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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Department of Medical Sciences, Laboratory for
Coagulation Research, Clinical Chemistry, University Hospital, Uppsala,
Sweden; the Ludwig Institute for Cancer Research,
Biomedical Centre, Uppsala, Sweden; and Tissue
Factor/Factor VIIa Research, Health Care Discovery, Novo Nordisk A/S,
Maaloev, Denmark.
Tissue factor (TF) is the cellular receptor for factor FVIIa
(FVIIa), and the complex is the principal initiator of blood coagulation. The effects of FVIIa binding to TF on cell migration and
signal transduction of human fibroblasts, which express high amounts of
TF, were studied. Fibroblasts incubated with FVIIa migrated toward a
concentration gradient of PDGF-BB at approximately 100 times lower
concentration than do fibroblasts not ligated with FVIIa. Anti-TF
antibodies inhibited the increase in chemotaxis induced by FVIIa/TF.
Moreover, a pronounced suppression of chemotaxis induced by PDGF-BB was
observed with active site-inhibited FVIIa (FFR-FVIIa). The possibility
that hyperchemotaxis was induced by a putative generation of FXa and
thrombin activity was excluded. FVIIa/TF did not induce increased
levels of PDGF Tissue factor (TF) is a transmembrane glycoprotein
with sequence homology to the class II cytokine/hematopoietic growth
factor receptor family that includes receptors for interferon- Human fibroblasts have a constitutive expression of TF.1
These cells also express receptors for platelet-derived growth factor
(PDGF).15,16 PDGF induces in its target cells
mitogenicity, actin reorganization, and directed cell migration
(chemotaxis) (for review, see Heldin and Westermark15). We
have previously shown that PDGF-BB is an efficient chemotactic factor
for human fibroblasts and that the chemotactic response is mediated by
the In this article we show for the first time a clear connection between
the signaling induced by FVIIa binding to TF and the cellular response
to a growth factor. We present data that in human fibroblasts the
FVIIa/TF complex leads to a hyperchemotactic response to PDGF-BB.
Cell cultures
Proteins
Flow cytometry The surface expression of TF was analyzed by immunofluorescence with a flow cytometer (Coulter Epics XL-MCL; Beckman Coulter, Fullerton, CA). The instrument was calibrated daily with Flow Check calibration beads (Coulter). For indirect immunofluorescence experiments, AG1518 or AG1523 fibroblasts and human vascular smooth muscle cells were washed twice with phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin, incubated for 30 minutes on ice with a fluorescein isothiocyanate (FITC)-labeled anti-human TF monoclonal antibody (4508CJ; American Diagnostica, Greenwich, CT). The anti-Aspergillus niger glucose oxidase monoclonal IgG1 (Dakopatts) was used as a negative control. Mean channel fluorescence intensity and percentage of positive cells were determined for each sample.Determination of TF activity The procoagulant activity of TF was determined as described by Lindmark et al.21 Briefly, aliquots containing 0.2 × 105 AG1518 or AG1523 fibroblasts were washed twice with PBS and placed in the wells of a 96-well microtiter plate (Nunc, Roskilde, Denmark). The procoagulant activity was measured in a 2-stage amidolytic assay in which a chromogenic substrate, S-2222 (Chromogenix, Mölndal, Sweden), is cleaved by FXa, which in turn is activated from FX by the FVIIa/TF complex. A reaction mixture containing final concentrations of 0.6 mmol/L S-2222, 2 mmol/L CaCl2, and coagulation factors from the factor concentrate Prothromplex-T TIM4 (Baxter, Vienna, Austria) at a final concentration of 1 U/mL FVII and 1.2 U/mL FX was added to the wells, and change in absorbance at 405 nm after a 30-minute incubation at 37°C was determined. Measurements were performed in triplicate.Chemotaxis assay The migration response of fibroblasts and vascular smooth muscle cells (SMCs) was assayed by means of the leading front technique in a modified Boyden chamber, as previously described.17,22 Nitrocellulose filters (pore size, 8 µm) were coated with a solution of type 1 collagen at room temperature overnight. The filters were air-dried for 30 minutes immediately before use. Human foreskin fibroblasts AG1523 were grown to confluence in EMEM supplemented with 10% FBS. The cells were detached by trypsinization (2.5 mg/mL for 10 minutes at 37°C) and suspended in EMEM with 10% FBS. The fibroblasts were incubated for 10 minutes with or without FVIIa, FFR-FVIIa, Fxa, or thrombin before assay. SMC were grown in SmGM-2 with 5% FBS, resuspended in the same medium, and incubated for 10 minutes with FVIIa or FFR-FVIIa before assay. One hundred microliters of the cell suspension (2 × 105 cells/mL) was added above the filter of the Boyden chamber. PDGF-BB was diluted in assay media (EMEM with 10% FBS or SmGM-2 with 5% FBS) and added below the filter in the chamber. The cells were incubated for 6 hours at 37°C in a humidified chamber containing 95% air/5% CO2. FVIIa, FFR-FVIIa, FXa, or thrombin was present during the entire experiment. The filters were then removed, fixed in ethanol, stained with Mayer hemalum, and mounted. Migration was measured as the distance of the 2 farthest migrating fibroblast nuclei of one high-power field (12.5 × 24) in focus. The migration distance in each filter was calculated as the mean of the readings of at least 3 different parts of the filter. Experiments were performed with 2 to 4 separate filters for each concentration of chemoattractant. For each set of experiments, the migration of cells toward the assay media served as control.When anti-TF monoclonal antibodies or inhibitors to coagulation factors TAP and Hirudin, were used, cells were preincubated for 10 minutes with these agents, then with or without FVIIa, FFR-FVIIa, FXa, or thrombin before the chemotaxis assay was performed. Antibodies, TAP, or Hirudin were also present during the entire chemotaxis experiment. Assay for release of inositol triphosphate Six-well plates with subconfluent cultures of AG1518 human fibroblasts were incubated overnight (approximately 20 hours) with 2 µCi of myo-[3H]-inositol (Amersham, Buckinghamshire, UK) in 2 mL Ham F12 with 0.1% FBS. Medium was changed to Ham F12 with 0.1% FBS (containing 2 mmol/L CaCl2) and 20 mmol/L LiCl, and the cells were incubated for 15 minutes at 37°C. Cells were then incubated in the absence or presence of 100 nmol/L FVIIa or 100 nmol/L FFR-FVIIa for 1 hour. PDGF-BB (0, 10, or 100 ng/mL) was added, and the incubation was continued for 10 minutes at 37°C. The IP3 assay was performed as previously described by Eriksson et al.23Assay for determination of PDGF Assay for agonist-induced PLC-  1 was precipitated, essentially as previously described,16 with
anti-PLC-1 antiserum generated by immunizing rabbits with a peptide
corresponding to the carboxy terminus of bovine
PLC-1.25 Samples were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
immunoblotted with the anti-phosphotyrosine antibody PY99.
Statistical analysis Data were analyzed using the Statistica for Windows package (StatSoft, Tulsa, OK). The Student t test for dependent samples was used to determine statistical significance between different data sets. P < .05 was considered statistically significant.
Effects of FVIIa and FFR-FVIIa on the chemotactic response of fibroblasts and vascular smooth muscle cells to PDGF-BB Fibroblasts expressing active TF (Figure 1) were incubated with 100 nmol/L FVIIa and seeded in the upper part of the modified Boyden chamber; and media containing 10% FBS and PDGF-BB at different concentrations were added below the 150 µm micropore filter. The migration of the cells under conditions in which medium containing 10% FBS without PDGF-BB was added below the filter was used as a measure of random migration and was calculated as 100% migration. A significant migration response was recorded at a concentration of 0.01 ng/mL PDGF-BB in cells stimulated by FVIIa compared to 1 ng/mL PDGF-BB for cells not ligated with FVIIa (ie, a 100-fold difference in concentration) (Figure 2A). At 0.01 to 0.1 ng/mL PDGF-BB, the migration response to FVIIa increased dose dependently, starting at 25 nmol/L with a maximal effect at 50 to 100 nmol/L FVIIa (Figure 3A-D). No enhancement of random migration was observed after activation with FVIIa. To test whether the proteolytically active FVIIa was mandatory for the hyperchemotactic response to PDGF-BB, fibroblasts were also incubated with 100 nmol/L FFR-FVIIa and assayed in the Boyden chamber in the same way (Figure 2A). No increased chemotaxis was observed with FFR-FVIIa at low concentrations of PDGF-BB, 0.01 to 1 ng/mL. In contrast, a pronounced suppression of chemotaxis induced by 10 to 50 ng/mL PDGF-BB was achieved by 100 nmol/L FFR-FVIIa (Figure 2A, 3A-D). To clarify whether these results are of a more general nature, migration experiments with 2 human vascular smooth muscle cell lines were performed under the same conditions. These cells have spontaneous expression of TF and PDGF -receptors (analyzed by flow cytometry; data not shown). Incubation
with 100 nmol/L FVIIa and FFR-FVIIa resulted in the same migration
pattern (Figure 2B).
When fibroblasts were preincubated with a mixture of 3 different TF
antibodies and then with FVIIa or FFR-FVIIa, the migration response to
PDGF-BB was identical to the response of fibroblasts without the
presence of ligand bonded to TF. An irrelevant monoclonal IgG antibody
prevented neither hyperchemotaxis induced by FVIIa nor inhibition of
the migration response induced by FFR-FVIIa (data not shown). The
presence of the IgG antibodies or the 3 TF antibodies did not change
the random migration of the fibroblasts (data not shown). To analyze
whether the enhanced chemotaxis was a result of increased numbers of
PDGF The hyperchemotactic response is not mediated by FXa or by thrombin Because FVIIa-induced signal transduction leading to the hyperchemotactic response to PDGF-BB was dependent on the catalytic activity of FVIIa, it was important to determine whether signaling occurred directly or through FXa or thrombin generated by the FVIIa/TF complex. The enhanced migration response transduced by FVIIa/TF was not blocked by 0.2 to 10 µmol/L Tick anticoagulant peptide (TAP), which specifically blocks the active site of FXa and prevents a further activation of the coagulation cascade leading to thrombin formation (Figure 4). Addition of 5 U/mL Hirudin, a specific thrombin inhibitor, had no effect on FVIIa/TF-induced hyperchemotaxis (data not shown). Neither TAP (Figure 4) nor Hirudin (data not shown) influenced the migration of fibroblasts in response to PDGF-BB without the presence of the ligand FVIIa. Stimulation of the fibroblasts with 1 to 100 nmol/L FXa or 0.1 to 100 nmol/L thrombin in the absence or the presence of TAP or Hirudin, respectively, resulted in identical chemotaxis toward PDGF-BB, as recorded with fibroblasts without ligands (Figure 5). Thus, it is unlikely that the effect of FVIIa on chemotaxis is mediated by the activation of FXa or thrombin.
FVIIa/TF-induced activation of PLC To investigate whether the FVIIa/TF-induced chemotactic response involved the activation of phosphatidylinositol-specific phospholipase C (PLC), we analyzed the direct effects of FVIIa/TF on PLC activity in fibroblasts. Activation of PLC leads to the production of 2 second messengers, inositol-1,4,5-trisphosphate (IP3) and diacylglycerol. Fibroblasts were incubated with myo-[3H]-inositol overnight and then with 100 nmol/L FVIIa or FFR-FVIIa for 60 minutes, followed by incubation with or without PDGF-BB at indicated concentrations. Treatment with 100 nmol/L FVIIa alone for 60 minutes induced IP3 release in fibroblasts at the same level as 10 ng/mL and 100 ng/mL PDGF-BB alone (Figure 6). Moreover, the combination of 100 nmol/L FVIIa and 10 ng/mL or 100 ng/mL PDGF-BB doubled the IP3 release. The active site-inhibited FVIIa did not induce the release of IP3. These results clearly show that PLC is activated on binding of FVIIa to TF.
Phosphorylation of PLC- 1 isoform, which is activated by
certain tyrosine kinase receptors, was responsible for the increased
PLC activity induced by FVIIa/TF, tyrosine phosphorylation of PLC-1
was studied. Fibroblasts were incubated in the absence or presence of
100 nmol/L FVIIa or FFR-FVIIa for 1 hour, followed by stimulation with
0, 2, 10, or 100 ng/mL PDGF-BB. After 5 minutes of incubation, the
cells were lysed and PLC-1 was immunoprecipitated, separated by
SDS-PAGE, and immunoblotted with anti-phosphotyrosine antibodies.
Whereas a significant increase in tyrosine phosphorylation of PLC-1
was recorded with increasing concentrations of PDGF-BB, the addition of
FVIIa alone to the fibroblasts did not induce any tyrosine
phosphorylation of PLC-1 (Figure 7).
Moreover, the combination of FVIIa and PDGF-BB at different
concentrations did not induce any further phosphorylation compared to
stimulation with PDGF-BB alone (Figure 7). FFR-FVIIa had no effect on
PLC-1 tyrosine phosphorylation (Figure 7). Thus, PLC isoforms other than PLC-1 are responsible for the increased PLC activity after FVIIa stimulation.
Tissue factor is constitutively expressed on the plasma membrane of many extravascular cells, such as stromal fibroblasts in vascular adventitia and in fibrous capsules of liver, spleen, and kidney.1 Thus, the expression of TF is found at sites physically separated from the circulating blood providing a hemostatic envelope. With injury this barrier is thought to protect the organism against bleeding. TF can, however, be induced in monocytes/macrophages, vascular smooth muscle cells, endothelial cells, and tumor cells by various agents, including cytokines and growth factors.1 Induction at the transcriptional level occurs rapidly after stimulation, identifying TF as a growth-related immediate-early gene.26 Data from studies in which the TF gene was inactivated in mice demonstrated that the deficiency of TF results in embryonic lethality because of the defective development of blood vessels.27-29 Insufficient accumulation and differentiation of peri-endothelial mesenchymal cells to pericytes/primitive smooth muscle cells occurred in the TF-deficient embryos.29 A role for TF in tumor angiogenesis has also been
proposed.30,31 However, contradictory results In this study we have investigated the role of TF as a signaling
receptor. We show that human fibroblasts with a constitutive expression
of TF on ligand binding of FVIIa migrate toward extremely low
concentrations of PDGF-BB. FVIIa/TF alone did not induce enhanced spontaneous migration (ie, random migration). Thus, a combination of
intracellular signal transduction by FVIIa/TF and the growth factor
PDGF-BB was necessary to achieve the motility response. Not only was
binding to TF mandatory, but so was the catalytic activity of FVIIa/TF
because active site-inhibited FVIIa did not elicit an enhanced
migration response. Furthermore, inhibitory monoclonal antibodies
prevented enhancement of the chemotactic response by FVIIa. We also
excluded that indirect signaling occurred as a result of FXa or
thrombin because TAP and hirudin had no effect on FVIIa/TF-induced
chemotaxis. Moreover, FXa and thrombin themselves did not enhance the
migration response to PDGF-BB. We instead found that increasing
concentrations of FFR-FVIIa actively inhibited PDGF-BB-induced
chemotaxis. Fibroblasts incubated with FFR-FVIIa showed completely
normal random migration. The inhibitory effect of FFR-FVIIa on
PDGF-BB-induced chemotaxis was not observed in the presence of the
combination of anti-TF antibodies, thereby ruling out the possibility
that FFR-FVIIa was toxic. The results suggest, rather, that in cells
expressing PDGF A chemoattractant property of FVIIa/TF complexes in solution has previously been demonstrated.36,37 In these experiments, aortic smooth muscle cells migrate toward a concentration gradient of 0.3 nmol/L FVIIa/TF complexes and thereby exclude signaling events transduced by the TF-intracellular domain. Our finding that FVIIa increases IP3 production, and the
previously reported data on FVIIa/TF-induced Ca++
oscillations, especially in MDCK cells, strongly support the notion
that PLC is activated by FVIIa/TF signaling in a number of
cells.7,8 We previously found a similar hyperchemotactic response to PDGF-BB in PDGF Lately, the connection between TF and the cytoskeleton was
identified.14,42 A molecular interaction between the
cytoplasmic domain of TF and the actin filament-binding protein ABP-280
was shown.14 Furthermore, TF was found to be in close
contact with actin and actin filament-binding proteins, such as
Chemotaxis plays a pivotal role in wound healing, angiogenesis, and metastasis. Chemotaxis is also an important component in the development of atherosclerotic plaques. In these processes various cells express TF, PDGF, and PDGF receptors. Restenosis is a major complication after interventional procedures to open obstructed arteries. PDGF has been implicated in vessel wall response (neointima formation) to mechanical injury by mediating the migration and proliferation of smooth muscle cells and fibroblasts. We have shown now, for the first time, that FVIIa binding to TF-expressing cells includes an increased chemotactic response to PDGF independent of coagulation. This finding can explain the efficacy of blocking FVIIa/TF activity in reducing neointima formation in animal models of restenosis.47 In addition to limiting thrombin generation, thrombus formation, and the cellular events caused by thrombin signaling, the inhibition of FVIIa/TF would locally inhibit the migration of TF-expressing cells at the site of injury. Our observations suggest that the inhibition of FVIIa may become clinically important for regulating these events.
We thank Dr Lars C. Petersen (Novo Nordisk A/S) for fruitful discussions.
Submitted June 23, 2000; accepted July 18, 2000.
Supported by the Swedish Cancer Society and the Swedish Medical Research Council.
A.S. and L.R. are Senior Researchers supported by the Swedish Medical Research Council.
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: Agneta Siegbahn, Department of Medical Sciences, Laboratory for Coagulation Research, Clinical Chemistry, University Hospital, SE-751 85 Uppsala, Sweden; e-mail: agneta.siegbahn{at}klinkem.uas.lul.se.
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Regulation of chemotaxis by the platelet-derived growth factor receptor- |
Top
Abstract
Introduction
,
-
shows the chemotactic
response of cells to different concentrations of PDGF-BB. Cells
incubated with 100 nmol/L FVIIa (
) or 100 nmol/L FFR-FVIIa (
)
migrated to different concentrations of PDGF-BB. Results are
means± SEM of 3 separate experiments (A) or mean± SD of 2 separate
experiments (B). *P < .05 was considered statistically
significant (Student t test); **P < .01; ***P < .001. The same migration response to
PDGF-BB was obtained with coronary artery SMC (data not
shown).
) of 200 nmol/L TAP and with 100 nmol/L thrombin in the
absence (
with no
essential role for TF in embryonic vascular development but the
requirement for uterine hemostasis
PLC isozymes. We cannot fully exclude that the PLC activation is
mediated by FXa or by thrombin. In the assay for IP3
release, the cell culture medium was supplemented with 0.1% FBS
containing only approximately 0.1 nmol/L FXa. We found that a
concentration of more than 20 nmol/L FXa is necessary to induce
IP3 production (data not shown). The mechanism by which