|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Department of Medical Oncology, University
Hospital "Vrije Universiteit," 1007 MB Amsterdam, The Netherlands,
and the Department of Internal Medicine, University Medical Center
Utrecht, Utrecht, The Netherlands.
Coagulation abnormalities, including an increased platelet
turnover, are frequently found in patients with cancer. Because platelets secrete angiogenic factors on activation, this study tested
the hypothesis that platelets contribute to angiogenesis. Stimulation
with vascular endothelial growth factor (VEGF, 25 ng/mL) of human
umbilical vein endothelial cells (HUVECs) promoted adhesion of
nonactivated platelets 2.5-fold. In contrast, stimulation of HUVECs
with basic fibroblast growth factor (bFGF) did not promote platelet
adhesion. By blocking tissue factor (TF) activity, platelet adhesion
was prevented and antibodies against fibrin(ogen) and the
platelet-specific integrin, Coagulation abnormalities occur in more than 80%
of patients with disseminated cancer.1 High concentrations
of circulating tissue factor (TF), thrombin-antithrombin complexes, and
an increased fibrinogen and platelet turnover have been found as
well.2 An important role for platelets in metastasis
formation and subsequent growth has been demonstrated in preclinical
studies.3 In addition, tumor growth and dissemination can
be inhibited by anticoagulants.4 It is not fully clear how
and to what extent the coagulation cascade is involved in tumor growth,
but growing evidence suggests that several coagulation factors are
important for tumor-induced angiogenesis. For example, it has been
shown in preclinical in vivo models that both TF and thrombin can
induce angiogenesis.5,6
Under normal physiologic circumstances, resting endothelial cells
(lining the vasculature) generate an active antithrombotic surface.7 Recent studies indicate that vascular
endothelial growth factor (VEGF), the major stimulus of angiogenesis,
activates endothelial cells to become prothrombotic. On VEGF
stimulation, endothelial cells increase TF expression on their
membranes and thereby generate thrombin activity from prothrombin
(activation of the coagulation cascade).8 In addition, TF
expression of tumor endothelium was found to correlate with VEGF
expression in breast cancer.9 Tumor-released VEGF may
change the antithrombotic surface of the vasculature into a
prothrombotic one, causing fibrin formation and platelet adhesion and
activation. In a previous study, we indeed found that activated
platelets were present in the microvessels of VEGF-producing soft
tissue sarcomas.10 It is well known that on activation,
platelets release their contents including angiogenic growth factors,
like VEGF and others.11 For example, platelet-derived
sphingosine-1-P has been found to protect endothelial cells from
apoptosis and to stimulate their DNA synthesis.12 Based on
these observations, we hypothesized that platelets contribute to
angiogenesis.13
In this study we investigated whether stimulation of endothelial cells
with angiogenic growth factors can cause adhesion and activation of
platelets and whether platelets can contribute to endothelial cell
proliferation in vitro, as an indication for their role in the
angiogenic process in vivo. We developed an adhesion assay for
fluorescence-labeled platelets on endothelial cells. In this assay,
stimulation with VEGF, but not with basic fibroblast growth factor
(bFGF), induced adhesion of nonactivated platelets on human umbilical
vein endothelial cells (HUVECs). An essential role for TF and the
subsequent activation of the coagulation pathway was found. Finally, we
demonstrated that physiologic concentrations of platelets stimulate
endothelial cell proliferation in vitro, indicative of a proangiogenic
effect in vivo.
Cell culture
Platelet preparation
Leukocyte preparation Leukocyte separation was performed according to standard procedures as previously described.16 Briefly, blood was drawn by venipuncture into a syringe containing ACD as described for the platelet isolation. Subsequently the blood was centrifuged at 1500g for 5 minutes. The buffy coat was separated, diluted 2 to 3 times in PBS, and carefully added on top of the Ficoll solution (Ficoll-Paque, Pharmacia Biotech AB Uppsala, Sweden). After centrifugation for 20 minutes at 540g, the supernatant was discarded and the interphase containing the leukocytes was selected. In this suspension, the erythrocytes were lysed with lysis buffer (0.15 mol/L NH4, 1 mmol/L KHCO3, 0.1 mol/L EDTA) and another centrifugation was performed at 380g for 5 minutes. This last step was repeated until all erythrocytes were lysed. Subsequently, cells were counted and labeled with calcein-AM as described for the platelets, but with a concentration of leukocytes of 2.5 × 106/mL.Adhesion assay of platelets and leukocytes The HUVECs were grown until confluence on precoated 96-well plates. Fifteen hours before stimulation, cells were gently washed twice with prewarmed M199 and cells were incubated with medium (100 µL/well) with 0.1% to 0.25% human pool serum without heparin. After overnight incubation, 100 µL medium with 0.10% to 0.25% human pool serum containing one of the stimuli or without a stimulus was added to the wells. Stimuli used were recombinant human bFGF and recombinant human VEGF (R&D Systems, Uithoorn, The Netherlands) and as a positive control tumor necrosis factor- (TNF-, 1000 U/mL; ICN Biomedicals,
Aurora, OH). After 6 or 18 hours of incubation, cells were washed once
with M199 and the fluorescence-labeled platelets or leukocytes were
added and incubated for 30 minutes at 37°C. When the experiment was
terminated, unbound platelets or leukocytes were washed away
extensively with PBS (3 ×). Subsequently, the remaining platelets or
leukocytes were lysed with 50 µL of the lysis buffer (1% NP40, 150 mmol/L NaCl, 10 mmol/L Tris-HCL, pH 7.5, 5 mmol/L EDTA, 1 mmol/L
phenylmethylsulfonyl fluoride, and 20 µg/mL soybean trypsin
inhibitor). Ten minutes later, fluorescence was measured with
an automatic plate reader (excitation filter 492 nm, emission filter
535 nm, TECAN Spectrafluor). Each condition was tested in the same
assay at least in triplicate.
Antibodies and a specific thrombin inhibitor To investigate platelet adhesion on endothelial cells, several blocking antibodies against membrane proteins of platelets or endothelial cells or against plasma proteins were used. The following antibodies were used in a blocking concentration of 40 µg/mL unless otherwise indicated17: C/70A (CD31, IgG1, DAKO, Glostrup, Denmark), A0080 (Fibrinogen, DAKO), E8 (Fibrin, IgG1, Immunotech, Marseille, France), SZ2 (CD42b [glycoprotein-Ib], IgG1, Immunotech), tissue factor (American Diagnostica, Greenwich, CT), LM609 (  3 integrin, Chemicon International,
Temecula, CA), P-selectin (Endogen, Woburn, MA), and reopro
(IIb 3 integrin and
3 integrin in a concentration of 50 µg/mL, Eli Lilly Nederland BV, Nieuwegein, The Netherlands). As a
negative control IgG was used obtained from DAKO.
In addition, the synthetic and specific thrombin inhibitor I2581 (Chromogenix Instrumentation Laboratory, Milan, Italy) was used in a concentration of 25 µmol/L. Determination of thrombin activity Determination of the thrombin activity is based on the enzymatic splitting reaction of p-nitroaniline (pNA) from the substrate S-2238 (Chromogenix Instrumentation Laboratory) by thrombin. After a 30-minute incubation of platelets on stimulated or unstimulated endothelial cells, samples of 50 µL were taken. As controls, platelet suspensions (kept for 30 minutes in a well without endothelial cells) and supernatant of stimulated or unstimulated endothelial cells were used. A working solution was composed of Millipore water with 0.5% bovine serum albumin (BSA) and 10% of a buffer (Tris 0.5 mol/L, EDTA 75 mmol/L, and 10% polyethyleneglycol 3640). The substrate S2238, dissolved in Millipore water (1 mmol/L) was diluted in this working solution (1:12). In preheated 37°C cuvets, 1 mL of the substrate-working solution and, subsequently, 50 µL of a sample and 50 µL water or 50 µL of the thrombin inhibitor I2581 (450 mmol/L) were added. Cuvets were mixed and the optical density was measured at 406 nm. After 30 minutes the measurement was repeated. Thrombin activity was calculated from a standard activity curve of purified thrombin in a concentration range of 0 to 0.5 U/mL. By measuring the activity of the samples in the presence or absence of the specific thrombin inhibitor, thrombin activity could be calculated.Flow cytometry To analyze the activation state of the isolated platelets, fluorescence-activated cell sorter (FACS) analysis was performed on the platelets in 3 independent experiments, as described by Simak and coworkers with only minor modifications.18 Platelets were isolated and prepared as if they were used for an adhesion assay and analyzed just before they should have been incubated with endothelial cells. As a positive control of platelet activation, platelets were stimulated with the weak activator adenosine diphosphate (ADP) in a concentration of 25 mmol/L for 15 minutes. In this concentration ADP activates platelets to some extent, but without complete aggregation and fibrin formation, because that interferes with the FACS analysis. After 15 minutes of incubation with ADP or no activator (similar to the incubation of 15 minutes in the adhesion assay with calcein-AM), platelets were fixated in 1% paraformaldehyde for 15 minutes. After washing, the platelets were stained with R-phycoerythrin (PE)-labeled P-selectin-antibody (mouse IgG, CLB, Amsterdam, The Netherlands) in a 1:50 dilution or unspecified IgG in a concentration of 1 µg/mL. Platelet activation was evaluated on the FACScan (FACS Calibur, Becton Dickinson, San Jose, CA) by measuring P-selectin expression. Experiments were performed in duplicate. The state of activation was estimated by the median fluorescence of 10 000 platelets.Fluorescence microscopy: localization of platelet adhesion The adhesion assay was performed in the same way as described above but now unlabeled platelets were used. At the end of these experiments, instead of lysing the cells and platelets, the wells were fixed with 4% paraformaldehyde for 15 minutes. After preincubation (10% FCS and glycine in PBS), platelets were stained with a monoclonal antibody against theIIb3 integrin
(M7057, DAKO) and endothelial cells with biotinylated ulex
europaeus agglutinin 1 (Vector Laboratories, Burlingame,
CA) for 30 minutes. As secondary fluorescence-labeled antibodies, we
used horse antimouse IgG-fluorescein isothiocyanate (FITC) for the
detection of the IIb3 integrin and
streptavidin-Texas red (Vector Laboratories) for the detection of the
biotinylated lectin. All antibodies were used in a 1:50 dilution for
this double staining. Thereafter the wells were mounted with
Vectashield mounting medium for fluorescence (H1200, Vector
Laboratories). All slides were examined within 1 week after staining on
a LEICA confocal microscope. The IIb3
integrin-stained platelets appeared green, the endothelial cells
appeared red, and areas of coincident labeling containing colocalized
antigens appeared yellow.
Proliferation assay The HUVECs were plated in a density of 3000 cells/well on precoated 96-well plates in endothelial cell medium containing 5% FCS and 5% newborn calf serum, with no endothelial cell growth factor added. Next day, 100 µL containing an increasing number of nonactivated platelets in suspension with or without 0.5 U/mL thrombin in endothelial cell growth medium without serum was added. After 72 hours, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay was performed according to standard procedures. Results were corrected for the platelet activity in the MTT assay. In each experiment, conditions were tested in triplicate and independent experiments have been performed 3 times.
Platelet activation state The FACS analysis demonstrated that the isolated platelets stained with fluorescent antibody against P-selectin had a median fluorescence of 3.3 ± 0.15% (n = 3), indicative of a very low activation state. ADP-activated platelets had a significantly higher median fluorescence of 5.6 ± 0.2% (n = 3, P < .0001).VEGF induces platelet adhesion The occurrence of platelet adhesion after 6 hours of stimulation of confluent endothelial cells with either VEGF or bFGF in a dose range from 0.01 to 50 ng/mL is shown in Figure 1. VEGF (25 ng/mL) induced a significant 2.5-fold increase of platelet adhesion on endothelial cells, whereas bFGF had no stimulatory effect on platelet adhesion. The effect was more pronounced using endothelial cells that were just preconfluent. Prolonged stimulation of the endothelial cells (up to 18 hours) did not (further) increase platelet adhesion (data not shown). VEGF induced platelet adhesion to a similar extent as 1000 U/mL TNF-. Whereas
TNF--stimulated endothelial cells promoted leukocyte adhesion,
neither VEGF-stimulated (25 ng/mL) nor bFGF-stimulated (12.5 ng/mL)
endothelial cells promoted leukocyte adhesion (data not
shown).
Blocking of VEGF-induced adhesion To explore the underlying mechanism, platelet adhesion was studied in the presence of blocking antibodies. Platelets or endothelial cells were incubated with blocking antibodies against TF, fibrinogen, fibrin, glycoprotein-Ib, CD31,3 integrin,
P-selectin, or reopro against the IIb3
and 3 integrins. In addition to the
antibodies that had no effect on platelet adhesion (glycoprotein-Ib, CD31, P-selectin), IgG was used as negative control and did not inhibit
platelet adhesion. Blocking of the TF activity inhibited adhesion of
platelets for 90% to 100%, independent of whether the antibody
against TF was preincubated with the platelets or with the endothelial
cells. In addition, incubation of the platelets with antibodies against
fibrinogen, fibrin, or IIb3 integrin (reopro) and 3 integrin (LM609 and
reopro) prevented the adhesion of platelets for 60% to 90%. These
antibodies did not inhibit platelet adhesion when incubated with the
endothelial cells. Blocking antibodies against glycoprotein-Ib, CD31,
and P-selectin incubated with platelet or endothelial cells had no significant effect on platelet adhesion (Table
1). These data suggest that adhering
platelets become activated by thrombin, which is generated by the
increased TF expression on VEGF-stimulated endothelial cells. This was
further confirmed by the fact that a specific thrombin inhibitor
(I1285), prevented platelet adhesion for 100% (Table 1). The selective
VEGF-receptor blocker SU5416 (obtained from Sugen, San Francisco, CA),
used in a concentration of 1 µmol/L (a concentration that inhibits
VEGF-stimulated endothelial cell proliferation for 80%19),
inhibited the platelet adhesion by 86% ± 7% (n = 5,
P < .001), when concomitantly added to the HUVECs with
VEGF for 6 hours. SU5416 in the same concentration had no inhibitory
effect on TNF--stimulated platelet adhesion (1.7% ± 1.7%,
n = 3, P = .4).
Thrombin activity Because the thrombin activity appeared to be essential for VEGF-induced platelet adhesion, we determined the thrombin activity of the platelet suspension after incubation for 30 minutes on either stimulated or control endothelial cells. A significant 5-fold increase in thrombin activity (0.11 ± 0.03 U/mL, n = 3) was detected in the platelet suspension incubated on stimulated endothelial cells compared with the platelet suspension after incubation on unstimulated endothelial cells (0.02 ± 0.01 U/mL, P = .05). No thrombin activity was detectable in the endothelial supernatant and the nonincubated platelet suspension.Fluorescence microscopy The localization of the adhering platelets was examined by immunofluorescence microscopy. Staining of platelets (in green) and endothelial cells (in red) reflected that the majority of the platelets adhere to the endothelial cells. Only a few platelets bound to the extracellular matrix. Although some background adhesion of platelets on unstimulated endothelial cells was found, a clear increase in adhesion on stimulated endothelial cells versus unstimulated cells was observed (Figure 2).
Platelet effect on endothelial cell proliferation To study whether platelets may contribute to angiogenesis, in vitro proliferation assays of endothelial cells incubated with platelets and activated platelets were performed. Compared to controls, a 2- to 3-fold increase in endothelial cell number could be reached by increasing the concentration of platelets from 0.5 to 500 × 106/mL. In this experiment, resting platelets became activated during the first 10 hours of incubation because they were not being gently moved. A representative experiment is shown in Figure 3.
This study was designed to investigate whether platelets, as carriers of angiogenic growth factors, are involved in angiogenesis-dependent diseases. We demonstrated that VEGF-stimulated, but not bFGF-stimulated, endothelial cells promote platelet adhesion and activation. This finding indicates that platelet adhesion can occur in the angiogenic microvascular sites of VEGF-producing tumors and other angiogenic diseases in which VEGF is involved. The importance of platelet adhesion and activation for angiogenesis is reflected by the stimulatory effect of platelets on endothelial cell proliferation in vitro. Taken together, these data support the hypothesis that platelets contribute to tumor-induced angiogenesis.13 In 1968, Gasic and coworkers demonstrated that platelets are involved in cancer biology. They found that in thrombopenic mice, experimental metastasis formation and tumor growth was reduced compared to normal controls; this inhibition could be recovered by platelet transfusion.3 In clinical studies, it was found that platelet turnover is increased in cancer patients (3-fold) compared to healthy controls.2 In addition, thrombocytosis (an increased platelet number) occurs frequently (1%-60%) in cancer patients and it has been described as a negative prognostic factor for survival for some tumor types.20,21 In a previous study, we demonstrated that platelets become activated in the microvasculature of soft tissue sarcomas.10 The results of the current study provide a possible explanation for these observations. Platelet adhesion and subsequent activation in tumor vasculature will lead to an increased platelet turnover in cancer patients. Angiogenesis is a complex multistep process consisting of the breakdown of the basal membrane, endothelial cell migration and proliferation, subsequent tube formation, and finally the establishment of connections between newly formed tubes and the initiation of blood flow.22 As is extensively studied, a variety of cells (tumor and stromal) play a role in angiogenesis. Both leukocytes and platelets transport several angiogenic growth factors, including VEGF, but it is unknown what the role of these cells is in angiogenesis-dependent diseases.23,24 It has been reported that platelets promote tube formation of endothelial cells in vitro.25 In addition, in an in vivo corneal neovascularization model a pellet of thrombin-activated platelets induced angiogenesis.26 As early as 1986, it was hypothesized that tumors are never-healing wounds without the involvement of platelets.27 However, no data were provided to confirm or to refute the assumption that platelets are not involved in these never-healing wounds. In contrast, we recently detected intratumoral platelet activation.10 Here we show that VEGF may stimulate platelet adhesion and activation and, subsequently, that these platelets can stimulate endothelial cell proliferation. These results provide a possible explanation for the clinical observation that thrombocytosis is a negative prognostic factor for survival of cancer patients, because this increased number of platelets may have an extra proangiogenic effect to the tumor-induced angiogenesis in these patients. The concomitant activation of angiogenesis and coagulation in cancer biology has been extensively described. Transfection of tissue factor enhanced the release of VEGF.5 VEGF, in turn, induced TF expression on endothelial cells.8 In breast cancer tissues, TF was highly expressed in the angiogenic tumor microvessels. In addition, it has been reported that thrombin is a proangiogenic factor.6 Fibrin, the converted form of fibrinogen, can also contribute to the formation of new vessels.28 In this study, platelet adhesion to endothelial cells, stimulated by VEGF, was TF and thrombin dependent, indicating that thrombin is responsible for the platelet activation in this assay. Our in vitro results suggest that the proangiogenic effects of TF, thrombin, and fibrin in vivo may be mediated by local adhesion and activation of platelets. Recently, Bombeli and colleagues reported that activated platelets
adhere to endothelial cells through bridging proteins like fibrin(ogen)
and von Willebrand factor.17 In this study we confirmed these results because prevention of fibrinogen bridging inhibited platelet adhesion. Integrins have been shown to play a major role in
binding matrix and plasma proteins to cell surfaces, forming bridges
between the underlying matrices or for cell-cell interactions directly.
Bombeli and coworkers demonstrated that the
Other stimulators of platelet adhesion to endothelial cells include the
proinflammatory cytokine TNF- The lack of platelet adhesion on bFGF stimulation of the HUVECs in the platelet adhesion assay might be explained by the fact that bFGF is not able to up-regulate TF expression on endothelial cells.34 It supports our observation that VEGF promotes adhesion through TF-expression on endothelial cells. Based on literature data and the results of this study, we conclude that platelet adhesion and activation in the tumor microvasculature might be induced by VEGF stimulation of endothelial cells. Subsequently, the stimulatory effect of platelets on endothelial cell proliferation in vitro is supportive for a proangiogenic activity of adhering and activated platelets in vivo. These findings indicate that intratumoral platelet trapping may be responsible for an increased platelet turnover in cancer patients. In addition, platelets may not only promote tumor-induced angiogenesis, but also other angiogenesis-dependent diseases in which VEGF is involved. Therefore, we believe that prolonged antiplatelet therapy may be beneficial for patients with angiogenesis-dependent diseases.
Submitted November 12, 1999; accepted August 22, 2000.
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: H. M. Pinedo, Department of Medical Oncology, University Hospital "Vrije Universiteit," Postbus 7057 MB, Amsterdam, The Netherlands; e-mail: hm.pinedo{at}azvu.nl.
1. Dvorak HF. Abnormalities of hemostasis in malignancy. In: Colman RW,Hirsch J,Marder VJ,Saltzman EW, eds. Hemostasis and Thrombosis. Basic Principles and Clinical Practice. 3rd ed. Philadelphia: JB Lippincott; 1994:1238. 2. Harker LA, Slichter SJ. Platelet and fibrinogen consumption in man. N Engl J Med. 1972;287:999.
3.
Gasic GJ, Gasic TB, Stewart CC.
Antimetastatic effects associated with platelet reduction.
Proc Natl Acad Sci U S A.
1968;61:46
4.
Hejna M, Raderer M, Zielinski CC.
Inhibition of metastases by anticoagulants.
J Natl Cancer Inst.
1999;91:22 5. Zhang Y, Deng Y, Luther T, et al. Tissue factor controls the balance of angiogenic and antiangiogenic properties of tumor cells in mice. J Clin Invest. 1994;94:1320.
6.
Tsopanoglou NE, Pipili-Synetos E, Maragoudakis ME.
Thrombin promotes angiogenesis by a mechanism independent of fibrin formation.
Am J Physiol.
1993;264:C1302
7.
Cines DB, Pollak ES, Buck CA, et al.
Endothelial cells in physiology and in the pathophysiology of vascular disorders.
Blood.
1998;91:3527 8. Zucker S, Mirza H, Conner CE, et al. Vascular endothelial growth factor induces tissue factor and matrix metalloproteinase production in endothelial cells: conversion of prothrombin to thrombin results in progelatinase-A activation and cell proliferation. Int J Cancer. 1998;75:780[Medline] [Order article via Infotrieve]. 9. Shoji M, Hancock WW, Abe K, et al. Activation of coagulation and angiogenesis in cancer. Am J Pathol. 1998;152:399[Abstract].
10.
Verheul HMW, Hoekman K, Lupu F, Broxterman HJ, Kakkar AK, Pinedo HM.
High VEGF concentration and activation of coagulation pathway, including platelets, in aspirated fluids of soft tissue sarcomas.
Clin Cancer Res.
2000;6:166
11.
Verheul HMW, Hoekman K, Luykx-de Bakker S, et al.
Platelet: transporter of vascular endothelial growth factor.
Clin Cancer Res.
1997;3:2187
12.
Hisano N, Yatomi Y, Satoh K, et al.
Induction and suppression of endothelial cell apoptosis by sphingolipids: a possible in vitro model for cell-cell interactions between platelets and endothelial cells.
Blood.
1999;93:4293 13. Pinedo HM, Verheul HMW, D'Amato RJ, Folkman J. Involvement of platelets in tumour angiogenesis? Lancet. 1998;352:1775[Medline] [Order article via Infotrieve]. 14. van Hinsbergh VWM, Draijer R. In vitro models and functional studies of endothelium. Culture, characterization and application of human endothelial cells. In: Shaw AJ, ed. Cell culture models of epithelial tissues. A practical approach. Oxford, UK: Oxford University Press; 1996:87.
15.
McNicol A.
Platelet preparation and estimation of functional responses. In:
Watson SP,Authi KS, eds.
Platelets 16. Schaefer U, Schneider A, Rixen D, Neugebauer E. Neutrophil adhesion to histamine stimulated cultured endothelial cells is primarily mediated via activation of phospholipase C and nitric oxide synthase isozymes. Inflamm Res. 1998;47:256[Medline] [Order article via Infotrieve].
17.
Bombeli T, Schwartz BR, Harlan JM.
Adhesion of activated platelets to endothelial cells: evidence for a GPIIbIIIa-dependent bridging mechanism and novel roles for endothelial intercellular adhesion molecule 1 (ICAM-1), 18. Simak J, Holada K, Janota J, Stranak Z. Surface expression of major membrane glycoproteins on resting and trap-activated neonatal platelets. Pediatr Res. 1999;46:445[Medline] [Order article via Infotrieve].
19.
Verheul HM, Hoekman K, Jorna AS, Smit EF, Pinedo HM.
Targeting vascular endothelial growth factor blockade: ascites and pleural effusion formation.
Oncologist.
2000;5(suppl 1):45 20. Pedersen LM, Milman N. Prognostic significance of thrombocytosis in patients with primary lung cancer. Eur Respir J. 1996;9:1826[Abstract].
21.
Monreal M, Fernandez-Llamazares J, Pinol M, et al.
Platelet count and survival in patients with colorectal cancer 22. Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumor angiogenesis. Cell. 1996;86:353[Medline] [Order article via Infotrieve]. 23. Banks RE, Forbes MA, Kinsey SE, et al. Release of the angiogenic cytokine vascular endothelial growth factor (VEGF) from platelets: significance for VEGF measurements and cancer biology. Br J Cancer. 1998;77:956[Medline] [Order article via Infotrieve].
24.
Gaudry M, Bregerie O, Andrieu V, El Benna J, Pocidalo MA, Hakim J.
Intracellular pool of vascular endothelial growth factor in human neutrophils.
Blood.
1997;90:4153 25. Pipili-Synetos E, Papadimitriou E, Maragoudakis ME. Evidence that platelets promote tube formation by endothelial cells on matrigel. Br J Pharmacol. 1998;125:1252[Medline] [Order article via Infotrieve]. 26. Knighton DR, Hunt TK, Thakral KK, Goodson WH III. Role of platelets and fibrin in the healing sequence: an in vivo study of angiogenesis and collagen synthesis. Ann Surg. 1982;196:379[Medline] [Order article via Infotrieve]. 27. Dvorak HF. Tumors: wounds that do not heal. N Engl J Med. 1986;315:1650[Medline] [Order article via Infotrieve]. 28. Kadish JL, Butterfield CE, Folkman J. The effect of fibrin on cultured vascular endothelial cells. Tissue Cell. 1979;11:99[Medline] [Order article via Infotrieve]. 29. Brock TA, Dvorak HF, Senger DA. Tumor secreted vascular permeability factor increases cytosolic Ca2+ and von Willebrand factor release in human endothelial cells. Am J Pathol. 1991;138:213[Abstract].
30.
Randolph GJ, Luther T, Albrecht S, Magdolen V, Muller WA.
Role of tissue factor in adhesion of mononuclear phagocytes to and trafficking through endothelium in vitro.
Blood.
1998;92:4167 31. Lou J, Donati YRA, Juillard P, et al. Platelets play an important role in TNF-induced microvascular endothelial cell pathology. Am J Pathol. 1997;151:1397[Abstract].
32.
Kirchhofer D, Sakariassen KS, Clozel M, et al.
Relationship between tissue factor expression and deposition of fibrin, platelets, and leukocytes on cultured endothelial cells under venous blood flow conditions.
Blood.
1993;81:2050 33. Melder RJ, Koenig GC, Witwer BP, Safabakhsh N, Munn LL, Jain RK. During angiogenesis, vascular endothelial growth factor and basic fibroblast growth factor regulate natural killer cell adhesion to tumor endothelium. Nat Med. 1996;2:992[Medline] [Order article via Infotrieve].
34.
Pendurthi UR, Williams JT, Rao LV.
Acidic and basic fibroblast growth factors suppress transcriptional activation of tissue factor and other inflammatory genes in endothelial cells.
Arterioscler Thromb Vasc Biol.
1997;17:940
© 2000 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  A. Thulin, M. Ringvall, A. Dimberg, K. Karehed, T. Vaisanen, M.-R. Vaisanen, O. Hamad, J. Wang, R. Bjerkvig, B. Nilsson, et al. Activated Platelets Provide a Functional Microenvironment for the Antiangiogenic Fragment of Histidine-Rich Glycoprotein Mol. Cancer Res., November 1, 2009; 7(11): 1792 - 1802. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Solanilla, J. Villeneuve, P. Auguste, M. Hugues, A. Alioum, S. Lepreux, J.-P. Ducroix, P. Duhaut, C. Conri, J.-F. Viallard, et al. The transport of high amounts of vascular endothelial growth factor by blood platelets underlines their potential contribution in systemic sclerosis angiogenesis Rheumatology, September 1, 2009; 48(9): 1036 - 1044. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. N. Gavrilovskaya, E. E. Gorbunova, N. A. Mackow, and E. R. Mackow Hantaviruses Direct Endothelial Cell Permeability by Sensitizing Cells to the Vascular Permeability Factor VEGF, while Angiopoietin 1 and Sphingosine 1-Phosphate Inhibit Hantavirus-Directed Permeability J. Virol., June 15, 2008; 82(12): 5797 - 5806. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. M.W. Verheul, M. P.J. Lolkema, D. Z. Qian, Y. H.A. Hilkes, E. Liapi, J.-W. N. Akkerman, R. Pili, and E. E. Voest Platelets Take Up the Monoclonal Antibody Bevacizumab Clin. Cancer Res., September 15, 2007; 13(18): 5341 - 5347. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. M.W. Verheul, H. Hammers, K. van Erp, Y. Wei, T. Sanni, B. Salumbides, D. Z. Qian, G. D. Yancopoulos, and R. Pili Vascular Endothelial Growth Factor Trap Blocks Tumor Growth, Metastasis Formation, and Vascular Leakage in an Orthotopic Murine Renal Cell Cancer Model Clin. Cancer Res., July 15, 2007; 13(14): 4201 - 4208. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. C. Kwaan Double Hazard of Thrombophilia and Bleeding in Leukemia Hematology, January 1, 2007; 2007(1): 151 - 157. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Chiodoni, M. Iezzi, C. Guiducci, S. Sangaletti, I. Alessandrini, C. Ratti, F. Tiboni, P. Musiani, D. N. Granger, and M. P. Colombo Triggering CD40 on endothelial cells contributes to tumor growth J. Exp. Med., October 30, 2006; 203(11): 2441 - 2450. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Shimamura, G. Matchett, H. Yatsushige, J. W. Calvert, H. Ohkuma, and J. Zhang Inhibition of Integrin {alpha}v{beta}3 Ameliorates Focal Cerebral Ischemic Damage in the Rat Middle Cerebral Artery Occlusion Model Stroke, July 1, 2006; 37(7): 1902 - 1909. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. J. Li and B. Young Karlan Androgen Mediation of Thrombocytosis in Epithelial Ovarian Cancer Biology Clin. Cancer Res., November 15, 2005; 11(22): 8015 - 8018. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. H. Im, W. Fu, H. Wang, S. K. Bhatia, D. A. Hammer, M. A. Kowalska, and R. J. Muschel Coagulation Facilitates Tumor Cell Spreading in the Pulmonary Vasculature during Early Metastatic Colony Formation Cancer Res., December 1, 2004; 64(23): 8613 - 8619. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Brill, H. Elinav, and D. Varon Differential role of platelet granular mediators in angiogenesis Cardiovasc Res, August 1, 2004; 63(2): 226 - 235. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Langlois, D. Gingras, and R. Beliveau Membrane type 1-matrix metalloproteinase (MT1-MMP) cooperates with sphingosine 1-phosphate to induce endothelial cell migration and morphogenic differentiation Blood, April 15, 2004; 103(8): 3020 - 3028. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Krotz, T. Riexinger, M. A. Buerkle, K. Nithipatikom, T. Gloe, H.-Y. Sohn, W. B. Campbell, and U. Pohl Membrane Potential-Dependent Inhibition of Platelet Adhesion to Endothelial Cells by Epoxyeicosatrienoic Acids Arterioscler Thromb Vasc Biol, March 1, 2004; 24(3): 595 - 600. [Abstract] [Full Text]
|
|
|
|
 |
|
 M. E. Daly, A. Makris, M. Reed, and C. E. Lewis Hemostatic Regulators of Tumor Angiogenesis: A Source of Antiangiogenic Agents for Cancer Treatment? J Natl Cancer Inst, November 19, 2003; 95(22): 1660 - 1673. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Jurasz, D. Alonso, S. Castro-Blanco, F. Murad, and M. W. Radomski Generation and role of angiostatin in human platelets Blood, November 1, 2003; 102(9): 3217 - 3223. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. M. W. Verheul and H. M. Pinedo The Importance of Platelet Counts and Their Contents in Cancer Clin. Cancer Res., August 1, 2003; 9(9): 3219 - 3221. [Full Text] [PDF]
|
|
|
|
|
|
R. T.-P. Poon, C. P.-Y. Lau, S.-T. Cheung, W.-C. Yu, and S.-T. Fan Quantitative Correlation of Serum Levels and Tumor Expression of Vascular Endothelial Growth Factor in Patients with Hepatocellular Carcinoma Cancer Res., June 15, 2003; 63(12): 3121 - 3126. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B.C. Kuenen, M. Levi, J.C.M. Meijers, V.W.M. van Hinsbergh, J. Berkhof, A.K. Kakkar, K. Hoekman, and H.M. Pinedo Potential Role of Platelets in Endothelial Damage Observed During Treatment With Cisplatin, Gemcitabine, and the Angiogenesis Inhibitor SU5416 J. Clin. Oncol., June 1, 2003; 21(11): 2192 - 2198. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Geimonen, S. Neff, T. Raymond, S. S. Kocer, I. N. Gavrilovskaya, and E. R. Mackow Pathogenic and nonpathogenic hantaviruses differentially regulate endothelial cell responses PNAS, October 15, 2002; 99(21): 13837 - 13842. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. M. H. de Groot, H. J. Broxterman, H. P. H. M. Adams, A. van Vliet, G. I. Tesser, Y. W. Elderkamp, A. J. Schraa, R. Jan Kok, G. Molema, H. M. Pinedo, et al. Design, Synthesis, and Biological Evaluation of a Dual Tumor-specific Motive Containing Integrin-targeted Plasmin-cleavable Doxorubicin Prodrug Mol. Cancer Ther., September 1, 2002; 1(11): 901 - 911. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B.C. Kuenen, M. Levi, J.C.M. Meijers, A.K. Kakkar, V.W.M. van Hinsbergh, P.J. Kostense, H.M. Pinedo, and K. Hoekman Analysis of Coagulation Cascade and Endothelial Cell Activation During Inhibition of Vascular Endothelial Growth Factor/Vascular Endothelial Growth Factor Receptor Pathway in Cancer Patients Arterioscler Thromb Vasc Biol, September 1, 2002; 22(9): 1500 - 1505. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Kou, D. Greif, and T. Michel Dephosphorylation of Endothelial Nitric-oxide Synthase by Vascular Endothelial Growth Factor. IMPLICATIONS FOR THE VASCULAR RESPONSES TO CYCLOSPORIN A J. Biol. Chem., August 9, 2002; 277(33): 29669 - 29673. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Chavakis, N. Boeckel, S. Santoso, R. Voss, I. Isordia-Salas, R. A. Pixley, E. Morgenstern, R. W. Colman, and K. T. Preissner Inhibition of Platelet Adhesion and Aggregation by a Defined Region (Gly-486-Lys-502) of High Molecular Weight Kininogen J. Biol. Chem., June 21, 2002; 277(26): 23157 - 23164. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Trikha, Z. Zhou, J. Timar, E. Raso, M. Kennel, E. Emmell, and M. T. Nakada Multiple Roles for Platelet GPIIb/IIIa and {alpha}v{beta}3 Integrins in Tumor Growth, Angiogenesis, and Metastasis Cancer Res., May 1, 2002; 62(10): 2824 - 2833. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2000 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
) or
by thrombin-activated (0.5 U/mL) platelets (
). Error bars represent
the SEM. A 2-fold increase in endothelial cell number, as measured by
the MTT assay, was found for both activated and nonactivated
platelets.
a practical approach. Oxford, UK: Oxford University Press; 1996:1.