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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Department of Immunology and Vascular Biology,
The Scripps Research Institute, La Jolla, CA; and the Department of
Medicine and Pharmacology, University of Pennsylvania, Philadelphia,
PA.
Cell signaling by coagulation factor Xa (Xa) contributes to
pro-inflammatory responses in vivo. This study characterizes the signaling mechanism of Xa in a HeLa cell line that expresses
protease-activated receptor 1 (PAR-1) but not PAR-2, -3, or -4. Xa
induced NF- In addition to maintaining normal hemostasis,
coagulation proteases play important roles in cell signaling. A family
of homologous G-protein-coupled receptors that are activated by
proteolytic cleavage rather than ligand binding have been identified in
recent years.1-5 Among the 4 currently known
protease-activated receptors (PARs), 3 are cleaved by thrombin, whereas
PAR-2 can be activated by trypsin2 and mast cell
tryptase.6 Only the recently identified thrombin receptor
PAR-4 is also activated by trypsin,4,5 indicating protease
selectivity in this receptor family. Studies have now clearly shown
that coagulation proteases upstream of thrombin can also elicit
cellular signals through proteolytic mechanisms. When allosterically
induced for full function on binding to its cellular receptor tissue
factor (TF), factor VIIa (VIIa) can induce Ca++
oscillations,7 activate the MAP kinase
pathway,8 and induce gene expression.9-11
Coagulation factor Xa (Xa) has been shown to be mitogenic for smooth
muscle cells12-14 and to elicit inflammatory responses in
endothelial cells, including nitric oxide-mediated hypotension15,16 and cytokine induction.15,17
Although it is controversial whether a cell surface receptor for Xa,
termed EPR-1,18 is involved in the Xa-dependent gene
induction,9,15,17,19 all recent studies concur that
blocking the proteolytic function of Xa with specific inhibitors
abolishes Xa signaling. Consistent with a requirement for proteolytic
function, a recent study20 indicated that the activation
of murine fibroblasts by Xa and VIIa can be mediated by PAR-2, though
other studies21,22 concluded that PAR-2 is not the
receptor activated by VIIa in different cell types.
Therapeutic intervention in animal models of gram-negative
septicemia indicates that the upstream coagulation proteases contribute significantly more than thrombin action to the inflammatory responses that lead to the lethality of septic shock. Inhibition of TF-initiated coagulation by anti-TF antibodies,23 tissue factor pathway
inhibitor,24 or active site-blocked VIIa25
all reduce lethality in a particular primate model, whereas blocking
prothrombin activation by the infusion of active site-blocked Xa was
ineffective in improving survival.26 These data emphasize
the importance of understanding the mechanism of signaling by upstream
coagulation serine proteases. Numerous inflammatory genes are regulated
by the nuclear factor kappa B (NF- Proteins and inhibitors
Cell culture
RNA isolation and Northern blot analysis Total RNA was prepared from 1 to 2 × 106 cells by using the Trizol reagent (Life Technologies, Gaithersburg, MD). Samples of RNA (10 µg) were separated on 1% agarose-formaldehyde gel and transferred to nylon membrane (GeneScreen; NEN, Boston, MA), according to standard procedures.36 The blots were hybridized to DNA probes corresponding to nucleotides 345-1622 for PAR-1,1 1-1451 for PAR-2,2 603-1003 for PAR-3,3 1-787 for PAR-4,5 589-1093 for Cyr61,37 and 1055-1467 for CTGF.38 The DNA probe for I B mRNA
was prepared from a 1.1-kb EcoRI fragment of the mouse IB cDNA.39 The probes were labeled by random priming using
32P-dCTP (ICN, Costa Mesa, CA), the DECAprime II DNA
labeling kit (Ambion, Austin, TX), and Sephadex G-50 mini-spin columns
(Worthington, Lakewood, NJ) for probe purification. Hybridization was
performed in QuikHyb hybridization solution (Stratagene, La Jolla, CA)
for 1 hour at 65°C, and the blots were washed to final stringencies of 0.2 × SSC, 0.1% sodium dodecyl sulfate (SDS), at 65°C. The probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was
prepared from synthetic oligonucleotides as described
previously.40
Nuclear extract preparation and EMSA HeLa cells were grown to subconfluence, incubated for 48 hours in growth medium without serum, and stimulated at 37°C as described. Nuclear extracts were prepared from approximately 106 cells and used for electrophoretic mobility shift assays (EMSA) as described previously.41 SP1- and NFB-specific probes for EMSA
were obtained from Promega (Madison, WI).
Microarray analysis Subconfluent HeLa cells in T150 flasks were serum-deprived for 48 hours, as described above. Quiescent cells were either not stimulated or stimulated by adding 50 nM thrombin or 100 nM hirudin and then 50 nM Xa. After 90 minutes at 37°C, total RNA was isolated from the control and protease-treated cells as described above, and poly(A) RNA was purified using 2 passes over Oligotex mRNA columns (Qiagen, Valencia, CA) according to the manufacturer's protocols. The mRNA samples were sent for high-density gene expression profiling on the UniGEM V microarray (Incyte Genomics, St Louis, MO) using the fluorogenic dyes Cy3 and Cy5 for control and protease-treated conditions, respectively.Analysis of MAP kinase phosphorylation Cells were grown and serum-deprived in 12 well plates as described above; after stimulation, proteins were extracted in 300 µL reducing SDS sample buffer. Aliquots (5 µL) were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE), and this was followed by transfer to nylon membrane for Western blotting. Phosphorylated MAP kinases p44/42 were reacted with a monoclonal antibody (New England Biolabs, Beverly, MA), and bound primary antibody was detected with horseradish peroxidase-conjugated secondary antibody (Amersham Pharmacia, Piscataway, NJ) and enhanced chemiluminescence. Signal intensities on the autoradiographs were quantified by laser densitometry using ImageQuant software (Molecular Dynamics, Piscataway, NJ).Analysis of PAR-1 cleavage by flow cytometry HeLa cells were grown, serum-deprived, and stimulated at 37°C in T25 flasks in the presence of 20 nM thrombin or 50 nM Xa/100 nM hirudin for 30 seconds or 5 minutes. The protease was inactivated by adding 100 nM hirudin or 1 µM NAP5, respectively, and the cells were detached with Enzyme-Free Cell Dissociation Buffer (Life Technologies) and kept on ice for all subsequent steps. Cells were pelleted in serum-free HeLa growth medium, washed, and resuspended in staining buffer (phosphate-buffered saline, 0.2% bovine serum albumin, pH 7.2). Cells were stained with a 1:300 dilution of ascites of monoclonal antibody SPAN11 for 30 minutes; this was followed by washing and a 30-minute incubation with a 1:50 dilution of FITC-labeled goat F(ab')2 antimouse IgG (Southern Biotechnology Associates, Birmingham, AL). After another washing step, cells were analyzed on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA).
Xa and thrombin induce NF- B,42 we
focused on this central pathway as a possible mechanism by which Xa can induce inflammatory responses. To identify Xa-responsive cell lines,
serum-starved cells were exposed to 50 nM Xa or thrombin for 45 minutes; this was followed by nuclear extract preparation for EMSA with
an NF-B consensus oligonucleotide probe. Although other cell lines
also showed some response to Xa stimulation, HeLa cells displayed a
similar induction of nuclear NF-B binding activity by thrombin and
Xa (Figure 1A). Because HeLa cells could be easily passaged without protease (trypsin) treatment for cell detachment, we focused on this cell line as a model system to study the
mechanism of Xa-dependent signaling. Induction of NF-B was specific
for thrombin and Xa because the serine proteases VIIa, activated
protein C (aPC), factor IXa, and trypsin did not induce nuclear
translocation of NF-B (Figure 1A). HeLa cells that were not
serum-starved and cells that were serum-starved under our experimental
conditions expressed similar levels of TF, as determined by a one-stage
clotting assay, but this excluded that the failure to respond to VIIa
resulted from the absence of the essential cofactor TF. Thus,
previously described protease cell signaling by VIIa7,43
likely involves a receptor that is distinct from the Xa receptor on
HeLa cells. These data demonstrate that HeLa cells are selectively
responsive to thrombin and Xa, indicating that a putative Xa-responsive
receptor is not activated by a broad range of homologous serine
proteases. More specifically, PAR-2, which has been previously
implicated as an Xa receptor,20 is presumably not the Xa
receptor on HeLa cells because PAR-2 can be activated by
trypsin2 and the TF-VIIa complex.20
NF- HeLa cells express only PAR-1 Xa induced the nuclear translocation of NF-B with similar
efficiency as the prototypical signaling of thrombin that can act through the previously identified protease activated receptors PAR-1,
-3, and -4. Expression of the known PAR transcripts in HeLa
cells was evaluated by Northern blotting (Figure
2A). Total RNA was prepared from
serum-starved and nonstarved HeLa cells and, as controls, from
non-serum-starved human umbilical vein endothelial cells and from the
megakaryocytic DAMI cell line.45 Only PAR-1 mRNA was
detected in HeLa cells, whereas endothelial cells also expressed
significant amounts of PAR-2 mRNA, consistent with previous
results.46 PAR-3 and PAR-4 mRNA were expressed in DAMI
cells but were not found at detectable levels in the HeLa cells. Like
serum-starved HeLa cells, non-serum-starved HeLa cells expressed only
PAR-1, excluding the possibility that PAR -2, -3, or -4 remained
expressed on the protein level after serum starvation because of slow
protein degradation. The expression of mRNA for PAR-1 is consistent
with the responsiveness of this cell type to thrombin. PAR -1, -2, and
-4 can be activated using small synthetic peptides that
correspond to the tethered ligand sequence of the specific PAR. To
analyze the cell surface expression of these PARs at the functional
level, HeLa cells were stimulated with agonist peptides (Figure 2B).
HeLa cells were responsive to the PAR-1 agonist peptides
SFLLRN1 and TFLLRNPNDK.47 Reduced induction
by agonist peptides, in comparison with that by proteases, might have
resulted from suboptimal peptide concentrations used to assure receptor
specificity. In contrast, the PAR-2-specific peptide
SLIGRL2 and the PAR-4-specific peptide
GYPGKF4,48 did not induce NFB translocation even at
high concentrations, corroborating mRNA data at the functional
level.
Microarray analysis identifies Cyr61 and CTGF as Xa- and thrombin-inducible genes Taken together, these data suggest that Xa acts either through PAR-1 or a novel member of the PAR family to induce NF-B in HeLa
cells. To define further more downstream cellular consequences of
thrombin versus Xa signaling in HeLa cells, we analyzed the effects of
thrombin and Xa on gene expression levels by high-density microarray
analysis. RNA was isolated from serum-starved cells after 90-minute
stimulation with either 50 nM thrombin or Xa. Hirudin (100 nM) was
included during stimulation with Xa to inhibit thrombin that might have
been generated from cell-associated prothrombin. mRNA samples from
stimulated and unstimulated control cells were labeled with the
fluorogenic dyes Cy5 and Cy3, respectively, and simultaneously
hybridized to the UniGEM V chip (Incyte Genomics). The UniGEM V array
contains a panel of approximately 7000 probes for different human
genes. The most prominent induction on the panel was observed for the
transcript of the angiogenesis-promoting genes Cyr61 and
connective tissue growth factor (CTGF). Expression of Cyr61 and CTGF compared to the unstimulated
cells was 12.6-fold and 5.2-fold higher for thrombin and 5.3-fold and
2.7-fold higher for Xa/hirudin. Cyr61 and CTGF are both members of the
CCN family of extracellular matrix-associated proteins that regulate
cellular processes such as adhesion, migration, proliferation, and
survival and that have been implicated in angiogenesis, wound healing, tumor biology, and fibrotic diseases.49,50 Additional
genes were induced to a much lesser extent by both thrombin and
Xa/hirudin eg, apolipoprotein D, retinoid X receptor gamma, apoptosis
inhibitor 1, macrophage scavenger receptor 1and several expressed
sequence tags for novel genes. No genes with significantly reduced
expression levels in response to Xa and thrombin were identified, and
overall no significant divergence in gene expression patterns in
response to the 2 coagulation proteases was seen. Northern blotting
confirmed the up-regulation of Cyr61 and CTGF
mRNA by thrombin as well as Xa in the presence of hirudin. Both genes
were highly induced after 15 minutes by thrombin and more slowly with
peak levels after 1 hour by Xa (Figure
3A). Dose titrations demonstrate that Xa
as low as 10 nM could induce Cyr61 and CTGF mRNA
(Figure 3B). Despite the differences in the kinetics of gene induction,
these data demonstrate that Xa and thrombin induce a similar set of genes in HeLa cells.
Xa activates cells through PAR-1 but independent of thrombin generation The cleavage of PAR-1 can be blocked with specific monoclonal antibodies,51 allowing us to analyze directly whether PAR-1 was a candidate receptor that mediated the Xa response of HeLa cells. In a first set of experiments, activation of the MAP kinase pathway in serum-starved HeLa cells was analyzed by Western blotting for the phosphorylated forms of p44/42. MAP kinase phosphorylation by 20 nM thrombin for 10 minutes was blocked to background levels in the presence of hirudin, whereas preincubation of the cells with cleavage-blocking anti-PAR-1 antibodies resulted in 98% ± 2% (n = 4) inhibition. Thus, hirudin can effectively block cell activation by exogenously added thrombin, and PAR-1 is the only thrombin receptor in these cells leading to MAP kinase phosphorylation. Xa also induced MAP kinase phosphorylation on HeLa cells. There was a slight reduction in MAP kinase phosphorylation on preincubation of the cells with hirudin, but Xa responses were completely abolished by anti-PAR-1 antibodies (Figure 4). The slight reduction of MAP kinase phosphorylation on hirudin inhibition indicated that some locally generated thrombin might have potentiated the Xa response. To test whether prothrombin, at concentrations that could contribute to cell signaling events, was associated with cells under our experimental conditions, we used the snake venom prothrombin activator Ecarin52,53 to convert prothrombin to thrombin in our assay system. Ecarin stimulated MAP kinase phosphorylation in the HeLa cells. This response was completely blocked by hirudin and by anti-PAR-1 antibody (Figure 4), providing evidence that the stimulation resulted from the conversion of prothrombin to thrombin, which, in turn, activated PAR-1. Notably, thrombin generated in situ from cell-associated prothrombin was efficiently blocked by hirudin. The failure of hirudin to inhibit the Xa response thus argues against thrombin as an intermediate in Xa signaling through PAR-1.
However, it was possible that thrombin generated by cell surface Xa
activates cells more effectively than Ecarin-generated thrombin. PAR-1
activation by Xa in the presence of hirudin required Xa at
concentrations of 10 nM or higher. We reasoned that indirect signaling
through prothrombin activation could occur at much lower concentrations
of Xa. Cells were supplemented with a defined amount of exogenous
prothrombin (10 nM) and were stimulated with increasing concentrations
of either Xa (Figure 5A) or Ecarin
(Figure 5B). Half-maximal MAP kinase phosphorylation was obtained with
0.1 nM Xa or 40 mU/mL Ecarin. Even when cells were maximally stimulated by thrombin generated in situ by 5 nM Xa or 500 mU/mL Ecarin, hirudin
was a potent and complete inhibitor of these signaling events. These
data exclude that the observed activation of PAR-1 by higher
concentrations of Xa in the presence of hirudin is indirect through
thrombin generation that escaped inhibition by hirudin. Another
difference between thrombin-dependent and thrombin-independent signaling of Xa is evident from the effect of factor Va (Va). As
expected from the role of this cofactor in Xa-dependent prothrombin conversion, the addition of Va shifted the dose-response curve of Xa in
indirect, thrombin-mediated signaling (Figure 5). However, the addition
of Va did not affect MAP kinase phosphorylation by higher
concentrations of Xa in the presence of hirudin (Figure 4), suggesting
that Va does not function as a cofactor in the thrombin-independent
activation of PAR-1 by Xa.
Figure 6 shows inhibition
experiments analogous to Figure 4 at the level of gene induction.
Activation of cell-associated prothrombin by Ecarin induced Cyr61
expression, which was completely inhibited by hirudin. Hirudin did
not block Cyr61 expression induced by Xa, but gene induction
was prevented by preincubation of the cells with anti-PAR-1
antibodies, consistent with the results obtained by measuring MAP
kinase phosphorylation. Taken together, these data provide conclusive
evidence that gene induction by Xa in HeLa cells is independent of
thrombin but dependent on PAR-1. Furthermore, the nuclear translocation
of NF-
The presented data demonstrate that Xa induces NF- The activation of PAR-1 by Xa differs from thrombin stimulation in 2 important aspects Platelets express PAR-1 along with PAR-4,48 but Xa fails to activate platelets in the presence of hirudin. Platelets are unique in their rapid change in procoagulant membrane properties after the generation of trace amounts of thrombin that lead to PAR-1 cleavage. Before this event, membrane-binding sites for Xa may be of insufficient affinity or availability to localize Xa in proximity to PAR-1. More important, membrane binding of Xa to platelets is mediated by Va,55 whereas tumor cells apparently have membrane-binding sites for factor X that are distinct from the procoagulant membrane environment that supports prothrombinase assembly.56 Va neither enhanced nor inhibited thrombin-independent Xa signaling in our HeLa cell model. However, this lack of inhibition in the presence of Va-independent membrane binding of Xa does not exclude that Va may inhibit Xa-mediated PAR-1 activation when the only cell surface binding site for Xa is Va. Indeed, Xa in the prothrombinase may be inhibited from activating PAR-1, and the unresponsiveness of platelets may be a consequence of these conformational effects of Va on the active site of Xa. Whether Xa signaling plays an important physiological role in vivo can be questioned in light of the fairly high concentrations of Xa required to elicit cellular responses. One must consider whether the in vitro analysis of cell signaling by Xa faithfully mimics concentration dependence during the activation of coagulation in vivo. Typically, the zymogen factor X assembles with the cell surface to become activated by the TF-VIIa complex. At the plasma concentration, the zymogen factor X likely saturates cell surface binding sites, eliminating a requirement for de novo binding of the protease. Zymogen conversion is the rate-limiting step for cell signaling under these conditions, and the cell surface half-life of newly generated Xa may be of sufficient duration to stimulate cells. Together with diffusion- or flow-dependent clearance of locally generated thrombin, a more stably membrane-localized protease, such as Xa, may provide sustained PAR activation in local activation of coagulation by the TF pathway. Furthermore, the conversion of approximately 5% to 10% of the plasma zymogen factor X is sufficient for gene induction. Massive activation of coagulation, such as during disseminated intravascular coagulopathy in gram-negative septic shock, could locally generate these relevant concentrations of Xa. Activation of coagulation within the vasculature typically
proceeds to the generation of thrombin that will be the primary activator of PAR-1. However, in specific vascular compartments, potent
anticoagulant mechanisms, such as high local concentrations of
thrombomodulin, can antagonize thrombin-mediated PAR-1 activation, and
Xa may serve to activate PAR-1 in these microenvironments. During
anticoagulant therapy with potent thrombin57 or
prothrombinase inhibitors,26 one can envision that the
excessive generation of Xa can propagate PAR-1 activation normally
achieved by thrombin. Xa signaling may be particularly important in
temporal or spatial separation from thrombin generation, when cells
exit into extravascular spaces that do not support downstream
activation of the coagulation cascade. Monocytes/macrophages are known
to express PAR-158 and the
Notwithstanding the experimental evidence that PAR-2, expressed in endothelial cells60 and vascular smooth muscle cells,61 is efficiently activated by Xa,20,62 endogenously expressed PAR-1 is here clearly shown to be a target for activation by Xa in a tumor cell line. The Xa-dependent activation of PAR-1 suggests that Xa may function as a more generalized proteolytic signal by bypassing the restricted expression of members of the PAR family in certain vascular (eg, monocytes/macrophages) and extravascular (eg, tumor cells) cell types that frequently stain positively for Xa.63 The apparent membrane-binding requirement for Xa signaling strongly suggests primarily autocrine functions of this protease, representing the most significant difference from thrombin that has a central role in cell-cell communication because of its free diffusion away from cell surfaces.
We thank Pablito Tejada and Aaron Donner for excellent technical assistance and Dr George Vlasuk (Corvas International, San Diego, CA) for the generous gift of the Xa inhibitors TAP and NAP5.
Submitted September 20, 2000; accepted January 19, 2001.
Supported by National Institutes of Health grants HL16411 (W.R.) and HL40387 (L.F.B.) and by an RPR/ISTH Thrombosis Research Fellowship Award (M.R.). W.R. is an Established Investigator of the American Heart Association.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Wolfram Ruf, Department of Immunology, C204, The Scripps Research Institute, 10550 North Torrey Pines Rd, La Jolla, CA 92037; e-mail: ruf{at}scripps.edu.
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  B. J. Wilson, R. Harada, L. LeDuy, M. D. Hollenberg, and A. Nepveu CUX1 Transcription Factor Is a Downstream Effector of the Proteinase-activated Receptor 2 (PAR2) J. Biol. Chem., January 2, 2009; 284(1): 36 - 45. [Abstract] [Full Text] [PDF]
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G. Bhattacharjee, J. Ahamed, R. Pawlinski, C. Liu, N. Mackman, W. Ruf, and T. S. Edgington Factor Xa Binding to Annexin 2 Mediates Signal Transduction via Protease-Activated Receptor 1 Circ. Res., February 29, 2008; 102(4): 457 - 464. [Abstract] [Full Text] [PDF]
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 K. Borensztajn, J. Stiekema, S. Nijmeijer, P. H. Reitsma, M. P. Peppelenbosch, and C. A. Spek Factor Xa Stimulates Proinflammatory and Profibrotic Responses in Fibroblasts via Protease-Activated Receptor-2 Activation Am. J. Pathol., February 1, 2008; 172(2): 309 - 320. [Abstract] [Full Text] [PDF]
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 J. W. Mitchell, N. Baik, F. J. Castellino, and L. A. Miles Plasminogen inhibits TNF{alpha}-induced apoptosis in monocytes Blood, June 1, 2006; 107(11): 4383 - 4390. [Abstract] [Full Text] [PDF]
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 Y.-J. Yin, V. Katz, Z. Salah, M. Maoz, I. Cohen, B. Uziely, H. Turm, S. Grisaru-Granovsky, H. Suzuki, and R. Bar-Shavit Mammary Gland Tissue Targeted Overexpression of Human Protease-Activated Receptor 1 Reveals a Novel Link to {beta}-Catenin Stabilization. Cancer Res., May 15, 2006; 66(10): 5224 - 5233. [Abstract] [Full Text] [PDF]
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R. Sood, S. Kalloway, A. E. Mast, C. J. Hillard, and H. Weiler Fetomaternal cross talk in the placental vascular bed: control of coagulation by trophoblast cells Blood, April 15, 2006; 107(8): 3173 - 3180. [Abstract] [Full Text] [PDF]
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 T. M. Hackeng, K. M. Sere, G. Tans, and J. Rosing Protein S stimulates inhibition of the tissue factor pathway by tissue factor pathway inhibitor PNAS, February 28, 2006; 103(9): 3106 - 3111. [Abstract] [Full Text] [PDF]
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D. R. Morris, Y. Ding, T. K. Ricks, A. Gullapalli, B. L. Wolfe, and J. Trejo Protease-Activated Receptor-2 Is Essential for Factor VIIa and Xa-Induced Signaling, Migration, and Invasion of Breast Cancer Cells Cancer Res., January 1, 2006; 66(1): 307 - 314. [Abstract] [Full Text] [PDF]
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 K. E. Welty-Wolf, M. S. Carraway, T. L. Ortel, A. J. Ghio, S. Idell, J. Egan, X. Zhu, J.-a. Jiao, H. C. Wong, and C. A. Piantadosi Blockade of tissue factor-factor X binding attenuates sepsis-induced respiratory and renal failure Am J Physiol Lung Cell Mol Physiol, January 1, 2006; 290(1): L21 - L31. [Abstract] [Full Text] [PDF]
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A. Hezi-Yamit, P. W. Wong, N. Bien-Ly, L. G. Komuves, K. S. S. Prasad, D. R. Phillips, and U. Sinha Synergistic induction of tissue factor by coagulation factor Xa and TNF: Evidence for involvement of negative regulatory signaling cascades PNAS, August 23, 2005; 102(34): 12077 - 12082. [Abstract] [Full Text] [PDF]
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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]
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 M. Steinhoff, J. Buddenkotte, V. Shpacovitch, A. Rattenholl, C. Moormann, N. Vergnolle, T. A. Luger, and M. D. Hollenberg Proteinase-Activated Receptors: Transducers of Proteinase-Mediated Signaling in Inflammation and Immune Response Endocr. Rev., February 1, 2005; 26(1): 1 - 43. [Abstract] [Full Text] [PDF]
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 G. Busch, I. Seitz, B. Steppich, S. Hess, R. Eckl, A. Schomig, and I. Ott Coagulation Factor Xa Stimulates Interleukin-8 Release in Endothelial Cells and Mononuclear Leukocytes: Implications in Acute Myocardial Infarction Arterioscler Thromb Vasc Biol, February 1, 2005; 25(2): 461 - 466. [Abstract] [Full Text] [PDF]
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L. V. M. Rao and U. R. Pendurthi Tissue Factor-Factor VIIa Signaling Arterioscler Thromb Vasc Biol, January 1, 2005; 25(1): 47 - 56. [Abstract] [Full Text] [PDF]
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M. Majumdar, T. Tarui, B. Shi, N. Akakura, W. Ruf, and Y. Takada Plasmin-induced Migration Requires Signaling through Protease-activated Receptor 1 and Integrin {alpha}9{beta}1 J. Biol. Chem., September 3, 2004; 279(36): 37528 - 37534. [Abstract] [Full Text] [PDF]
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N. Mackman Role of Tissue Factor in Hemostasis, Thrombosis, and Vascular Development Arterioscler Thromb Vasc Biol, June 1, 2004; 24(6): 1015 - 1022. [Abstract] [Full Text] [PDF]
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J. Ahamed and W. Ruf Protease-activated Receptor 2-dependent Phosphorylation of the Tissue Factor Cytoplasmic Domain J. Biol. Chem., May 28, 2004; 279(22): 23038 - 23044. [Abstract] [Full Text] [PDF]
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G. M. Hjortoe, L. C. Petersen, T. Albrektsen, B. B. Sorensen, P. L. Norby, S. K. Mandal, U. R. Pendurthi, and L. V. M. Rao Tissue factor-factor VIIa-specific up-regulation of IL-8 expression in MDA-MB-231 cells is mediated by PAR-2 and results in increased cell migration Blood, April 15, 2004; 103(8): 3029 - 3037. [Abstract] [Full Text] [PDF]
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R. Pawlinski, B. Pedersen, G. Schabbauer, M. Tencati, T. Holscher, W. Boisvert, P. Andrade-Gordon, R. D. Frank, and N. Mackman Role of tissue factor and protease-activated receptors in a mouse model of endotoxemia Blood, February 15, 2004; 103(4): 1342 - 1347. [Abstract] [Full Text] [PDF]
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B.-H. Koo and D.-S. Kim Factor Xa Induces Mitogenesis of Vascular Smooth Muscle Cells via Autocrine Production of Epiregulin, J. Biol. Chem., December 26, 2003; 278(52): 52578 - 52586. [Abstract] [Full Text] [PDF]
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P. J. O'Brien, H. Koi, S. Parry, L. F. Brass, J. F. Strauss III, L.-P. Wang, J. E. Tomaszewski, and L. K. Christenson Thrombin Receptors and Protease-Activated Receptor-2 in Human Placentation: Receptor Activation Mediates Extravillous Trophoblast Invasion in Vitro Am. J. Pathol., October 1, 2003; 163(4): 1245 - 1254. [Abstract] [Full Text] [PDF]
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C. D. Major, R. J. Santulli, C. K. Derian, and P. Andrade-Gordon Extracellular Mediators in Atherosclerosis and Thrombosis: Lessons From Thrombin Receptor Knockout Mice Arterioscler Thromb Vasc Biol, June 1, 2003; 23(6): 931 - 939. [Abstract] [Full Text] [PDF]
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M. Moser and C. Patterson Thrombin and Vascular Development: A Sticky Subject Arterioscler Thromb Vasc Biol, June 1, 2003; 23(6): 922 - 930. [Abstract] [Full Text] [PDF]
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 H. H. Versteeg, M. P. Peppelenbosch, and C. A. Spek Tissue factor signal transduction in angiogenesis Carcinogenesis, June 1, 2003; 24(6): 1009 - 1013. [Abstract] [Full Text] [PDF]
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 N. Asokananthan, P. T. Graham, D. J. Stewart, A. J. Bakker, K. A. Eidne, P. J. Thompson, and G. A. Stewart House Dust Mite Allergens Induce Proinflammatory Cytokines from Respiratory Epithelial Cells: The Cysteine Protease Allergen, Der p 1, Activates Protease-Activated Receptor (PAR)-2 and Inactivates PAR-1 J. Immunol., October 15, 2002; 169(8): 4572 - 4578. [Abstract] [Full Text] [PDF]
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U. R. Pendurthi, M. Ngyuen, P. Andrade-Gordon, L. C. Petersen, and L. V. M. Rao Plasmin Induces Cyr61 Gene Expression in Fibroblasts Via Protease-Activated Receptor-1 and p44/42 Mitogen-Activated Protein Kinase-Dependent Signaling Pathway Arterioscler Thromb Vasc Biol, September 1, 2002; 22(9): 1421 - 1426. [Abstract] [Full Text] [PDF]
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E. Camerer, H. Kataoka, M. Kahn, K. Lease, and S. R. Coughlin Genetic Evidence That Protease-activated Receptors Mediate Factor Xa Signaling in Endothelial Cells J. Biol. Chem., May 3, 2002; 277(18): 16081 - 16087. [Abstract] [Full Text] [PDF]
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M. Riewald and W. Ruf Mechanistic coupling of protease signaling and initiation of coagulation by tissue factor PNAS, July 3, 2001; 98(14): 7742 - 7747. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
(20 ng/mL) for the indicated times, followed by nuclear extract
preparation for EMSA using oligonucleotide probes specific for NF-
2 integrin (Mac1) that is a receptor for
the zymogen factor X.