Activation of the coagulation system in cancerogenesis and metastasation

Biomedicine & Pharmacotherapy 59 (2005) 70–75 http://france.elsevier.com/direct/BIOPHA/

Dossier: drug efficacy and drug resistance

Activation of the coagulation system in cancerogenesis and metastasation W.Z. Xie a,b, M. Leibl a, M.R. Clark a, P. Dohrmann c, T. Kunze d, F. Gieseler a,* a

Department of Internal Medicine, Section Hematology/Oncology, University of Kiel, Germany b First Affıliated Hospital, Medical College of Zhejiang University, Hangzhou, China c Department of Surgery (PD), University of Kiel, Germany d Department of Pharmacy (T.K.), University of Kiel, Germany Received 10 September 2004

Abstract The activation of the coagulation system in cancer patients is a well-known phenomenon responsible for recurrent clinical problems. A number of fascinating molecular mechanisms have been recognized showing that the tumor not only activates the coagulation system, but vice versa, activated coagulation proteins are able to induce molecular effects in tumor cells. The molecular basis is the expression of defined membrane receptors by tumor cells that are activated, for example, by thrombin. As the liberation of thrombin from prothrombin is one of the key events in coagulation, it’s impact upon biological processes, such as cancerogenesis and metastasation, seems to be a regular pathophysiological consequence. These perceptions are not only interesting for the comprehension of cancerogenesis, metastasation, and clinical phenomena, but they also have a high impact upon modern strategies of tumor therapy. Especially, the development of clinically useful coagulation inhibitors, such as modern low molecular weight heparins or melagatran, created the possibility of therapies that combine cell biological approaches with apoptosis-inducing principals such as chemotherapy. Several clinical studies that demonstrate the implication of these strategies have already been published recently. In this article the cell biological basics for these approaches are reviewed. © 2005 Elsevier SAS. All rights reserved. Keywords: Coagulation system; Cancerogenesis; Metastasation

Thrombosis is a significant problem for cancer patients. Blood tests for hemostatic hypercoagulability markers, such as d-dimers, reveal abnormally high levels in as many as 90% of cancer patients. The incidence of manifest thrombosis can be up to 28% depending upon the cancer type including lung, colorectal, ovarian, pancreas, breast, stomach, and prostate cancer [1–6]. Also, a correlation of thrombosis with stageprogression of the malignant disease has been observed. For example, the thrombotic risk for stage II breast cancer patients undergoing chemotherapy is estimated 5–10%, with thrombotic events occurring predominately in post-menopausal women [7,8], and up to 17.6% in stage IV breast cancer patients [9]. A multitude of factors, both genetic and acquired, contribute to the hypercoagulable and thrombophilic state of cancer * Corresponding author. Department of Internal Medicine, Section Hematology/Oncology, University of Kiel, Schittenhelmstr. 12, 24105 Kiel, Germany. E-mail address: [email protected] (F. Gieseler). 0753-3322/$ - see front matter © 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.biopha.2005.01.001

patients. Besides host- and tumor-derived mechanisms, including stasis due to immobilization or blood flow obstruction, surgery, infections, endothelium damage due to chemotherapeutic agents or central catheters, and abnormalities of blood coagulation, are involved [8,10]. Besides these direct factors, thrombosis is a major complication within paraneoplastic syndromes in malignant disease. Trousseau has described this already in 1865 [4]. In 10.9% of patients deep vein thrombosis is the first symptom of the malignoma [5]; within 2 years after unexplained pulmonary embolism 6.8% of the patients develop malignant diseases [6]. The central biochemical event resulting in hypercoagulability during cancer development and metastasis is the generation of thrombin. Thrombin (EC 3.4.21.5) is a trypsin-like serine protease produced, thus activated, by the proteolytic cleavage of its precursor molecule prothrombin by coagulation factor Xa. Physiologically, thrombin generation is restricted to sites of vascular injury. The clotting actions of thrombin are central to hemostasis mainly through catalyzing the conversion of soluble fibrinogen into insoluble

W.Z. Xie et al. / Biomedicine & Pharmacotherapy 59 (2005) 70–75

fibrin even at thrombin concentrations as low as 5–10 nM [11,12]. One of the pathological consequences of the impaired regulation of thrombin is thrombosis.

71

smooth muscle cells [29]. Notably, human platelets do not express PAR-3 [14]. 1.2. Thrombomodulin

1. Molecular basis of cellular thrombin-effects In addition to its central role in coagulation, thrombin is able to induce biological effect in cancer cells by several molecular mechanisms: the activation of protease-activated receptors (PAR) and the binding to thrombomodulin or glycoprotein Iba. 1.1. Protease-activated receptors The interplay between malignancy and hemostasis including its central protease thrombin has long been recognized. Recently, the identification of protease-activated receptors (PARs) provides mechanistic explanations at the molecular level. PARs are G-protein coupled, seven-transmembrane segment receptors that require the cleavage of their extracellular N-terminus by proteinases such as thrombin, trypsin, or tryptase [13]. The first receptor being described was PAR-1, a prototypic functional thrombin receptor. From PAR-1 a fourmember family comprising PAR-1, PAR-2, PAR-3, and PAR4 has emerged [14–17]. Besides PAR-2, which is activated by trypsin and mast cell tryptase [18,19], the other three receptors are proteolytically activated by thrombin. Physiologically, PAR-1 and PAR-4 are expressed on human platelets, either of which independently mediates thrombin signaling, PAR-1 at low while PAR-4 at high thrombin concentrations [20]. It has been suggested that PAR-4 may act as a “backup” mechanism in human platelets in the absence of PAR-1 [21]. Additionally, PAR-1 expression has been found in blood vessel cells [22], as well as in the central nervous system [23]. It plays an essential regulatory role in inflammation as it modulates the scenario of platelet aggregation, vasodilatation, increased vascular permeability, granulocyte chemotaxis, and calcium-dependent chloride secretion in intestinal epithelial cells [13,24]. Thrombin inhibited both Bim (Bcl-2-interacting mediator of cell death) protein and Bim mRNA expression and prevented apoptosis induced by serum withdrawal via PAR-1. Thrombin activation of RhoA (Ras homolog A) was necessary for thrombin-induced cell death suggesting a sequential linkage of cellular events, thus leading to a proposed model for a second messenger cascade induced by thrombin that ultimately results in apoptosis [25]. It has also been shown that PAR-1 was partly required for the anti-apoptotic effects of activated protein C in staurosporineinduced apoptosis [26]. PAR-2 is also expressed by a variety of cell types, including epithelial cells, and has also been implicated in inflammation [27]. Although the coagulation factors VIIa and Xa can activate PAR-2, it is not considered to be a thrombin receptor [28]. PAR-3 has been detected on mouse platelets, rat brain capillary endothelial cells, astrocytes, and human airway

In addition to protease-activated receptors, thrombomodulin, and glycoprotein Iba have been identified as cellular thrombin-binding sites. Thrombomodulin is a glycoprotein with endothelial growth factor like domains presented on the luminal vascular endothelial cell surface. It plays a functional role as endothelial cell anticoagulant converting thrombin from a procoagulant protease to an anticoagulant and inducing its interaction with Protein-C [30]. Following exposure of endothelial cells to vascular endothelial growth factor (VEGF) for 24 h, the increase in thrombin-dependent activation of human protein C generation correlated with a proportional and concentration dependent increase in the level of cell surface thrombomodulin antigen. The VEGF regulation of thrombomodulin may contribute to mechanisms that would maintain local hemostasis during angiogenesis and revascularization and could play a role in minimizing loss of vessel anticoagulant function during inflammatory processes [31]. As a consequence of the described events, thrombomodulin precludes thrombin from activating PAR-1 [32]. Expression of thrombomodulin seems to be restricted to certain types of malignancies including transitional cell carcinoma [33], squamous cell carcinoma [34], ovarian cancer [35], and pancreatic cancer [36]; although in A549 lung cancer cells thrombomodulin constitutes about 90% of thrombin binding sites [37]. 1.3. Glycoprotein Ib␣ Glycoprotein Iba (GPIba) contains several ligand-binding domains for von-Willebrand factor (vWF), Mac-1, P-selectin, coagulation factor XII, and thrombin. Although it was a longheld belief that the expression of GPIba is restricted to cells of megakaryocytic lineage, GPIba has also been detected in human breast cancer cell lines and primary breast tumors; in addition, it may play a role in tumor-induced platelet aggregation and in the metastasation of breast cancer cells [38–40]. The GPIb/IX complex contains three transmembranous leucine-rich repeat polypeptides (GpIba, GpIbb, and GpIX) that form the platelet vWF receptor. GpIb/IX functions to effect platelet adhesion, activation, and aggregation under conditions of high shear stress [38]. It was found that GpIba could cause growth arrest in the G1 phase of the cell cycle associated with the induction of the cyclin-dependent kinase inhibitor p21. Also, the GpIb complex plays a role in megakaryocyte growth regulation [38]. 2. Cellular effects of thrombin 2.1. Physiological effects A number of thrombin-interacting cell surface structures have been described from which the activation of protease

72

W.Z. Xie et al. / Biomedicine & Pharmacotherapy 59 (2005) 70–75

activated receptors (PAR) results in G-protein triggered cellular reactions. Thus, thrombin participates in a variety of biological processes such as the induction of mitogenesis and differentiation, the production of cytokines and growth factors, and apoptosis. Thrombin regulates microvascular permeability by the formation of interendothelial gaps, induces mitogenesis of several cell types such as fibroblasts, smooth muscle cells, and malignant cells, and demonstrates proinflammatory action by the stimulation of transvascular leukocyte migration [41,42]. Additionally, thrombin inhibited myoblast fusion and functions as a survival factor for myoblasts [43]. While PAR1 and PAR-3 mediated anti-apoptotic signaling from two divergent inducers of apoptosis (N-methyl-D-aspartate and staurosporine) in neurons [44], loss of PAR-4 reduces the ability of tumor necrosis factor-alpha (TNF-alpha) to induce apoptosis by increased activation of NF-kappaB [45]. In summary, thrombin is implicated in the growth, progression, and metastasis of cancer through the activation of PARs, the initiation of angiogenesis, tumor growth, and metastasis 1. 2.2. Pathophysiological effects on cancer cells Whereas the mitogenic effect of thrombin on smooth muscle cells and fibroblasts has long been appreciated [46], thrombin has recently been demonstrated to act as a mitogen for endothelial and astrocytoma cells by inducing polyphosphoinositide hydrolysis, Ca2+ mobilization, and DNA synthesis [47]. Obviously, thrombin stimulation of tumor growth requires the participation of Ras protein, protein kinase C (PKC), and the mitogen-activated protein kinase (MAPK) pathway [47]. Consequently, mitogenic activities of thrombin and PAR1 activation could be abolished by the PKC inhibitor bisindolylmaleimide in human epidermoid carcinoma cells [48]. Also, the MEK inhibitor PD98059 suppressed thrombininduced cell proliferation in human colon cancer cells [49]. PAR-1 has been detected on the surface of a number of established malignant cell lines and a variety of cancer tissues suggesting an association between the expression level, tumorigenesis, cancer progression, and tumor biology including differentiation and metastasis. For instance, PAR-1 protein was not detected in the epithelium of normal pancreatic tissue whereas immunofluorescence staining and Western blots of pancreatic cancer cell lines revealed a correlation between PAR-1 expression intensity and the grade of differentiation. The level of PAR-1 mRNA was lower in normal pancreas compared to pancreatic cancer tissue with mRNA levels differ up to 25-fold between different pancreatic cancer cell lines [50]. Interestingly, the cellular effects are not necessarily correlated to fibrin formation or the induction of coagulation [51]. Recently, Tellez and Bar-Eli [52] proposed that loss of AP-2 results in increased expression of the thrombin receptor, which subsequently contributes to the metastatic phenotype of melanoma by up-regulating the expression of adhesion molecules, proteases, and angiogenic

molecules. Also, PAR-1 was expressed in oral squamous cell carcinomas with PAR-1 protein levels being lower in nonmetastatic when compared to metastatic cells [53]. Recent studies observed dose-dependent dual effects of thrombin on the proliferation kinetics of certain types of tumor cells in vitro, leading to increased cell proliferation with low concentrations, while high concentrations of thrombin inhibited tumor cell growth and/or lead to apoptosis [10]. Very little has been published about PAR-3 and PAR4 expression in cancer cells and tissues. Human astrocytoma cells presented both PAR-1 and PAR-4, while renal carcinoma cells co-expressed PAR-1 and PAR-3 [54]. Similarly, a complex PAR expression profile was reported for breast cancer cells: very high levels of functional PAR-1, PAR-4 and trypsin receptor PAR-2 have been found in highly invasive breast cancer cells MDA-MB-231, while in minimally invasive MCF7 cells only traces of PAR-1 and low levels of PAR4 and PAR-2 have been described [55]. In addition to the expression of PARs, thrombomodulin might play an active role in cancer invasion and metastasis. Thrombomodulin expression might serve as a new prognostic factor e.g., in invasive breast cancer as low thrombomodulin expression correlated significantly with a high relapse rate [56]. Also, patients with intense thrombomodulin expression in oral squamous cell carcinoma showed a significantly lower frequency of lymph node metastasis and more favorable survival rates than those with negative thrombomodulin expression; the thrombomodulin expression was a better marker than the other prognostic factors, such as differentiation degree, tumor size, and invasion mode 34. Similar results were found for lung squamous cell carcinoma patients with thrombomodulin-negative expression in primary tumors [57]. Furthermore, the injection of human urine thrombomodulin suppressed the formation of lung metastasis in mice [58]. 2.3. Pathophysiological effects on tumor-surrounding cells and angiogenesis Besides the direct interaction of thrombin with tumor cells, additional effects upon non-malignant cells might be decisive for the clinical course of a malignant disease. Thrombin has been reported to initiate the expression of various adhesion molecules and the stimulation of cellular adhesion of tumor cells to endothelial cells, platelets, and extra-cellular matrix components including fibronectin, laminin, collagen, and vWF [53]. Pre-incubation of either tumor cells or platelets with thrombin increased the adhesion of tumor cells to platelets [59]. Thrombin enhanced adhesion of B16F10 melanoma cells transfected with full-length PAR-1 cDNA to fibronectin compared to mocktransfected cells [59]. PAR1 up-regulation may also be critically involved in prostate carcinoma metastasis to bone [60]. The adhesion of tumor cells to platelets, vascular endothelium, and extra cellular matrix enhances their invasive and metastatic capacity. PAR1 has also been found on stromal cells surrounding malignant tissue. As the expression was up-regulated in cancer stro-

W.Z. Xie et al. / Biomedicine & Pharmacotherapy 59 (2005) 70–75

mal fibroblasts, aberrant PAR-1 expression in and/or around tumor tissue has been suggested [61]. PAR-1 expression in stromal cells might contribute to abnormal cellular communication during malignancy since tumor progression requires bidirectional communication with its microenvironment. A number of evidences indicate that thrombin is able to activate angiogenesis. Physiologically, angiogenesis takes place in embryogenesis, wound healing, and the proliferative phase of the female reproductive cycle. As a solid tumor cannot grow beyond a critical size of 1–2 mm in diameter without gaining access to the host circulatory system that supplies the tumor with nutrients and waste-disposal capacity, angiogenesis is an essential process during tumor growth and metastasis. Tumor cells are able initiate vascularization by the secretion of an array of angiogenic factors, such as VEGF, interleukin-8 (IL-8), basic fibroblast growth factor (bFGF), and angiopoietins. Various actions of thrombin at the cellular level contribute to the initiation of angiogenesis: • Thrombin decreases the ability of endothelial cells to attach to basement membrane proteins; • Thrombin greatly enhances VEGF induced endothelial cell proliferation. This enhancement is accompanied by upregulation of VEGF receptor mRNA expression and increased secretion of VEGF. It also enhanced the expression and protein synthesis of matrix metalloprotease- 9 and integrin in human prostate cancer PC-3 cells [51,62]; • Thrombin increases the mRNA and protein levels of alpha(v)beta [3] integrin and serves as a ligand to this receptor [51,63]. In contrast, Chan et al. reported that thrombin’s proteolytic activity could also be anti-angiogenic under distinctive circumstances. Prothrombin significantly inhibited endothelial cell tube formation in vitro at 10 mg/ml and bFGF-induced angiogenesis in matrigel-plug assays performed in mice. The proteolytic activity of thrombin appeared to be critical for the antiangiogenic activity of prothrombin [64]. The angiogenic and tumor-promoting effect of thrombin provides the basis for the development of thrombin receptor mimetics or receptor-antagonists for therapeutic application [65]. These research results indicate the value of a comprehension of the coherency between the frequently activated coagulation system, the resulting clinical problems, and the impact upon the cell biology of tumor cells. These interrelations offer new therapeutical options in clinical oncology and provide explanation for the results of clinical studies with coagulation inhibitors in clinical oncology [66–75].

[4]

[5]

[6] [7] [8]

[9]

[10]

[11] [12] [13]

[14]

[15] [16]

[17]

[18]

[19]

[20]

[21]

[22]

References [1] [2] [3]

Francis JL, Biggerstaff J, Amirkhosravi A. Hemostasis and malignancy. Semin Thromb Hemost 1998;24:93–109. van Wersch JW, Tjwa MK. Coagulation/fibrinolysis balance and lung cancer. Haemostasis 1991;21:117–23. Gabazza E, Taguchi O, Yamakami T, Machishi M, Ibata H, Suzuki S. Correlation between increased granulocyte elastase release and activation of blood coagulation in patients with lung cancer. Cancer 1993;72:2134–40.

[23]

[24]

73

van Duijnhoven EM, Lustermans FA, van Wersch JW. Evaluation of the coagulation/fibrinolysis balance in patients with colorectal cancer. Haemostasis 1993;23:168–72. von Tempelhoff GF, Dietrich M, Niemann F, Schneider D, Hommel G, Heilmann L. Blood coagulation and thrombosis in patients with ovarian malignancy. Thromb Haemost 1997;77:456–61. Lip GY, Chin BS, Blann AD. Cancer and the prothrombotic state. Lancet Oncol 2002;3:27–34. Lee AY, Levine MN. The thrombophilic state induced by therapeutic agents in the cancer patient. Semin Thromb Hemost 1999;25:137–45. von Tempelhoff GF, Dietrich M, Hommel G, Heilmann L. Blood coagulation during adjuvant epirubicin/cyclophosphamide chemotherapy in patients with primary operable breast cancer. J Clin Oncol 1996;14:2560–8. Goodnough LT, Saito H, Manni A, Jones PK, Pearson OH. Increased incidence of thromboembolism in stage IV breast cancer patients treated with a five-drug chemotherapy regimen. A study of 159 patients. Cancer 1984;54:1264–8. Schiller H, Bartscht T, Arlt A, et al. Thrombin as a survival factor for cancer cells: thrombin activation in malignant effusions in vivo and inhibition of idarubicin-induced cell death in vitro. Int J Clin Pharmacol Ther 2002;40:329–35. Stubbs MT, Bode W. A model for the specificity of fibrinogen cleavage by thrombin. Semin Thromb Hemost 1993;19:344–51. Mann KG, Brummel K, Butenas S. What is all that thrombin for? J Thromb Haemost 2003;1:1504–14. Dery O, Corvera CU, Steinhoff M, Bunnett NW. Proteinase-activated receptors: novel mechanisms of signaling by serine proteases. Am J Physiol 1998;274:C1429–C1452. Nakanishi-Matsui M, Zheng YW, Sulciner DJ, Weiss EJ, Ludeman MJ, Coughlin SR. PAR3 is a cofactor for PAR4 activation by thrombin. Nature 2000;404:609–13. Macfarlane SR, Seatter MJ, Kanke T, Hunter GD, Plevin R. Proteinase-activated receptors. Pharmacol Rev 2001;53:245–82. Cupit LD, Schmidt VA, Bahou WF. Proteolytically activated receptor-3. A member of an emerging gene family of protease receptors expressed on vascular endothelial cells and platelets. Trends Cardiovasc Med 1999;9:42–8. Xu WF, Andersen H, Whitmore TE, et al. Cloning and characterization of human proteaseactivated receptor 4. Proc Natl Acad Sci USA 1998;95:6642–6. Nystedt S, Emilsson K, Wahlestedt C, Sundelin J. Molecular cloning of a potential proteinase activated receptor. Proc Natl Acad Sci USA 1994;91:9208–12. Chinni C, de Niese MR, Jenkins AL, Pike RN, Bottomley SP, Mackie EJ. Protease-activated receptor-2 mediates proliferative responses in skeletal myoblasts. J Cell Sci 2000;113(Pt 24):4427–33. Kahn ML, Nakanishi-Matsui M, Shapiro MJ, Ishihara H, Coughlin SR. Protease-activated receptors 1 and 4 mediate activation of human platelets by thrombin. J Clin Invest 1999;103:879–87. Major CD, Santulli RJ, Derian CK, Andrade-Gordon P. Extracellular mediators in atherosclerosis and thrombosis: lessons from thrombin receptor knockout mice. Arterioscler Thromb Vasc Biol 2003;23: 931–9. Hou L, Howells GL, Kapas S, Macey MG. The protease-activated receptors and their cellular expression and function in blood-related cells. Br J Haematol 1998;101:1–9. Weinstein JR, Gold SJ, Cunningham DD, Gall CM. Cellular localization of thrombin receptor mRNA in rat brain: expression by mesencephalic dopaminergic neurons and codistribution with prothrombin mRNA. J Neurosci 1995;15:2906–19. Buresi MC, Schleihauf E, Vergnolle N, et al. Protease-activated receptor-1 stimulates Ca(2+)-dependent Cl(–) secretion in human intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 2001;281: G323–G332.

74

W.Z. Xie et al. / Biomedicine & Pharmacotherapy 59 (2005) 70–75

[25] Donovan FM, Pike CJ, Cotman CW, Cunningham DD. Thrombin induces apoptosis in cultured neurons and astrocytes via a pathway requiring tyrosine kinase and RhoA activities. J Neurosci 1997;17: 5316–26. [26] Mosnier LO, Griffin JH. Inhibition of staurosporine-induced apoptosis of endothelial cells by activated protein C requires proteaseactivated receptor-1 and endothelial cell protein C receptor. Biochem J 2003;373:65–70. [27] Vliagoftis H, Befus AD, Hollenberg MD, Moqbel R. Airway epithelial cells release eosinophil survival-promoting factors (GM-CSF) after stimulation of proteinase-activated receptor 2. J Allergy Clin Immunol 2001;107:679–85. [28] Camerer E, Huang W, Coughlin SR. Tissue factor- and factor X-dependent activation of protease-activated receptor 2 by factor VIIa. Proc Natl Acad Sci USA 2000;97:5255–60. [29] Bartha K, Domotor E, Lanza F, Adam-Vizi V, Machovich R. Identification of thrombin receptors in rat brain capillary endothelial cells. J Cereb Blood Flow Metab 2000;20:175–82. [30] Wienhard J, Bielska B, Munstedt K, Lang U, Zygmunt M. Increased endothelial thrombomodulin (TM) expression in pregnancies complicated by IUGR. J Perinat Med 2002;30:322–8. [31] Calnek DS, Grinnell BW. Thrombomodulin-dependent anticoagulant activity is regulated by vascular endothelial growth factor. Exp Cell Res 1998;238:294–8. [32] Ruf W. PAR1 signaling: more good than harm? Nat Med 2003;9:258– 60. [33] Ordonez NG. Thrombomodulin expression in transitional cell carcinoma. Am J Clin Pathol 1998;110:385–90. [34] Tabata M, Sugihara K, Yonezawa S, Yamashita S, Maruyama I. An immunohistochemical study of thrombomodulin in oral squamous cell carcinoma and its association with invasive and metastatic potential. J Oral Pathol Med 1997;26:258–64. [35] Wilhelm S, Schmitt M, Parkinson J, Kuhn W, Graeff H, Wilhelm OG. Thrombomodulin, a receptor for the serine protease thrombin, is decreased in primary tumors and metastases but increased in ascitic fluids of patients with advanced ovarian cancer FIGO IIIc. Int J Oncol 1998;13:645–51. [36] Oikawa T, Kushuhara M, Ishikawa S, et al. Production of endothelin1 and thrombomodulin by human pancreatic cancer cells. Br J Cancer 1994;69:1059–64. [37] Maruyama I, Majerus PW. The turnover of thrombin–thrombomodulin complex in cultured human umbilical vein endothelial cells and A549 lung cancer cells. Endocytosis and degradation of thrombin. J Biol Chem 1985;260:15432–8. [38] Feng S, Christodoulides N, Kroll MH. The glycoprotein Ib/IX complex regulates cell proliferation. Blood 1999;93:4256–63. [39] Kaushansky K. The mpl ligand: molecular and cellular biology of the critical regulator of megakaryocyte development. Stem Cells 1994; 12(Suppl 1):91–6 discussion 96–97. [40] Suter CM, Hogg PJ, Price JT, Chong BH, Ward RL. Identification and characterisation of a platelet GPIb/V/IX-like complex on human breast cancers: implications for the metastatic process. Jpn J Cancer Res 2001;92:1082–92. [41] Guttridge DC, Lau A, Tran L, Cunningham DD. Thrombin causes a marked delay in skeletal myogenesis that correlates with the delayed expression of myogenin and p21CIP1/WAF1. J Biol Chem 1997;272: 24117–20. [42] Derian CK, Eckardt AJ. Thrombin receptor-dependent prostaglandin E2 synthesis in hamster fibroblasts: synergistic interactions with interleukin-1beta. Exp Cell Res 1997;232:1–7. [43] Chinni C, de Niese MR, Tew DJ, Jenkins AL, Bottomley SP, Mackie EJ. Thrombin, a survival factor for cultured myoblasts. J Biol Chem 1999;274:9169–74. [44] Guo H, Liu D, Gelbard H, et al. Activated protein C prevents neuronal apoptosis via protease activated receptors 1 and 3. Neuron 2004;41: 563–72.

[45] Garcia-Cao I, Lafuente MJ, Criado LM, Diaz-Meco MT, Serrano M, Moscat J. Genetic inactivation of Par4 results in hyperactivation of NF-kappaB and impairment of JNK and p38. EMBO Rep 2003;4: 307–12. [46] Glenn KC, Carney DH, Fenton II JW, Cunningham DD. Thrombin active site regions required for fibroblast receptor binding and initiation of cell division. J Biol Chem 1980;255:6609–16. [47] LaMorte VJ, Kennedy ED, Collins LR, et al. A requirement for Ras protein function in thrombin-stimulated mitogenesis in astrocytoma cells. J Biol Chem 1993;268:19411–5. [48] Kaufmann R, Schafberg H, Rudroff C, Nowak G. Thrombin receptor activation results in calcium signaling and protein kinase C-dependent stimulation of DNA synthesis in HEp-2g laryngeal carcinoma cells. Cancer 1997;80:2068–74. [49] Darmoul D, Gratio V, Devaud H, Lehy T, Laburthe M. Aberrant expression and activation of the thrombin receptor protease-activated receptor-1 induces cell proliferation and motility in human colon cancer cells. Am J Pathol 2003;162:1503–13. [50] Rudroff C, Seibold S, Kaufmann R, et al. Expression of the thrombin receptor PAR-1 correlates with tumour cell differentiation of pancreatic adenocarcinoma in vitro. Clin Exp Metastasis 2002;19:181–9. [51] Tsopanoglou NE, Maragoudakis ME. Role of thrombin in angiogenesis and tumor progression. Semin Thromb Hemost 2004;30:63–9. [52] Tellez C, Bar-Eli M. Role and regulation of the thrombin receptor (PAR-1) in human melanoma. Oncogene 2003;22:3130–7. [53] Liu Y, Gilcrease MZ, Henderson Y, Yuan XH, Clayman GL, Chen Z. Expression of protease activated receptor 1 in oral squamous cell carcinoma. Cancer Lett 2001;169:173–80. [54] Kaufmann R, Patt S, Zieger M, et al. The two-receptor system PAR1/PAR-4 mediates alpha thrombin-induced [Ca(2+)](i) mobilization in human astrocytoma cells. J Cancer Res Clin Oncol 2000;126:91–4. [55] Kamath L, Meydani A, Foss F, Kuliopulos A. Signaling from protease-activated receptor-1 inhibits migration and invasion of breast cancer cells. Cancer Res 2001;61:5933–40. [56] Kim SJ, Shiba E, Ishii H, et al. Thrombomodulin is a new biological and prognostic marker for breast cancer: an immunohistochemical study. Anticancer Res 1997;17:2319–23. [57] Ogawa H, Yonezawa S, Maruyama I, et al. Expression of thrombomodulin in squamous cell carcinoma of the lung: its relationship to lymph node metastasis and prognosis of the patients. Cancer Lett 2000;149:95–103. [58] Hosaka Y, Higuchi T, Tsumagari M, Ishii H. Inhibition of invasion and experimental metastasis of murine melanoma cells by human soluble thrombomodulin. Cancer Lett 2000;161:231–40. [59] Nierodzik ML, Chen K, Takeshita K, et al. Protease-activated receptor 1 (PAR-1) is required and rate-limiting for thrombin-enhanced experimental pulmonary metastasis. Blood 1998;92:3694–700. [60] Cooper CR, Chay CH, Gendernalik JD, et al. Stromal factors involved in prostate carcinoma metastasis to bone. Cancer 2003;97:739–47. [61] D’Andrea MR, Derian CK, Santulli RJ, Andrade-Gordon P. Differential expression of protease-activated receptors-1 and -2 in stromal fibroblasts of normal, benign, and malignant human tissues. Am J Pathol 2001;158:2031–41. [62] Yamahata H, Takeshima H, Kuratsu J, et al. The role of thrombin in the neo-vascularization of malignant gliomas: an intrinsic modulator for the up-regulation of vascular endothelial growth factor. Int J Oncol 2002;20:921–8. [63] Tsopanoglou NE, Maragoudakis ME. On the mechanism of thrombininduced angiogenesis: inhibition of attachment of endothelial cells on basement membrane components. Angiogenesis 1998;1:192–200. [64] Chan B, Merchan JR, Kale S, Sukhatme VP. Antiangiogenic property of human thrombin. Microvasc Res 2003;66:1–14. [65] Maragoudakis ME, Tsopanoglou NE, Andriopoulou P. Mechanism of thrombin-induced angiogenesis. Biochem Soc Trans 2002;30:173–7. [66] Brodsky SV. Coagulation, fibrinolysis and angiogenesis: new insights from knockout mice. Exp Nephrol 2002;10:299–306.

W.Z. Xie et al. / Biomedicine & Pharmacotherapy 59 (2005) 70–75 [67] Blot E, Gutman F, Thannberger A. Dalteparin compared with an oral anticoagulant for thromboprophylaxis in patients with cancer. N Engl J Med 2003;349:1385–7 author reply 1385–7. [68] Lee AY, Levine MN, Baker RI, et al. Low-molecular-weight heparin versus a coumarin for the prevention of recurrent venous thromboembolism in patients with cancer. N Engl J Med 2003;349:146–53. [69] Altinbas M, Coskun HS, Er O, et al. A randomized clinical trial of combination chemotherapy with and without low-molecular-weight heparin in small cell lung cancer. J Thromb Haemost 2004;2:1266–71. [70] Mousa SA. Low-molecular-weight heparins in thrombosis and cancer: emerging links. Cardiovasc Drug Rev 2004;22:121–34. [71] Kakkar AK, Levine MN, Kadziola Z, et al. Low molecular weight heparin, therapy with dalteparin, and survival in advanced cancer: the fragmin advanced malignancy outcome study (FAMOUS). J Clin Oncol 2004;22:1944–8.

75

[72] Mousa SA. Antithrombotics in thrombosis and cancer. Expert Rev Cardiovasc Ther 2003;1:283–91. [73] Unsal E, Atalay F, Atikcan S, Yilmaz A. Prognostic significance of hemostatic parameters in patients with lung cancer. Respir Med 2004; 98:93–8. [74] Akashi T, Furuya Y, Ohta S, Fuse H. Tissue factor expression and prognosis in patients with metastatic prostate cancer. Urology 2003; 62:1078–82. [75] Robert F, Busby E, Marques MB, Reynolds RE, Carey DE. Phase II study of docetaxel plus enoxaparin in chemotherapy-naive patients with metastatic non-small cell lung cancer: preliminary results. Lung Cancer 2003;42:237–45.