On the mechanism of thrombin-induced angiogenesis: involvement of alpha vbeta 3-integrin

Nikos E. Tsopanoglou, Paraskevi Andriopoulou, and Michael E. Maragoudakis

Department of Pharmacology, Medical School, University of Patras, 25110 Patras, Greece


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Thrombin has been reported to be a potent angiogenic factor both in vitro and in vivo, and many of the cellular effects of thrombin may contribute to activation of angiogenesis. In this report we show that thrombin-treatment of human endothelial cells increases mRNA and protein levels of alpha vbeta 3-integrin. This thrombin-mediated effect is specific, dose dependent, and requires the catalytic site of thrombin. In addition, thrombin interacts with alpha vbeta 3 as demonstrated by direct binding of alpha vbeta 3 protein to immobilized thrombin. This interaction of thrombin with alpha vbeta 3-integrin, which is an angiogenic marker in vascular tissue, is of functional significance. Immobilized thrombin promotes endothelial cells attachment, migration, and survival. Antibody to alpha vbeta 3 or a specific peptide antagonist to alpha vbeta 3 can abolish all these alpha vbeta 3-mediated effects. Furthermore, in the chick chorioallantoic membrane system, the antagonist peptide to alpha vbeta 3 diminishes both basal and the thrombin-induced angiogenesis. These results support the pivotal role of thrombin in activation of endothelial cells and angiogenesis and may be related to the clinical observation of neovascularization within thrombi.

attachment; migration; apoptosis; reverse transcription-polymerase chain reaction


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE FREQUENCY OF BLOOD COAGULATION in cancer patients, known for more than 130 years, is supported by clinical, laboratory, and histopathological evidence. This is explained at the molecular and cellular level by the thromboplastic activity of circulating tumor cells, the existence of "a cancer coagulative factor," the activation of factor X, the generation of prothrombinase by tumor cells, and the encircling of cancerous tissue by fibrin deposits (38, 50). In addition, the possibility of a relation between blood clotting mechanisms and tumor progression and development of metastases was postulated as early as 1878 by Billroth (7) on the basis of the observation that cancer cells exist within thrombi. This finding was interpreted as evidence that tumor cells spread by thromboembolism. More recently, large epidemiological studies have provided evidence that the standardized incidence ratio for certain types of cancer is as high as 6.7 within a year following a thromboembolic episode (3, 41). These clinical data are in line with animal experiments where thrombin-treated B16 melanoma cells show a dramatic increase in their metastatic potential in the lung of rats (37). These observations have led to experimental use of heparin, aspirin, and warfarin for the prevention and treatment of tumors in animal models and humans (23, 50).

We proposed earlier (33, 47) that the tumor-promoting effect of thrombin/thrombosis may be related to our finding that thrombin is a potent promoter of angiogenesis, a process essential for tumor growth and metastasis. The angiogenic action of thrombin was shown to be receptor-mediated and independent of fibrin formation. The importance of thrombin and its receptors in embryonic development and angiogenesis is also supported by the findings of Griffin et al. (21), who showed that the expression of protease-activated receptor-1 (PAR-1) by endothelial cells rescues the fetal vessel fragility and bleeding of mouse embryo engineered to lack PAR-1. Recently, we have shown that thrombin has a synergistic effect with the vascular endothelial growth factor (VEGF) by upregulating its receptors in cultured endothelial cells (46). This finding, in connection to data showing that thrombin increases the secretion of VEGF from human prostate cancer cells (32), may result in mutual stimulation of endothelial and cancer cells for activation of angiogenesis and tumor progression. The role of thrombin receptors in angiogenesis and tumor progression is also supported by the findings of Even-Ram et al. (19), who showed that the metastatic ability of human breast cancer cells is related to the number of thrombin receptors on these cells.

In this paper we present evidence that thrombin interacts with alpha vbeta 3-integrin at the molecular and cellular level in endothelial cells. Integrin alpha vbeta 3 is known to be expressed in vascular cells during angiogenesis and remodeling and in tumor cells, where it contributes to malignant phenotype (18). We demonstrate that thrombin-treatment of endothelial cells upregulates the expression of mRNA and protein of alpha vbeta 3-integrin, resulting in the potentiation of cell migration toward vitronectin, the alpha vbeta 3 ligand. In addition, we show that immobilized thrombin, like vitronectin, can serve as a ligand for alpha vbeta 3-integrin. This interaction of thrombin with alpha vbeta 3 is shown to be of functional significance in vitro and in vivo. Immobilized thrombin facilitates endothelial cell attachment, migration, and survival via alpha vbeta 3-integrin interaction. In addition, we show that alpha vbeta 3-integrin is involved in the thrombin-promoting angiogenesis in the chick chorioallantoic membrane system.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

Endothelial cell culture. Human umbilical vein endothelial cells (HUVECs) were obtained by established methods (25) from freshly delivered umbilical cords from caesarean births. Cells were cultured as described previously (46) and used for experiments from passages 3-5.

RNA isolation and semiquantitative RT-PCR. After reaching confluence and 3 days after the last medium change, HUVECs were incubated with serum-free M199 medium containing 0.5% bovine serum albumin (SFM199-0.5% BSA) alone or with thrombin (kindly provided by Dr. J. Fenton II, Albany, NY) or with hirudin (Sigma) or DIP-thrombin (thrombin that is chemically inactivated at the active site by diisopropylphosphofluoridate; kindly provided by Dr. J. Fenton II). After the indicated time periods, total cellular RNA was purified by the guanidinium thiocyanate-phenol-chloroform method (12). RT-PCR was performed by using the Promega access RT-PCR system (Madison, WI) according to the manufacturer's protocol. Primer sequences (all synthesized by Research and Technology Institute, Heraclion, Greece) were as follows: beta 3 (24) (sense, 5'-GTGCTGACGCTAACTGACC-3'; antisense, 5'-CATGGTAGTGGAGGCAGAGT-3'; expected size of PCR product, 284 bp) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal control (sense, 5'-TGAAGGTCGGAGTCAACGGATTTG-3'; antisense, 5'-CATGTGGGCCATGAGGTCCACCAC-3'; expected size of PCR product, 967 bp). The RT-PCR profile consisted of 45 min at 48°C for reverse transcription and 5 min of initial denaturation at 94°C, followed by 26 cycles of 1 min of denaturation at 94°C, 1 min of annealing at 54°C, 1 min of polymerization at 72°C, and, finally, 10 min of extension at 72°C. Ten microliters of the RT-PCR products were separated in 1% (wt/vol) agarose gels and stained with ethidium bromide. The gels were then photographed and scanned to quantitate the obtained RT-PCR products. Densitometry analysis was performed by using image analysis software (ImagePC; Scion), and the ratio of beta 3 to GAPDH in each lane was calculated. Results are representative of three independent experiments and are expressed as means ± SE. Statistical analysis was performed with Student's t-test.

Immunoprecipitation and Western blot analysis. Three days after the last medium change, confluent endothelial monolayers on gelatin were incubated with SFM199-0.5% BSA alone or with thrombin for 24 h. Viability of endothelial cells was tested by trypan blue exclusion, and live cell number was estimated for each experimental group. Cells were then lysed at 4°C by scraping in lysis buffer containing 10 mM Tris · HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 mM N-ethylmaleimide, 0.1 U/ml aprotinin, and 10 µg/ml leupeptin (all from Sigma). For immunoprecipitation, cell extracts were precleared with normal mouse IgG (Sigma) for 1 h and immunoprecipitated with protein A-Sepharose conjugated with mouse monoclonal antibody against human alpha vbeta 3-integrin (clone LM609; Chemicon International). The supernatants were then reimmunoprecipitated with protein A-Sepharose conjugated with goat polyclonal antibody against human actin (Santa Cruz Biotechnology), a highly conserved protein that is expressed in all eukaryotic cells. Immunoprecipitated protein (alpha vbeta 3, actin) were then separated on 8% SDS-PAGE under nonreducing conditions. After electrophoresis, proteins on gels were transferred on nitrocellulose membranes. The membranes obtained after blocking with 10% skim milk were first incubated with anti-beta 3 mouse monoclonal antibody CD61 (Chemicon International) or the anti-actin goat polyclonal antibody for 2 h at room temperature and with a rabbit anti-mouse IgG or rabbit anti-goat IgG conjugated with horseradish peroxidase for 1 h. The blots were then developed by using enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech) according to the manufacturer's protocol. Films were then scanned to quantitate the obtained beta 3-subunit and actin bands, and the ratio of beta 3 to actin was calculated. Results are representative of three independent experiments and expressed as means ± SE. Statistical analysis was performed with Student's t-test.

Cell attachment assay. Forty-eight-well plates were coated overnight with the indicated concentrations of thrombin or DIP-thrombin. The wells were blocked for 60 min with 0.5% BSA in PBS at 37°C. HUVECs were briefly trypsinized, followed by washes with serum-free M199 medium. Cells were suspended 5 × 105 ml-1 in SFM199-0.5% BSA and incubated in the presence or absence of the cyclic peptide EMD 121974 (c-RGD), cyclic peptide EMD 135981 (c-RAD), or mouse anti-alpha vbeta 3 (LM609) antibody for 15 min at 37°C. The c-RGD peptide (cycled Arg-Gly-Asp-D-Phe-Nme-Val) is a highly active and specific alpha vbeta 3-integrin antagonist (17), whereas the c-RAD (cycled Arg-beta -Ala-Asp-D-Phe-Val) peptide is inactive. Cell suspensions (1 × 105 cells/well) were then added to the wells, and the plates were incubated at 37°C for 60 min. The nonadherent cells were aspirated, and the adhered cells were rinsed twice with cold PBS before being fixed and stained with Diff-Quick (Baxter Healthcare). The average area covered by adhered cells was measured in triplicate wells with a computerized digital image analyzer (ver. 4.12, MCID software, Brock University, St Catherines, ON, Canada). Each experiment was repeated at least two times. Results are means ± SE expressed as pixels area × 10-3.

Cell migration. HUVECs migration was assessed by the modified Boyden's chamber assay, i.e., in Transwell cell culture chambers (Corning and Costar). Polycarbonate filters with 8-µm pores were used to separate the upper and the lower chamber. When chemotaxis (directional motility) analysis was performed, the indicated proteins were added to the lower chamber. Endothelial cells were added to the upper compartment of the chamber at density of 1 × 105 cells/100 µl in SFM199-0.5% BSA and incubated for 6 h at 37°C, allowing cells to migrate in the lower chamber. In some experiments, HUVECs were pretreated with thrombin (1 IU/ml) for 12 h and then washed, trypsinized, and added to the upper chambers. In haptotactic cell motility assay, the undersurface of the membrane filter was precoated with the indicated concentrations of thrombin, DIP-thrombin, the ECM proteins vitronectin or gelatin, or heat-denatured BSA. To modulate the migration toward the immobilized proteins, lower chambers were filled with SFM199-0.5% BSA containing the c-RGD or c-RAD peptide or anti-alpha vbeta 3 antibody (LM609). After incubation for 6 h, the cells on the filters were fixed and stained with Diff-Quick reagents. The nonmigrated cells (cells in upper surface) were removed by wiping with cotton swabs. The filters then with the migrated cells were cut off and mounted on glass slides. To examine the effect of soluble thrombin on the migration toward various immobilized matrix proteins (vitronectin or gelatin), we added thrombin (1 IU/ml) to the lower chambers. The cells on the lower surface were counted manually under microscope in six predetermined fields at high magnification. Each experiment was repeated at least two times, and results are means ± SE expressed in terms of number of cells per high-magnification microscopic field (cells/HMMF). Statistical analysis was performed with Student's t-test.

Cell survival. To determine the capability of immobilized thrombin to support cell survival, we plated HUVECs suspended in SFM199-0.5% BSA on dishes that had been precoated with thrombin, DIP-thrombin, or 0.5% heat-denatured BSA. Where indicated, soluble c-RGD or c-RAD peptides or anti-alpha vbeta 3 antibody was added to cells. Apoptotic cell death was monitored 6 h later by measuring DNA fragmentation using the Cell Death Detection ELISA kit (Roche Molecular Biochemicals). Each experiment was repeated at least two times, and results are means ± SE expressed as optical density at 402 nm (OD402). Statistical analysis was performed with Student's t-test.

Solid-phase ligand-binding assay. Microtiter wells were coated with thrombin or DIP-thrombin in PBS overnight at room temperature. The wells were blocked with 1% BSA in Ca2+/Mg2+ TBS (150 mM NaCl, 25 mM Tris · HCl, pH 7.4, 1 mM CaCl2, 1 mM MgCl2) at room temperature for 2 h. The alpha vbeta 3 protein (Chemicon International) was overlaid in Ca2+/Mg2+ TBS with 10 mM octyl glucoside (Sigma) and incubated with rotation at 4°C overnight. To modify the binding of the purified integrin to thrombin, we preincubated the alpha vbeta 3 protein with c-RGD peptide, anti-alpha vbeta 3 antibody (LM609), or EDTA for 30 min at 4°C. Unbound integrin molecules were removed by three washes with Ca2+/Mg2+ TBS-0.05% Tween 20. The bound alpha vbeta 3 was incubated with monoclonal mouse anti-alpha vbeta 3 antibody (LM609) for 2 h at room temperature. After extensive washes with Ca2+/Mg2+ TBS-0.05% Tween 20, the bound antibodies were detected by using rabbit anti-mouse IgG (Chemicon International) conjugated with horseradish peroxidase. Substrate solution consisting of hydrogen peroxide and tetramethylbenzidine was added to the wells. The reactions were stopped with 2 N sulfuric acid, and absorbance was measured at 492 nm (OD492). Background absorbance observed in the wells coated with BSA was deducted from the values obtained. Each experiment was repeated at least two times, and results are means ± SE expressed as OD492. Statistical analysis was performed with Student's t-test.

Chick chorioallantoic membrane assay. The in vivo chick chorioallantoic membrane (CAM) angiogenesis model was used as described previously (31). Briefly, biochemical evaluation of angiogenesis was performed by determining the extent of collagenous protein biosynthesis in the CAM lying directly under the disks applied at day 9 of chick embryo development. Both control and test disks contained radiolabeled proline (0.5 µCi/disk), and test disks also contained thrombin or c-RGD or c-RAD peptides, or the combination. After 48 h, the tissue under the disks was subjected to collagenase digestion. The resulting radiolabeled tripeptides, corresponding to basement membrane collagen and other collagenous material synthesized by the CAM, were counted and expressed as cpm/mg protein. For each egg, collagenous protein biosynthesis under the disk containing the test material was expressed as a percentage of that under the control disk in the same egg. Results are means ± SE expressed as % of control. Statistical analysis was performed with Student's t-test.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Thrombin upregulates alpha vbeta 3 mRNA and protein levels in HUVECs. We employed a sensitive quantitative RT-PCR technique to examine beta 3 gene expression in HUVECs. Primers for beta 3 and GAPDH were chosen so that they would correspond to the regions where sequence homologies between the two primers are relatively low and would generate products of differing lengths. This allowed us to perform RT-PCR with the housekeeping gene GAPDH into the same reaction tubes with beta 3. Results presented in this report show that each set of primers worked well in specifying their corresponding mRNA. Single bands at about 284 and 967 bp were obtained for beta 3 and GAPDH mRNA, respectively. Titration curves of RT-PCR products were employed for determining the quantitative range in which the reactions proceeded exponentially (Fig. 1A). Signal intensities of the products obtained were plotted as functions of RNA template amount and cycle number. Thus we established the optimal conditions for RT-PCR, which were performed with 500 ng of total RNA for 26 cycles. As shown in Fig. 1B, treatment of HUVECs with thrombin (1.0 IU/ml) resulted in a significant increase in the message for beta 3 compared with untreated cells. The upregulation of beta 3 mRNA expression was evident 8 h after thrombin stimulation. In addition, thrombin increased beta 3 mRNA in a dose-dependent fashion. As shown in Fig. 1C, thrombin at 1 IU/ml increased beta 3 mRNA expression to maximal levels. At 3 IU/ml, the stimulatory effect of thrombin declined to control levels. This bell-shaped effect of thrombin is observed in many of the cellular actions of thrombin, including angiogenesis (47). The specificity of thrombin was examined with the use of hirudin, which inhibits thrombin by binding both the catalytic and the anion-binding exocite. When hirudin (1 IU/ml) combined with thrombin (1 IU/ml), the thrombin-upregulating effect was abolished (Fig. 1D). In addition, DIP-thrombin, which is catalytically inactive, had no effect on beta 3 mRNA levels (Fig. 1D).


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Fig. 1.   Thrombin upregulates alpha vbeta 3 mRNA and protein levels in endothelial cells. A: titration curves of RT-PCR. Signal intensities of obtained beta 3 and GAPDH mRNA products were plotted as a function of cycle number and RNA template amount. B: time course of beta 3 mRNA upregulation by thrombin. Human umbilical vein endothelial cells (HUVECs) were treated with thrombin (Thr, 1 IU/ml), and total RNA was extracted from the cells at indicated times. RNA was also extracted from control cells receiving vehicle but not thrombin from the same intervals of incubation. Quantitative RT-PCR analysis was performed by using 500 ng of total RNA and 26 amplification cycles. The mRNA amounts were quantified by densitometric analysis, and the ratio of beta 3 to GAPDH in each lane was calculated. C: upregulation of beta 3 by thrombin is dose dependent. HUVECs were incubated for 8 h with the indicated concentrations of thrombin. Total RNA was extracted and RT-PCR was performed. D: upregulation of beta 3 by thrombin is specific and requires an active catalytic site. HUVECs were incubated for 8 h with vehicle (Cont), thrombin (1 IU/ml), DIP-thrombin (DIP, 25 µg/ml), or the combination of thrombin with hirudin (Thr+Hir, 1 IU/ml). Total RNA was extracted and RT-PCR was performed. E: thrombin increases the alpha vbeta 3 protein synthesis. HUVECS were treated with vehicle (control) or thrombin (1 IU/ml) for 24 h, and the lysed protein extracts were immunoprecipitated with the anti-alpha vbeta 3 monoclonal antibody (LM609), separated by 8% SDS-PAGE, and transferred to nitrocellulose membranes. Filters were then immunoblotted with anti-beta 3 monoclonal antibody, and a major band (~95 kDa) was detected. Results are representative of 3 independent experiments. Data are means ± SE expressed as beta 3/GAPDH and beta 3/actin ratios. ***P < 0.01; ns, not significant.

To determine whether the increase in beta 3 mRNA was accompanied by an increase in protein synthesis, we immunoprecipitated total endothelial cell lysates using the anti-alpha vbeta 3 mouse monoclonal antibody (LM609), which recognizes human alpha vbeta 3-integrin. Immunoprecipitates were electrophoresed, transferred onto nitrocellulose membranes, and immunoblotted with mouse monoclonal anti-beta 3 antibody. A major band of ~95 kDa was detected (Fig. 1E). After 24 h of thrombin treatment, beta 3 protein increased by ~94% over that of control (Fig. 1E). No signal was detectable if the antibodies used were preadsorbed with purified alpha vbeta 3 protein, thus demonstrating the specificity of the band (data not shown).

The transduction mechanisms involved in the upregulation of alpha vbeta 3 by thrombin and the participation of thrombin receptors are under study.

Endothelial cell adhesion on immobilized thrombin is mediated by alpha vbeta 3-integrin. The upregulation of alpha vbeta 3 by thrombin raises the question as to whether this thrombin-induced angiogenic phenotype of endothelial cells is manifested in other integrin-mediating effects related to angiogenesis such as attachment, migration, and survival. Quantitative cell attachment assays were used to characterize the endothelial cell-thrombin interactions. As shown in Fig. 2A, immobilized thrombin supported endothelial cell adhesion in a concentration-dependent manner up to 1 µg/well. Above this concentration we observed the characteristic bell-shaped curve, known to occur in many of the actions of thrombin. DIP-thrombin (the chemically inactivated thrombin at the active site) also increased cell attachment when immobilized on a solid surface. This excluded the involvement of the catalytic site of thrombin and the requirement for a proteolytic activation of thrombin receptors on endothelial cells. Furthermore, it was demonstrated that cell attachment on immobilized thrombin or DIP-thrombin was alpha vbeta 3 dependent. When HUVECs were pretreated with 10 µg/ml LM609 antibody or 10 µg/ml c-RGD, the attachment of endothelial cells on thrombin or DIP-thrombin was abolished (Fig. 2B). The inactive c-RAD peptide had no effect (Fig. 2B).


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Fig. 2.   Thrombin mediates endothelial cell adhesion through alpha vbeta 3 integrin. A: dose dependence of cell adhesion to thrombin. HUVECs (1 × 105 cells/well) were plated onto wells coated with the indicated concentrations of thrombin (alpha -thr) or DIP-thrombin (DIP-thr), and cell attachment was evaluated as described in METHODS. B: cell adhesion to thrombin is alpha vbeta 3 dependent. HUVECs were incubated in the presence or absence of the cyclic peptide c-RGD (10 µg/ml), inactive cyclic peptide c-RAD (10 µg/ml), or mouse anti-alpha vbeta 3 (LM609) antibody (10 µg/ml) for 15 min. Cells were then plated on wells coated with 1 µg/well thrombin, DIP-thrombin, or BSA alone for 60 min. Results are representative of 3 independent experiments. Data are means ± SE expressed as pixel area. ***P < 0.01.

Immobilized thrombin promotes alpha vbeta 3-dependent endothelial cell migration. The alpha vbeta 3-integrin is known to have a critical role in cell migration and survival (28, 34). Furthermore, alpha vbeta 3 antagonists have been shown to exert their antiangiogenic effects in vivo by blocking survival signals mediated by this integrin (8, 44). In view of the above findings, we examined whether thrombin affects alpha vbeta 3-integrin-dependent endothelial cell migration and survival.

HUVECs readily migrated in an haptotactic Boyden chamber assay through a microporous membrane toward immobilized thrombin or DIP-thrombin in a dose-dependent manner up to 1 µg/filter. Above that concentration the stimulatory effect is diminished, and we have the characteristic bell-shaped curve (Fig. 3A). This migration was completely blocked by the anti-alpha vbeta 3 antibody (LM609) or the c-RGD peptide but not by inactive c-RAD peptide (Fig. 3B).


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Fig. 3.   Thrombin promotes endothelial cell migration in an alpha vbeta 3-dependent manner. A: dose dependence of cell migration toward immobilized thrombin. The undersurface of the membrane filters of the Boyden chamber were coated with the indicated concentrations (µg/filter) of thrombin or DIP-thrombin. HUVECs (1 × 105 cells/well) were added to the upper compartment of the chamber and incubated for 6 h, allowing migration to the lower chamber. The haptotactic cell motility was analyzed as described in METHODS. B: cell migration toward immobilized thrombin is alpha vbeta 3 dependent. To modulate the migration toward the immobilized thrombin (1 µg/ml) or DIP-thrombin (1 µg/ml), lower chambers were filled with SFM199-0.5% BSA containing the c-RGD peptide (10 µg/ml), c-RAD peptide (10 µg/ml), or anti-alpha vbeta 3 antibody (10 µg/ml). The number of cells migrating to BSA-coated membrane filters is very low. Results are representative of 3 independent experiments. Data are means ± SE expressed as no. of cells per high-magnification microscopic field. ***P < 0.01.

Endothelial cell haptotactic migration is effectively prevented by the presence of thrombin in solution. As shown in Fig. 4A, soluble thrombin (1 IU/ml) in the lower chamber inhibits cell migration toward immobilized vitronectin and gelatin, two reported alpha vbeta 3-integrin ligands (18, 14). In similar experiments, anti-alpha vbeta 3 antibody had the same inhibitory effect on endothelial cell motility toward vitronectin and gelatin (data not shown). On the other hand, when HUVECs preincubated with thrombin (1 IU/ml) for 12 h were detached with EDTA and subsequently exposed to vitronectin or gelatin in chemotaxis analysis or in haptotactic cell motility assay, an increase of ~40% in migration was observed (Fig. 4B). These findings are in agreement with the results presented in Fig. 1 and further support the notion that thrombin upregulates alpha vbeta 3 expression and synthesis.


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Fig. 4.   Soluble thrombin modulates endothelial cell migration toward vitronectin or gelatin. A: thrombin inhibits cell haptotactic migration to immobilized vitronectin or gelatin. The undersurface of the membrane filters was coated with vitronectin (2.5 µg/filter) or gelatin (5 µg/filter). Vehicle (alone) or thrombin (1 IU/ml) was added to the lower compartment of the chamber and HUVECs (1 × 105 cells/well) to the upper compartment. After 6 h of incubation, the apoptotic cell motility was evaluated as described in METHODS. B: pretreatment of endothelial cells with thrombin potentiates their migratory effect toward vitronectin or gelatin. HUVECs were pretreated with thrombin (1 IU/ml) for 12 h and subsequently added to upper chamber and allowed to migrate toward immobilized vitronectin (2.5 µg/filter) and gelatin (5 µg/filter) (haptotaxis) or toward soluble vitronectin (10 µg/ml) and gelatin (10 µg/ml) (chemotaxis). Results are representative of 3 independent experiments. Data are means ± SE expressed as no. of cells per high-magnification microscopic field. **P < 0.05.

Immobilized thrombin promotes alpha vbeta 3-dependent endothelial cell survival. When HUVECs were plated on immobilized BSA under serum-free conditions, significant levels of apoptosis were detected after 6 h of incubation (Fig. 5). In contrast, and similar to what was reported previously (44), plating of HUVECs on immobilized anti-alpha vbeta 3 antibody protect cells from apoptosis (data not shown). We found that immobilized thrombin or DIP-thrombin mimics the effects of anti-alpha vbeta 3 antibody and similarly promoted endothelial cell survival. Decreased levels of apoptosis were detected in cells adherent on plates coated with thrombin or DIP-thrombin (Fig. 5). When soluble anti-alpha vbeta 3 antibody or c-RGD peptide was present, a significant increase in the apoptosis of cells plated on immobilized thrombin was observed (Fig. 5). In contrast, the inactive peptide c-RAD was without effect on thrombin-induced cell survival (Fig. 5).


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Fig. 5.   Immobilized thrombin promotes endothelial cell survival in an alpha vbeta 3-dependent manner. HUVECs were plated under serum-free condition on a BSA-coated surface or on immobilized thrombin (10 µg/ml) or DIP-thrombin (10 µg/ml) for 6 h. Where indicated, soluble c-RGD (10 µg/ml), c-RAD (10 µg/ml), or anti-alpha vbeta 3 antibody (10 µg/ml) was added to the cells before they were plated on thrombin or DIP-thrombin. DNA fragmentation was measured as an indication of apoptosis. Results are representative of 3 independent experiments. Data are means ± SE presented as absorbance at 402 nm (OD402). ***P < 0.05.

Thrombin binds to purified alpha vbeta 3-integrin. We demonstrated a direct thrombin-alpha vbeta 3-integrin interaction in a solid-phase ligand binding assay, using a commercially available purified alpha vbeta 3 preparation. As shown in Fig. 6A, soluble alpha vbeta 3-integrin can bind to immobilized thrombin or DIP-thrombin in a concentration-dependent manner, and the binding is saturable. The specificity of the interaction was established by the fact that when alpha vbeta 3 was neutralized by anti-alpha vbeta 3 monoclonal antibody (LM609) or antagonized by c-RGD peptide, the binding was abolished. When EDTA was present, the binding was also abolished, suggesting that this process was cation dependent (Fig. 6B).


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Fig. 6.   Thrombin binds to purified alpha vbeta 3-integrin in a concentration-specific manner in a solid-phase ligand binding assay. A: indicated concentrations of alpha vbeta 3-integrin were added to wells coated with thrombin (1 µg/well) or DIP-thrombin (1 µg/ml) and incubated at 4°C overnight. Bound integrin was detected as described in METHODS. B: alpha vbeta 3-integrin was preincubated with soluble c-RGD (10 µg/ml), anti-alpha vbeta 3 antibody (LM609, 10 µg/ml), or EDTA (10 mM) for 30 min at 4°C to block the interaction between thrombin and alpha vbeta 3-integrin. Results are representative of 3 independent experiments. Data are means ± SE presented as OD492.

Integrin alpha vbeta 3 is involved in thrombin-induced angiogenesis. We have used collagenous protein biosynthesis (CPB) as a biochemical index of angiogenesis (31). As shown in Fig. 7, thrombin (1 IU/disk) caused 100% increase of CPB, which is in line with our previous results (47). The c-RGD (100 µg/disk) peptide when used alone caused an inhibition of 30% compared with controls. The combination of thrombin with c-RGD peptide decreased the thrombin-promoting angiogenesis to 28%. The inactive c-RAD peptide had no effect. These results suggest that alpha vbeta 3 antagonists can modulate the overall angiogenic effect of thrombin.


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Fig. 7.   Involvement of alpha vbeta 3-integrin in thrombin-induced angiogenesis. Biochemical evaluation of angiogenesis in vivo was performed by determining the extent of collagenous protein biosynthesis (CPB) in the chorioallantoic membrane lying directly under the disks applied at day 9 of chick embryo development. Both control and test disks contained radiolabeled proline (0.5 µCi/disk), and test disks contained thrombin (1 IU/disk), c-RGD peptide (100 µg/disk), c-RAD peptide (100 µg/disk), or combinations as indicated. For each egg, CPB under the disk containing the test material was expressed as a percentage of that under the control disk for the same egg. In each group at least 12 eggs were used. Results are means ± SE presented as %control. **P < 0.05; ***P < 0.01.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this report we have shown in endothelial cells that thrombin increases the mRNA and protein levels of alpha vbeta 3-integrin in a time- and concentration-dependent fashion. The transduction mechanisms involved for this upregulation are unknown. We are in the process of exploring these mechanisms and the requirement of thrombin receptor activation. Upregulation of alpha vbeta 3 by thrombin has also been demonstrated previously in human colon adenocarcinoma cells (11) and in cultured smooth muscle cells (43). Furthermore, it has been shown that thrombin induces adhesion of tumor cells, neutrophils, and monocytes to endothelial cell by increasing the expression of the integrin alpha IIbbeta 3 in these cells (27, 36, 48).

We have also demonstrated that thrombin serves as a ligand to alpha vbeta 3. In a cell-free system, immobilized thrombin binds to purified human alpha vbeta 3-integrin. Soluble anti-alpha vbeta 3 antibody, c-RGD, or EDTA blocked this interaction. These findings are in line with the results of Byzova and Plow (9), who have shown that prothrombin can serve as a ligand for activated alpha vbeta 3 on vascular endothelial cells and smooth muscle cells. This was proposed as an explanation for a previously unrecognized interface between the adhesive and procoagulant properties of these cells.

The interaction of thrombin with the alpha vbeta 3-integrin of endothelial cells is of functional significance. As shown in Fig. 2, immobilized thrombin supported endothelial cell attachment. This adhesion apparently was mediated via RGD sequence of the thrombin molecule, and the chemically inactivated DIP-thrombin behaves the same as, if not better than, native thrombin probably because DIP-thrombin has more of the RGD sequence exposed. The endothelial cell attachment to thrombin or DIP-thrombin was specific and dependent on alpha vbeta 3. A selective antagonist peptide to alpha vbeta 3 (c-RGD), but not the inactive analog (c-RAD), as well as a monoclonal antibody against alpha vbeta 3 (LM609) prevented the adhesion to thrombin. Thrombin as well as prothrombin contains an RGD sequence within its catalytic domain. It was shown by Stubbs and Bode (45) by analysis of the crystal structure of thrombin that the RGD sequence is part of its active site and lies at the bottom of the S1 specificity pocket. On the basis of that finding, it was postulated that the RGD domain is not accessible on the surface of the thrombin molecule. This proposal was further supported by the work of Bar-Shavit and colleagues (5, 6), who reported that cleavage products of thrombin or chemically modified thrombin, but not native thrombin, increased the adhesion of endothelial cells in an RGD-dependent manner. Contrary to these results, we found that endothelial cells adhered to native thrombin. The source of our thrombin preparation was the same as that used in the work of Bar-Shavit and colleagues (5, 6). Similar results were also obtained when a commercially available purified native alpha -thrombin (Sigma) was used (data not shown). The preparation of thrombin used was fully active in two assay systems: upregulating alpha vbeta 3-integrin and promoting angiogenesis in the CAM assay. Both thrombin preparations supported endothelial cell adhesion in a dose-dependent manner up to 1 µg/well. Above that concentration of thrombin, the effect is diminished. This dose-dependent effect of thrombin may explain the discrepancy with the results of Bar-Shavit and colleagues (5, 6), although in their work the thrombin concentrations used were not obvious. In their work, they found that DIP-thrombin, gamma -thrombin, and N-alpha -tosyl-L-lysylchloromethylketone-thrombin were poorly active in endothelial cell attachment. On the contrary, NO2-thrombin is active and its action is antagonized by soluble native thrombin, which they claimed to be inactive.

The bell-shaped effect of thrombin is a general phenomenon for many of the actions of thrombin and is not well understood. Receptor-mediated effects of thrombin may be related to desensitization and recycling of thrombin receptors by thrombin (30). In our experiments of endothelial cell attachment and migration to thrombin, one possible explanation might be that increasing thrombin concentration favors thrombin-thrombin interactions, thus hiding binding sites. Another explanation might be that thrombin at higher concentrations binds to low-affinity sites in the cell surface and activates intracellular mechanisms that antagonize the effects of high-affinity sites. This proposal is supported by the results of Sower et al. (42). They have shown that increased concentrations of the thrombin peptide TP508 (corresponding to the binding region of thrombin) stimulated a nonproteolytically activated receptor component. This caused an increased expression of annexin V, which has been shown to inhibit protein kinase C (40). Protein kinase C inhibition may be the explanation for the effects of high thrombin concentrations. It has also been shown that various types of cells respond to thrombin in a biphasic way. Zain et al. (49) and Ahmad et al. (2) have shown that a low concentration of thrombin induces mitogenesis in tumor cells, whereas a high concentration impairs cell growth. The apoptosis-inducing effects of thrombin in neurons and astrocytes were only visible at high concentrations. On the contrary, at low concentrations, thrombin was mitogenic for these nervous tissue cells and protected them from oxidative stress, cytotoxicity of beta -amploid, hypoxia, and withdrawal of growth factors (15, 16). Opposing concentration-dependent effects of thrombin were also shown in rat glioma cells by Schafberg et al. (39).

Thrombin also acts as an haptotactic factor for endothelial cells. As shown in Fig. 3, endothelial cells migrated toward immobilized thrombin or DIP-thrombin, and this effect was also specific and alpha vbeta 3 dependent. The presence of c-RGD or LM609 (but not the inactive c-RAD peptide) cancelled out the haptotactic effect of thrombin. Furthermore, thrombin mimics the effect of other matrix proteins such as vitronectin and gelatin in enhancing endothelial cells migration. However, when soluble thrombin is present, this binding is abolished, most likely because of saturation of alpha vbeta 3 binding sites of endothelial cells by thrombin. The fact that the promotion of endothelial cells migration by thrombin is an alpha vbeta 3-mediated event is also supported by the results of the experiments of Fig. 4B. When endothelial cells are pretreated with thrombin for 12 h, for upregulation of alpha vbeta 3 expression, their ability to migrate toward vitronectin or gelatin is substantially increased. We cannot rule out the possibility that thrombin may also interact with other members of the integrin family, because it occurs in human melanoma cells in which thrombin interacts with alpha vbeta 5 integrin (20). Experiments are in progress to assess this possibility. The association of endothelial cells with thrombin via alpha vbeta 3 integrin supports their survival under serum-free conditions. The data presented in Fig. 5 indicate that the protection from apoptosis caused by thrombin or DIP-thrombin is cancelled out when c-RGD or LM609 is present. This is in line with in vivo results by others (8), who have shown that integrin alpha vbeta 3 antagonists promote tumor regression by inducing apoptosis in angiogenic blood vessels.

The intracellular signaling events triggered by alpha vbeta 3-thrombin interaction is under investigation. Recent reports showing that integrin engagement leads to activation of MAP kinases and that this pathway is mediated by the stimulation of p125FAK tyrosine phosphorylation (10, 35). Thrombin, like VEGF, increases pp125FAK tyrosine phosphorylation in platelets, mesangial cells, and endothelial cells (1, 13, 22, 29). It is likely, therefore, that the stimulation of endothelial cells attachment, migration, and survival by thrombin via alpha vbeta 3 involves an extensive network of signaling events distal to p125FAK. Components of focal adhesions and of endothelial cell-to-cell junctions are both linked to the actin of cytoskeleton and may be functionally integrated through common signaling events in the migration and survival of endothelial cells.

The aforementioned alpha vbeta 3-mediated effects of thrombin are most likely contributing to angiogenic action of thrombin, providing an explanation for the angiogenesis par excellence occurring within thrombi. A very common clinical observation is that after thrombosis in a large vein, the thrombus is recanalized with new vessels seen with angiography. It is known that while thrombin in the plasma is rapidly inactivated by anti-thrombin, the thrombin trapped within the thrombi is protected and is slowly released during thrombolysis. Most likely, this trapped thrombin acts as angiogenic factor by attracting endothelial cells, mediating their angiogenic phenotype. In addition, thrombin is also known to interact with various constituents of the ECM. ECM-immobilized thrombin is protected from inactivation by its circulating inhibitors and induces many cellular responses (26). Binding of thrombin to the subendothelial ECM through a short anchorage binding site leaves the majority of the molecule functional and available for cellular interaction (4).

The experimental findings discussed here provide evidence at the molecular and cellular level that many processes involved in angiogenesis are promoted by thrombin through alpha vbeta 3-integrin. In the CAM system, which is used as a model for studying angiogenesis in vivo by many investigators, we have shown that alpha vbeta 3 antagonist can downregulate the angiogenesis-promoting effect of thrombin. The results present in this paper along with our previous findings (46, 47) establish the role of thrombin as an angiogenic factor. Many of the actions of thrombin on endothelial cells can contribute to their angiogenic phenotype and provide an explanation for the long-known association between thrombosis and tumor progression.


    ACKNOWLEDGEMENTS

We thank Dr. Simon Goodman and Dr. Alfred Jonczyk (Merck, Germany) for providing the cyclic peptides EMD 121974 and EMD 135981.


    FOOTNOTES

This work was supported by grant from the Greek Ministry of Research and Technology.

Address for reprint requests and other correspondence: M. E. Maragoudakis, Dept. of Pharmacology, Medical School, Univ. of Patras, 25110 Patras, Greece (E-mail: maragoud{at}med.upatras.gr).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

July 17, 2002;10.1152/ajpcell.00162.2002

Received 10 April 2002; accepted in final form 11 July 2002.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abedi, H, and Zachary I. Vascular endothelial growth factor stimulates tyrosine phosphorylation and recruitment to new focal adhesions of focal adhesion kinase and paxillin in endothelial cells. J Biol Chem 272: 15442-15451, 1994[Abstract/Free Full Text].

2.   Ahmad, R, Knafo L, Xu J, Sindhu STAK, Menezes J, and Ahmad A. Thrombin induces apoptosis in human tumor cells. Int J Cancer 87: 707-715, 2000[ISI][Medline].

3.   Baron, JA, Gridley G, Weiderpass E, Nyren O, and Linet M. Venous thromboembolism and cancer. Lancet 351: 1077-1080, 1998[ISI][Medline].

4.   Bar-Shavit, R, Eldor A, and Vlodavsky I. Binding of thrombin to subendothelial extracellular matrix: protection and expression of functional properties. J Clin Invest 84: 1096-1104, 1989[ISI][Medline].

5.   Bar-Shavit, R, Eskohjido Y, Fenton JW, II, Esko JD, and Vlodavsky I. Thrombin adhesive properties: induction by plasmin and heparan sulfate. J Cell Biol 123: 1279-1287, 1993[Abstract].

6.   Bar-Shavit, R, Sabbah V, Lampugnani MG, Marchisio PC, Fenton JW, 2nd, Vlodavsky I, and Dejana E. An Arg-Gly-Asp sequence within thrombin promotes endothelial cell adhesion. J Cell Biol 112: 335-344, 1991[Abstract].

7.   Billroth, T. Lectures on Surgical Pathology And Therapeutics: A Handbook For Students And Practitioners (translated from 8th ed.). London: New Syndenhan Soc, 1878.

8.   Brooks, PC, Montgomery AM, Rosenfeld M, Reisfeld RA, Hu T, Klier G, and Cheresh DA. Integrin alpha vbeta 3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell 79: 1157-1164, 1994[ISI][Medline].

9.   Byzova, TV, and Plow EF. Activation of alpha vbeta 3 on vascular cells controls recognition of prothrombin. J Cell Biol 143: 2081-2092, 1998[Abstract/Free Full Text].

10.   Chen, Q, Kinch MS, Lin TH, Burridge K, and Juliano RL. Integrin-mediated cell adhesion activates mitogen-activated protein kinases. J Biol Chem 269: 26602-26605, 1994[Abstract/Free Full Text].

11.   Chiang, HS, Yang RS, and Huang TF. Thrombin enhances the adhesion and migration of human colon adenocarcinoma cells via increased beta 3-integrin on the tumor cell surface and their inhibition by the snake venom peptide, rhodostomin. Br J Cancer 73: 902-908, 1996.

12.   Chomczynski, P, and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156-159, 1987[ISI][Medline].

13.   Choudhury, GG, Marra F, and Abboud HE. Thrombin stimulates association of src homology domain containing adaptor protein Nck with pp125FAK. Am J Physiol Renal Fluid Electrolyte Physiol 270: F295-F300, 1996[Abstract/Free Full Text].

14.   Davis, GE. Affinity of integrins for damaged extracellular matrix: alpha vbeta 3 binds to denatured collagen type I through RGD sites. Biochem Biophys Res Commun 182: 1025-1031, 1992[ISI][Medline].

15.   Debeir, T, Benavides J, and Vige X. Dual effects of thrombin and a 14-amino acid peptide agonist of the thrombin receptor on septal cholinergic neurons. Brain Res 708: 159-166, 1996[ISI][Medline].

16.   Debeir, T, Gueugnon J, Vige X, and Benavides J. Transduction mechanisms involved in thrombin receptor-induced nerve growth factor secretion and cell division in primary cultures of astrocytes. J Neurochem 99: 2320-2328, 1996.

17.   Dechantsreiter, MA, Planker E, Matha B, Lohof E, Holzemann G, Jonczyk A, Goodman SL, and Kessler H. N-methylated cyclic RGD peptides as highly active and selective alpha Vbeta 3 integrin antagonists. J Med Chem 42: 3033-3040, 1999[ISI][Medline].

18.   Eliceiri, BP, and Cheresh DA. The role of alpha v integrins during angiogenesis: insights into potential mechanisms of action and clinical development. J Clin Invest 103: 1227-1230, 1999[Free Full Text].

19.   Even-Ram, S, Usiely B, Cohen P, Grisaru-Granovsky S, Maoz M, Ginzburg Y, Reich R, Vlodavsky I, and Bar-Shavit R. Thrombin receptor overexpression in malignant and physiological invasion processes. Nat Med 4: 909-914, 1998[ISI][Medline].

20.   Even-Ram, SC, Maoz M, Pokroy E, Reich R, Katz BZ, Gutwein P, Altevogt P, and Bar-Shavit R. Tumor cell invasion is promoted by activation of protease activated receptor-1 in cooperation with the alpha vbeta 5 integrin. J Biol Chem 276: 10952-10962, 2001[Abstract/Free Full Text].

21.   Griffin, CT, Srinivasan Y, Zheng YW, Huang W, and Coughlin SR. A role for thrombin receptor signaling in endothelial cells during embryonic development. Science 293: 1666-1670, 2001[Abstract/Free Full Text].

22.   Guinebault, C, Payrastre B, Racaud-Sultan C, Mazarguil H, Breton M, Mauco G, Plantavid M, and Chap H. Integrin-dependent translocation of phosphoinositide 3-kinase to the cytoskeleton of thrombin-activated platelets involves specific interactions of p85 alpha with actin filaments and focal adhesion kinase. J Cell Biol 129: 831-842, 1995[Abstract].

23.   Hejna, M, Radener M, and Zielinski CC. Inhibition of metastases by anticoagulants. J Natl Cancer Inst 91: 22-36, 1999[Abstract/Free Full Text].

24.   Hoshiga, M, Alpers CE, Smith LL, Giachelli CM, and Schwartz SM. Alpha-v beta-3 integrin expression in normal and atherosclerotic artery. Circ Res 77: 1129-1135, 1995[Abstract/Free Full Text].

25.   Jaffe, EA, Nachman RL, Becker CG, and Minick CR. Culture of human endothelial cells derived from umbilical veins. Identification by morphological and immunological criteria. J Clin Invest 52: 2745-2756, 1973[ISI][Medline].

26.   Kanthou, C, Kakkar VV, and Benzakour O. Cellular and molecular effects of thrombin in the vascular system. In: Angiogenesis Models, Modulators and Clinical Applications, edited by Maragoudakis ME.. New York: Plenum, 1998, p. 263-282.

27.   Kelpfish, A, Greco MA, and Karpatkin S. Thrombin stimulates melanoma tumor cell binding to endothelial cells and subendothelial matrix. Int J Cancer 53: 978-982, 1993[ISI][Medline].

28.   Leavesley, DI, Ferguson GD, Wayner EA, and Cheresh DA. Requirement of the integrin beta 3 subunit for carcinoma cell spreading or migration on vitronectin and fibrinogen. J Cell Biol 117: 1101-1107, 1992[Abstract].

29.   Lipfert, L, Haimovich B, Schaller MD, Cobb BS, Parsons JT, and Brugge JS. Integrin-dependent phosphorylation and activation of the protein tyrosine kinase pp125FAK in platelets. J Cell Biol 119: 905-912, 1992[Abstract].

30.   Macfarlane, SR, Seatter MJ, Kanke T, Hunter GD, and Plevin R. Proteinase-activated receptors. Pharmacol Rev 53: 245-282, 2001[Abstract/Free Full Text].

31.   Maragoudakis, ME, Haralabopoulos G, Tsopanoglou NE, and Pipili-Synetos E. Validation of collagenous protein synthesis as an index for angiogenesis with the use of morphological methods. Microvasc Res 50: 215-222, 1995[ISI][Medline].

32.   Maragoudakis, ME, Tsopanoglou NE, and Andriopoulou P. Mechanism of thrombin-induced angiogenesis. Biochem Soc Trans 30: 173-177, 2002[ISI][Medline].

33.   Maragoudakis, ME, Tsopanoglou NE, Sakkoula E, and Pipili-Synetos E. On the mechanism of promotion of angiogenesis by thrombin (Abstract). FASEB J 9: A587, 1995.

34.   Montgomery, AM, Reisfeld RA, and Cheresh DA. Integrin alpha v beta 3 rescues melanoma cells from apoptosis in three-dimensional dermal collagen. Proc Natl Acad Sci USA 91: 8856-8860, 1994[Abstract].

35.   Morino, N, Mimura T, Hamasaki K, Tobe K, Ueki K, Kikuchi K, Takehara K, Kadowaki T, Yazaki Y, and Nojima Y. Matrix/integrin interaction activates the mitogen-activated protein kinase, p44erk-1 and p42erk-2. J Biol Chem 270: 269-273, 1995[Abstract/Free Full Text].

36.   Nierodzik, ML, Chen K, Takeshita K, Li JJ, Huang YQ, Feng XS, D'Andrea MR, Andrade-Gordon P, and Karpatkin S. Protease-activated receptor 1 (PAR-1) is required and rate-limiting for thrombin-enhanced experimental pulmonary metastasis. Blood 92: 3694-3700, 1998[Abstract/Free Full Text].

37.   Nierodzik, ML, Kajumo F, and Karpatkin S. Effects of thrombin treatment of tumor cells on adhesion of tumor cells to platelets in vitro and tumor metastasis in vivo. Cancer Res 52: 3267-3272, 1992[Abstract].

38.   Rickles, FR, and Edwards RL. Activation of blood coagulation in cancer: Trousseaeu's syndrome revisited. Blood 64: 14-31, 1983.

39.   Schafberg, H, Nowak G, and Kaufmann R. Thrombin has a bimodal effect on glioma cell growth. Br J Cancer 76: 1592-1595, 1997[ISI][Medline].

40.   Schlaepfer, DD, Jones J, and Haigler HT. Inhibition of protein kinase C by annexin V. Biochemistry 31: 1886-1891, 1992[ISI][Medline].

41.   Sorensen, HT, Mellem KL, Steffensen FH, Olsen JH, and Nielsen GH. The risk of a diagnosis of cancer after primary deep venous thrombosis or pulmonary embolism. N Engl J Med 338: 1169-1173, 1998[Abstract/Free Full Text].

42.   Sower, LE, Payne DA, Meyers R, and Carney DH. Thrombin peptide, TP508, induces differential gene expression in fibroblasts a nonproteolytic activation pathway. Exp Cell Res 247: 422-431, 1999[ISI][Medline].

43.   Stouffer, GA, Hu Z, Sajid M, Li H, Jin G, Nakada MT, Hanson SR, and Runge MS. Beta 3 integrins are upregulated after vascular injury and modulate thrombospondin- and thrombin-induced proliferation of cultured smooth muscle cells. Circulation 97: 907-915, 1998[Abstract/Free Full Text].

44.   Stromblad, S, Becker JC, Yebr M, Brooks PC, and Cheresh DA. Suppression of p53 activity and p21WAF1/CIP1 expression by vascular cell integrin alpha v beta 3 during angiogenesis. J Clin Invest 98: 426-433, 1996[Abstract/Free Full Text].

45.   Stubbs, MT, and Bode W. A player of many parts: the spotlight falls on thrombin's structure. Thromb Res 69: 1-58, 1993[ISI][Medline].

46.   Tsopanoglou, NE, and Maragoudakis ME. On the mechanism of thrombin-induced angiogenesis. Potentiation of vascular endothelial growth factor activity on endothelial cells by up-regulation of its receptors. J Biol Chem 274: 23969-23976, 1999[Abstract/Free Full Text].

47.   Tsopanoglou, NE, Pipili-Synetos E, and Maragoudakis ME. Thrombin promotes angiogenesis by a mechanism independent of fibrin formation. Am J Physiol Cell Physiol 264: C1302-C1307, 1993[Abstract/Free Full Text].

48.   Wojtukiewicz, MZ, Tang DG, Nelson KK, Walz DA, Diglio CA, and Honn KV. Thrombin enhances tumor cell adhesive and metastatic properties via increased alpha IIb beta 3 expression on the cell surface. Thromb Res 68: 233-245, 1992[ISI][Medline].

49.   Zain, J, Huang YQ, Feng XS, Nierodzik ML, Li JJ, and Karpatkin S. Concentration-dependent dual effect of thrombin on impaired growth/apoptosis or mitogenesis in tumor cells. Blood 95: 3133-3138, 2000[Abstract/Free Full Text].

50.   Zielinski, CC, and Hejna M. Warfarin for cancer prevention. N Engl J Med 342: 1991-1993, 2000[Free Full Text].


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