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.

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.

scholarly journals Tissue factor activation: is disulfide bond switching a regulatory mechanism?

Blood ◽  
2007 ◽  
Vol 110 (12) ◽  
pp. 3900-3908 ◽  
Author(s):  
Usha R. Pendurthi ◽  
Samit Ghosh ◽  
Samir K. Mandal ◽  
L. Vijaya Mohan Rao
Keyword(s):  
Cell Surface ◽  
Cell Signaling ◽  
Tissue Factor ◽  
Disulfide Bond ◽  
Annexin V ◽  
Disulfide Exchange ◽  
Anionic Phospholipids ◽  
Coagulant Activity ◽  
Novel Concept ◽  
Cysteine Thiols

AbstractA majority of tissue factor (TF) on cell surfaces exists in a cryptic form (ie, coagulation function inactive) but retains its functionality in cell signaling. Recent studies have suggested that cryptic TF contains unpaired cysteine thiols and that activation involves the formation of the disulfide bond Cys186-Cys 209 and that protein disulfide isomerase (PDI) regulates TF coagulant and signaling activities by targeting this disulfide bond. This study was carried out to investigate the validity of this novel concept. Although treatment of MDA 231 tumor cells, fibroblasts, and stimulated endothelial cells with the oxidizing agent HgCl2 markedly increased the cell-surface TF coagulant activity, the increase is associated with increased anionic phospholipids at the cell surface. Annexin V, which binds to anionic phospholipids, attenuated the increased TF coagulant activity. It is noteworthy that treatment of cells with reducing agents also increased the cell surface TF activity. No evidence was found for either detectable expression of PDI at the cell surface or association of TF with PDI. Furthermore, reduction of PDI with the gene silencing had no effect on either TF coagulant or cell signaling functions. Overall, the present data undermine the recently proposed hypothesis that PDI-mediated disulfide exchange plays a role in regulating TF procoagulant and cell signaling functions.

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.

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.

β2-glycoprotein I Binding to Platelet Microparticle Membrane Specifically Reduces Immunoreactivity of Glycoproteins IIb/IIIa

2001 ◽  
Vol 85 (02) ◽  
pp. 314-319 ◽  
Author(s):  
Laurent Vallar ◽  
Véronique Regnault ◽  
Véronique Latger-Cannard ◽  
Thomas Lecompte
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Annexin V ◽  
Human Platelets ◽  
Resonance Spectroscopy ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Anionic Phospholipids ◽  
Glycoprotein I ◽  
Specific Influence

SummaryWe have investigated β2-glycoprotein I (β2GPI) binding to platelet-derived microparticles (PMP) and its effect on GPIIb/IIIa. PMP were isolated from washed human platelets after stimulation with A23187, and analyzed by surface plasmon resonance spectroscopy. β2GPI as well as activated protein C (APC) or annexin V bound to PMP-coated sensorchips, demonstrating exposure of anionic phospholipids on immobilized PMP. β2GPI binding was impaired by calcium and occurred in a concentration-dependent manner with apparent kon = 2.6104 M-1.s-1 and koff = 4.410-3 s-1, corresponding to a KD value of 1.710-7 M. When analyzed by flow cytometry, the binding of certain mAbs specific for GPIIb and/or GPIIIa was reduced in the presence of β2GPI but not of APC or annexin V, whereas the binding of anti-GPIb or anti-P-selectin mAbs, or of soluble fibrinogen remained unchanged. These results suggest a broad but specific influence of β2GPI on GPIIb/IIIa immunoreactivity, and indicate that β2GPI may act as a modulator of GPIIb/IIIa-dependent functions of PMP.

Antibodies from patients with heparin-induced thrombocytopenia stimulate monocytic cells to express tissue factor and secrete interleukin-8

Blood ◽  
2001 ◽  
Vol 98 (4) ◽  
pp. 1252-1254 ◽  
Author(s):  
Gowthami M. Arepally ◽  
Irene M. Mayer
Keyword(s):  
Tissue Factor ◽  
Human Platelet ◽  
Interleukin 8 ◽  
Dependent Manner ◽  
Monocyte Activation ◽  
Monocytic Cells ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Factor Expression ◽  
Life Threatening

Thrombosis is a life-threatening complication that occurs in a subset of patients with heparin-induced thrombocytopenia (HITT). The pathogenic mechanisms underlying the variable occurrence of thrombosis in HITT is poorly understood. It was hypothesized that monocyte activation leading to tissue factor expression may play a role in promoting a thrombogenic state in HITT. This study demonstrates that a human platelet factor 4 (PF4)/heparin-specific murine monoclonal antibody (KKO) binds to peripheral blood-derived human monocytes in a PF4-dependent manner. KKO and antibodies from patients with HITT induce monocytes to synthesize and secrete interleukin-8 and induce cell-surface procoagulant activity, which is abrogated following treatment with antihuman tissue factor antibody. The findings suggest a novel mechanism by which PF4/heparin antibodies may promote a hypercoagulable state in patients with HITT.

scholarly journals Fusion proteins comprising annexin V and Kunitz protease inhibitors are highly potent thrombogenic site-directed anticoagulants

Blood ◽  
2005 ◽  
Vol 105 (10) ◽  
pp. 3902-3909 ◽  
Author(s):  
Hsiu-Hui Chen ◽  
Cristina P. Vicente ◽  
Li He ◽  
Douglas M. Tollefsen ◽  
Tze-Chein Wun
Keyword(s):  
Tissue Factor ◽  
Fusion Proteins ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Amyloid Β ◽  
Annexin V ◽  
Amyloid Β Protein ◽  
Anionic Phospholipid ◽  
Tissue Factor Pathway

AbstractThe anionic phospholipid, phosphatidyl-l-serine (PS), is sequestered in the inner layer of the plasma membrane in normal cells. Upon injury, activation, and apoptosis, PS becomes exposed on the surfaces of cells and sheds microparticles, which are procoagulant. Coagulation is initiated by formation of a tissue factor/factor VIIa complex on PS-exposed membranes and propagated through the assembly of intrinsic tenase (factor VIIIa/factor IXa), prothrombinase (factor Va/factor Xa), and factor XIa complexes on PS-exposed activated platelets. We constructed a novel series of recombinant anticoagulant fusion proteins by linking annexin V (ANV), a PS-binding protein, to the Kunitz-type protease inhibitor (KPI) domain of tick anticoagulant protein, an aprotinin mutant (6L15), amyloid β-protein precursor, or tissue factor pathway inhibitor. The resulting ANV-KPI fusion proteins were 6- to 86-fold more active than recombinant tissue factor pathway inhibitor and tick anticoagulant protein in an in vitro tissue factor–initiated clotting assay. The in vivo antithrombotic activities of the most active constructs were 3- to 10-fold higher than that of ANV in a mouse arterial thrombosis model. ANV-KPI fusion proteins represent a new class of anticoagulants that specifically target the anionic membrane-associated coagulation enzyme complexes present at sites of thrombogenesis and are potentially useful as antithrombotic agents.

Lipopolysaccharide activation of the MEK-ERK1/2 pathway in human monocytic cells mediates tissue factor and tumor necrosis factor α expression by inducing Elk-1 phosphorylation and Egr-1 expression

Blood ◽  
2001 ◽  
Vol 98 (5) ◽  
pp. 1429-1439 ◽  
Author(s):  
Mausumee Guha ◽  
Maria A. O'Connell ◽  
Rafal Pawlinski ◽  
Angela Hollis ◽  
Patricia McGovern ◽  
...  
Keyword(s):  
Gene Expression ◽  
Tumor Necrosis Factor ◽  
Tissue Factor ◽  
Tumor Necrosis ◽  
Nuclear Translocation ◽  
Mapk Pathway ◽  
Dependent Manner ◽  
Monocytic Cells ◽  
Tnf Α ◽  
Necrosis Factor

Lipopolysaccharide (LPS) induces human monocytes to express many proinflammatory mediators, including the procoagulant molecule tissue factor (TF) and the cytokine tumor necrosis factor alpha (TNF-α). The TF and TNF-α genes are regulated by various transcription factors, including nuclear factor (NF)-κB/Rel proteins and Egr-1. In this study, the role of the MEK-ERK1/2 mitogen-activated protein kinase (MAPK) pathway in LPS induction of TF and TNF-α gene expression in human monocytic cells was investigated. The MAPK kinase (MEK)1 inhibitor PD98059 reduced LPS induction of TF and TNF-α expression in a dose-dependent manner. PD98059 did not affect LPS-induced nuclear translocation of NF-κB/Rel proteins and minimally affected LPS induction of κB-dependent transcription. In contrast, PD98059 and dominant-negative mutants of the Ras-Raf1-MEK-ERK (extacellular signal–regulated kinase) pathway strongly inhibited LPS induction of Egr-1 expression. In kinetic experiments LPS induction of Egr-1 expression preceded induction of TF expression. In addition, mutation of the Egr-1 sites in the TF and TNF-α promoters reduced expression of these proinflammatory genes. It was demonstrated that LPS induction of the Egr-1 promoter was mediated by 3 SRE sites, which bound an LPS-inducible complex containing serum response factor and Elk-1. LPS stimulation transiently induced phosphorylation of Elk-1 and increased the functional activity of a GAL4–Elk-1TA chimeric protein via the MEK-ERK1/2 pathway. The data indicate that LPS induction of Egr-1 gene expression is required for maximal induction of the TNF-α and TF genes in human monocytic cells.

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