Cellular Effects of Heparin on the Production and Release of Tissue Factor Pathway Inhibitor in Human Endothelial Cells in Culture

Lupu, Cristina; Poulsen, E.; Roquefeuil, Sylvie B.; Westmuckett, Andrew D.; Kakkar, Vijay V.; Lupu, Florea

doi:10.1161/01.atv.19.9.2251

Cited by 112 publications

(123 citation statements)

References 61 publications

Supporting

Mentioning

113

Contrasting

Unclassified

Order By: Relevance

“…Proteolytic Activity of Cell Surface TF-FVIIa-Confluent monolayers in 24-well plates were tested with a two-stage chromogenic assay (12,16). 50 g/ml cycloheximide was added to prevent de novo protein synthesis.…”

Section: Methodsmentioning

confidence: 99%

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

Lupu

2005

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

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.Tissue factor (TF) 1 is a transmembrane protein that triggers blood coagulation in vivo. Assembly of TF with factor VIIa (FVIIa) on cell surfaces initiates limited proteolysis of factors IX and X (FX), leading to thrombin generation. TF elicits thrombogenic responses in septicemia, cancer, and atherosclerosis (1-4), promotes metastasis, angiogenesis, and intima hyperplasia after arterial injury (5, 6), and acts as signaling receptor upon binding of FVIIa (7).Tissue factor pathway inhibitor (TFPI) is the endogenous regulator of TF-FVIIa activity. TFPI is a Kunitz-type protease inhibitor, which binds FVIIa via Kunitz-1 and FXa via Kunitz-2. Formation of the TF-FVIIa-FXa-TFPI complex provides sustained repression of the TF pathway (8). TFPI was first described as a soluble plasma protein that binds through its C terminus to yet uncharacterized anionic sites on the cell surface. Nevertheless, the majority of TFPI circulating in plasma is C-terminal truncated and bound to plasma lipoproteins, and as such, has significantly less inhibitory activity than the full-length TFPI (9).TFPI in vivo is mainly produced by endothelial cells (EC), has an intact C terminus, and associates with the plasma membrane through mechanisms that are not fully identified (3, 10 -13). Heparin releases a portion of the full-length, functionally active cell-associated TFPI, either from cell surface-binding sites or from intracellular stores (14, 15). However, the bulk of heparin-resistant cellular TFPI is released by phosphatidylinositol-phospholipase C in vitro, which indicates that most cellular TFPI associates with the cell surface via a glycosylphosphatidylinositol link (10 -12, 16, 17). Furthermore, endogenous TFPI in resting endothelium (10), monocytes (18), and the ECV304 cell line (11, 16) partitions in low-density fractions insoluble in cold detergent (lipid rafts). In monocytes and ECV304 endogenous TFPI inhibits efficiently FVIIa-TF...

show abstract

Section: Methodsmentioning

confidence: 99%

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

Lupu

2005

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…Heparin induces TFPI synthesis and secretion by endothelial cells [44,45], and causes the displacement of TFPI bound to cell membranes. The inhibitory effects of TFPI on the Fxa-TF-FVIIa complex are enhanced significantly in the presence of heparin [46].…”

Section: ó 2004 Blackwell Publishing Ltdmentioning

confidence: 99%

Tissue factor and tissue factor pathway inhibitor

Price

Thompson²,

Kam

2004

Anaesthesia

View full text Add to dashboard Cite

SummaryThe classical 'cascade ⁄ waterfall' hypothesis formulated to explain in vitro coagulation organised the amplification processes into the intrinsic and extrinsic pathways. Recent molecular biology and clinical data indicate that tissue factor ⁄ factor-VII interaction is the primary cellular initiator of coagulation in vivo. The process of blood coagulation is divided into an initiation phase followed by a propagation phase. The discovery of tissue factor pathway inhibitor further supports the revised theory of coagulation. Tissue factor is also a signalling receptor. Recent evidence has shown that blood-borne tissue factor has an important procoagulant function in sepsis, atherosclerosis and cancer, and other functions beyond haemostasis such as immune function and metastases.

show abstract

“…11,12 The form of TFPI that is released is full-length TFPI␣. 13 Heparin also increases the release of TFPI from endothelial cells in culture, 6,14 even though treatment with heparin does not perceptibly reduce surface TFPI. 6,15,16 The endothelium appears to be the major source of TFPI in vivo.…”

Section: Introductionmentioning

confidence: 99%

TFPIβ is the GPI-anchored TFPI isoform on human endothelial cells and placental microsomes

Girard

Tuley

Broze

2012

Blood

View full text Add to dashboard Cite

native C-terminus that directs the attachment of a glycosylphosphatidylinositol (GPI) anchor, but whether TFPI␤ protein is actually expressed is not clear. Moreover, previous studies have suggested that the predominant form of TFPI released from cells by phosphatidylinositolspecific phospholipase C (PIPLC) treatment is TFPI␣, implying it is bound at cell surfaces to a separate GPI-anchored coreceptor. Our studies show that the form of TFPI released by PIPLC treatment of cultured endothelial cells and placental microsomes is actually TFPI␤ based on (1) migration on SDS-PAGE before and after deglycosylation, (2) the lack of a Kunitz-3 domain, and ( IntroductionTissue factor pathway inhibitor (TFPI) is the key endogenous regulator of TF-initiated coagulation. TFPI inhibits factor Xa (FXa) directly, and, in a FXa-dependent manner, inhibits factor VIIa/tissue factor (FVIIa/ TF) activity. 1 The human TFPI gene encodes for 2 dominant messages that are generated by alternative intron/exon splicing (GenBank NM_006287.4, GenBank NM_001032281.2). TFPI␣, the originally described message, encodes a signal peptide that is removed by processing, followed by a 276-amino acid protein that consists of an acidic N-terminal region, 3 tandem Kunitz-type protease inhibitor domains, and a basic C-terminal region. 2 Functional studies reveal that the Kunitz-2 domain mediates binding to and inhibition of FXa, whereas the Kunitz-1 domain is responsible for recognition and inhibition of the TF/FVIIa complex. 3 The Kunitz-3 domain lacks protease inhibitor activity; however, it and the C-terminal basic region of TFPI␣ are required for protein S enhancement of inhibition of FXa. 4,5 The TFPI␤ message lacks the Kunitz-3 and C-terminal exons of TFPI␣, and in their place is an exon that encodes a 42-amino acid C-terminal sequence following residue 181 of TFPI␣. This new C-terminal sequence is predicted to direct the proteolytic cleavage of the peptide following N193 with the attachment of a GPI anchor. The protein mass of TFPI␤ is less than that of TFPI␣, but it migrates on SDS-PAGE at a molecular weight similar to TFPI␣ (46 kDa) apparently because of greater sialylation of its O-linked carbohydrate. 6 In humans, TFPI circulates in plasma at approximately 70 ng/mL (1.6nM), with 80% being a C-terminal truncated form that is lipoprotein associated and the remaining 20% being full-length and near full-length TFPI␣ forms. 7,8 Platelets contain TFPI␣ and, at the site of injury TFPI␣ levels, increase dramatically. 9,10 The parenteral administration of heparin to humans increases the circulating levels of total TFPI in plasma to approximately 2.5-fold baseline levels. 11,12 The form of TFPI that is released is full-length TFPI␣. 13 Heparin also increases the release of TFPI from endothelial cells in culture, 6,14 even though treatment with heparin does not perceptibly reduce surface TFPI. 6,15,16 The endothelium appears to be the major source of TFPI in vivo. In human endothelial cell cultures, the ratio of TFPI␣/TFPI␤ mRNA varies between 5 and 10. 1...

show abstract

Cellular Effects of Heparin on the Production and Release of Tissue Factor Pathway Inhibitor in Human Endothelial Cells in Culture

Cited by 112 publications

References 61 publications

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

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

Tissue factor and tissue factor pathway inhibitor

TFPIβ is the GPI-anchored TFPI isoform on human endothelial cells and placental microsomes

Contact Info

Product

Resources

About