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Clinical and Applied... 2023The procoagulant effect of microparticles (MPs) contributes to hypercoagulability-induced thrombosis. We provide preliminary findings of the MPs-Activated Clotting Time...
The procoagulant effect of microparticles (MPs) contributes to hypercoagulability-induced thrombosis. We provide preliminary findings of the MPs-Activated Clotting Time (MPs-ACT) assay to determine the procoagulant activity of MPs. MPs-rich plasma was obtained and recalcified. Changes in plasma viscoelasticity were evaluated and the time to the peak viscoelastic changes was defined as the MPs-ACT. MPs concentration was measured by flow cytometry. Coagulation products produced during plasma clotting were identified by fibrin and fibrinopeptide A. MPs were prepared in vitro and added to standard plasma to simulate pathological samples. In addition, reproducibility and sensitivity were evaluated. We confirmed the linear relationship between MPs-ACT and MP concentrations. Dynamic changes in fibrin production were depicted. We simulated the correlation between MPs-ACT and standard plasma containing MPs prepared in vitro. The reproducibility of high-value and low-value samples was 6.0% and 10.8%, respectively. MPs-ACT sensitively detected hypercoagulable samples from patients with pre-eclampsia, hip fractures, and lung tumors. MPs-ACT largely reflects the procoagulant effect of MPs. MPs-ACT sensitively and rapidly detects hypercoagulability with MPs-rich plasma. It may be promising for the diagnosis of hypercoagulable states induced by MPs.
Topics: Female; Humans; Reproducibility of Results; Phosphatidylserines; Blood Coagulation; Thrombophilia; Cell-Derived Microparticles; Fibrin
PubMed: 36843474
DOI: 10.1177/10760296231159374 -
Journal of Virology Sep 2021Ebola virus (EBOV) attaches to target cells using two categories of cell surface receptors: C-type lectins and phosphatidylserine (PS) receptors. PS receptors typically...
Ebola virus (EBOV) attaches to target cells using two categories of cell surface receptors: C-type lectins and phosphatidylserine (PS) receptors. PS receptors typically bind to apoptotic cell membrane PS and orchestrate the uptake and clearance of apoptotic debris. Many enveloped viruses also contain exposed PS and can therefore exploit these receptors for cell entry. Viral infection can induce PS externalization in host cells, resulting in increased outer PS levels on budding virions. Scramblase enzymes carry out cellular PS externalization; thus, we targeted these proteins in order to manipulate viral envelope PS levels. We investigated two scramblases previously identified to be involved in EBOV PS levels, transmembrane protein 16F and Xk-related protein 8 (XKR8), as possible mediators of cellular and viral envelope surface PS levels during the replication of recombinant vesicular stomatitis virus containing its native glycoprotein (rVSV/G) or the EBOV glycoprotein (rVSV/EBOV-GP). We found that rVSV/G and rVSV/EBOV-GP virions produced in XKR8 knockout cells contain decreased levels of PS on their surfaces, and the PS-deficient rVSV/EBOV-GP virions are 70% less efficient at infecting cells through PS receptors. We also observed reduced rVSV and EBOV virus-like particle (VLP) budding in ΔXKR8 cells. Deletion of XKR8 in HAP1 cells reduced rVSV/G and rVSV/EBOV-GP budding by 60 and 65%, respectively, and reduced Ebola VLP budding more than 60%. We further demonstrated that caspase cleavage of XKR8 is required to promote budding. This suggests that XKR8, in addition to mediating virion PS levels, may also be critical for enveloped virus budding at the plasma membrane. Within the last decade, countries in western and central Africa have experienced the most widespread and deadly Ebola outbreaks since Ebola virus was identified in 1976. While outbreaks are primarily attributed to zoonotic transfer events, new evidence is emerging outbreaks may be caused by a combination of spillover events and viral latency or persistence in survivors. The possibility that Ebola virus can remain dormant and then reemerge in survivors highlights the critical need to prevent the virus from entering and establishing infection in human cells. Thus far, host cell scramblases TMEM16F and XKR8 have been implicated in Ebola envelope surface phosphatidylserine (PS) and cell entry using PS receptors. We assessed the contributions of these proteins using CRISPR knockout cells and two EBOV models: rVSV/EBOV-GP and EBOV VLPs. We observed that XKR8 is required for optimal EBOV envelope PS levels and infectivity and particle budding across all viral models.
Topics: Cell Line; Ebolavirus; Glycoproteins; Hemorrhagic Fever, Ebola; Humans; Phosphatidylserines; Phospholipid Transfer Proteins; Viral Envelope Proteins; Virion; Virus Assembly; Virus Release
PubMed: 34319156
DOI: 10.1128/JVI.01165-21 -
Cellular & Molecular Biology Letters Jul 2023The dynamics of phosphatidylserine in the plasma membrane is a tightly regulated feature of eukaryotic cells. Phosphatidylserine (PS) is found preferentially in the...
BACKGROUND
The dynamics of phosphatidylserine in the plasma membrane is a tightly regulated feature of eukaryotic cells. Phosphatidylserine (PS) is found preferentially in the inner leaflet of the plasma membrane. Disruption of this asymmetry leads to the exposure of phosphatidylserine on the cell surface and is associated with cell death, synaptic pruning, blood clotting and other cellular processes. Due to the role of phosphatidylserine in widespread cellular functions, an efficient phosphatidylserine probe is needed to study them. Currently, a few different phosphatidylserine labelling tools are available; however, these labels have unfavourable signal-to-noise ratios and are difficult to use in tissues due to limited permeability. Their application in living tissue requires injection procedures that damage the tissue and release damage-associated molecular patterns, which in turn stimulates phosphatidylserine exposure.
METHODS
For this reason, we developed a novel genetically encoded phosphatidylserine probe based on the C2 domain of the lactadherin (MFG-E8) protein, suitable for labelling exposed phosphatidylserine in various research models. We tested the C2 probe specificity to phosphatidylserine on hybrid bilayer lipid membranes by observing surface plasmon resonance angle shift. Then, we analysed purified fused C2 proteins on different cell culture lines or engineered AAVs encoding C2 probes on tissue cultures after apoptosis induction. For in vivo experiments, neurotropic AAVs were intravenously injected into perinatal mice, and after 2 weeks, brain slices were collected to observe C2-SNAP expression.
RESULTS
The biophysical analysis revealed the high specificity of the C2 probe for phosphatidylserine. The fused recombinant C2 proteins were suitable for labelling phosphatidylserine on the surface of apoptotic cells in various cell lines. We engineered AAVs and validated them in organotypic brain tissue cultures for non-invasive delivery of the genetically encoded C2 probe and showed that these probes were expressed in the brain in vivo after intravenous AAV delivery to mice.
CONCLUSIONS
We have demonstrated that the developed genetically encoded PS biosensor can be utilised in a variety of assays as a two-component system of C2 and C2m2 fusion proteins. This system allows for precise quantification and PS visualisation at directly specified threshold levels, enabling the evaluation of PS exposure in both physiological and cell death processes.
Topics: Animals; Mice; Phosphatidylserines; Cell Membrane; Lipid Bilayers; Biosensing Techniques; Cell Line
PubMed: 37501184
DOI: 10.1186/s11658-023-00472-7 -
IUBMB Life Mar 2022Indole propionic acid (IPA) which majorly influences the modulation of cellular respiration is a metabolite generated by gut microbiota. The antimicrobial effects of IPA...
Indole propionic acid (IPA) which majorly influences the modulation of cellular respiration is a metabolite generated by gut microbiota. The antimicrobial effects of IPA have not been previously demonstrated. Therefore, this study focused on investigating the antimicrobial activity of IPA. Initially, antifungal activity of IPA against Candida albicans was observed, accompanied by variations in mitochondrial respiration indicating modulation of NAD /NADH ratios. Consumption of O contributes to the respiratory regulation and triggered by Ca overloading. After treatment with IPA, the cells were monitored, and Ca increases leading to membrane depolarization and reactive oxygen species (ROS) accumulation in mitochondria were noted. Depolarization of mitochondria membrane induced release of proapoptotic proteins in mitochondria. Oxidative stress exerted by ROS contributed to glutathione depletion and oxidation of glutathione (GSH). Fragmentation of DNA is a characteristic event leading to apoptosis and accompanies major hallmarks of apoptosis including phosphatidylserine exposure and metacaspase activation. In addition, phosphatidylserine exposure and metacaspase activation were detected in the cell treated with IPA. In conclusion, IPA triggered apoptosis in C. albicans under the influence of Ca .
Topics: Antifungal Agents; Apoptosis; Candida albicans; Glutathione; Indoles; Membrane Potential, Mitochondrial; Phosphatidylserines; Propionates; Reactive Oxygen Species
PubMed: 34779568
DOI: 10.1002/iub.2579 -
Molecules (Basel, Switzerland) Dec 2009In the 1970s, morphological evidence collected by electron microscopy linked mineral deposition ("calcification" or "mineralization") in newly-forming bone to... (Review)
Review
In the 1970s, morphological evidence collected by electron microscopy linked mineral deposition ("calcification" or "mineralization") in newly-forming bone to membrane-encapsulated particles of a diameter of approximately 100 nm (50-200 nm) that were called "matrix vesicles". As the characterisation of these vesicles progressed towards their biochemical composition, the role of lipids in the biomineralization process appeared to be crucial. In particular, a group of cell-membrane phospholipids were identified as major players in the crystal formation process. Indeed, in the 1980s it became clear that phosphatidylserine, together with proteins of the annexin family, was among the most important molecules in binding calcium ions and that this phospholipid was involved in the regulation of the early stages of mineralization in vivo. During the same period of time, the number of surgical implantations of orthopaedic, dental and maxilo-facial devices requiring full integration with the treated bone prompted the study of new functionalization molecules able to establish a stable bonding with the mineral phase of the host tissue. In the late 1990 s studies started that aimed at exploiting the potential of calcium-binding phospholipids and, in particular, of the phosphatidylserine as functionalization molecules to improve the osteointegration of artificial implants. Later, papers have been published that show the potential of the phophatidylserine and phosphatidylserine-mimicking coating technology to promote calcification both in vitro and in vivo. The promising results support the future clinical application of these novel osteointegrative biomaterials.
Topics: Animals; Bone Remodeling; Calcification, Physiologic; Microscopy, Electron; Models, Molecular; Phosphatidylserines; Rabbits
PubMed: 20032899
DOI: 10.3390/molecules14125367 -
Proceedings of the Japan Academy.... 2012By using "our devised up-to-the-second technique" over 30 years ago, we succeeded in the first isolation in the world of the three different kinds of mammalian cell... (Review)
Review
By using "our devised up-to-the-second technique" over 30 years ago, we succeeded in the first isolation in the world of the three different kinds of mammalian cell mutants defective in the biosynthesis on each of phosphatidylserine (PS), cardiolipin (CL) and sphingomyelin (SM) from the parental CHO cells. As the results, we found that during the biosyntheses of PS and SM, the biosynthetic precursor or the final lipids are transported from their synthesized intracellular organelles to the plasma membranes via the other intracellular organelles. We further clarified the presence of the reversed routes for PS and SM from the plasma membranes to their synthesized organelles too. Our first epoch-making finding is not only the cycling inter-conversion reactions between PS and PE catalyzed by PSS-II and PSD but also their simultaneous transferring between MAM and Mit (found by O. Kuge). Our second finding is "the ceramide-trafficking protein (CERT)" working as the specific transfer protein of ceramide from the ER to the Golgi apparatus, during the SM biosynthesis (by K. Hanada). As for their new biological roles, we clarified possible contribution of PS and/or PE to the fusion process between viral envelope and endosomal membrane, releasing the genetic information of the virus to the host cytoplasm. CL is contributing to the functional NADH-ubiquinone reductase activity by keeping the right structure of Coenzyme Q9 for its functioning. SM and cholesterol form the microdomain within the plasma membrane, so-called "the raft structure" where the GPI-anchored proteins are specifically located for their functioning.
Topics: Animals; Cardiolipins; Cell Line; History, 20th Century; History, 21st Century; Humans; Phosphatidylserines; Phospholipids; Sphingolipids
PubMed: 23229749
DOI: 10.2183/pjab.88.536 -
Biochimica Et Biophysica Acta Jun 2008In normal healthy cells phosphatidylserine is located in the inner leaflet of the plasma membrane. However, on activated platelets, dying cells and under specific... (Review)
Review
In normal healthy cells phosphatidylserine is located in the inner leaflet of the plasma membrane. However, on activated platelets, dying cells and under specific circumstances also on various types of viable leukocytes phosphatidylserine is actively externalized to the outer leaflet of the plasma membrane. Annexin A5 has the ability to bind in a calcium-dependent manner to phosphatidylserine and to form a membrane-bound two-dimensional crystal lattice. Based on these abilities various functions for extracellular annexin A5 on the phosphatidylserine-expressing plasma membrane have been proposed. In this review we describe possible mechanisms for externalization of annexin A5 and various processes in which extracellular annexin A5 may play a role such as blood coagulation, apoptosis, phagocytosis and formation of plasma membrane-derived microparticles. We further highlight the recent discovery of internalization of extracellular annexin A5 by phosphatidylserine-expressing cells.
Topics: Annexin A5; Apoptosis; Cell Membrane; Crystallization; Humans; Phagocytosis; Phosphatidylserines
PubMed: 18334229
DOI: 10.1016/j.bbamcr.2008.01.030 -
The Journal of Biological Chemistry Jan 2018Bone-resorbing multinucleated osteoclasts that play a central role in the maintenance and repair of our bones are formed from bone marrow myeloid progenitor cells by a...
Bone-resorbing multinucleated osteoclasts that play a central role in the maintenance and repair of our bones are formed from bone marrow myeloid progenitor cells by a complex differentiation process that culminates in fusion of mononuclear osteoclast precursors. In this study, we uncoupled the cell fusion step from both pre-fusion stages of osteoclastogenic differentiation and the post-fusion expansion of the nascent fusion connections. We accumulated ready-to-fuse cells in the presence of the fusion inhibitor lysophosphatidylcholine and then removed the inhibitor to study synchronized cell fusion. We found that osteoclast fusion required the dendrocyte-expressed seven transmembrane protein (DC-STAMP)-dependent non-apoptotic exposure of phosphatidylserine at the surface of fusion-committed cells. Fusion also depended on extracellular annexins, phosphatidylserine-binding proteins, which, along with annexin-binding protein S100A4, regulated fusogenic activity of syncytin 1. Thus, in contrast to fusion processes mediated by a single protein, such as epithelial cell fusion in , the cell fusion step in osteoclastogenesis is controlled by phosphatidylserine-regulated activity of several proteins.
Topics: Animals; Annexins; Bone Resorption; Bone and Bones; Cell Differentiation; Cell Fusion; Cell Line; Cell Membrane; Gene Products, env; Hematopoiesis; Humans; Membrane Fusion; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Nerve Tissue Proteins; Osteoclasts; Osteogenesis; Phosphatidylserines; Pregnancy Proteins; S100 Calcium-Binding Protein A4
PubMed: 29101233
DOI: 10.1074/jbc.M117.809681 -
International Journal of Molecular... Jul 2023Circulating extracellular microvesicles (cEVs) are characterised by presenting surface antigens of parental cells. Since their biogenesis involves the translocation of...
Circulating extracellular microvesicles (cEVs) are characterised by presenting surface antigens of parental cells. Since their biogenesis involves the translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane, exposed PS has been considered as a recognition hallmark of cEVs. However, not all cEVs externalise PS. In this study, we have phenotypically and quantitatively characterised cEVs by flow cytometry, paying special attention to the proportions of PS in chronic heart failure patients (cHF; = 119) and a reference non-HF group ( = 21). PS-cEVs were predominantly found in both groups. Parental markers showed differential pattern depending on the PS exposure. Endothelium-derived and connexin 43-rich cEVs were mainly PS-cEVs and significantly increased in cHF. On the contrary, platelet-derived cEVs were mostly PS and were increased in the non-HF group. We observed similar levels of PS- and PS-cEVs in non-HF subjects when analysing immune cell-derived Evs, but there was a subset-specific difference in cHF patients. Indeed, those cEVs carrying CD45, CD29, CD11b, and CD15 were mainly PS-cEVs, while those carrying CD14, CD3, and CD56 were mainly PS-cEVs. In conclusion, endothelial and red blood cells are stressed in cHF patients, as detected by a high shedding of cEVs. Despite PS-cEVs and PS-cEVs representing two distinct cEV populations, their release and potential function as both biomarkers and shuttles for cell communication seem unrelated to their PS content.
Topics: Humans; Phosphatidylserines; Erythrocytes; Extracellular Vesicles; Endothelium; Heart Failure
PubMed: 37511585
DOI: 10.3390/ijms241411824 -
The Journal of Biological Chemistry Dec 2022Brain-specific angiogenesis inhibitor 1 (BAI1; also called ADGRB1 or B1) is an adhesion G protein-coupled receptor known from studies on macrophages to bind to...
Brain-specific angiogenesis inhibitor 1 (BAI1; also called ADGRB1 or B1) is an adhesion G protein-coupled receptor known from studies on macrophages to bind to phosphatidylserine (PS) on apoptotic cells via its N-terminal thrombospondin repeats. A separate body of work has shown that B1 regulates postsynaptic function and dendritic spine morphology via signaling pathways involving Rac and Rho. However, it is unknown if PS binding by B1 has any effect on the receptor's signaling activity. To shed light on this subject, we studied G protein-dependent signaling by B1 in the absence and presence of coexpression with the PS flippase ATP11A in human embryonic kidney 293T cells. ATP11A expression reduced the amount of PS exposed extracellularly and also strikingly reduced the signaling activity of coexpressed full-length B1 but not a truncated version of the receptor lacking the thrombospondin repeats. Further experiments with an inactive mutant of ATP11A showed that the PS flippase function of ATP11A was required for modulation of B1 signaling. In coimmunoprecipitation experiments, we made the surprising finding that ATP11A not only modulates B1 signaling but also forms complexes with B1. Parallel studies in which PS in the outer leaflet was reduced by an independent method, deletion of the gene encoding the endogenous lipid scramblase anoctamin 6 (ANO6), revealed that this manipulation also markedly reduced B1 signaling. These findings demonstrate that B1 signaling is modulated by PS exposure and suggest a model in which B1 serves as a PS sensor at synapses and in other cellular contexts.
Topics: Humans; Phosphatidylserines; Receptors, G-Protein-Coupled; Signal Transduction; Thrombospondins; HEK293 Cells
PubMed: 36370845
DOI: 10.1016/j.jbc.2022.102685