This Article Abstract Full Text (PDF) All Versions of this Article: 108/5/623    most recent 01.CIR.0000078642.45127.7Bv1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zhang, J. Articles by Broze, G. J. Search for Related Content PubMed PubMed Citation Articles by Zhang, J. Articles by Broze, G. J., Jr Related Collections Coagulation inhibitors

(Circulation. 2003;108:623.)
© 2003 American Heart Association, Inc.

Basic Science Reports

Glycosyl Phosphatidylinositol Anchorage of Tissue Factor Pathway Inhibitor

Jing Zhang, PhD; Orlando Piro, MD; Lan Lu; George J. Broze, Jr, MD

From the Division of Hematology, Barnes-Jewish Hospital at Washington University Medical Center, St Louis, Mo.

Correspondence to George J. Broze, Jr, MD, BJH 90-20-66, 216 South Kingshighway Blvd, St Louis, MO 63110. E-mail gbroze{at}im.wustl.edu

Received January 30, 2003; revision received April 7, 2003; accepted April 19, 2003.

	Abstract

Top Abstract Introduction Methods Results and Discussion References

 
Background— The endothelium is a major source of tissue factor pathway inhibitor (TFPI), the endogenous regulator of TF-induced coagulation, and a significant proportion of the expressed TFPI remains associated with the endothelial surface.

Methods and Results— Phosphatidylinositol-specific phospholipase C (PI-PLC) treatment reduced TFPI at the surface of cultured endothelial cells by 80%, and at least a portion of the TFPI released by PI-PLC contained an intrinsic glycosylphosphatidylinositol (GPI) anchor that is recognized by anti-crossreactive determinant antibodies. Endothelial cells express both of the alternatively spliced forms of TFPI mRNA at a ratio of TFPIß/TFPI mRNA of 0.1 to 0.2. In Chinese hamster ovary (CHO) cells, TFPI is predominantly secreted, whereas TFPIß is a GPI-anchored membrane protein. Like TFPIß, the small proportion of the TFPI expressed by CHO cells that remains surface associated is also released by PI-PLC treatment, suggesting that it is bound to a separate GPI-anchored protein(s) at the surface of the cells.

Conclusions— Both direct (TFPIß) and indirect (TFPI) GPI-mediated membrane anchorage is involved in localizing TFPI to the surface of cells.

Key Words: coagulation • inhibitors • endothelium-derived factors

	Introduction

Top Abstract Introduction Methods Results and Discussion References

 
Tissue factor pathway inhibitor (TFPI) is the endogenous regulator of TF-induced coagulation. TFPI directly binds and inhibits activated factor X (FXa), and in an FXa-dependent fashion, produces feedback inhibition of the FVIIa/TF catalytic complex. TFPI is an 43-kDa glycoprotein that contains an acidic N-terminal region followed by 3 tandem, Kunitz-type protease inhibitor domains and a basic C-terminal region.¹ TFPIß is an alternatively spliced form of TFPI that lacks the Kunitz-3 and C-terminus of TFPI and instead contains an unrelated C-terminal amino acid sequence.² TFPIß mRNA in the mouse has a similar tissue distribution as mRNA for TFPI, but the encoded TFPIß protein has not been detected.² FXa-dependent feedback inhibition of FVIIa/TF by TFPI involves the formation of a quaternary FXa-TFPI-FVIIa/TF complex. It is the second Kunitz domain of TFPI that mediates the binding and inhibition of FXa, whereas the first Kunitz domain is responsible for the inhibition of FVIIa bound to TF. Presumably by localizing the molecule to cell surfaces through interactions with anionic phospholipids, glycosaminoglycans, or surface proteins, the C-terminus of TFPI plays an important role in its inhibitory functions, and C-terminally truncated forms of TFPI are less potent inhibitors of FXa and FVIIa/TF.^3,4

There is a broad range of TFPI concentrations in the plasma of normal individuals (mean, 2.0 nmol/L), and platelets carry 8% of the total blood TFPI. Most of the plasma TFPI circulates as a C-terminally truncated form(s) that is bound to plasma lipoproteins. The major form carried by LDL has a molecular weight of 34 kDa and lacks at least a large portion of the Kunitz-3 domain and the C-terminus of TFPI. The 41-kDa form of TFPI that circulates with HDL appears to be a similar C-truncated form of TFPI that is disulfide linked to monomeric apolipoprotein-AII. Additional forms of TFPI with less extensive C-terminal truncation and a small amount of full-length TFPI also circulate in plasma.⁵

The endothelium is presumed to be the major source of TFPI in vivo.⁶ A significant fraction of the TFPI produced by cultured endothelial cells and present in placental tissue remains membrane associated.^7–11 In vitro heparin induces the release of a portion of this cell-associated TFPI,^7–9,11 and in vivo heparin treatment increases the plasma level of TFPI by 1.5- to 2.5-fold.¹² This process could represent the release of TFPI from cell-surface binding sites or the secretion of TFPI from intracellular storage sites.^13,14 The bulk of the heparin-resistant, cell-associated TFPI is released by phosphatidylinositol-specific phospholipase C (PI-PLC) treatment in vitro, suggesting that this form of TFPI contains a glycosyl phosphatidylinositol (GPI) membrane anchor or is tightly bound to a surface GPI-containing protein.^9,11,15,16 Because cell-bound TFPI is believed to play a critical role in regulating surface FVIIa/TF and FXa activity, the following experiments were undertaken to further define the mechanism(s) responsible for the association of TFPI with cell membranes.^7,9,11,17,18

	Methods

Top Abstract Introduction Methods Results and Discussion References

 
Materials
Human mammalian gene collection clone 9251 (mgc:9251; image:3902987; gb:BC015514; gi:15930155) containing TFPIß cDNA and the human umbilical vein cell (HUVEC)–derived line ECV304 were obtained from American Tissue Culture Collection (ATCC, Manassas, Va). The HUVEC-derived cell line EA.hy926 was a kind gift from Cora-Jean Edgell (University of North Carolina, Chapel Hill), and HUVECs were purchased from Clonetics BioWhittaker (Walkersville, Md). The Expand high-fidelity polymerase chain reaction (PCR) system and the Lumi-Light Western blotting substrate were from Roche. Dulbecco’s modified Eagle’s medium (DMEM), nonessential amino acids, fetal bovine serum (FBS), and Chinese hamster ovary (CHO)-S cells were from Invitrogen (Carlsbad, Calif). PI-PLC, horseradish peroxidase–labeled goat anti-rabbit immunoglobulin G (IgG) antibodies, Tris, EDTA, and Triton X-114 were from Sigma Chemical. Prestained molecular-weight standards and Affigel-10 were from Bio-Rad. Oligonucleotides were synthesized by the Protein Chemistry Laboratory at Washington University School of Medicine. Anti–cross-reactive determinant (anti-CRD) antiserum was a kind gift from Paul Englund (Johns Hopkins University, Baltimore, Md). The production and characterization of monoclonal anti-TFPI antibodies Mab2H8 and Mab2B12 and of rabbit polyclonal anti-TFPI antisera have been previously described.⁵

Cell Culture
HUVECs were cultured in essential growth medium-2 (BioWhittaker) supplemented with 2% FBS and studied between passages 3 and 6. ECV304 cells were cultured in medium 199 (BioWhittaker) supplemented with 1 mmol/L pyruvate and 10% FBS, and EA.hy926 cells were cultured in DMEM (BioWhittaker) with 1 mmol/L pyruvate and 10% FBS. CHO-S cells were routinely maintained in DMEM supplemented with 0.1 mmol/L nonessential amino acids and 10% FBS. All cells were cultured at 37°C with 5% CO₂, and all media contained penicillin (50 U/mL) and streptomycin (50 µg/mL).

Expression of TFPI and TFPIß in CHO-S Cells
Human TFPI cDNA was amplified from plasmid pBS-SK/TFPI¹⁹ by using forward primer 5'-CAC CAT GAT TTA CAC AAT GAA GAA AGT ACA-3' and reverse primer 5'-TCA CAT ATT TTT AAC AAA AAT TTC TTC ATA-3'. Human TFPIß cDNA was amplified from the mgc:9251 clone by using forward primer 5'-CAC CAT GAT TTA CAC AAT GAA GAA AGT ACA-3' and reverse primer 5'-TTA ACA TAG GCA TGA AAT GCT ATC CCA TCC-3'. The TFPI and TFPIß cDNA fragments were cloned into pcDNA3.1D/V5-His-TOPO (Invitrogen) in a manner to prevent expression of the C-terminal V5 and His tags. The direction and sequence of the cDNAs were confirmed by double-strand sequencing. CHO-S cells were transiently transfected with the TFPI and TFPIß pcDNA3.1 constructs by using LipofectAMINE 2000 in a 24-well plate, according to the manufacturer’s protocol (Invitrogen). Stable clones expressing TFPI (CHO-TFPI) and TFPIß (CHO-TFPIß) were isolated in a similar fashion by using G418 selection.

Triton X-114 Fractionation of Membrane-Associated Protein
Forty-eight hours after transfection, CHO-S cells cultured in CD CHO serum-free medium supplemented with hypoxanthine and thymidine (Invitrogen) were detached with cell dissociation buffer (Invitrogen) and washed with phosphate-buffered saline (PBS). Cells (5x10⁵/mL) were lysed with 1% Triton X-114 in 0.1 mol/L Tris-HCl, pH 7.5, and 10 mmol/L EDTA with repeated mixing on ice for 15 minutes. Cell debris was removed by centrifugation at 14 000g x2 minutes. at 4°C. Phase separation was induced by incubation of the cleared detergent lysate for 5 minutes at 37°C, followed by brief centrifugation at 14 000g at room temperature to separate the detergent and aqueous phases. The detergent pellicle was extracted with acetone, and the membrane proteins were resuspended in reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. The detergent extract derived from 2.5x10⁵ cells was loaded in each lane for separation by 10% SDS-PAGE and Western blotting with rabbit polyclonal anti-TFPI antibodies with chemiluminescent detection.

Quantitative RT-PCR–Based Gene Expression Analysis
Real-time reverse transcription (RT)-PCR was used to determine the relative amount of TFPI and TFPIß mRNA in HUVE, ECV304, and EA.hy926 cells. Primers for human TFPI and TFPIß were designed by using Primer Express software (Applied Biosystems). For TFPI, the forward and reverse primers were 5'-TAT GGA ACC CAG CTC AAT GCT-3' and 5'-ACA CCA TGA GGG ACC GTG AA-3', respectively. For TFPIß, the forward and reverse primers were 5'-CCA AGG TTC CAA GCC TTT TTG-3' and 5'-ATG AAT GCA GAA GGC GTT CAG-3', respectively. Total RNA was isolated from the cells with the RNeasy mini-kit and QIAShredder (Qiagen) and treated with reagents from the RNase-free DNase set (Qiagen). For each sample, 2 µg total RNA was reverse-transcribed by using the SuperScript first-strand synthesis system (Invitrogen) with oligo-dT primers. TFPI and TFPIß real-time PCR was performed in separate tubes in triplicate with 1 µL of the 20-µL RT reaction products by using the SYBR green dye kit in an ABI Prism 5700 sequence detector (Applied Biosystems). RT-PCR and subsequent calculations with the average threshold cycle method were performed, following the manufacturer’s protocols (Applied Biosystems).

Determination of Cellular TFPI Expression
CHO-TFPI, CHO-TFPIß, HUVE, ECV304, and EA.hy926 endothelial cells were allowed to grow to 90% confluence in their appropriate media. Complete medium was then withdrawn, and the cells were incubated overnight in the appropriate serum-free medium. The cells were washed 3 times in PBS, harvested in unicellular suspension by treatment with cell dissociation buffer (Invitrogen), washed again 3 times in PBS, and resuspended at 1x10⁶ cell/mL in appropriate serum-free medium. Cell suspensions (0.5 mL) were then treated at 37°C in different ways: (1) incubated in serum-free medium for 1 hour; (2) incubated with heparin (5 U/mL) for the last 20 minutes of a 1-hour incubation; (3) incubated with PI-PLC (1 U/mL) for 1 hour; and (4) incubated with heparin (5 U/mL) for 20 minutes after the cells had been incubated 1 hour with PI-PLC (1 U/mL) and washed twice. At the end of the incubations, cells were collected by centrifugation (250g x5 minutes.) for flow cytometric analysis. In the case of HUVECs, supernatants were centrifuged a second time to remove cell debris, concentrated 2.5-fold (Centricon YM10, Millipore), and stored at -20°C for subsequent TFPI immunoassay.

Flow Cytometry
Cells (5x10⁵) were resuspended in 200 µL of ice-cold PBS containing 0.08% (wt/vol) sodium azide, 25 mmol/L EDTA, and 3% (wt/vol) bovine serum albumin and incubated with mouse monoclonal anti-TFPI Kunitz-1 (Mab2H8, 5 µg/mL) for 30 minutes. After being washed 3 times with the same buffer, the cells were incubated for an additional 30 minutes on ice in the dark with phycoerythrin-conjugated F(ab)₂ sheep anti-mouse IgG (Sigma). The labeled cells were washed twice and then analyzed by fluorescence-activated cell sorting flow cytometry (FACScan, Becton Dickinson). Regions appropriate for each of the tested cell types were defined by using forward light scatterxside-angle light intensity dot plots. All measurements were performed with the same instrument settings, and at least 5x10³ events were analyzed by using CellQuest software (Becton Dickinson). Results were expressed as mean fluorescence intensity (MFI), and the difference between the surface TFPI of cells that had been treated in various ways was assessed by using Kolmogorov-Smirnov statistical analysis (CellQuest, Becton Dickinson). Cells incubated with isotype control IgG, with the primary antibody alone, with the secondary antibody alone, and with neither were used as negative controls.

Immunoaffinity Isolation of TFPI Released From ECV304 by PI-PLC
ECV304 cells that had reached confluence in a T75 culture flask were washed twice with PBS and then treated with PI-PLC (1 U/mL) in 4 mL serum-free medium at room temperature. After 1 hour, the medium was collected by filtration (0.22 µm) to remove debris and passed through a 200-µL column of Mab2H8-Affigel (2 mg/mL). After the column was washed with 0.1 mol/L NaCl and 0.05 mol/L Tris-HCl, pH 7.5, the bound TFPI was eluted with 8 mol/L urea and 0.02 mol/L Tris-HCl, pH 7.5. Twenty microliters of reduced samples was evaluated by 12% SDS-PAGE and Western blotting with chemiluminescent detection with a rabbit polyclonal anti-TFPI and anti-CRD antisera.

TFPI Immunoassay
Wells of a microtiter plate (Nunc, Fisher Scientific) were incubated overnight at room temperature with 150 µL mouse monoclonal anti-TFPI Kunitz-2 (Mab2B12, 20 µg/mL) and then washed (200 µL x4) with PBS containing 0.05% Tween 20 (PBST). Duplicate samples (100 µL) were applied to the wells and incubated for 2 hours. After the wells were washed with PBST (200 µL x4), a solution (150 µL) containing biotin-conjugated (EZ-link-Sulfo-NHS-LC-Biotin, Pierce) Mab2H8 (4 µg/mL) was incubated in the wells for 1.5 hours. The wells were washed with PBST (200 µL x4), and 150 µL of a 1:100 dilution of streptavidin–horseradish peroxidase complex (Pierce) in PBST was applied. After 1 hour, the wells were washed with PBST (200 µL x4), and 200 µL of TMB liquid substrate (Sigma) was added. After 5 minutes, 0.5 mol/L H₂SO₄ (100 µL) was added to stop the reaction, and the absorbance at 450 nm was measured with a Vmax microtiter plate reader and Spectramax Pro software (Molecular Devices). Purified, recombinant TFPI expressed in Escherichia coli²⁰ was used as the standard and produced a linear response between concentrations of 0 and 40 ng/mL.

	Results and Discussion

Top Abstract Introduction Methods Results and Discussion References

 
Endothelial Cell–Associated TFPI
The association of TFPI with endothelial cells was investigated in initial experiments (Figure 1). Heparin (5 U/mL) treatment of HUVECs increased the TFPI concentration in the medium modestly but did not reduce cell surface TFPI significantly. Thus, the TFPI released by heparin comes from intracellular stores¹³ or is removed from untreated cells during flow cytometric analysis. Previous studies have shown that this heparin-releasable TFPI is TFPI.²¹ PI-PLC (1 U/mL) treatment released substantial TFPI into the medium and reduced cell surface TFPI by 80% (P<0.001). Subsequent exposure of the cells to heparin (5 U/mL) after PI-PLC treatment released a greater amount of TFPI than did heparin treatment alone and further reduced cell surface TFPI (P<0.001). This suggests that a portion of the TFPI released by PI-PLC treatment remains associated with the cell surface in a manner that is sensitive to heparin.¹¹ Studies with ECV304 and EA.hy926 cells produced the same results (not shown), and our work with cultured endothelial cells largely mirrors that of Mast et al¹¹ using placental tissue.

View larger version (43K): [in this window] [in a new window]   Figure 1. Release of TFPI from HUVECs. HUVECs were left untreated (Cont), heparin treated (Hep), PI-PLC treated (PI-PLC), or heparin treated after PI-PLC exposure (PLC/Hep; see Methods). Open bars depict surface levels of TFPI as determined by flow cytometry. Filled bars depict TFPI released into media, as determined by immunoassay. Figure shows 1 of 5 experiments that produced same results.

To determine whether the TFPI released by PI-PLC contains a GPI anchor or is bound by a separate GPI-linked protein, ECV304 cells were washed and treated with PI-PLC (1 U/mL). The released TFPI was immunoaffinity-isolated and evaluated by Western blotting with the use of polyclonal anti-TFPI and anti-crossreactive-determinant (anti-CRD) antibodies (Figure 2A). The PI-PLC–cleaved TFPI migrated with an apparent molecular weight of 45 kDa and was recognized by both anti-TFPI and anti-CRD antibodies. The major epitope involved in anti-CRD recognition is the inositol 1,2-cyclic monophosphate that is generated on PLC cleavage of a GPI anchor.²² Recognition of a protein by anti-CRD antiserum after PI-PLC cleavage is virtually unequivocal evidence for the presence of a GPI anchor.²² Thus, at least a portion of the TFPI produced by endothelial cells contains an intrinsic GPI anchor.

View larger version (45K): [in this window] [in a new window]   Figure 2. TFPI expressed by endothelial and CHO cells. A, Western blot, with anti-TFPI (left) and anti-CRD (right) antibodies of TFPI released from ECV304 cells by PI-PLC. B, Western blot with anti-TFPI antibodies of TX114 membrane fractions from CHO cells transiently transfected with TFPI (left) and TFPIß (right) cDNAs (see Methods). Lower-molecular-weight band detected on Western blot of TFPIß might represent degradation product. Positions of prestained molecular-weight markers in kDa are also shown.

TFPIß Is an GPI-Anchored Protein
A search of the National Center for Biotechnology GenBank showed that the cDNA sequences for mouse (gb:AF016313) and human (gb:AF021834) TFPIß had been deposited by Chang et al² and that the TFPI gene (Hs2 5422, gi:2204416) contains an exon encoding the alternative C-terminal amino acid sequence of TFPIß. This exon precedes that encoding the Kunitz-3 domain of TFPI. The algorithm of A.L. Veuthey from the Swiss Institute of Bioinformatics (http://us.expasy.org/tools; select DGPI) predicts that the C-terminal amino acid sequence of TFPIß (but not of TFPI) following residue 181 of mature TFPI contains the appropriate signal to direct attachment of a GPI anchor: VTKEGTN-DGWKN*AAHIYQVFLNAFCIHASMFFLGLDSISCLC (* indicates site of GPI attachment; Figure 3). This prediction was evaluated by expressing hTFPI and hTFPIß in CHO cells. Consistent with its GPI anchorage, TFPIß, but not TFPI, was found in the Triton X-114–isolated membrane fraction of CHO cells (Figure 2B; see Methods). TFPI is secreted by CHO-TFPI cells, whereas the TFPIß produced by CHO-TFPIß cells is not, with levels of TFPI in overnight-conditioned medium of 60 and <1 ng/mL, respectively. TFPIß on the surface of CHO-TFPIß cells is unaffected by heparin exposure, and it is nearly completely released by PI-PLC (P<0.001) without an apparent effect of subsequent heparin treatment (Figure 4). Only a small amount of the TFPI expressed by CHO-TFPI cells remains at the cell surface. The TFPI that is cell associated, however, is also resistant to heparin treatment and reduced by PI-PLC (P<0.001). In contrast to TFPIß, the surface TFPI of CHO-TFPI cells is further reduced by heparin treatment after PI-PLC (P<0.001; Figure 4, insert).

View larger version (10K): [in this window] [in a new window]   Figure 3. Schematic of structures of TFPI and TFPIß. Shaded boxes denote Kunitz domains 1, 2, and 3. Amino acid sequences of TFPI and TFPIß are identical through residue 181 of mature proteins. Thereafter, TFPIß contains alternative C-terminal sequence that directs appropriate cleavage and attachment of GPI anchor.

View larger version (48K): [in this window] [in a new window]   Figure 4. Surface TFPI and TFPIß expressed in CHO cells. Stable clones of CHO-TFPI (open bars) and CHO-TFPIß (filled bars) cells were left untreated (Cont), heparin treated (Hep), PI-PLC treated (PI-PLC), or heparin treated after PI-PLC exposure (PLC/Hep) in parallel (see Methods). Concentrations of TFPI in media of CHO-TFPI and CHO-TFPIß cells after overnight culture in serum-free media were 60 ng/mL and <1 ng/mL, respectively. MFI values in figure are means of 2 experiments. Insert, Expanded view of surface TFPI and TFPIß after various treatments, normalized by assigning surface MFI of control cells value of 100%.

GPI-Linked Endothelial TFPI
HUVE, ECV304, and EA.hy926 cells contain both TFPI and TFPIß mRNA by quantitative RT-PCR at a ratio of TFPIß/TFPI mRNA of 0.08, 0.17, and 0.20, respectively. The extent to which TFPI and TFPIß contribute to the PI-PLC–releasable TFPI on these endothelial cells, however, is unclear. Both TFPI, presumably bound to a separate GPI-linked protein(s), and TFPIß are released by PI-PLC treatment (Figure 4). The proteins cannot be distinguished by size, because the TFPI and TFPIß expressed by CHO cells and the TFPI released by PI-PLC from HUVE, ECV304, and EA.hy926 endothelial cells all migrate with the same apparent molecular weight (45 kDa) on SDS-PAGE gels (Figure 2 and not shown). Only a small amount of the TFPI produced by CHO-TFPI cells remains at the cell surface, but the putative GPI-linked protein(s) with which TFPI associates might be expressed at a much higher concentration on endothelial cells than on CHO cells. Previous publications have reported the recognition of endothelial surface and placental TFPI by anti-peptide antibodies against the Kunitz-3 domain²³ and the C-terminus¹¹ of TFPI, respectively. Thus, at least some of the endothelial surface TFPI appears to be TFPI. Our studies, however, suggest that TFPIß might also contribute significantly to the surface TFPI of endothelial cells (Figure 2A).

In endothelial cells, post–PI-PLC treatment with heparin further decreased cell surface TFPI (Figure 1). This phenomenon was seen in CHO-TFPI cells, in which only a small quantity of TFPI remained cell associated, but not in CHO-TFPIß cells (Figure 4). At first glance, this result would suggest that the Kunitz-3 domain and/or C-terminus present in TFPI, but not TFPIß, are required for the heparin-inhibitable interaction of TFPI with the cell surface after PI-PLC treatment. A firm conclusion, however, cannot be drawn because of the dramatically different surface levels of TFPI and TFPIß and the possibility that the CHO-TFPIß cells might have expressed additional surface TFPI during the course of the experiment.

Deletion of the TFPI Kunitz-1 domain in mice, which affects the function of both TFPI and TFPIß, leads to a consumptive coagulopathy and intrauterine lethality.²⁴ The relative physiological importance of TFPI and TFPIß, however, is not known. Ongoing studies characterizing mice individually lacking TFPI or TFPIß should address this issue.

	Acknowledgments

 
This work was supported in part by a grant (HL60782) from the National Heart, Lung, and Blood Institute (Bethesda, Md).

	References

Top Abstract Introduction Methods Results and Discussion References

 

1. Wun T-C, Kretzmer KK, Girard TJ, et al. Cloning and characterization of a cDNA coding for the lipoprotein-associated coagulation inhibitor shows that it consists of three tandem Kunitz-type inhibitory domains. J Biol Chem. 1988; 263: 6001–6004.[Abstract/Free Full Text]
   
2. Chang JY, Monroe DM, Oliver JA, et al. TFPIß, a second product from the mouse tissue factor pathway inhibitor (TFPI) gene. Thromb Haemost. 1999; 81: 45–49.[Medline] [Order article via Infotrieve]
   
3. Wesselschmidt R, Likert K, Girard T, et al. Tissue factor pathway inhibitor: the carboxy-terminus is required for optimal inhibition of factor Xa. Blood. 1992; 79: 2004–2010.[Abstract/Free Full Text]
   
4. Lindhout T, Franssen J, Willems G. Kinetics of the inhibition of tissue factor-factor VIIa by tissue factor pathway inhibitor. Thromb Haemost. 1995; 74: 910–915.[Medline] [Order article via Infotrieve]
   
5. Broze GJ Jr, Lange GW, Duffin KL, et al. Heterogeneity of plasma tissue factor pathway inhibitor. Blood Coagulation Fibrinol. 1994; 5: 551–559.[Medline] [Order article via Infotrieve]
   
6. Werling RW, Zacharski LR, Kisiel W, et al. Distribution of tissue factor pathway inhibitor in normal and malignant human tissues. Thromb Haemost. 1993; 69: 366–369.[Medline] [Order article via Infotrieve]
   
7. Sevinsky JR, Rao LV, Ruff W. Ligand induced protease receptor translocation into caveolae: a mechanism for regulating cell surface proteolysis of the tissue factor-dependent coagulation pathway. J Cell Biol. 1996; 133: 293–304.[Abstract/Free Full Text]
   
8. Lupu C, Lupu F, Dennehy U, et al. Thrombin induces the redistribution and acute release of tissue factor pathway inhibitor from specific granules within human endothelial cells in culture. Arterioscler Thromb. 1995; 15: 2055–2062.[Abstract/Free Full Text]
   
9. Ott I, Miyagi Y, Miyazaki K, et al. Reversible regulation of tissue factor–induced coagulation by glycosyl phosphatidylinositol-anchored tissue factor pathway inhibitor. Arterioscler Thromb Vasc Biol. 2000; 20: 874–882.[Abstract/Free Full Text]
   
10. Crawley J, Lupu F, Westmuckett AD, et al. Expression, localization, and activity of tissue factor pathway inhibitor in normal and atherosclerotic human vessels. Arterioscler Thromb Vasc Biol. 2000; 20: 1362–1373.[Abstract/Free Full Text]
    
11. Mast AE, Acharya N, Malecha MJ, et al. Characterization of the association of tissue factor pathway inhibitor with human placenta. Arterioscler Thromb Vasc Biol. 2002; 22: 2099–2104.[Abstract/Free Full Text]
    
12. Sandset PM, Abildgaard U, Larsen ML. Heparin induces release of extrinsic coagulation pathway inhibitor (EPI). Thromb Res. 1988; 50: 803–813.[CrossRef][Medline] [Order article via Infotrieve]
    
13. Lupu C, Poulsen E, Roquefeuil S, et al. Cellular effects of heparin on the production and release of tissue factor pathway inhibitor in human endothelial cells in culture. Arterioscler Thromb Vasc Biol. 1999; 19: 2251–2262.[Abstract/Free Full Text]
    
14. Hansen JB, Svensson B, Olsen R, et al. Heparin induces synthesis and secretion of tissue factor pathway inhibitor from endothelial cells in vitro. Thromb Haemost. 2000; 83: 937–943.[Medline] [Order article via Infotrieve]
    
15. Lupu C, Goodwin CA, Westmuckett AD, et al. Tissue factor pathway inhibitor in endothelial cells collocalizes with glycolipid microdomains/caveolae. Arterioscler Thromb Vasc Biol. 1997; 17: 2964–2974.[Abstract/Free Full Text]
    
16. Mast AE, Higuchi DA, Huang Z-F, et al. Glypican-3 is a binding protein on the HepG2 cell surface for tissue factor pathway inhibitor. Biochem J. 1997; 327: 577–583.[Medline] [Order article via Infotrieve]
    
17. Ho G, Broze GJ Jr, Schwartz AL. Role of heparan sulfate proteoglycans in the uptake and degradation of tissue factor pathway inhibitor-coagulation factor Xa complexes. J Biol Chem. 1997; 272: 16838–16844.[Abstract/Free Full Text]
    
18. Iakhiaev A, Pendurthi U, Voigt J, et al. Catabolism of factor VIIa bound to tissue factor in fibroblasts in the presence and absence of tissue factor pathway inhibitor. J Biol Chem. 1999; 274: 36995–37003.[Abstract/Free Full Text]
    
19. Girard TJ, Warren LA, Novotny WF, et al. Identification of the 1.4 kb and 4.0 kb messages for the lipoprotein-associated coagulation inhibitor and expression of the encoded protein. Thromb Res. 1989; 55: 37–50.[CrossRef][Medline] [Order article via Infotrieve]
    
20. Warshawsky I, Broze GJ Jr, Schwartz AL. The low density lipoprotein receptor-related protein mediates the cellular degradation of tissue factor pathway inhibitor. Proc Natl Acad Sci U S A. 1994; 91: 6664–6668.[Abstract/Free Full Text]
    
21. Novotny WF, Palmier M, Wun T-C, et al. Purification and properties of heparin-releasable LACI. Blood. 1991; 78: 393–400.
    
22. Hooper NM. Determination of glycosyl-phosphatidylinositol membrane protein anchorage. Proteomics. 2001; 1: 748–755.[CrossRef][Medline] [Order article via Infotrieve]
    
23. Tiemann C, Brinkmann T, Kleesiek K. Detection of the three Kunitz-type single domains of membrane bound tissue factor pathway inhibitor (TFPI) by flow cytometry. Eur J Clin Chem Clin Biochem. 1997; 35: 855–860.[Medline] [Order article via Infotrieve]
    
24. Huang Z-F, Higuchi D, Lasky, N et al. Tissue factor pathway inhibitor gene disruption produces intrauterine lethality in mice. Blood. 1997; 90: 944–951.[Abstract/Free Full Text]
    

This article has been cited by other articles:

				P. E.R. Ellery, K. Hardy, R. Oostryck, and M. J. Adams Further Insight Into the Heparin-Releasable and Glycosylphosphatidylinositol-Lipid-- Anchored Forms of Tissue Factor Pathway Inhibitor Clinical and Applied Thrombosis/Hemostasis, July 1, 2008; 14(3): 267 - 278. [Abstract] [PDF]

				J. T.B. Crawley and D. A. Lane The Haemostatic Role of Tissue Factor Pathway Inhibitor Arterioscler. Thromb. Vasc. Biol., February 1, 2008; 28(2): 233 - 242. [Abstract] [Full Text] [PDF]

				H. Tang, L. Ivanciu, N. Popescu, G. Peer, E. Hack, C. Lupu, F. B. Taylor Jr, and F. Lupu Sepsis-Induced Coagulation in the Baboon Lung Is Associated with Decreased Tissue Factor Pathway Inhibitor Am. J. Pathol., September 1, 2007; 171(3): 1066 - 1077. [Abstract] [Full Text] [PDF]

				H. H. Versteeg and W. Ruf Tissue Factor Coagulant Function Is Enhanced by Protein-disulfide Isomerase Independent of Oxidoreductase Activity J. Biol. Chem., August 31, 2007; 282(35): 25416 - 25424. [Abstract] [Full Text] [PDF]

				L. Ye, H. K. Haider, and E. K. W. Sim Adult Stem Cells for Cardiac Repair: A Choice Between Skeletal Myoblasts and Bone Marrow Stem Cells Experimental Biology and Medicine, January 1, 2006; 231(1): 8 - 19. [Abstract] [Full Text] [PDF]

				N. Mackman Tissue-Specific Hemostasis in Mice Arterioscler. Thromb. Vasc. Biol., November 1, 2005; 25(11): 2273 - 2281. [Abstract] [Full Text] [PDF]

				C. Lupu, X. Hu, and F. Lupu Caveolin-1 Enhances Tissue Factor Pathway Inhibitor Exposure and Function on the Cell Surface J. Biol. Chem., June 10, 2005; 280(23): 22308 - 22317. [Abstract] [Full Text] [PDF]

				H.-H. Chen, C. P. Vicente, L. He, D. M. Tollefsen, and T.-C. Wun Fusion proteins comprising annexin V and Kunitz protease inhibitors are highly potent thrombogenic site-directed anticoagulants Blood, May 15, 2005; 105(10): 3902 - 3909. [Abstract] [Full Text] [PDF]

				B. Pedersen, T. Holscher, Y. Sato, R. Pawlinski, and N. Mackman A balance between tissue factor and tissue factor pathway inhibitor is required for embryonic development and hemostasis in adult mice Blood, April 1, 2005; 105(7): 2777 - 2782. [Abstract] [Full Text] [PDF]

				J. Ahamed, M. Belting, and W. Ruf Regulation of tissue factor-induced signaling by endogenous and recombinant tissue factor pathway inhibitor 1 Blood, March 15, 2005; 105(6): 2384 - 2391. [Abstract] [Full Text] [PDF]

				O. Piro and G. J. Broze Jr Role for the Kunitz-3 Domain of Tissue Factor Pathway Inhibitor-{alpha} in Cell Surface Binding Circulation, December 7, 2004; 110(23): 3567 - 3572. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 108/5/623    most recent 01.CIR.0000078642.45127.7Bv1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zhang, J. Articles by Broze, G. J. Search for Related Content PubMed PubMed Citation Articles by Zhang, J. Articles by Broze, G. J., Jr Related Collections Coagulation inhibitors