© 1998 American Heart Association, Inc.
Vascular Biology |
Expression of Tissue Factor Pathway Inhibitor in Vascular Smooth Muscle Cells and Its Regulation by Growth Factors
From the Divisions of Cardiovascular Diseases and Biochemistry and Molecular Biology (N.M.C., C.S.M., L.S.K., T.E.P., R.D.S.), Molecular Medicine Program, Mayo Clinic and Foundation, Rochester, Minn; and the Department of Medicine, Washington University Medical Center (G.J.B.), St. Louis, Mo.
Correspondence to Robert D. Simari, MD, 200 1st St SW, Rochester, MN 55904. E-mail simari.robert{at}mayo.edu
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Abstract |
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Abstract—Tissue factor pathway inhibitor (TFPI) in vivo is thought to be synthesized mainly by endothelial cells. To date, no significant regulator of TFPI synthesis has been described. Vascular smooth muscle cells (VSMC) express tissue factor in vitro and in vivo, which may contribute to vascular thrombosis. We hypothesized that VSMC might also express TFPI. To determine this, we examined growth-arrested coronary VSMC in culture and found that VSMC secreted an amount of TFPI similar to that seen in endothelial cells. Immunohistochemistry of normal human coronary arteries showed TFPI staining throughout the media and intima of the vessel with localization to VSMC and endothelial cells. To determine regulation of TFPI expression in VSMC, we examined the effects of serum stimulation on TFPI secretion and found that FBS induced a 5-fold increase in TFPI antigen and activity levels in conditioned medium at 48 hours (P<0.001) when compared with serum-free conditions. A similar stimulatory effect was seen with 10% pooled human serum. Moreover, epidermal growth factor and platelet-derived growth factor-B increased TFPI secretion by 4- to 5-fold and 2- to 3-fold, respectively (P<0.05), and these growth factors accounted for
50% of the TFPI secretion
effects of human serum. The serum effect was associated with a 3-fold
increase in TFPI mRNA 24 hours after release from growth arrest and a
50% decrease in TFPI secretion after treatment with actinomycin D.
Taken together, this study suggests that there is significant TFPI
expression in VSMC in culture and in VSMC within the intima and media
of the normal coronary artery wall. We present the first
evidence for TFPI regulation by serum in VSMC and more specifically by
its constituent growth factors, epidermal growth factor and
platelet-derived growth factor-B.
Key Words: tissue factor • inhibitor • smooth muscle • regulation
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Arterial thrombosis is a significant complication of primary atherosclerosis. The complex of tissue factor (TF) and factor VIIa is one of the most potent initiators of in vivo arterial thrombosis, leading to activation of factors IX and X.1 Several studies have shown that TF is expressed in the adventitia of normal human arteries,2 3 intima of human atherosclerotic plaque, and neointima of the balloon-injured rat arteries.4 An important inhibitor of TF-mediated coagulation is tissue factor pathway inhibitor (TFPI), a multivalent protease inhibitor with 3 Kunitz-type domains that inactivates factor Xa generation by binding initially via its Kunitz II domain to factor Xa and subsequently by its Kunitz I domain to TF-VIIa catalytic complex.5 The formation of a quaternary TF-VIIa-TFPI-Xa complex dampens ongoing coagulation and may allow modulation of thrombosis in vivo. TFPI was purified initially from conditioned medium of a hepatoma cell line (HepG2)6 and since has been described in platelets, microvascular endothelium, and stimulated blood monocytes.7 8 9 10 The endothelium currently is thought to be the principle site of intravascular TFPI synthesis.7 Vascular smooth muscle cells (VSMC) are known to express TF in vivo, which may contribute to vascular thrombosis.4 11 Little is known about TFPI expression within VSMC and, in particular, within coronary VSMC. Bajaj and coworkers7 previously showed that human VSMC synthesized only small quantities of TFPI, but coronary VSMC were not examined. Furthermore, previous studies have failed to identify any factors that might significantly regulate TFPI expression in either monocyte/macrophages or endothelial cells.10 12 The purpose of the current study was to examine the expression and regulation of TFPI in human coronary VSMC under culture conditions and within the normal vessel wall. TFPI expression within coronary VSMC in culture was compared with that of endothelial and HepG2 cells, and in vivo expression of TFPI within normal human coronary arteries was studied. Finally, regulation of TFPI expression by serum and its constituent growth factors was examined.
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Materials and Methods |
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Cell Culture
Human coronary VSMC (Clonetics Inc) were cultured initially on 90-mm dishes in the presence of basal media (SmBm, Clonetics Inc) and supplemented with 10% FBS (Gibco BRL), 100 u/mL penicillin, 100 mg/mL streptomycin, and glutamine (Sigma Chemical Co). HepG2 cells (American Type Culture Collection) were grown in MEM with 10% FBS, antibiotic supplements, and 1 mmol/L sodium pyruvate. Human coronary artery endothelial cells (Clonetics Inc) were grown in basal media (EcBm, Clonetics Inc) supplemented with 5% FBS, bFGF, vascular endothelial growth factor, and antibiotics. For all experiments, coronary VSMC were used between passages 4 and 8, HepG2 cells between passages 4 and 10, and coronary artery endothelial cells between passages 2 and 4. Immunostaining of these coronary VSMC for smooth muscle
-actin was similar to that
of primary cell cultures, suggesting that their phenotype was
similar to early passage primary cultured VSMC. Human embryonic kidney
cells (293, American Type Culture Collection) were grown in DMEM (Gibco
BRL) supplemented with 10% FBS and antibiotics.
TFPI Antigen Measurement
A sandwich enzyme immunoassay was carried out using a commercial
ELISA kit (American Diagnostica), which identifies the
Kunitz I domain of the human TFPI antigen.13 Duplicate
samples were examined in 3 separate experiments.
Tissue Processing
Three human coronary arteries obtained at the time of
cardiac transplantation or at the time of autopsy (within 24 hours)
were either freshly frozen in liquid nitrogen or snap frozen in
Cryoform (Intermediate Equipment Co) in liquid nitrogen–cooled
isopentane. Human tissue was obtained according to a protocol approved
by the Institutional Review Board. Thawed tissue was
homogenized subsequently in lysis buffer (50 mmol/L
Tris HCl, pH 8.0, 150 mmol/L NaCl, 0.02% sodium azide, 0.1% SDS,
100 µg/mL PMSF, 1 µg/mL aprotinin, 1% NP-40, 0.5% sodium
deoxycholate) using a tissue homogenizer. A portion of
each tissue underwent endothelial denuding and
adventitial removal before analysis. The supernatant then was
analyzed for protein content using a Bradford assay
(BioRad).
Immunohistochemistry
Freshly frozen sections (5 µm) from normal human
coronary arteries were fixed in acetone at -20°C for 10
minutes. Sections were washed in TBS, blocked with 10% normal donkey
serum, and incubated for 1 hour at room temperature with a polyclonal
rabbit anti-human TFPI antibody (gift from Dr L.V.M. Rao, University of
Texas, San Antonio, Tex)14 at 1:100 dilution. Control
sections were incubated with rabbit IgG (Sigma Chemical Co) at a
similar dilution. Sections were washed and incubated with an
anti-rabbit biotinylated antibody (Amersham Life Sciences) at 1:200
dilution for 30 minutes and then with a streptavidin alkaline
phosphatase antibody (Vector Laboratories) at 1:300 dilution for an
additional 45 minutes. Visualization of reaction product was
carried out using Fast Red (Sigma Chemical Co) and counterstained with
hematoxylin. Further serial sections were immunolabeled with -actin
(HHF 35, Dako Co) and anti-VWF (Dako Co) primary antibodies. These
antibodies were labeled with secondary anti-mouse biotinylated
antibodies at a dilution similar to that described previously. Anti-VWF
was visualized with an alkaline phosphatase system using Vector Blue
stubstrate (Vector Laboratories). Anti–-actin was visualized with a
horseradish peroxidase system using diaminobenzidine
substrate.
Serum and Growth Factor Stimulation of Cultured VSMC
Human coronary VSMC were seeded at a density of
1x105 cells per well on a 6-well dish. After
overnight attachment, cells were growth-arrested for 24 hours in
serum-free medium. BrdU incorporation experiments confirmed negligible
DNA synthesis consistent with growth arrest after 24 hours in
serum-free conditions. Cells then were stimulated with medium
containing 15% FBS, and conditioned media and cell lysates were
processed in duplicate at 12, 24, and 48 hours after release from
growth arrest. Control cells at similar density, treated with
serum-free medium, were processed at similar time points. At each time
point, cells were counted using a hemocytometer before lysis. In other
experiments, cells were stimulated with 10% pooled human (AB) serum
(Sigma Chemical Co), and conditioned media were collected at 24 hours
after release from growth arrest.
In separate experiments, cells were growth-arrested for 24 hours and then stimulated with platelet-derived growth factor (PDGF)-A, PDGF-B, aFGF, bFGF, epidermal growth factor (EGF), and transforming growth factor (TGF)-ß1 (all from R&D Systems) over a concentration range of 2 log-folds on either side of the mitogenic ED50 as determined by the manufacturer for that particular growth factor. Conditioned medium was processed and cell counts determined as previously described at 48 hours after release from growth arrest. Duplicate wells were measured from at least 3 separate experiments.
Measurement of TFPI Activity
Functional TFPI activity in conditioned medium from serum-free
and serum-treated VSMC was measured using a commercial Actichrome TFPI
activity assay (American Diagnostica). This assay measures
the inhibition by TFPI of factor Xa generation after activation by
TF/VIIa catalytic complex and was carried out at 37°C.13
Duplicate samples were measured from 3 separate experiments.
Western Blot Analysis
TFPI protein in conditioned media and tissue
homogenates was analyzed using Western blot
analysis. After protein determination, 1 mmol/L DTT (Sigma
Chemical Co) and lysis buffer were added to equal amounts of protein,
and samples were denatured by boiling for 5 minutes and resolved by
electrophoresis on a 12% SDS-polyacrylamide gel. Transfer of
protein to a nitrocellulose membrane was carried out over 3 hours at
4°C. Immunoblotting was performed using the
previously described polyclonal rabbit anti-human TFPI
antibody14 at 1:200 dilution in nonfat milk/TBS buffer.
After washes, the membrane was probed subsequently with a rabbit
secondary antibody conjugated to horseradish peroxidase (Amersham Life
Sciences) at 1:5000 dilution and developed with chemoluminescence
(Supersignal, Pierce). The membrane then was exposed to x-ray film
(Kodak) and subsequently developed.
Growth Factor Neutralization
Human VSMC were growth-arrested for 24 hours on 6-well plates as
previously described. Cells then were stimulated with medium enriched
with 10% pooled human serum in the presence or absence of neutralizing
antibodies to EGF and PDGF-B (R&D Systems). Each neutralizing antibody
was used at a 100-fold excess (20 µg/mL) compared with each growth
factor concentration in 10% human serum. The antibodies also were used
in combination. An isotype-matched IgG at the same concentration was
used as a control. The 10% human serum-enriched medium was
preincubated with the neutralizing antibody for 2 hours at 37°C
before incubation with the cells in culture. Conditioned medium then
was collected at 48 hours after release from growth arrest. In each
case, TFPI activity of the conditioned medium was measured in duplicate
in at least 3 separate experiments.
Northern Blot Analysis
Human coronary VSMC were grown in 150-mm dishes and
growth-arrested for 48 hours in serum-free medium. Cells then were
stimulated with medium containing 15% FBS and harvested at 0, 4, 8,
12, and 24 hours after release from growth arrest. Total RNA was
isolated using RNAZOL B (Tel-Test Inc). Northern blot analysis
was performed using standard techniques (Northern Max, Ambion Inc).
Briefly, 20 µg of total RNA was electrophoresed on a 1% denaturing
formamide gel and transferred to a nitrocellulose membrane using the
Turbo Blot (Schleicher and Schuell) method of capillary transfer.
Membranes then were crosslinked and prehybridized for 2 hours at
42°C, followed by hybridization overnight at 42°C with a random
prime–labeled (-32P dCTP) cDNA probe (Prime
It II Random Prime Labeling Kit, Stratagene). TFPI mRNA bands
subsequently were visualized on x-ray film. To control for variations
in RNA loading, membranes were stripped of previously labeled TFPI
probe by boiling in 1% SDS solution and rehybridized with a cDNA probe
to mouse GAPDH (Ambion Inc).
Inhibition of Transcription With Actinomycin D
Human coronary VSMC, after 24 hours in serum-free
medium, were released from growth arrest as previously described with
addition of 15% FBS in the presence or absence of 2 µg/mL
actinomycin D (Boehringer Mannheim). Conditioned medium then
was processed at 12, 24, and 48 hours after serum stimulation, and cell
counts were obtained from each well as previously described. Wells were
measured in duplicate in 3 separate experiments.
Transfection of 293 Cells With pCMV-TFPI
Transfection of 293 cells, grown to 90% confluence on 90-mm
dishes, was carried out with 10 µg of pCMV-TFPI plasmid (a
eukaryotic expression plasmid that expresses full-length
human TFPI from the cytomegalovirus immediate-early promoter/enhancer)
in the presence of 10 µg of liposome (GAP DLRIE-DOPE, kindly provided
by Gibco BRL) in OPTI-MEM (Gibco BRL) according to standard
methods.15 The conditioned medium from these cells was
collected at 48 hours after transfection and used as a positive control
for TFPI in human artery homogenate Western blot
experiments.
Statistical Analysis
Data are presented as mean±SEM. Comparisons were made
using unpaired Student t test (2-tailed) or ANOVA with
Fisher post hoc test. P<0.05 was considered statistically
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TFPI Antigen Levels in VSMC, Endothelial Cells, and HepG2 Cells
To quantify TFPI expression in human coronary VSMC, the levels of TFPI antigen in VSMC, endothelial cell, and HepG2 cell lysates and conditioned media assessed by ELISA were compared (Figure 1
). HepG2 cells
synthesized and secreted 3-fold greater amounts of TFPI per cell than
either endothelial or VSMC. Similar amounts of TFPI
were secreted from coronary VSMC compared with coronary
endothelial cells. Approximately 10% of TFPI was
cell-associated in endothelial cells and VSMC. A
nonvascular cell line (293 cells) had undetectable TFPI in cell lysates
or conditioned medium under basal cell culture conditions.
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Immunolocalization of TFPI in Human Coronary Arteries
To determine whether TFPI is expressed by human coronary
VSMC in vivo, immunostaining of TFPI was performed on
normal coronary arteries (n=3) with diffuse intimal thickening.
TFPI staining was found within cells in the
subendothelial intima and throughout the media of the
coronary artery (Figure 2A) and
in association with adventitial microvessels (Figure 2E). To
examine whether VSMC were the cells associated with TFPI staining,
serial sections were stained with -smooth muscle actin antibody
(Figure 2B and 2F) and VWF antibody (Figure 2C).
This showed that the predominant cells within the coronary
artery staining positive for TFPI were smooth muscle cells and luminal
and microvessel endothelial cells. No staining was seen
using a rabbit IgG control antibody at a concentration similar to that
of the TFPI antibody (Figure 2D). Staining appeared to be both
cell-associated and extracellular (Figure 2G).
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Western blot analysis of homogenized
coronary artery with the same polyclonal TFPI antibody revealed
a protein of similar in size to that expressed in conditioned medium of
cultured coronary VSMC, suggesting that the TFPI seen within
the coronary vessel was the full-length form (Figure 2H
). In addition, nanogram quantities of TFPI were found in all
coronary artery homogenates (denuded and
nondenuded) by ELISA (data not shown).
Regulation of TFPI Synthesis and Activity
Serum-Induced TFPI Secretion
The effects of serum stimulation on growth-arrested VSMC were
examined at 12, 24, and 48 hours after stimulation. Conditioned medium
was analyzed for TFPI antigen, activity, and protein size using
an ELISA, an activity assay, and Western blot analysis,
respectively. TFPI antigen secretion from human VSMC in culture over
12, 24, and 48 hours in the presence of serum measured by ELISA
increased 5-fold when compared with serum-free treatment (Figure 3A). TFPI activity as measured by
inhibition of factor X activation by TF-VIIa catalytic complex was
increased similarly at 48 hours after serum treatment (Figure 3B). On Western blot analysis, a protein
consistent with full-length human TFPI was identified in
serum-treated conditioned medium over the 48-hour time period (Figure 3C). This protein was not detected in serum-free conditioned
medium.
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) or absence (
)
of 15% FBS. Data represent the mean of 3 experiments performed
in duplicate. *P<0.05 for serum-treated vs serum-free
cells. B, Time course of TFPI activity in human VSMC–conditioned media
in the presence (
TFPI antigen also was measured in conditioned medium of cells after 24
hours of stimulation with 10% pooled human serum. Pooled human
serum–treated cells showed an 8-fold increase in TFPI antigen at 24
hours after serum stimulation (Figure 3D).
Growth Factor–Induced TFPI Secretion
To evaluate whether growth factors within human or FBS might be
contributing to TFPI secretion, concentration response experiments were
performed using several serum-derived growth factors. EGF and PDGF-B
significantly increased TFPI secretion by VSMC 24 hours after release
from growth arrest (Figure 4A).
Furthermore, 30% and 40% of the human serum's stimulatory effect
on TFPI secretion could be abolished by using neutralizing antibodies
to PDGF-B and EGF, respectively (Figure 4B). An irrelevant
isotype-matched control antibody at a similar concentration had no
effect on TFPI activity. Combining EGF– and PDGF-B–neutralizing
antibodies caused a 50% reduction in the serum effect, suggesting a
nonadditive antibody effect perhaps because of convergent intracellular
signaling from these growth factors.16 None of the other
growth factors studied including PDGF-A, aFGF, bFGF, or TGF-ß, had
any significant effect on TFPI secretion (data not shown).
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P<0.001 compared with
control.
Quantitation of Serum-Induced mRNA for TFPI
To determine whether serum stimulation of VSMC is associated with
an increase in steady-state levels of TFPI mRNA, quantitative
assessment of Northern blots (n=4) was performed (Figure 5). Analysis of RNA at baseline
and 4, 8, 12, and 24 hours after serum stimulation with 15% FBS
demonstrated a 3-fold increase in TFPI mRNA (normalized to GAPDH) 24
hours after stimulation (P=0.0019).
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Inhibition of Transcription With Actinomycin D
To determine the contributions of transcription to serum-induced
increases in TFPI antigen, cells were stimulated by serum in the
absence and presence of actinomycin D, and the secreted antigen was
measured in the conditioned medium by ELISA. Exposure to actinomycin D
resulted in a 50% reduction (P<0.01) in TFPI antigen
secretion within the conditioned medium over the 24-hour time
period.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we show that human coronary VSMC in culture synthesize and secrete an amount of TFPI similar to that by coronary endothelial cells. We have shown for the first time TFPI immunostaining in vivo within VSMC throughout the intima and media of the coronary artery wall. We present experimental evidence for significant serum-derived regulation of TFPI synthesis in VSMC in the form of EGF and PDGF-B. In addition, serum-induced stimulation of TFPI synthesis is associated with an increase in steady-state levels of mRNA and is inhibited partially by actinomycin D, suggesting that TFPI is regulated, in part, at the mRNA level.
Endothelial cells previously have been thought to be the major source of TFPI within the vasculature.7 We show that coronary VSMC in culture synthesize similar amounts of TFPI when compared with coronary endothelial cells. In this study, TFPI predominantly was secreted with only 10% being cell-associated in culture. The relative levels of TFPI secretion by endothelial and HepG2 cells in the current study are similar to those previously reported for these cell types.7 However, the levels of TFPI antigen in VSMC are much greater in the current study than previously reported by Bajaj and colleagues.7 This difference might be explained by different sources of VSMC or different methods used to measure TFPI antigen levels.
Few data currently exist on the distribution of TFPI within large- to medium-sized vessels. Drew et al17 recently showed TFPI immunostaining within the adventitia of normal human aorta and renal artery but not within the medial layer or luminal endothelium. However, Werling and colleagues9 found no evidence of TFPI within larger vessels, although they found TFPI in the endothelium throughout the microvasculature. In our study, TFPI staining was found throughout the media of normal coronary arteries and in the subendothelial VSMC within diffuse intimal thickenings of these arteries. TFPI staining also was seen along the endothelium of these vessels. The current study differs from the other studies in that we examined TFPI staining patterns in isolated coronary arteries, whereas Werling and coworkers9 looked at smaller vessels within several organs, and Drew and coworkers17 did not specifically examine the coronary circulation. In the current study, the presence of TFPI protein in coronary artery homogenates on Western blot analysis and ELISA using both polyclonal and monoclonal antibodies to TFPI is consistent with the presence of significant amounts and a wider intracellular and extracellular distribution of TFPI within the coronary vasculature.
To date, no significant regulator of TFPI expression has been identified in either endothelial cells12 or monocyte/macrophages10 despite the use of a range of inflammatory cytokines, PMA, LPS, and human serum. In the current study, we have shown that FBS and human serum caused a significant increase in TFPI protein secretion from smooth muscle cells after their release from growth arrest. This increase in TFPI antigen within the culture medium was accompanied by a similar increase in TFPI anticoagulant activity. Furthermore, Western blot analysis showed that full-length TFPI was secreted after serum stimulation. The association of full-length TFPI with high levels of anticoagulant activity is consistent with previous studies.18 By Northern analysis and transcription-inhibition experiments, we also showed that the serum-induced increase in TFPI protein secretion at least partly was regulated at the RNA level with peaking of steady-state TFPI mRNA at 24 hours after release from growth arrest. Serum is also known to rapidly induce transcriptional activation of TF in VSMC within 1 hour of release from growth arrest, with mRNA returning to baseline at 8 hours.4 It is interesting to speculate whether an "immediate early" effect of serum on TF mRNA induction within VSMC is related to the later induction of TFPI mRNA seen in the present study.
Ameri and colleagues12 previously showed a modest
(1.5-fold) upregulation of TFPI in endothelial cells by
whole blood serum over that of plasma-derived serum and suggested that
the increase might be due to TGF-ß from platelets. Two
TGF-ß–like response elements are present in the 5' end of the
TFPI gene.19 20 21 In the current study, we found that
TGF-ß had no effect on TFPI secretion by VSMC, whereas other
serum-derived growth factors, PDGF-B and EGF, had significant effects
on TFPI secretion. These data suggest there are differences in
regulatory mechanisms for TFPI secretion between VSMC and other cells
within the vessel wall. However, consistent with other studies
on endothelial cells,12 we found that
tumor necrosis factor- did not appear to significantly affect TFPI
secretion by VSMC (data not shown).
Taken together, our data suggest that VSMC are a significant in vitro and in vivo source of TFPI. The levels of TFPI synthesized by VSMC under culture conditions in our study are equivalent to those of endothelial cells previously thought to be the major cellular pool of vascular TFPI. The evidence from our study of regulation of TFPI by serum and its constituents PDGF-B and EGF and the presence of TFPI staining within the media and intima of coronary arteries may have significant and related in vivo implications. Several serum-derived growth factors are known to be upregulated locally and released in atherosclerosis22 23 24 and after coronary angioplasty in human subjects.25 It is possible that growth factors within the vessel wall might locally regulate TFPI synthesis and secretion. Future studies will need to assess regulation of TFPI in vascular disease states where TF/factor VII activation plays a major role in thrombosis formation.
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Acknowledgments |
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This study was supported in part by grants from the Bruce and Ruth Rappaport Program in Vascular Biology at the Mayo Clinic, the National Institutes of Health (HL-03473), the Miami Heart Institute, and the American Heart Association, Minnesota Affiliate, Inc. Dr Caplice is a recipient of the Bracco Diagnostic Research Fellowship. We gratefully acknowledge Drs Whyte Owen, William Fay, and David R. Holmes, Jr, for their valuable suggestions; James Tarara and Ross Aleff for their technical assistance; and Maureen Craft for preparation of the manuscript.
Received August 14, 1998; accepted September 23, 1998.
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 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]
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 J. Golledge, S. Mangan, and P. Clancy Effects of Peroxisome Proliferator-Activated Receptor Ligands in Modulating Tissue Factor and Tissue Factor Pathway Inhibitor in Acutely Symptomatic Carotid Atheromas Stroke, May 1, 2007; 38(5): 1501 - 1508. [Abstract] [Full Text] [PDF]
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 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]
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 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]
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 S. V. Pislaru, C. Pislaru, R. R. Kinnick, R. Singh, R. Gulati, J. F. Greenleaf, and R. D. Simari Optimization of ultrasound-mediated gene transfer: comparison of contrast agents and ultrasound modalities Eur. Heart J., September 2, 2003; 24(18): 1690 - 1698. [Abstract] [Full Text] [PDF]
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 X. Yin, C. Yutani, Y. Ikeda, K. Enjyoji, H. Ishibashi-Ueda, S. Yasuda, Y. Tsukamoto, H. Nonogi, Y. Kaneda, and H. Kato Tissue factor pathway inhibitor gene delivery using HVJ-AVE liposomes markedly reduces restenosis in atherosclerotic arteries Cardiovasc Res, December 1, 2002; 56(3): 454 - 463. [Abstract] [Full Text] [PDF]
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H. Kato Regulation of Functions of Vascular Wall Cells by Tissue Factor Pathway Inhibitor: Basic and Clinical Aspects Arterioscler Thromb Vasc Biol, April 1, 2002; 22(4): 539 - 548. [Abstract] [Full Text] [PDF]
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 N. M. Caplice, C. Panetta, T. E. Peterson, L. S. Kleppe, C. S. Mueske, G. M. Kostner, G. J. Broze Jr, and R. D. Simari Lipoprotein (a) binds and inactivates tissue factor pathway inhibitor: a novel link between lipoproteins and thrombosis Blood, November 15, 2001; 98(10): 2980 - 2987. [Abstract] [Full Text] [PDF]
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 P. Golino, P. Cirillo, P. Calabro', M. Ragni, D. D'Andrea, E. V. Avvedimento, F. Vigorito, N. Corcione, F. Loffredo, and M. Chiariello Expression of exogenous tissue factor pathway inhibitor in vivo suppresses thrombus formation in injured rabbit carotid arteries J. Am. Coll. Cardiol., August 1, 2001; 38(2): 569 - 576. [Abstract] [Full Text] [PDF]
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I. Ott, M. Andrassy, D. Zieglgansberger, S. Geith, A. Schomig, and F.-J. Neumann Regulation of monocyte procoagulant activity in acute myocardial infarction: role of tissue factor and tissue factor pathway inhibitor-1 Blood, June 15, 2001; 97(12): 3721 - 3726. [Abstract] [Full Text] [PDF]
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J. Crawley, F. Lupu, A. D. Westmuckett, N. J. Severs, V. V. Kakkar, and C. Lupu Expression, Localization, and Activity of Tissue Factor Pathway Inhibitor in Normal and Atherosclerotic Human Vessels Arterioscler Thromb Vasc Biol, May 1, 2000; 20(5): 1362 - 1373. [Abstract] [Full Text] [PDF]
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U. R. Pendurthi, L. V. M. Rao, J. T. Williams, and S. Idell Regulation of Tissue Factor Pathway Inhibitor Expression in Smooth Muscle Cells Blood, July 15, 1999; 94(2): 579 - 586. [Abstract] [Full Text] [PDF]
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R. Singh, S. Pan, C. S. Mueske, T. Witt, L. S. Kleppe, T. E. Peterson, A. Slobodova, J.-Y. Chang, N. M. Caplice, and R. D. Simari Role for Tissue Factor Pathway in Murine Model of Vascular Remodeling Circ. Res., July 6, 2001; 89(1): 71 - 76. [Abstract] [Full Text] [PDF]
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H. Kato Regulation of Functions of Vascular Wall Cells by Tissue Factor Pathway Inhibitor: Basic and Clinical Aspects Arterioscler Thromb Vasc Biol, April 1, 2002; 22(4): 539 - 548. [Abstract] [Full Text] [PDF]
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