Functional Regulation of TFPI in Membrane Lipid Rafts.

Abstract The assembly of tissue factor-factor VIIa (TF-FVIIa) complex results in the proteolytic activation of factors X (FX) and IX and ultimate thrombin generation. The inhibition of this pathway by the Kunitz-type inhibitor, tissue factor pathway inhibitor (TFPI), involves the formation of a stable TF-FVIIa-FXa-TFPI complex. TFPI in endothelial cells (EC) locates primarily in rafts and caveolae, which are membrane microdomains enriched in cholesterol, glycosphingolipids (GSL) and caveolins, and which regulate the function of TFPI. Since caveolin-1 supports the TFPI-dependent inhibition of TF-FVIIa, we aimed to decipher the role played by the individual components of rafts in the anticoagulant function of cell surface TFPI. To this end, we studied the distribution of TFPI, TF and caveolin-1 by immunofluorescence microscopy, and we assayed the functional activity of TFPI after cholesterol-complexation on EC (EA. hy926 and HUVEC) and HEK293 expressing TFPI or TFPI+caveolin-1, or we used GSL-deficient CHO mutant cell lines. In EC, cholesterol complexation with filipin led to patching of TFPI over the cell surface and reduced inhibition of TF-FVIIa. Extraction of cholesterol from the external leaflet of the membrane with methyl-β-cyclodextrin (M-β-CD) shifted the partition of TFPI from predominantly raft-associated to the non-raft cellular fractions isolated through temperature-induced phase separation of Triton X-114 lysates. Although activation of FX by TF-FVIIa was significantly enhanced by M-β-CD and reversed after cholesterol replenishment, the effect was only modestly affected by the TFPI activity reduction. By immunofluorescence we observed that M-β-CD produced redistribution of both TFPI and TF over the EC and 293 cell surface with apparent segregation into separate domains and complete lack of co-localization. Such accumulations of TF will likely promote strong procoagulant activity when not inhibited by TFPI. Since M-β-CD selectively disrupts the glycerophospholipid-rich regions of the membrane while leaving the caveolar cholesterol virtually intact, we also tested progesterone, which extracts cholesterol specifically from caveolae. Treatment of HEK293 cells with progesterone for 2 hrs reduced significantly the inhibition of TF-FVIIa-dependent activation of FX by TFPI for TFPI+Cav+ cells but not for TFPI+ cells, suggesting that the process was specific for cells that have caveolae. To study the role of GSL for the activity of TFPI, we used Ly-B cells, a GSL-deficient mutant derived from CHO-K1, which have a defect in the LCB1 subunit of serine palmitoyltransferase. Characterization of endogenous TFPI in CHO-K1, Ly-B and its genetically corrected revertant Ly-B/cLCB1 (cLCB) revealed strong similarities between CHOs and EC with regard to the expression and function of TFPI. Whereas not affecting cLCB cells, incubation of Ly-B for 2 days in sphingolipid-deficient medium shifted the partition of cellular TFPI from the detergent-soluble (rafts) to the water-soluble (non-raft) fraction, which suggests that GSL play a major role in the distribution and function of the membrane TFPI. The fundamental knowledge developed by these studies will improve our understanding of the mechanisms by which TFPI functions against TF-FVIIa procoagulant activity on cell surfaces. In the long term, they may guide novel therapeutic approaches to prevent inflammation and thrombosis.

Download Full-text

Factor VIIa Regulates the Level of Cell-Surface Tissue Factor through Separate but Cooperative Mechanisms

Cancers ◽

10.3390/cancers13153718 ◽

2021 ◽

Vol 13 (15) ◽

pp. 3718

Author(s):

Yahya Madkhali ◽

Araci M. R. Rondon ◽

Sophie Featherby ◽

Anthony Maraveyas ◽

John Greenman ◽

...

Keyword(s):

Endothelial Cells ◽

Cell Surface ◽

Tissue Factor ◽

Lipid Rafts ◽

Human Blood ◽

Factor Viia ◽

Procoagulant Activity ◽

Early Release ◽

Caveolin 1 ◽

Proliferative Signal

Procoagulant activity of tissue factor (TF) in response to injury or inflammation is accompanied with cellular signals which determine the fate of cells. However, to prevent excessive signalling, TF is rapidly dissipated through release into microvesicles, and/or endocytosis. To elucidate the mechanism by which TF signalling may become moderated on the surface of cells, the associations of TF, fVII/fVIIa, PAR2 and caveolin-1 on MDA-MB-231, BxPC-3 and 786-O cells were examined and compared to that in cells lacking either fVII/fVIIa or TF. Furthermore, the localisation of labelled-recombinant TF with cholesterol-rich lipid rafts was explored on the surface of primary human blood dermal endothelial cells (HDBEC). Finally, by disrupting the caveolae on the surface of HDBEC, the outcome on TF-mediated signalling was examined. The association between TF and PAR2 was found to be dependent on the presence of fVIIa. Interestingly, the presence of TF was not pre-requisite for the association between fVII/fVIIa and PAR2 but was significantly enhanced by TF, which was also essential for the proliferative signal. Supplementation of HDBEC with exogenous TF resulted in early release of fVII/fVIIa from caveolae, followed by re-sequestration of TF-fVIIa. Addition of labelled-TF resulted in the accumulation within caveolin-1-containing cholesterol-rich regions and was also accompanied with the increased assimilation of cell-surface fVIIa. Disruption of the caveolae/rafts in HDBEC using MβCD enhanced the TF-mediated cellular signalling. Our data supports a hypothesis that cells respond to the exposure to TF by moderating the signalling activities as well as the procoagulant activity of TF, through incorporation into the caveolae/lipid rafts.

Download Full-text

Cellular localization and trafficking of tissue factor

Blood ◽

10.1182/blood-2005-11-4674 ◽

2006 ◽

Vol 107 (12) ◽

pp. 4746-4753 ◽

Cited By ~ 53

Author(s):

Samir K. Mandal ◽

Usha R. Pendurthi ◽

L. Vijaya Mohan Rao

Keyword(s):

Cell Surface ◽

Tissue Factor ◽

Cellular Localization ◽

Factor Viia ◽

Coagulation Cascade ◽

Clotting Factor ◽

Cell Surfaces ◽

Caveolin 1 ◽

Intracellular Compartments ◽

Resultant Increase

AbstractTissue factor (TF) is the cellular receptor for clotting factor VIIa (FVIIa). The formation of TF-FVIIa complexes on cell surfaces triggers the activation of coagulation cascade and cell signaling. In the present study, we characterized the subcellular distribution of TF and its transport in fibroblasts by dual immunofluorescence confocal microscopy and biochemical methods. Our data show that a majority of TF resides in various intracellular compartments, predominantly in the Golgi. Tissue factor at the cell surface is localized in cholesterol-rich lipid rafts and extensively colocalized with caveolin-1. FVIIa binding to TF induces the internalization of TF. Of interest, we found that TF-FVIIa complex formation at the cell surface leads to TF mobilization from the Golgi with a resultant increase in TF expression at the cell surface. This process is dependent on FVIIa protease activity. Overall, the present data suggest a novel mechanism for TF expression at the cell surface by FVIIa. This mechanism could play an important role in hemostasis in response to vascular injury by increasing TF activity where and when it is needed.

Download Full-text

Caveolin-1 Enhances Tissue Factor Pathway Inhibitor Exposure and Function on the Cell Surface

Journal of Biological Chemistry ◽

10.1074/jbc.m503333200 ◽

2005 ◽

Vol 280 (23) ◽

pp. 22308-22317 ◽

Cited By ~ 18

Author(s):

Cristina Lupu ◽

Xiaohong Hu ◽

Florea Lupu

Keyword(s):

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Factor Viia ◽

Protease Production ◽

Extrinsic Pathway ◽

Caveolin 1 ◽

Tissue Factor Pathway ◽

Quaternary Complex ◽

And Function

Tissue factor pathway inhibitor (TFPI) blocks tissue factor-factor VIIa (TF-FVIIa) activation of factors X and IX through the formation of the TF-FVIIa-FXa-TFPI complex. Most TFPI in vivo associates with caveolae in endothelial cells (EC). The mechanism of this association and the anticoagulant role of caveolar TFPI are not yet known. Here we show that expression of caveolin-1 (Cav-1) in 293 cells keeps TFPI exposed on the plasmalemma surface, decreases the membrane lateral mobility of TFPI, and increases the TFPI-dependent inhibition of TF-FVIIa. Caveolae-associated TFPI supports the co-localization of the quaternary complex with caveolae. To investigate the significance of these observations for EC we used RNA interference to deplete the cells of Cav-1. Functional assays and fluorescence microscopy revealed that the inhibitory properties of TFPI were diminished in EC lacking Cav-1, apparently through deficient assembly of the quaternary complex. These findings demonstrate that caveolae regulate the inhibition by cell-bound TFPI of the active protease production by the extrinsic pathway of coagulation.

Download Full-text

Lipid rafts are necessary for tonic inhibition of cellular tissue factor procoagulant activity

Blood ◽

10.1182/blood-2003-07-2399 ◽

2004 ◽

Vol 103 (8) ◽

pp. 3038-3044 ◽

Cited By ~ 43

Author(s):

Dennis J. Dietzen ◽

Keith L. Page ◽

Tina A. Tetzloff

Keyword(s):

Tissue Factor ◽

Lipid Rafts ◽

Factor Viia ◽

Cellular Tissue ◽

Procoagulant Activity ◽

Hek293 Cells ◽

Factor Activity ◽

Tissue Factor Activity ◽

Factor Antigen

Abstract A fraction of total cellular tissue factor procoagulant activity remains masked or “encrypted” in intact cells. Decryption of this activity partly involves the extracellular exposure of anionic phospholipids such as phosphatidylserine. Because of the potential association of tissue factor and phospholipid scramblase activity with lipid rafts, we have explored the role of lipid rafts in regulating factor VIIa/tissue factor activity. In HEK293 cells, tissue factor antigen was not stably associated with lipid rafts, yet disruption of rafts with methyl-β-cyclodextrin resulted in a 3-fold stimulation of tissue factor procoagulant activity. Treatment with methyl-β-cyclodextrin was not associated with cytotoxicity and did not result in the exposure of additional tissue factor antigen. Factor VIIa/tissue factor activity decrypted with methyl-β-cyclodextrin was quantitatively similar to that obtained by using lytic concentrations of octyl glucoside but more sensitive to inhibition by cell surface tissue factor pathway inhibitor and the phospholipid binding protein, annexin V. Partial decryption of tissue factor was achieved with methyl-β-cyclodextrin prior to complete disruption of lipid rafts, suggesting the role of an enzyme localized to lipid rafts in the transbilayer transport of phosphatidylserine. We conclude that lipid rafts are required for the maintenance of cellular tissue factor in an encrypted state. (Blood. 2004;103:3038-3044)

Download Full-text

Novel protein ADTRP regulates TFPI expression and function in human endothelial cells in normal conditions and in response to androgen

Blood ◽

10.1182/blood-2011-05-355370 ◽

2011 ◽

Vol 118 (16) ◽

pp. 4463-4471 ◽

Cited By ~ 39

Author(s):

Cristina Lupu ◽

Hua Zhu ◽

Narcis I. Popescu ◽

Jonathan D. Wren ◽

Florea Lupu

Keyword(s):

Tissue Factor ◽

Factor Viia ◽

Dependent Manner ◽

Uncharacterized Protein ◽

Over Expression ◽

Caveolin 1 ◽

Tissue Factor Pathway ◽

And Function ◽

Triton X ◽

Expression And Activity

Abstract Thrombosis and cardiovascular disease (CVD) represent major causes of morbidity and mortality. Low androgen correlates with higher incidence of CVD/thrombosis. Tissue Factor Pathway Inhibitor (TFPI) is the major inhibitor of tissue factor-factor VIIa (TF-FVIIa)–dependent FXa generation. Because endothelial cell (EC) dysfunction leading to vascular disease correlates with low EC-associated TFPI, we sought to identify mechanisms that regulate the natural expression of TFPI. Data mining of NCBI's GEO microarrays revealed strong coexpression between TFPI and the uncharacterized protein encoded by C6ORF105, which is predicted to be multispan, palmitoylated and androgen-responsive. We demonstrate that this protein regulates both the native and androgen-enhanced TFPI expression and activity in cultured ECs, and we named it androgen-dependent TFPI-regulating protein (ADTRP). We confirm ADTRP expression and colocalization with TFPI and caveolin-1 in ECs. ADTRP-shRNA reduces, while over-expression of ADTRP enhances, TFPI mRNA and activity and the colocalization of TF-FVIIa–FXa-TFPI with caveolin-1. Imaging and Triton X-114–extraction confirm TFPI and ADTRP association with lipid rafts/caveolae. Dihydrotestosterone up-regulates TFPI and ADTRP expression, and increases FXa inhibition by TFPI in an ADTRP- and caveolin-1-dependent manner. We conclude that the ADTRP-dependent up-regulation of TFPI expression and activity by androgen represents a novel mechanism of increasing the anticoagulant protection of the endothelium.

Download Full-text

The Factor VIII Bypassing Activity of Prothrombin Complex Concentrates: The Roles of Factor VIla and of Endothelial Cell Tissue Factor

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646459 ◽

1991 ◽

Vol 66 (05) ◽

pp. 559-564 ◽

Cited By ~ 4

Author(s):

Jerome M Teitel

Keyword(s):

Factor Viii ◽

Tissue Factor ◽

Factor Viia ◽

Factor Xa ◽

Procoagulant Activity ◽

Tissue Factor Expression ◽

Prothrombin Complex Concentrates ◽

Cell Tissue ◽

Factor Expression ◽

Necrosis Factor

SummaryAn experimental model incorporating cultured endothelial cells (EC) was used to study the "factor VIII bypassing" activity of prothrombin complex concentrates (PCC), a property exploited in the treatment of hemophiliacs with alloantibodies to factor VIII. Two PCC preparations were ineffective as stimuli of tissue factor expression by EC. However, incubation with a combination of PCC plus endotoxin (lipopolysaccharide, LPS) or tumor necrosis factor (TNF) induced much greater tissue factor expression than was seen in response to either substance alone. PCC expressed an additional direct procoagulant activity at the EC surface, which could not be attributed to either thrombin or factor Xa, and which was diminished by an anti-tissue factor antibody. Therefore factor VIIa, which was detectable in both PCC preparations, likely provided this additional direct procoagulant activity at the EC surface. We also excluded the possibility that coagulation proteases contained in or generated in the presence of PCC are protected from inactivation by AT III. Therefore, PCC can indirectly bypass factor VIII by enhancing induced endothelial tissue factor expression, and also possess direct procoagulant activity, probably mediated by factor VIIa.

Download Full-text

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

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648973 ◽

1994 ◽

Vol 72 (06) ◽

pp. 848-855 ◽

Cited By ~ 34

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.

Download Full-text

Endotoxin-induced tissue factor messenger RNA in human monocytes is negatively regulated by a cyclic AMP-dependent mechanism

Blood ◽

10.1182/blood.v81.4.973.973 ◽

1993 ◽

Vol 81 (4) ◽

pp. 973-979 ◽

Cited By ~ 1

Author(s):

V Ollivier ◽

S Houssaye ◽

C Ternisien ◽

A Leon ◽

H de Verneuil ◽

...

Keyword(s):

Cyclic Amp ◽

Tissue Factor ◽

Messenger Rna ◽

Factor Viia ◽

Functional Expression ◽

Mrna Levels ◽

Proteolytic Activation ◽

Protein Levels ◽

Tf Gene ◽

Camp Levels

Abstract Tissue factor (TF) is a transmembrane receptor that serves as the major cofactor for factor VIIa-catalyzed proteolytic activation of factors IX and X. In response to bacterial lipopolysaccharide (LPS), monocytes transcribe, synthesize, and express TF on their surface, thereby conveying to activated monocytes the ability to initiate the blood coagulation protease cascades. Agents that elevate cellular cyclic AMP (cAMP) inhibit the functional expression of TF by LPS-stimulated monocytes. In this study, we investigated the mechanism of this suppression. Northern blot analysis of total RNA from LPS-stimulated monocytes showed a concentration-dependent decrease in TF messenger RNA (mRNA) levels in response to dibutyryl-cAMP (dBt-cAMP). TF mRNA and procoagulant activity were inhibited as early as 1 hour after the addition of dBt-cAMP and the inhibition persisted through 4 hours. Suppression of specific mRNA abundance was also observed with agents, including forskolin and iso-butyl-methyl-xanthine (IBMX), that increase cAMP levels by independent mechanisms. Flow immunocytometric analysis confirmed that cell-surface TF protein levels declined in parallel with TF functional activity. The rate of decay of TF mRNA after the arrest of transcription by actinomycin D was not altered by the addition of dBt-cAMP, IBMX, or forskolin, thus excluding effects on TF mRNA stability. We conclude that elevated cAMP levels suppress TF mRNA by reducing the rate of TF gene transcription.

Download Full-text

Modulation of Tissue Factor-Factor VIIa Signaling by Lipid Rafts and Caveolae.

Blood ◽

10.1182/blood.v108.11.1744.1744 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1744-1744

Author(s):

Vineet Awasthi ◽

Samir Mandal ◽

Veena Papanna ◽

L. Vijaya Mohan Rao ◽

Usha Pendurthi

Keyword(s):

Cell Signaling ◽

Lipid Rafts ◽

G Proteins ◽

Intracellular Signaling ◽

Factor Viia ◽

Membrane Microdomains ◽

Caveolin 1 ◽

Intracellular Signaling Pathways ◽

Plasma Membrane Microdomains

Abstract Tissue factor (TF) is a cellular receptor for clotting factor VIIa (VIIa) and the formation of TF-VIIa complexes on cell surfaces not only triggers the coagulation cascade but also transduces cell signaling via activation of protease-activated receptors (PARs), particularly PAR2. Although a number of recent studies provide valuable information on intracellular signaling pathways that are activated by TF-VIIa, the role of various cell surface components in mediating the interaction of TF-VIIa with PARs, and the subsequent signal transmittance are unknown. Unlike thrombin and trypsin, VIIa has to bind to its cellular receptor (TF) to activate PARs. The inability of TF-VIIa to effectively activate Ca2+ signaling and failure to desensitize the signaling to subsequently added trypsin suggest that the TF-VIIa is a poor activator of PAR2. Despite this, a number of studies have shown that VIIa is as effective as trypsin or PAR2 agonist peptide in activating intracellular signaling pathways and gene expression in cells expressing TF. Although the potential mechanism for this phenomenon is unknown, compartmentalization of TF, PAR2, and G-proteins in plasma membrane microdomains could facilitate a robust TF-VIIa-induced PAR2-mediated cell signaling. Although certain G-protein coupled receptors and G-proteins are known to be segregated into specialized membrane microdomains, lipid rafts and caveolae, little is known whether PARs are segregated into lipid rafts and caveolae, and how such segregation might influence their activation by TF-VIIa and the subsequent coupling to G-proteins. To obtain answers to some of these questions, in the present study, we have characterized TF and PAR2 distribution on tumor cell surfaces and investigated the role of lipid raft/caveolae in modulating the TF-VIIa signaling in tumor cells. Detergent extraction of cells followed by fractionation on sucrose gradient centrifugation showed that TF and PAR2 were distributed both in lipid rafts (low-density) and soluble fractions. Immunofluorescence confocal microscopy revealed that TF at the cell surface is localized in discrete plasma membrane microdomains, and colocalized with caveolin-1, a structural integral protein of caveolae, indicating caveolar localization of TF. Similar to TF, PAR2 also displayed significant punctuate staining and colocalization with caveloin-1. Further, a substantial fraction of TF and PAR2 was colocalized in caveolae. Disruption of lipid rafts/caveolae by ß-methyl cyclodextrin or filipin treatments reduced TF association with PAR2 in lipid rafts and caveolar fractions and impaired the TF-VIIa-induced cell signaling (PI hydrolysis and IL-8 gene expression). Additional studies showed that both mßCD and filipin treatments specifically impaired TF-VIIa cleavage of PAR2 and but had no significant effect on trypsin cleavage of PAR2. Disruption of caveolae with caveolin-1 silencing had no effect on the TF-VIIa coagulant activity but inhibited the TF-VIIa-induced cell signaling. In summary, the data presented herein demonstrate that TF localization at the cell membrane could influence different functions of TF differently. While caveolar localization of TF had no influence in propagating the procoagulant activity of TF, it is essential in supporting the TF-VIIa-induced cell signaling.

Download Full-text