Ligand-regulated secretion of recombinant annexin V from cultured thyroid epithelial cells

2002 ◽  
Vol 282 (6) ◽  
pp. C1313-C1321 ◽  
Author(s):  
Xiuqiong Wang ◽  
Marcia A. Kaetzel ◽  
Sung E. Yoo ◽  
Paul S. Kim ◽  
John R. Dedman
Keyword(s):  
Mammalian Cells ◽  
Secretory Pathway ◽  
External Surface ◽  
Annexin V ◽  
Therapeutic Agents ◽  
Anionic Phospholipid ◽  
Thyroid Cells ◽  
Tissue Ischemia ◽  
Anionic Phospholipids ◽  
Activated Platelets

The exposure of anionic phospholipids on the external surface of injured endothelial cells and activated platelets is a primary biological signal to initiate blood coagulation. Disease conditions that promote the formation of ectopic thrombi result in tissue ischemia. Annexins, Ca2+-dependent anionic phospholipid binding proteins, are potential therapeutic agents for the inhibition of coagulation. We have designed a transgene that targets secretion of annexin V from cultured thyroid cells under the control of doxycycline. Our results indicate that annexin V in the endoplasmic reticulum (ER)/Golgi lumen does not affect the synthesis, processing, and secretion of thyroglobulin. ER luminal Ca2+was moderately increased and can be released by inositol 1,4,5-trisphosphate. Our study demonstrates that targeting and secretion of annexin V through the secretory pathway of mammalian cells does not adversely affect cellular function. Regulated synthesis and release of annexin V may exert anticoagulatory and anti-inflammatory effects systemically and may prove useful in further developing therapeutic strategies for conditions including antiphospholipid syndrome.

scholarly journals Oyxsterols induce membrane procoagulant activity in monocytic THP-1 cells

1996 ◽  
Vol 314 (3) ◽  
pp. 1027-1033 ◽  
Author(s):  
Karine AUPEIX ◽  
Florence TOTI ◽  
Nathalie SATTA ◽  
Pierre BISCHOFF ◽  
Jean-Marie FREYSSINET
Keyword(s):  
Tissue Factor ◽  
Annexin V ◽  
Antigen Expression ◽  
Resting Cells ◽  
Dependent Manner ◽  
Anionic Phospholipid ◽  
Monocytic Cells ◽  
Anionic Phospholipids ◽  
Phosphatidylserine Exposure ◽  
And Control

Oxidized cholesterol compounds or oxysterols are thought to be potent membrane-destabilizing agents. Anionic phospholipids, chiefly phosphatidylserine, have a procoagulant potential due to their ability to favour the membrane assembly of the characteristic clotting enzyme complexes including the tissue factor-dependent initiating complex. However, in resting cells, phosphatidylserine is sequestered in the inner leaflet of the plasma membrane. When THP-1 monocytic cells were cultured in the presence of 7β-hydroxycholesterol (7β-OH) or 25-hydroxycholesterol (25-OH), prothrombinase, which reflects anionic phospholipid exposure and tissue factor (TF) procoagulant activities, increased in a time- and dose-dependent manner. 7β-OH appeared 1.5- to 2-fold more potent than 25-OH. Interestingly, no effect of cholesterol itself could be detected on procoagulant activities. Nevertheless, no difference in TF activity could be detected between oxysterol-treated and control cells after disruption. TF antigen expression was the same in oxysterol-treated and control cells as shown by flow cytometry. In contrast, the use of labelled annexin V, a protein probe of anionic phospholipids, revealed an elevated number of cells with exposed phosphatidylserine. Because the latter also constitutes a signal for phagocyte recognition of apoptotic cells and fragments, and a proportion of cells displayed altered morphology with condensed chromatin and membrane blebs, analysis of DNA was performed and indicated apoptosis in oxysterol-treated cells. Hence, oxysterol-induced phosphatidylserine exposure and enhanced TF activity may result from apoptosis. These results suggest relationships between oxysterol and the amplification of coagulation reactions by monocytic cells resulting from induced phosphatidylserine exposure.

Anticoagulation Targeting Membrane-Bound Anionic Phospholipids Improved Outcomes of Traumatic Brain Injury in Mice

Blood ◽  
2021 ◽  
Author(s):  
Xinlong Dong ◽  
Wei Liu ◽  
Yu Shen ◽  
Katie L Houck ◽  
Mengchen Yang ◽  
...  
Keyword(s):  
Traumatic Brain Injury ◽  
Endothelial Cells ◽  
Brain Injury ◽  
Extracellular Vesicles ◽  
Annexin V ◽  
Hypercoagulable State ◽  
Anionic Phospholipid ◽  
Anionic Phospholipids ◽  
Fluid Percussion Injury ◽  
Tissue Edema

Severe traumatic brain injury (TBI) often causes an acute systemic hypercoagulable state that rapidly develops into consumptive coagulopathy. We have recently demonstrated that TBI-induced coagulopathy (TBI-IC) is initiated and disseminated by brain-derived extracellular vesicles (BDEVs) and propagated by extracellular vesicles (EVs) from endothelial cells and platelets. Here, we present results from a study designed to test the hypothesis that anticoagulation targeting anionic phospholipid-expressing EVs prevents TBI-IC and improves the outcomes of mice subjected to severe TBI. We evaluated the effects of a fusion protein (ANV-6L15) for improving the outcomes of TBI. ANV-6L15 combines the phosphatidylserine (PS)-binding annexin V with a peptide anticoagulant modified to preferentially target extrinsic coagulation. We found that ANV-6L15 reduced intracranial hematoma by 70.2%, improved neurological function, and reduced death by 56.8% in mice subjected to fluid percussion injury at 1.9 atm. It protected the TBI mice by preventing vascular leakage, tissue edema, and the TBI-induced hypercoagulable state. We further showed that the extrinsic tenase complex was formed on the surfaces of circulating EVs, with the highest level found on BDEVs. Phospholipidomic analysis detected the highest levels of PS on BDEVs, as compared to EVs from endothelial cells and platelets (79.1, 15.2, and 3.5 nM/mg of protein, respectively). These findings demonstrate that TBI-IC results from a trauma-induced hypercoagulable state and may be treated by anticoagulation targeting on the anionic phospholipid-expressing membrane of EVs from the brain and other cells.

Antiphospholipid antibody-mediated interference with annexin-V anticoagulant activity

2001 ◽  
Vol 21 (02) ◽  
pp. 50-53 ◽  
Author(s):  
X.-X. Wu ◽  
J. H. Rand
Keyword(s):  
Antiphospholipid Syndrome ◽  
Protein Complexes ◽  
Anticoagulant Activity ◽  
Annexin V ◽  
Phospholipid Bilayer ◽  
Antiphospholipid Antibody ◽  
Anionic Phospholipid ◽  
Anionic Phospholipids ◽  
Coagulation Proteins ◽  
Recurrent Pregnancy Losses

SummaryThe antiphospholipid (aPL) syndrome is a disorder in which vascular thrombosis and/or recurrent pregnancy losses occur together with serologic and coagulation evidence for antibodies directed against anionic phospholipid-protein complexes. Evidence has been developed for the idea that thrombosis in this syndrome may result from disruption of the binding of annexin-V to the phospholipids which line the placental and systemic vasculatures. We hypothesize that annexin-V, a protein known to have high affinity for anionic phospholipids, plays a thromboregulatory role at the vascular-blood interface by shielding anionic phospholipids from complexation with coagulation proteins in circulating blood. We propose that the thrombotic manifestations of the antiphospholipid syndrome are due to disruption of this annexin-V shield by antiphospholipid antibodies, thereby resulting in a net increase of thrombogenic phospholipids exposed to circulating blood. The accumulated data from tissue immunohistochemistry, trophoblast and endothelial cell culture studies, coagulation studies using noncellular phospholipids, and competition studies on artificial phospholipid bilayer are consistent with the hypothesis that the interference with the binding of annexin-V to anionic phospholipid surfaces plays an important role in the mechanism of thrombosis and in pregnancy loss in the antiphospholipid syndrome.

Studies of the Mechanism for Enhanced Cell Surface Factor VIIa/Tissue Factor Activation of Factor X on Fibroblast Monolayers after Their Exposure to N-Ethylmaleimide

1994 ◽  
Vol 72 (06) ◽  
pp. 848-855 ◽  
Author(s):  
Dzung The Le ◽  
Samuel I Rapaport ◽  
L Vijaya Mohan Rao
Keyword(s):  
Cell Surface ◽  
Tissue Factor ◽  
Factor Viia ◽  
Cell Damage ◽  
Specific Binding ◽  
Surface Membrane ◽  
Annexin V ◽  
Factor X ◽  
Binding Studies ◽  
Anionic Phospholipid

SummaryFibroblast monolayers constitutively expressing surface membrane tissue factor (TF) were treated with 0.1 mM N-ethylmaleimide (NEM) for 1 min to inhibit aminophospholipid translocase activity without inducing general cell damage. This resulted in increased anionic phospholipid in the outer leaflet of the cell surface membrane as measured by the binding of 125I-annexin V and by the ability of the monolayers to support the generation of prothrombinase. Specific binding of 125I-rVIIa to TF on NEM-treated monolayers was increased 3- to 4-fold over control monolayers after only brief exposure to 125I-rVIIa, but this difference progressively diminished with longer exposure times. A brief exposure of NEM-treated monolayers to rVIIa led to a maximum 3- to 4-fold enhancement of VIIa/TF catalytic activity towards factor X over control monolayers, but, in contrast to the binding studies, this 3- to 4-fold difference persisted despite increasing time of exposure to rVIIa. Adding prothrombin fragment 1 failed to diminish the enhanced VIIa/TF activation of factor X of NEM-treated monolayers. Moreover, adding annexin V, which was shown to abolish the ability of NEM to enhance factor X binding to the fibroblast monolayers, also failed to diminish the enhanced VIIa/TF activation of factor X. These data provide new evidence for a possible mechanism by which availability of anionic phospholipid in the outer layer of the cell membrane limits formation of functional VIIa/TF complexes on cell surfaces.

Autophagy Modulators and Neuroinflammation

2020 ◽  
Vol 27 (6) ◽  
pp. 955-982 ◽  
Author(s):  
Kyoung Sang Cho ◽  
Jang Ho Lee ◽  
Jeiwon Cho ◽  
Guang-Ho Cha ◽  
Gyun Jee Song
Keyword(s):  
Neurodegenerative Disorders ◽  
Neurological Disorders ◽  
Mammalian Cells ◽  
Critical Role ◽  
Therapeutic Agents ◽  
Mechanisms Of Action ◽  
Scientific Interest ◽  
Therapeutic Tools ◽  
Underlying Mechanisms

Background: Neuroinflammation plays a critical role in the development and progression of various neurological disorders. Therefore, various studies have focused on the development of neuroinflammation inhibitors as potential therapeutic tools. Recently, the involvement of autophagy in the regulation of neuroinflammation has drawn substantial scientific interest, and a growing number of studies support the role of impaired autophagy in the pathogenesis of common neurodegenerative disorders. Objective: The purpose of this article is to review recent research on the role of autophagy in controlling neuroinflammation. We focus on studies employing both mammalian cells and animal models to evaluate the ability of different autophagic modulators to regulate neuroinflammation. Methods: We have mostly reviewed recent studies reporting anti-neuroinflammatory properties of autophagy. We also briefly discussed a few studies showing that autophagy modulators activate neuroinflammation in certain conditions. Results: Recent studies report neuroprotective as well as anti-neuroinflammatory effects of autophagic modulators. We discuss the possible underlying mechanisms of action of these drugs and their potential limitations as therapeutic agents against neurological disorders. Conclusion: Autophagy activators are promising compounds for the treatment of neurological disorders involving neuroinflammation.

scholarly journals Size-Tunable Paclitaxel Nanoparticles Stabilized by Anionic Phospholipids for Transdermal Delivery Applications

2020 ◽  
Vol 15 (3) ◽  
pp. 1934578X1990068
Author(s):  
Noriyuki Uchida ◽  
Masayoshi Yanagi ◽  
Hiroki Hamada
Keyword(s):  
Stratum Corneum ◽  
Transdermal Delivery ◽  
Skin Tissue ◽  
Composite Nanoparticles ◽  
Rat Skin ◽  
Cooling Process ◽  
Anionic Phospholipid ◽  
Anionic Phospholipids ◽  
Subsequent Heating

Composite nanoparticles composed of an anionic phospholipid of 1,2-dipalmitoyl-sn-glycero-3-phosphorylglycerol (DPPG) and paclitaxel (PTX) were successfully prepared by mixing them in water followed by a subsequent heating/cooling process. The size of DPPG-PTX nanoparticle could be easily tuned by ultrasonic fragmentation. Upon addition of small-sized fluorescently labeled paclitaxel (FLPTX) nanoparticles with DPPG (DPPG-FLPTX) to rat skin tissue, part of the FLPTX molecules permeated to the stratum corneum.

scholarly journals Extension of the Scope of Anionic Phospholipid-Based Nanoformulation to Kaempferol and Indometacin

2021 ◽  
Vol 16 (3) ◽  
pp. 1934578X2110026
Author(s):  
Noriyuki Uchida ◽  
Masayoshi Yanagi ◽  
Kei Shimoda ◽  
Hiroki Hamada
Keyword(s):  
Phosphatidic Acid ◽  
Anionic Phospholipid ◽  
Anionic Phospholipids ◽  
Dispersion Agent

In this work, resveratrol was dispersed with anionic phospholipids of 1,2-dipalmitoyl-sn-glycero-3-phosphorylglycerol (DPPG), 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid, and 1,2-distearoyl-sn-glycero-3-phosphoglycerol. Moreover, small-sized nanoparticles of kaempferol and indometacin were successfully prepared by using DPPG as a dispersion agent.

scholarly journals Rab1b regulates vesicular transport between the endoplasmic reticulum and successive Golgi compartments.

1991 ◽  
Vol 115 (1) ◽  
pp. 31-43 ◽  
Author(s):  
H Plutner ◽  
A D Cox ◽  
S Pind ◽  
R Khosravi-Far ◽  
J R Bourne ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Essential Role ◽  
Secretory Pathway ◽  
Binding Protein ◽  
Vesicular Transport ◽  
Initial Step ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Golgi Compartment

We report an essential role for the ras-related small GTP-binding protein rab1b in vesicular transport in mammalian cells. mAbs detect rab1b in both the ER and Golgi compartments. Using an assay which reconstitutes transport between the ER and the cis-Golgi compartment, we find that rab1b is required during an initial step in export of protein from the ER. In addition, it is also required for transport of protein between successive cis- and medial-Golgi compartments. We suggest that rab1b may provide a common link between upstream and downstream components of the vesicular fission and fusion machinery functioning in early compartments of the secretory pathway.

scholarly journals Hierarchical regulation of mRNA partitioning between the cytoplasm and the endoplasmic reticulum of mammalian cells

2011 ◽  
Vol 22 (14) ◽  
pp. 2646-2658 ◽  
Author(s):  
Qiang Chen ◽  
Sujatha Jagannathan ◽  
David W. Reid ◽  
Tianli Zheng ◽  
Christopher V. Nicchitta
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Signal Recognition Particle ◽  
Signal Recognition ◽  
Endomembrane System ◽  
Genomic Analyses ◽  
Specific Manner ◽  
Signal Encoding ◽  
Mrna Transcriptome

The mRNA transcriptome is currently thought to be partitioned between the cytosol and endoplasmic reticulum (ER) compartments by binary selection; mRNAs encoding cytosolic/nucleoplasmic proteins are translated on free ribosomes, and mRNAs encoding topogenic signal-bearing proteins are translated on ER-bound ribosomes, with ER localization being conferred by the signal-recognition particle pathway. In subgenomic and genomic analyses of subcellular mRNA partitioning, we report an overlapping subcellular distribution of cytosolic/nucleoplasmic and topogenic signal-encoding mRNAs, with mRNAs of both cohorts displaying noncanonical subcellular partitioning patterns. Unexpectedly, the topogenic signal-encoding mRNA transcriptome was observed to partition in a hierarchical, cohort-specific manner. mRNAs encoding resident proteins of the endomembrane system were clustered at high ER-enrichment values, whereas mRNAs encoding secretory pathway cargo were broadly represented on free and ER-bound ribosomes. Two distinct modes of mRNA association with the ER were identified. mRNAs encoding endomembrane-resident proteins were bound via direct, ribosome-independent interactions, whereas mRNAs encoding secretory cargo displayed predominantly ribosome-dependent modes of ER association. These data indicate that mRNAs are partitioned between the cytosol and ER compartments via a hierarchical system of intrinsic and encoded topogenic signals and identify mRNA cohort-restricted modes of mRNA association with the ER.

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