Phosphatidylserine Exposure Research Articles

Hemin promotes platelet activation and plasma membrane disintegration regulated by the subtilisin-like proprotein convertase furin.

Platelet activation plays a critical role in thrombosis and hemostasis. Several pathophysiological situations lead to hemolysis, resulting in the liberation of free ferric iron-containing hemin. Hemin has been shown to activate platelets and induce thrombo-inflammation. Classical antiplatelet therapy failed to prevent hemin-induced platelet activation. Thus, the aim of the present study was to characterize the mechanism of hemin-induced platelet death (ferroptosis). We evaluated the invitro effect of hemin on platelet activation, signaling, oxylipins, and plasma membrane destruction using light transmission aggregometry, exvivo thrombus formation, multiparametric flow cytometry, micro-UHPLC mass spectrometry for oxylipin profiling, and scanning ion conductance microscopy (SICM). We found that hemin induces platelet cell death indicated by increased ROS levels, phosphatidyl serine (PS) exposure, and loss of mitochondrial membrane potential (ΔΨm). Further, hemin causes lipid peroxidation and generation of distinct oxylipins, which strongly affects plasma membrane integrity leading to generation of platelet-derived microvesicles. Interestingly, hemin-dependent platelet death (ferroptosis) is specifically regulated by the subtilisin-like proprotein convertase furin. In summary, platelet undergo a non-apoptotic cell death mediated by furin. Inhibition of furin may offer a therapeutic strategy to control hemin-induced thrombosis and thrombo-inflammation at a site of hemolysis.

Blood circulation effect of fermented citrus bioconversion product (FCBP) in EA.hy926 endothelial cells and high-fat diet-fed mouse model.

The escalating global burden of cardiovascular diseases, largely driven by unhealthy lifestyle choices and dietary patterns, has intensified the search for effective and safe interventions. With current treatments often marred by significant side effects, the exploration of natural compounds such as flavonoids presents a compelling alternative. This study investigated the effects of fermented citrus bioconversion product (FCBP), a fermented citrus bioflavonoid, on various markers of cardiovascular health in the context of a high-fat diet. In vivo, a high-fat diet-induced mouse model was used to assess the effects of FCBP on body weight, serum nitric oxide (NO) levels, activated partial thromboplastin time (aPTT), phosphatidylserine (PS) exposure on red blood cells, and the expression of inflammatory markers Intercellular Adhesion Molecule (ICAM)-1 and Vascular Cell Adhesion Molecule (VCAM)-1 in the thoracic aorta. In vitro, EA.hy926 endothelial cells were used to evaluate the compound's effects on cell viability, NO production, endothelial nitric oxide synthase (eNOS) expression, and cell adhesion molecule (CAM) levels to further understand the mechanisms behind the in vivo findings. In vivo, FCBP supplementation led to a dose-dependent reduction in weight gain, a significant decrease in serum NO levels at 10 mg/kg, and reduced ICAM-1 and VCAM-1 expressions in the thoracic aorta, indicating anti-inflammatory properties. PS exposure on red blood cells was also reduced, suggesting decreased procoagulant activity, while aPTT remained unchanged. In vitro, FCBP was non-cytotoxic to endothelial cells, showed a trend toward increased NO production and eNOS expression, and reduced the expression of ICAM-1 and VCAM-1, supporting its potential anti-inflammatory effects. FCBP demonstrates potential as a bioactive compound for managing cardiovascular health by reducing inflammation, mitigating weight gain, and influencing blood circulation-related parameters under high-fat diet conditions. Further studies, including diverse models and human trials, are warranted to elucidate its mechanisms and compare its efficacy with established cardiovascular therapeutics.

Three new spirocyclic terpenoids from Euphorbia amygdaloides exhibit cytotoxicity against cancerous cell lines through early and late apoptosis

Bioassay-guided fractionation led to the isolation of three new spirocyclic terpenoid compounds from Euphorbia amygdaloides L., named Zagrosin I–III. Their structures were identified by 1D and 2D NMR (1H NMR, 13C NMR, DEPT 135, HMBC, and HSQC-TOCSY) and LC-MS-MS spectrometry. The cytotoxicity of the isolated spirocyclic terpenoids (Zagrosin I-III) was assessed against human breast cancer (MCF-7), human fibrosarcoma (HT1080), and normal human foreskin fibroblast cells with MTT assays (24, 48, and 72 h treatments). The FITC-Annexin V apoptosis flow cytometry assays and cell cycle analysis were performed for Zagrosin I–III.These isolated compounds were identified as: (9)-8a-((benzoyloxy)methyl)-2-methoxy-4,9-dimethyltetrahydro-4H,5H-2,4a-methanobenzo[d] [1,3] dioxine-4-carboxylate (Zagrosin I), ((9)-4-hydroxy-2-methoxy-4,9-dimethyltetrahydro-4H,8aH-2,4a-methanobenzo[d] [1,3] dioxin-8a-yl) methyl benzoate (Zagrosin II), and (9)-2-methoxy-4,9-dimethyl-8a-(phenoxy methyl) tetrahydro-4H,5H-2,4a-methanobenzo[d][1,3]dioxin-4-yl 4-methylpentanoate (Zagrosin III).The IC50 of Zagrosin I on 48-h-treated MCF-7 was calculated as 1.5 μg/mL. Zagrosin II and III exhibited cytotoxicity on 48-h-treated MCF-7 with IC50s of 14.04 and 12.50 μg/mL, respectively. The IC50 of Zagrosin I on human fibrosarcoma (HT1080) was 115.5 μg/mL, Zagrosin III, 16.81 μg/mL (48 h treatment), and Zagrosin II, 142.7 μg/mL (72 h treatment). Zagrosin I-III exhibited significant cytotoxicity against the MCF-7 cell line and human fibrosarcoma (HT1080), with the mechanism of early and late apoptosis affecting cells mostly in G0/G1 fallowed by S and G2 phases. MCF-7 had a higher rate of phosphatidyl serine exposure on the cell membrane than two other studied cells. The cytotoxicity on normal human foreskin fibroblasts was low. Zagrosin I-III can be considered an effective chemical backbone for anticancer drug development.Abbreviations: 2D NMR: Two-Dimensional Nuclear Magnetic Resonance Spectroscopy; API: Atmospheric Pressure Ionization; DEPT: Distortionless Enhancement by Polarization Transfer; ELISA: Enzyme-Linked Immunosorbent assay; ERK: Extracellular signal-regulated kinase; ESI: Electrospray ionization; FBS: Fetal Bovine Serum; FITC: Fluorescein isothiocyante; fr: fraction; HMBC: Heteronuclear Multiple Bond Correlation; HPLC: High-Performance Liquid Chromatography; HSQC: Heteronuclear Single Quantum Coherence; HT1080: Human fibrosarcoma cell line; IC50: Half-maximal inhibitory concentration; MCF-7: Human breast cancer cell line; MHz: Megahertz; MTT: 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide; NMR: Nuclear Magnetic Resonance; PBS: Phosphate Buffered Saline; PI: Propidium Iodide; ppm: Part Per Million; Rf: Retention Factor; TLC: Thin-layer chromatography; TOCSY: Total Correlation Spectroscopy; VLC: Vacuum Liquid Chromatography.

Phosphatidylserine-Positive Live Sperm as a Biomarker of Fertility Status: Evidence from Patients Undergoing Varicocele Treatment

Purpose: Phosphatidylserine exposed on live and motile sperm is necessary for sperm:egg fusion, and emerging data suggest it is a biomarker for fertilization competent sperm. Men with varicocele often display infertility, and one of the known causes is poor sperm quality. Here, we sought to determine whether the percentage of phosphatidylserine-positive live sperm (PS+ live sperm) could serve as a reliable indicator of male fertility. Materials and Methods: Semen samples from cryo-banked pregnancy-proven donors, from self-identified pregnancy-reported donors, and from men seeking infertility treatment for varicocele were analyzed for phosphatidylserine exposure on live sperm. Semen samples were also analyzed premicrosurgical and postmicrosurgical varicocele repair to determine whether varicocele repair led to a change in the percentage of PS+ live sperm. Results: Semen samples from “pregnancy-proven” donors (n = 31) had 82% ± 2% PS+ live sperm and samples from “self-identified pregnancy-reported” donors (n = 34) had 87% ± 1%. Men presenting with a varicocele (n = 80) had significantly lower PS+ live sperm with 68% ± 3%. PS+ live sperm correlated with reported pregnancies in patients with varicocele, whereas total motile sperm count did not. Varicocele repair (n = 10) led to a significant increase in PS+ live sperm, and 2 of the patients reported pregnancies after varicocele repair. Conclusions: These results suggest that the percentage of PS+ live sperm in an ejaculate correlates with male fertility status and could help in guiding treatment options for patients with varicoceles.

In vivo imaging of ferroptosis through nanodynamic changes in lipid membranes.

Ferroptosis emerged as a cell death modality for drug resistant cancer cells, but there are currently no available biomarkers for imaging ferroptosis based therapies. To address this gab, we evaluated the nanodynamic changes in lipid membranes occurring during cell death to explore potential targeting opportunities to image cell death. We nano-sized gaps at late stages of ferroptosis can serve as entry points for dyes that can bind to cellular structures. These changes were accompanied with cellular signaling components similar to platelet activation, with phosphatidyl serine emerging on the surface of the cells and therefore as a potential target for imaging of programed cell death, including ferroptosis. Taking advantage of these changes in cell membrane dynamics, we employed a novel tumor-seeking dye CJ215 that can label apoptotic cells as recently described by us. We show that CJ215 accumulates in ferroptotic cells both in vitro and in vivo by binding to phosphatidyl serine, a process that is prevented by inhibition of ferroptosis. Since phosphatidyl serine exposure also occurs during apoptosis, CJ215 can serve to image both apoptosis and ferroptosis based therapy.

Flow cytometry for RBC damage and complement activation

Abstract Introduction Red blood cells (RBCs) express anionic phospholipids such as phosphatidylserine (PS) in their inner cell membrane layer. As RBCs age, physiochemical changes in their cell membranes, particularly exposure of PS on the outer membrane layer, lead to phagocytosis and clearance from circulation. PS in RBCs induces activation of the complement system, increasing binding of C3 to RBCs. Thus, quantifying PS-expressing (PS+) RBCs and complement deposition on RBCs (C3+) is a potential measure of RBC injury and may improve transfusion quality when used for predicting post-transfusion RBC recovery. Our goal was to develop a flow cytometry assay that quantifies PS+ and C3+RBCs and to assess how this assay correlates with the direct antiglobulin test for C3 (C3-DAT). Methods Using the Cytoflex S flow cytometer, we measured PS+ and C3+ RBCs using anti-annexin-PE (PS+RBC) and biotin-labeled anti-C3 with streptravidin-PE-Cy5 (C3+RBC). RBCs from healthy donors (NL-RBCs) with negative direct antiglobulin tests for C3 (C3-DAT) (n = 4) were analyzed. To assess the sensitivity of the assay and correlation with C3-DATs, complement control cells (CCC; Immucor) were used as a positive control for both PS+RBCs and C3+RBCs and diluted to different concentrations by mixing CCCs with NL-RBCs. Aliquots of diluted RBCs were analyzed blinded for C3-DAT. Differences between means for PS+RBC and C3+RBC were compared using two-sample t-test, and one-way ANOVA and Tukey’s HSD test were used to determine differences in C3+RBCs among a C3-DAT range of reactivities (0+ to 3+) in diluted CCC samples. P values less than 0.05 were statistically significant. Results In healthy, DAT-negative individuals, the mean positivity for PS+RBCs was 0.28% and for C3+RBCs was 0.34%, indicating that healthy individuals have very low percentages of RBCs with PS and C3 on their cell membrane surface. In CCC diluted samples, the mean C3+RBC percentage was 1.4% in negative C3-DAT samples, 5.9% in weak+ C3-DAT samples, 28.1% in 1+ C3-DAT samples, 67.2% in 2+ C3-DAT samples, and 99.0% in 3+ C3-DAT samples. One-way ANOVA revealed a statistically significant difference in C3+RBCs amongst all C3-DAT reactivities in diluted CCC samples (P<0.001), and Tukey’s HSD test found a mean difference in C3+RBCs percentage of 26.7% between 0+ and 1+ samples (P<0.001), 39.2% between 1+ and 2+ samples (P<0.001), and 31.8% between 2+ and 3+ samples (P<0.01). Undiluted CCC samples had a mean PS+RBC percentage of 7.0% and was statistically different from the PS+RBC percentage in NL-RBCs (P<0.01). Conclusion We describe a feasible assay to measure both PS+RBCs and C3+RBCs using flow cytometry in which CCC can be used as a positive control for PS+RBCs. This assay could serve to evaluate the quality of transfused RBC units and provide insights into the role of phosphatidylserine expression and complement deposition for RBC clearance.

Suppressed ORAI1-STIM1-dependent Ca2+ entry by protein kinase C isoforms regulating platelet procoagulant activity

Agonist-induced rises in cytosolic Ca2+ control most platelet responses in thrombosis and hemostasis. In human platelets we earlier demonstrated that the ORAI1-STIM1 pathway is a major component of extracellular Ca2+ entry, in particular when induced via the ITAM-linked collagen receptor, glycoprotein VI (GPVI). In the present paper, using functionally defective platelets from patients with a loss-of-function mutation in ORAI1 or STIM1, we show that Ca2+ entry induced by the endoplasmic reticulum ATPase inhibitor, thapsigargin, fully relies on this pathway. We demonstrate that both the GPVI-induced and thapsigargin-induced Ca2+ entry is strongly suppressed by protein kinase C (PKC) activation, while leaving intracellular Ca2+ mobilization unchanged. Comparing effects of a PKC inhibitory panel pointed to redundant roles of beta and theta PKC isoforms in Ca2+-entry suppression. In contrast, tyrosine kinases positively regulated GPVI-induced Ca2+ entry and mobilization. Label-free and stable isotope phosphoproteome analysis of GPVI-stimulated platelets suggested a regulatory role of bridging integrator-2 (BIN2), known as important mediator of the ORAI1-STIM1 pathway in mouse platelets. Identified were 25-45 regulated phospho- sites in BIN2 and 16-18 in STIM1. Five of these were characterized as direct substrates of the expressed PKC isoforms alpha, beta delta and theta. Functional platelet testing indicated that the downregulation of Ca2+ entry by PKC resulted in suppressed phosphatidylserine exposure and plasmatic thrombin generation. Conclusively, our results indicate that in platelets multiple PKC isoforms constrain the store-regulated Ca2+ entry via ORAI1-BIN2-STIM1, and hence downregulate platelet-dependent coagulation.

Reduced platelet activation in triple-negative essential thrombocythemia compared with JAK2V617F-mutated essential thrombocythemia.

Triple-negative (TN) essential thrombocytopenia (ET) is characterized by the absence of driver mutations while retaining histological and phenotypic characteristics sufficient for an ET diagnosis. Our understanding of TN-ET and its platelet activation remains incomplete. We carried out a large-scale multi-center clinical analysis to analyze the clinical and molecular characteristics, thrombotic complications of TN-ET. We also related the above characteristics to platelet activation to further explore the thrombosis mechanism of TN-ET. A retrospective multicenter study was conducted on 138 TN-ET and 759 ET patients with driver mutations from 1 March 2012 to 1 December 2021. The clinical and molecular characteristics of the TN-ET were summarized. Additionally, platelet activation, apoptosis, and reactive oxygen species (ROS) levels were analyzed in 73 TN-ET patients from this cohort and compared to 41 age- and sex-matched healthy donors. Compared to patients with the JAK2V617F mutation, those with triple-negative mutation were younger (P < 0.001) and exhibited fewer thrombotic events before diagnosis (P < 0.001) and during follow-up (P = 0.039). Patients with triple-negative mutation also presented with significantly reduced CD62P expression in platelets (P = 0.031), slightly reduced calcium concentration in platelets (P = 0.063), increased mitochondrial membrane potential (P = 0.011), reduced phosphatidylserine exposure (P = 0.015), reduced levels of ROS (P = 0.043) and MitoSOX in platelets (P = 0.047). In comparison to JAK2V617F-mutated ET, TN-ET is associated with lower platelet ROS levels, which leads to reduced platelet activation and consequently a lower risk of thrombosis.

Circulating extracellular vesicles as novel biomarkers for pulmonary arterial hypertension in patients with systemic lupus erythematosus.

Pulmonary arterial hypertension (PAH) is a serious complication of systemic lupus erythematosus (SLE) with increased mortality. A prothrombotic state may contribute to pathogenesis of SLE-PAH. Extracellular vesicles (EVs) are known to be associated with thrombosis. Here, we investigated circulating EVs and their associations with SLE-PAH. Eighteen SLE-PAH patients, 36 SLE-non-PAH patients, and 36 healthy controls (HCs) were enrolled. Flow cytometry was used to analyze circulating EVs from leukocytes (LEVs), red blood cells (REVs), platelets (PEVs), endothelial cells (EEVs), and Annexin V+ EVs with membrane phosphatidylserine (PS) exposure. Plasma levels of all EV subgroups were elevated in SLE patients with or without PAH compared to HCs. Furthermore, plasma Annexin V+ EVs, LEVs, PEVs, REVs, EEVs, and Annexin V+ REVs were significantly elevated in SLE-PAH patients compared to SLE-non-PAH patients. Additionally, PAH patients with moderate/high SLE showed a significant increase in LEVs, PEVs, REVs, Annexin V+ EVs, and Annexin V+ REVs compared to SLE-non-PAH patients. However, PAH patients with inactive/mild SLE only exhibited elevations in Annexin V+ EVs, REVs, and Annexin V+ REVs. In the SLE-PAH patients, EEVs were positively correlated with pulmonary arterial systolic pressure, while PEVs and EEVs were positively correlated with right ventricular diameter. Moreover, the receiver operating characteristic curve indicated that Annexin V+ EVs, LEVs, PEVs, REVs, EEVs and Annexin V+ REVs could predict the presence of PAH in SLE patients. Importantly, multivariate logistic regression analysis showed that circulating levels of LEVs or REVs, anti-nRNP antibody, and serositis were independent risk factors for PAH in SLE patients. Findings reveal that specific subgroups of circulating EVs contribute to the hypercoagulation state and the severity of SLE-PAH. Higher plasma levels of LEVs or REVs may serve as biomarkers for SLE-PAH.

NADPH oxidase 1/4 dual inhibitor setanaxib suppresses platelet activation and thrombus formation

AimsThe production of reactive oxygen species (ROS) by NADPH oxidase (NOX) is able to induce platelet activation, making NOX a promising target for antiplatelet therapy. In this study, we examined the effects of setanaxib, a dual NOX1/4 inhibitor, on human platelet function and ROS-related signaling pathways. Materials and methodsIn collagen-stimulated human platelets, aggregometry, assessment of ROS and Ca2+, immunoblotting, ELISA, flow cytometry, platelet adhesion assay, and assessment of mouse arterial thrombosis were performed in this study. Key findingsSetanaxib inhibited both intracellular and extracellular ROS production in collagen-activated platelets. Additionally, setanaxib significantly inhibited collagen-induced platelet aggregation, P-selectin exposure from α-granule release, and ATP release from dense granules. Setanaxib blocked the specific tyrosine phosphorylation-mediated activation of Syk, LAT, Vav1, and Btk within collagen receptor signaling pathways, leading to reduced activation of PLCγ2, PKC, and Ca2+ mobilization. Setanaxib also inhibited collagen-induced activation of integrin αIIbβ3, which is linked to increased cGMP levels and VASP phosphorylation. Furthermore, setanaxib suppressed collagen-induced p38 MAPK activation, resulting in decreased phosphorylation of cytosolic PLA2 and reduced TXA2 generation. Setanaxib also inhibited ERK5 activation, affecting the exposure of procoagulant phosphatidylserine. Setanaxib reduced thrombus formation under shear conditions by preventing platelet adhesion to collagen. Finally, in vivo administration of setanaxib in animal models led to the inhibition of arterial thrombosis. SignificanceThis study is the first to show that setanaxib suppresses ROS generation, platelet activation, and collagen-induced thrombus formation, suggesting its potential use in treating thrombotic or cardiovascular diseases.

The effects of complement-independent, autoantibody-induced apoptosis of platelets in immune thrombocytopenia (ITP).

Autoantibodies that cause platelet apoptosis may play a role in the development of immune thrombocytopenia (ITP), specifically antibodies that target GPIIbIIIa and GPIbα. Our research aims to compare the impact of the antigen specificity of antiplatelet antibodies on normal platelets under conditions that do not rely on complement. Using a modified monoclonal antibody-specific immobilization of platelet antigen (MAIPA) assay, we detected the levels of autoantibodies against specific platelet membrane glycoproteins (GPIIb/IIIa, GPIb/IX) in the plasma of 36 patients diagnosed with chronic ITP. IgG was isolated and purified using a protein A agarose affinity chromatography column, and their concentrations were measured using spectrophotometry. We obtained normal platelets and treated them with the purified IgG anti-GPIIb/IIIa and/or anti-GPIb/IX antibodies, as well as an IgG-free buffer and healthy control IgG. Flow cytometry was used to analyze markers of apoptosis, including phosphatidylserine (PS) exposure, mitochondrial inner membrane potential (ΔΨm), and platelet particle formation. Our results indicate that ITP patients with GPIb/IX-specific autoantibodies can induce platelet apoptosis and platelet particle formation through complement-independent pathways, which are not associated with platelet activation, while GPIIb/IIIa-specific autoantibodies did not have this effect. This suggests that specific autoantibodies may serve as a valuable predictive tool to identify patients who could potentially benefit from complement-inhibiting therapy in the future.

Exposed Phosphatidylserine as a Biomarker for Clear Identification of Breast Cancer Brain Metastases in Mouse Models.

Brain metastasis is the most common intracranial malignancy in adults. The prognosis is extremely poor, partly because most patients have more than one brain lesion, and the currently available therapies are nonspecific or inaccessible to those occult metastases due to an impermeable blood-tumor barrier (BTB). Phosphatidylserine (PS) is externalized on the surface of viable endothelial cells (ECs) in tumor blood vessels. In this study, we have applied a PS-targeting antibody to assess brain metastases in mouse models. Fluorescence microscopic imaging revealed that extensive PS exposure was found exclusively on vascular ECs of brain metastases. The highly sensitive and specific binding of the PS antibody enables individual metastases, even micrometastases containing an intact BTB, to be clearly delineated. Furthermore, the conjugation of the PS antibody with a fluorescence dye, IRDye 800CW, or a radioisotope, 125I, allowed the clear visualization of individual brain metastases by optical imaging and autoradiography, respectively. In conclusion, we demonstrated a novel strategy for targeting brain metastases based on our finding that abundant PS exposure occurs on blood vessels of brain metastases but not on normal brain, which may be useful for the development of imaging and targeted therapeutics for brain metastases.

A novel role for thioredoxin-related transmembrane protein TMX4 in platelet activation and thrombus formation

BackgroundThe functions of critical platelet proteins are controlled by thiol-disulfide exchanges, which are mediated by the protein disulfide isomerase (PDI) family. It has been shown that some PDI family members are important in platelet activation and thrombosis with distinct functions. TMX4, a membrane-type PDI family member, is expressed in platelets, but whether it has a role in platelet activation remains unknown. ObjectivesTo determine the role of TMX4 in platelet activation and thrombosis. MethodsThe phenotypes of TMX4-deficient mice were evaluated in tail bleeding time assay and laser-induced and FeCl3-induced arterial injury models. The functions of TMX4 in platelets were assessed in vitro using TMX4-null platelets, recombinant TMX4 protein, and anti-TMX4 antibody. ResultsCompared with the control mice, Tie2-Cre/TMX4fl/fl mice deficient of hematopoietic and endothelial TMX4 exhibited prolonged tail bleeding times and reduced platelet thrombus formation. Pf4-Cre/TMX4fl/fl mice deficient of platelet TMX4 also had prolonged tail bleeding times and decreased thrombus formation, which was rescued by injection of recombinant TMX4 protein. Consistently, TMX4 deficiency inhibited platelet aggregation, integrin αIIbβ3 activation, P-selectin expression, phosphatidylserine exposure, and thrombin generation, without affecting tyrosine phosphorylation of intracellular signaling molecules Syk, LAT, PLCγ2 and calcium mobilization. Recombinant TMX4 protein enhanced platelet aggregation and reduced integrin αIIbβ3 disulfide bonds, and TMX4 deficiency decreased free thiols of integrin αIIbβ3, consistent with a potent reductase activity of TMX4. In contrast, an inactive TMX4 protein and a specific anti-TMX4 antibody inhibited platelet aggregation. ConclusionTMX4 is a novel PDI family member that enhances platelet activation and thrombosis.

Effect of a thiohydantoin salt derived from l-Arginine on Leishmania amazonensis and infected cells: Insights from biological effects to molecular docking interactions

Leishmaniasis is a neglected tropical disease caused by parasites of the genus Leishmania and is responsible for more than 1 million new cases and 70,000 deaths annually worldwide. Treatment has high costs, toxicity, complex and long administration time, several adverse effects, and drug-resistant strains, therefore new therapies are urgently needed. Synthetic compounds have been highlighted in the medicinal chemistry field as a strong option for drug development against different diseases. Organic salts (OS) have multiple biological activities, including activity against protozoa such as Leishmania spp. This study aimed to investigate the in vitro leishmanicidal activity and death mechanisms of a thiohydantoin salt derived from l-arginine (ThS) against Leishmania amazonensis. We observed that ThS treatment inhibited promastigote proliferation, increased ROS production, phosphatidylserine exposure and plasma membrane permeabilization, loss of mitochondrial membrane potential, lipid body accumulation, autophagic vacuole formation, cell cycle alteration, and morphological and ultrastructural changes, showing parasites death. Additionally, ThS presents low cytotoxicity in murine macrophages (J774A.1), human monocytes (THP-1), and sheep erythrocytes. ThS in vitro cell treatment reduced the percentage of infected macrophages and the number of amastigotes per macrophage by increasing ROS production and reducing TNF-α levels. These results highlight the potential of ThS among thiohydantoins, mainly related to the arginine portion, as a leishmanicidal drug for future drug strategies for leishmaniasis treatment. Notably, in silico investigation of key targets from L. amazonensis, revealed that a ThS compound from the l-arginine amino acid strongly interacts with arginase (ARG) and TNF-α converting enzyme (TACE), suggesting its potential as a Leishmania inhibitor.

Role of Piezo1 in Terminal Density Reversal of Red Blood Cells.

Density reversal of senescent red blood cells has been known for a long time, yet the identity of the candidate ion transporter(s) causing the senescent cells to swell is still elusive. While performing fractionation of RBCs from healthy individuals in Percoll density gradient and characterization of the separated fractions, we identified a subpopulation of cells in low-density fraction (1.02% ± 0.47) showing signs of senescence such as loss of membrane surface area associated with a reduction in band 3 protein abundance, and Phosphatidylserine (PS) exposure to the outer membrane. In addition, we found that these cells are overloaded with Na+ and Ca2+. Using a combination of blockers and activators of ion pumps and channels, we revealed reduced activity of Plasma membrane Ca2+ ATPase and an increase in Ca2+ and Na+ leaks through ion channels in senescent-like cells. Our data revealed that Ca2+ overload in these cells is a result of reduced PMCA activity and facilitated Ca2+ uptake via a hyperactive Piezo1 channel. However, we could not exclude the contribution of other Ca2+-permeable ion channels in this scenario. In addition, we found, as a universal mechanism, that an increase in intracellular Ca2+ reduced the initially high selectivity of Piezo1 channel for Ca2+ and allowed higher Na+ uptake, Na+ accumulation, and swelling.

Carbamylation Is Instrumental in End-Stage Kidney Disease Coagulopathies: The Impact on von Willebrand Factor and Platelet Functionality.

Chronic kidney disease (CKD) is a progressive, irreversible, and incurable condition characterized by high morbidity and mortality, affecting approximately one-tenth of the global population. Rise of urea-derived cyanate levels in CKD patients, severalfold higher in comparison to those found in healthy individuals, leads to an increased rate of carbamylation of lysine residues of proteins and peptides. This posttranslational modification plays an important role in the progression of kidney failure but also in the onset of CKD-related complications, including previously reported coagulopathies. In this study, we have explored the impact of carbamylation on the functionality of von Willebrand factor (vWF), a pivotal player in hemostasis, and its implications for platelet adhesion. We have explored carbamylated vWF's interactions with its partner proteins via ELISA. Mass spectrometry was employed to identify modified lysine residues. Blood platelets isolated from healthy donors were carbamylated, and their activation, binding to endothelium and thromboxane release were evaluated using flow cytometry, adhesion assays and ELISA, respectively. Using mass spectrometry we detected the vWF's lysine residue smost susceptible to carbamylation. This modification has in turn affected vWF's interactions with its key binding partners: decreased binding to collagen types I/III but increased the affinity to factor FVIII, while its binding to fibrinogen remained unchanged. Carbamylation of vWF impeded vWF-blood platelet binding, but carbamylation of platelets led to their increased thrombin-dependent activation as observed by enhanced phosphatidylserine exposure, improved their binding to vascular endothelium, at the same time decreasing the production of the prothrombotic mediator, thromboxane A2. Our findings highlight the multifaceted impact of carbamylation on vWF and platelets, disturbing the delicate balance of coagulation cascade. These alterations could contribute to the complex hemostatic imbalance in ESKD, underscoring the need for further research to fully understand these mechanisms and their clinical implications.

Biomedical application of TiO2NPs can cause arterial thrombotic risks through triggering procoagulant activity, activation and aggregation of platelets.

Titanium dioxide nanoparticles (TiO2NPs) are widely used in medical application. However, the relevant health risk has not been completely assessed, the potential of inducing arterial thrombosis (AT) in particular. Alterations in platelet function and susceptibility to arterial thrombosis induced by TiO2NPs were examined using peripheral blood samples from healthy adult males and an in vivo mouse model, respectively. Here, using human platelets (hPLTs) freshly isolated from health volunteers, we demonstrated TiO2NP treatment triggered the procoagulant activity of hPLTs through phosphatidylserine exposure and microvesicles generation. In addition, TiO2NP treatment increased the levels of glycoprotein IIb/IIIa and P-selectin leading to aggregation and activation of hPLTs, which were exacerbated by providing physiology-mimicking conditions, including introduction of thrombin, collagen, and high shear stress. Interestingly, intracellular calcium levels in hPLTs were increased upon TiO2NP treatment, which were crucial in TiO2NP-induced hPLT procoagulant activity, activation and aggregation. Moreover, using mice in vivo models, we further confirmed that TiO2NP treatment a reduction in mouse platelet (mPLT) counts, disrupted blood flow, and exacerbated carotid arterial thrombosis with enhanced deposition of mPLT. Together, our study provides evidence for an ignored health risk caused by TiO2NPs, specifically TiO2NP treatment augments procoagulant activity, activation and aggregation of PLTs via calcium-dependent mechanism and thus increases the risk of AT.

Immunization of recombinant NS3 protein (protease region) of dengue virus induces high levels of CTLA-4 and apoptosis in splenocytes of BALB/c mice.

DENV infection outcomes depend on the host's variable expression of immune receptors and mediators, leading to either resolution or exacerbation. While the NS3 protein is known to induce robust immune responses, the specific impact of its protease region epitopes remains unclear. This study investigated the effect of recombinant NS3 protease region proteins from all four DENV serotypes on splenocyte activation in BALB/c mice (n = 5/group). Mice were immunized with each protein, and their splenocytes were subsequently stimulated with homologous antigens. We measured the expression of costimulatory molecules (CD28, CD80, CD86, CD152) by flow cytometry, along with IL-2 production, CD25 expression, and examined the antigen-specific activation of CD4 + and CD8 + T cells. Additionally, the expression of IL-1, IL-10, and TGF-β1 in splenocytes from immunized animals was assessed. Apoptosis was evaluated using Annexin V/PI staining and DNA fragmentation analysis. Stimulation of splenocytes from immunized mice triggered apoptosis (phosphatidylserine exposure and caspase 3/7 activation) and increased costimulatory molecule expression, particularly CD152. Low IL-2 production and low CD25 expression, as well as sustained expression of the IL-10 gene. These results suggest that these molecules might be involved in mechanisms by which the NS3 protein contributes to viral persistence and disease pathogenesis.

RNF135 Promotes Human Osteosarcoma Cell Growth and Inhibits Apoptosis by Upregulating the PI3K/AKT Pathway.

Ring finger protein 135 (RNF135) is an E3 ubiquitin ligase that has been implicated in the tumorigenesis of multiple human malignancies. However, whether RNF135 plays a role in the development of human osteosarcoma (OS) remains unknown. RNF135 expression in 20 human OS and 20 human osteochondroma specimens were evaluated by means of immunohistochemistry staining. The effects of shRNA-mediated RNF135 knockdown on human OS cell growth and apoptosis were evaluated through a panel of in vitro studies on cell proliferation, colony formation, exposure of phosphatidylserine on the cell surface, and caspase 3/7 activation. The protein levels of PI3K, AKT, and p-AKT were determined by western blot analysis. We detected significantly higher RNF135 levels in human OS tissues than human osteochondroma tissues. In in vitro studies, shRNA-mediated RNF135 knockdown in human OS cells inhibited proliferation and induced apoptosis. In addition, RNF135 knockdown reduced PI3K and p-AKT protein levels and activated caspase 3 and 7. These results supported that RNF135 contributes to human OS development through PI3K/AKT-dependent mechanisms. Targeting RNF135 may provide a new therapeutic approach for treating this human malignancy.

Neutrophil extracellular traps induce intrahepatic thrombotic tendency and liver damage in cholestatic liver disease.

Cholestatic liver diseases induce local and systemic hypercoagulation, with neutrophil extracellular traps (NETs) serving as major drivers. These NETs have been linked to decreased liver function in patients with obstructive jaundice. However, the impact of NETs on liver hypercoagulation in cholestatic liver disease remains unknown. We utilized bile duct ligation to create experimental mice and analyzed NETs formation in the liver. Fibrin deposition, tissue factor expression, and inflammation in the liver were visualized through western blot and immunohistochemical techniques. LSECs were incubated with isolated NETs, and we detected endothelial procoagulant activity using coagulation protein production assays and measuring endothelial permeability. In both in vivo and in vitro settings, DNase I was applied to clarify the effect of NETs on intrahepatic hypercoagulability, hepatotoxicity, LSEC, and macrophage activation or injury. Bile duct ligation mice exhibited significantly increased levels of NETs in liver tissue, accompanied by neutrophil infiltration, tissue necrosis, fibrin deposition, and thrombophilia compared to sham mice. Notably, NETs resulted in phosphatidylserine and tissue factor exposure on LSEC, enhancing coagulation Factor Xa and thrombin production. The enhanced procoagulant activity could be reversed by degrading NETs with DNase I. Additionally, NETs-induced permeability changes in LSECs, characterized by increased VE-cadherin expression and F-actin retraction, which could be rescued by DNase I. Meanwhile, NET formation is associated with KC activation and the formation of inflammatory factors. NETs promote intrahepatic activation of coagulation and inflammation, leading to liver tissue injury. Strategies targeting NET formation may offer a potential therapeutic approach for treating cholestatic liver disease.