obtained by inducing stronger mitochondrial depolarization or more intense phosphatidylserine exposure than miltefosine, highlighting compound 8 with moreover 9-times better selectivity index. In addition, the new five molecules activated the apoptosis-like process in the parasite. All the signals observed were indicative of the death process that the parasites were undergoing.