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Journal of Thrombosis and Haemostasis :... Dec 2023Our recent studies showed that activated factor (F) VII (FVIIa) releases extracellular vesicles (EVs) from the endothelium. FVIIa-released EVs were found to be enriched...
BACKGROUND
Our recent studies showed that activated factor (F) VII (FVIIa) releases extracellular vesicles (EVs) from the endothelium. FVIIa-released EVs were found to be enriched with phosphatidylserine (PS) and contribute to the hemostatic effect of FVIIa in thrombocytopenia and hemophilia.
OBJECTIVE
To investigate mechanisms by which FVIIa induces EV biogenesis and enriches EVs with PS.
METHODS
FVIIa activation of acid sphingomyelinase (aSMase) was evaluated by its translocation to the cell surface. The role of aSMase in the biogenesis of FVIIa-induced EVs and their enrichment with PS was investigated using specific siRNAs and inhibitors of aSMase and its downstream metabolites. Wild-type and aSMase mice were injected with a control vehicle or FVIIa. EVs released into circulation were quantified by nanoparticle tracking analysis. EVs hemostatic potential was assessed in a murine thrombocytopenia model.
RESULTS
FVIIa activation of aSMase is responsible for both the externalization of PS and the release of EVs in endothelial cells. FVIIa-induced aSMase activation led to ceramide generation and de novo expression of transmembrane protein 16F. Inhibitors of ceramidases, sphingosine kinase, or sphingosine-1-phosphate receptor modulator blocked FVIIa-induced expression of transmembrane protein 16F and PS externalization without interfering with FVIIa release of EVs. In vivo, FVIIa release of EVs was markedly impaired in aSMase mice compared with wild-type mice. Administration of a low dose of FVIIa, sufficient to induce EVs release, corrected bleeding associated with thrombocytopenia in wild-type mice but not in aSMase mice.
CONCLUSION
Our study identifies a novel mechanism by which FVIIa induces PS externalization and releases PS-enriched EVs.
Topics: Animals; Mice; Endothelial Cells; Extracellular Vesicles; Factor VIIa; Hemostatics; Phosphatidylserines; Sphingomyelin Phosphodiesterase; Thrombocytopenia
PubMed: 37875382
DOI: 10.1016/j.jtha.2023.08.025 -
Trends in Cell Biology Nov 2015Apoptosis and the clearance of apoptotic cells are essential processes in animal development and homeostasis. For apoptotic cells to be cleared, they must display an... (Review)
Review
Apoptosis and the clearance of apoptotic cells are essential processes in animal development and homeostasis. For apoptotic cells to be cleared, they must display an 'eat me' signal, most likely phosphatidylserine (PtdSer) exposure, which prompts phagocytes to engulf the cells. PtdSer, which is recognized by several different systems, is normally confined to the cytoplasmic leaflet of the plasma membrane by a 'flippase'; apoptosis activates a 'scramblase' that quickly exposes PtdSer on the cell surface. The molecules that flip and scramble phospholipids at the plasma membrane have recently been identified. Here we discuss recent findings regarding the molecular mechanisms of apoptotic PtdSer exposure and the clearance of apoptotic cells.
Topics: Animals; Apoptosis; Humans; Phagocytosis; Phosphatidylserines; Signal Transduction
PubMed: 26437594
DOI: 10.1016/j.tcb.2015.08.003 -
Transfusion Aug 2014
Topics: Blood Platelets; Blood Preservation; Cell-Derived Microparticles; Cryopreservation; Hemostasis; Humans; Membrane Lipids; Phosphatidylserines
PubMed: 25130330
DOI: 10.1111/trf.12758 -
Science Advances Apr 2023Cells maintain a constant dialog between the extracellular matrix and their plasma membrane to fine tune signal transduction processes. We found that the receptor kinase...
Cells maintain a constant dialog between the extracellular matrix and their plasma membrane to fine tune signal transduction processes. We found that the receptor kinase FERONIA (FER), which is a proposed cell wall sensor, modulates phosphatidylserine plasma membrane accumulation and nano-organization, a key regulator of Rho GTPase signaling in Arabidopsis. We demonstrate that FER is required for both Rho-of-Plant 6 (ROP6) nano-partitioning at the membrane and downstream production of reactive oxygen species upon hyperosmotic stimulus. Genetic and pharmacological rescue experiments indicate that phosphatidylserine is required for a subset of, but not all, FER functions. Furthermore, application of FER ligand shows that its signaling controls both phosphatidylserine membrane localization and nanodomains formation, which, in turn, tunes ROP6 signaling. Together, we propose that a cell wall-sensing pathway controls via the regulation of membrane phospholipid content, the nano-organization of the plasma membrane, which is an essential cell acclimation to environmental perturbations.
Topics: Arabidopsis Proteins; Phosphatidylserines; Signal Transduction; Arabidopsis; Phosphotransferases; Cell Membrane; Plants
PubMed: 37027473
DOI: 10.1126/sciadv.add4791 -
Cell Communication and Signaling : CCS Jan 2020Phosphatidylserine (PS) is normally located in the inner leaflet of the membrane bilayer of healthy cells, however it is expressed at high levels on the surface of... (Review)
Review
Phosphatidylserine (PS) is normally located in the inner leaflet of the membrane bilayer of healthy cells, however it is expressed at high levels on the surface of cancer cells. This has allowed for the development of selective therapeutic agents against cancer cells (without affecting healthy cells). SapC-DOPS is a PS-targeting nanovesicle which effectively targets and kills several cancer types including pancreatic, lung, brain, and pediatric tumors. Our studies have demonstrated that SapC-DOPS selectively induces apoptotic cell death in malignant and metastatic cells, whereas untransformed cells remain unaffected due to low surface PS expression. Furthermore, SapC-DOPS can be used in combination with standard therapies such as irradiation and chemotherapeutic drugs to significantly enhance the antitumor efficacy of these treatments. While the PS-targeting nanovesicles are a promising selective therapeutic option for the treatment of cancers, more preclinical studies are needed to fully understand the mechanisms leading to non-apoptotic PS expression on the surface of viable cancer cells and to determine the effectiveness of SapC-DOPS in advanced metastatic disease. In addition, the completion of clinical studies will determine therapeutic effects and drug safety in patients. A phase I clinical trial using SapC-DOPS has been completed on patients with solid tumors and has demonstrated compelling patient outcomes with a strong safety profile. Results from this study are informing future studies with SapC-DOPS. Abstract video.
Topics: Animals; Clinical Trials as Topic; Humans; Nanoparticles; Neoplasms; Phosphatidylserines; Saposins
PubMed: 31918715
DOI: 10.1186/s12964-019-0476-6 -
International Urology and Nephrology Nov 2022Anemia in patients with chronic kidney disease (CKD) is the result of reduced erythropoietin, disturbed erythropoiesis and decreased lifespan of circulating...
OBJECTIVE
Anemia in patients with chronic kidney disease (CKD) is the result of reduced erythropoietin, disturbed erythropoiesis and decreased lifespan of circulating erythrocytes. Excessive eryptosis or premature suicidal erythrocyte death is characterized by cell shrinkage and phosphatidylserine externalization. This study aimed to explore accelerated eryptosis and accompanying biochemical alterations in CKD patients.
PATIENTS AND METHODS
A total of 106 CKD patients (59 predialysis [PreD] patients, 26 haemodialysis [HD] patients and 21 peritoneal dialysis [PD] patients) and a control group composed of 29 healthy volunteers were included in this study. Data on superoxide dismutase (SOD) activity (U/mL), annexin-V binding (mean fluorescent intensity, MFI) and intracellular calcium ([Ca]i; MFI) as well as the hematologic and biochemical parameters were recorded.
RESULTS
The [Ca]i levels were 3.05 ± 1.66 MFI, 2.24 ± 0.99 MFI, 2.38 ± 0.87 MFI and 1.71 ± 0.46 MFI in the PreD, HD, PD and control groups, respectively. Other than significantly higher [Ca]i levels in the PreD group than in the control group (p < 0.001), no significant difference was noted between study groups in terms of [Ca]i. Annexin-V binding was 1.05 ± 0.99 MFI in PreD group, 1.15 ± 0.56 MFI in HD group, 1.06 ± 0.87 MFI in PD group, and 0.88 ± 0.86 MFI in controls. Annexin-V binding was significantly higher in PreD, HD and PD groups compared with the control group (p < 0.001 for each). SOD activity was 0.07 ± 0.07 in the PreD group, 0.13 ± 0.08 in the HD group, 0.14 ± 0.07 in the PD group, and 0.03 ± 0.01 in the control group. SOD activity in both HD and PD groups were significantly higher than control and PreD groups (p < 0.001 for each). Lower albumin, higher ferritin, and higher parathormon levels were found to be correlated with eryptosis biomarkers. Patients treated vs. non-treated with calcium channel blockers had significantly lower annexin-V binding levels (p = 0.013). Patients treated vs. non-treated with erythropoietin (EPO) had elevated annexin-V binding level (p < 0.001) and lower [Ca]i (p = 0.014).
CONCLUSION
In conclusion, our findings revealed the presence accelerated eryptosis, as a potential contributing factor to development of anemia, in patients with CKD stages 3-5D. Inflamation and parathormon can also accelerate eryptosis. Favorable effect of CCB and EPO on eryptosis needs to be confirmed in larger scale studies.
Topics: Albumins; Anemia; Annexin A5; Calcium; Calcium Channel Blockers; Eryptosis; Erythropoietin; Ferritins; Humans; Phosphatidylserines; Renal Insufficiency, Chronic; Superoxide Dismutase
PubMed: 35469112
DOI: 10.1007/s11255-022-03207-3 -
Cell Communication and Signaling : CCS Oct 2019The exposure of phosphatidylserine (PS) on the outer plasma membrane has long been considered a unique feature of apoptotic cells. Together with other "eat me" signals,... (Review)
Review
The exposure of phosphatidylserine (PS) on the outer plasma membrane has long been considered a unique feature of apoptotic cells. Together with other "eat me" signals, it enables the recognition and phagocytosis of dying cells (efferocytosis), helping to explain the immunologically-silent nature of apoptosis. Recently, however, PS exposure has also been reported in non-apoptotic forms of regulated inflammatory cell death, such as necroptosis, challenging previous dogma. In this review, we outline the evidence for PS exposure in non-apoptotic cells and extracellular vesicles (EVs), and discuss possible mechanisms based on our knowledge of apoptotic-PS exposure. In addition, we examine the outcomes of non-apoptotic PS exposure, including the reversibility of cell death, efferocytosis, and consequent inflammation. By examining PS biology, we challenge the established approach of distinguishing apoptosis from other cell death pathways by AnnexinV staining of PS externalization. Finally, we re-evaluate how PS exposure is thought to define apoptosis as an immunologically silent process distinct from other non-apoptotic and inflammatory cell death pathways. Ultimately, we suggest that a complete understanding of how regulated cell death processes affect the immune system is far from being fully elucidated.
Topics: Animals; Apoptosis; Cell Death; Humans; Inflammation; Phagocytosis; Phosphatidylserines
PubMed: 31665027
DOI: 10.1186/s12964-019-0437-0 -
Cell Communication and Signaling : CCS May 2023Extracellular vesicles (EVs) are important mediators of intercellular communication in the tumour microenvironment. Many studies suggest that cancer cells release higher...
Extracellular vesicles (EVs) are important mediators of intercellular communication in the tumour microenvironment. Many studies suggest that cancer cells release higher amounts of EVs exposing phosphatidylserine (PS) at the surface. There are lots of interconnections between EVs biogenesis and autophagy machinery. Modulation of autophagy can probably affect not only the quantity of EVs but also their content, which can deeply influence the resulting pro-tumourigenic or anticancer effect of autophagy modulators. In this study, we found that autophagy modulators autophinib, CPD18, EACC, bafilomycin A1 (BAFA1), 3-hydroxychloroquine (HCQ), rapamycin, NVP-BEZ235, Torin1, and starvation significantly alter the composition of the protein content of phosphatidylserine-positive EVs (PS-EVs) produced by cancer cells. The greatest impact had HCQ, BAFA1, CPD18, and starvation. The most abundant proteins in PS-EVs were proteins typical for extracellular exosomes, cytosol, cytoplasm, and cell surface involved in cell adhesion and angiogenesis. PS-EVs protein content involved mitochondrial proteins and signalling molecules such as SQSTM1 and TGFβ1 pro-protein. Interestingly, PS-EVs contained no commonly determined cytokines, such as IL-6, IL-8, GRO-α, MCP-1, RANTES, and GM-CSF, which indicates that secretion of these cytokines is not predominantly mediated through PS-EVs. Nevertheless, the altered protein content of PS-EVs can still participate in the modulation of the fibroblast metabolism and phenotype as p21 was accumulated in fibroblasts influenced by EVs derived from CPD18-treated FaDu cells. The altered protein content of PS-EVs (data are available via ProteomeXchange with identifier PXD037164) also provides information about the cellular compartments and processes that are affected by the applied autophagy modulators. Video Abstract.
Topics: Phosphatidylserines; Extracellular Vesicles; Autophagy; Cytokines; Exosomes
PubMed: 37226246
DOI: 10.1186/s12964-023-01126-z -
Methods in Molecular Biology (Clifton,... 2022This chapter describes a simple, nondestructive, annexin V apoptosis detection method that can be employed in real time over a 48-h test exposure. The real-time...
This chapter describes a simple, nondestructive, annexin V apoptosis detection method that can be employed in real time over a 48-h test exposure. The real-time functionality allows for temporal resolution of apoptotic and cell death responses during the test exposure and obviates the need for onerous sample preparation and time course protocols associated with other annexin V methods. Further, this technique is eminently accessible to a wide range of laboratories because it does not require flow cytometry or other cytometric methods. It was developed for use with a variety of microplate well densities and with standard multimodal plate readers. The central feature of this assay is that it continuously reports the residency status of phosphatidylserine (PS) on the exofacial surface of a cell as it is translocated from the inner membrane leaflet during the apoptotic process. This homogenous, no-wash assay is made possible by two optimized and distinct annexin V fusion proteins which contain complementing NanoBiT™ luciferase enzyme subunits, a time-released luciferase substrate, and a fluorescent membrane integrity reagent. During an apoptotic event, the luminescent signal arising from an assay well is proportional to the number of cells with PS exposure, and fluorescence intensity correlates with the degree of cell death (secondary necrosis). Conversely, untreated cells contribute negligible luminescent or fluorescent signals throughout the time course. The data collected from these assay measures provide for both standard potency determinations and kinetic characterization of dose- and agent-dependent apoptotic responses, from early through late phases.
Topics: Annexin A5; Apoptosis; Coloring Agents; Flow Cytometry; Humans; Necrosis; Phosphatidylserines
PubMed: 36087254
DOI: 10.1007/978-1-0716-2553-8_1 -
Life Science Alliance Aug 2022The evolutionarily conserved extended synaptotagmin (E-Syt) proteins are calcium-activated lipid transfer proteins that function at contacts between the ER and plasma...
The evolutionarily conserved extended synaptotagmin (E-Syt) proteins are calcium-activated lipid transfer proteins that function at contacts between the ER and plasma membrane (ER-PM contacts). However, roles of the E-Syt family members in PM lipid organisation remain incomplete. Among the E-Syt family, the yeast tricalbin (Tcb) proteins are essential for PM integrity upon heat stress, but it is not known how they contribute to PM maintenance. Using quantitative lipidomics and microscopy, we find that the Tcb proteins regulate phosphatidylserine homeostasis at the PM. Moreover, upon heat-induced membrane stress, Tcb3 co-localises with the PM protein Sfk1 that is implicated in PM phospholipid asymmetry and integrity. The Tcb proteins also control the PM targeting of the known phosphatidylserine effector Pkc1 upon heat-induced stress. Phosphatidylserine has evolutionarily conserved roles in PM organisation, integrity, and repair. We propose that phospholipid regulation is an ancient essential function of E-Syt family members required for PM integrity.
Topics: Cell Membrane; Homeostasis; Membrane Proteins; Phosphatidylserines; Phospholipids; Synaptotagmins
PubMed: 35440494
DOI: 10.26508/lsa.202201430