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(Chest. 2003;124:58S-68S.)
© 2003 American College of Chest Physicians

Tissue Factor, Thrombin, and Cancer*

Frederick R. Rickles, MD; Steven Patierno, PhD and Patricia M. Fernandez, PhD

* From the Departments of Medicine, Pediatrics, Pharmacology, and Urology, The George Washington University, Washington, DC.

Correspondence to: Frederick R. Rickles, MD, Executive Director, Federation of American Societies for Experimental Biology, 9650 Rockville Pike, Bethesda, MD 20814-3998


   Abstract
TOP
 Abstract
Introduction
 Cancer and Thrombosis;...
 TF
 Thrombin
 Fibrin
 Therapeutic Applications
 Concluding Remarks
 References
 
In addition to its primary role in hemostasis and blood coagulation, thrombin is a potent mitogen capable of inducing cellular functions. Therefore, it should come as no surprise that thrombin has proved to be of importance in the behavior of cancer. In this review, we focus on the ability of tissue factor (TF) and thrombin to influence tumor angiogenesis. Both exert their influence on angiogenesis through clotting-dependent and clotting-independent mechanisms: (1) directly affecting signaling pathways that mediate cell functions, and (2) mediating clot formation, thereby providing a growth media for tumor cells. Therefore, anticoagulant drugs may prove efficacious in cancer treatment due to their ability to reduce the characteristic hypercoagulability of cancer and alter the fundamental biology of cancer.

Key Words: angiogenesis • cancer • fibrinogen • thrombosis • tissue factor


   Introduction
TOP
 Abstract
 Introduction
Cancer and Thrombosis;...
 TF
 Thrombin
 Fibrin
 Therapeutic Applications
 Concluding Remarks
 References
 
The association of thrombosis and cancer owes its origin to the observations of Professor Armand Trousseau, who noted that patients who present with idiopathic venous thromboembolism (VTE) frequently harbor an occult cancer. Trousseau also observed that patients with known cancer have an increased propensity to acquire VTE. Although Professor Trousseau first recognized this link between coagulation and malignancy in 1865,1 the mechanisms underlying this association are only recently starting to unravel. We now believe that there are two key mediators of this link: (1) the enzyme thrombin, whose broad substrate specificity supports a variety of cellular effects relevant to tumor growth and metastasis; and (2) tissue factor (TF), the primary initiator of the coagulation cascade, whose rather ubiquitous presence as a transmembrane receptor on a variety of nucleated cells confers responsibility for the generation of cell-surface thrombin in many pathologic situations.

Via both clotting-dependent and -independent pathways, TF and thrombin are capable of inducing angiogenesis, the process of generating new blood vessels from preexisting vessels, which is essential for tumor growth and metastasis. The principal pathways are schematically represented in Figure 1 . In this article, we will focus on the links between thrombin and cancer biology. We hope that the reader will find provocative the hypothesis that within the clotting cascade are several novel and promising targets for drug discovery for cancer therapeutics. Indeed, we suggest that new therapeutic approaches to down-regulating thrombin generation in cancer may accomplish two goals: prevention of thrombosis, and prevention of tumor growth and metastasis.



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  		Figure 1. Coagulation cascade, angiogenesis, and tumor growth and metastasis. The coagulation cascade is linked to tumor growth and metastasis via its induction of angiogenesis. Both clotting-dependent and -independent mechanisms induce angiogenesis. TF induces nonclotting-dependent mechanisms via phosphorylation of its cytoplasmic tail and subsequent signal transduction cascades. TF induces angiogenesis via clotting-dependent mechanisms by downstream generation of the serine protease thrombin. Thrombin also induces angiogenesis via clotting-independent and -dependent mechanisms. Clotting-independent mechanisms are thought to be mediated via proteolytic cleavage of the PARs and subsequent activation of G-protein–coupled signal transduction cascades that induce angiogenesis-related genes. Clotting-dependent mechanisms are mediated via fibrin deposition and platelet activation. Fibrin induces angiogenesis via clot formation. XLF matrices provide provisional, proangiogenic matrices that facilitate endothelial infiltration and tubule formation. Degradation of the fibrin matrix exposes cryptic sites that facilitate endothelial cell adhesion and migration. Byproducts of fibrinolysis can also elicit proangiogenic effects. Reproduced with permission from Fernandez et al.9

 

   Cancer and Thrombosis; Thrombosis and Cancer
TOP
 Abstract
 Introduction
 Cancer and Thrombosis;...
TF
 Thrombin
 Fibrin
 Therapeutic Applications
 Concluding Remarks
 References
 
That cancer patients are at high risk for the development of VTE has been recognized since the original observations of Trousseau, but investigators have only recently attempted to quantify the risk. Indeed, while it is difficult to estimate the risk of de novo VTE in patients with cancer, particularly since the risk rate in patients without cancer is not precisely known (best estimate is approximately 100/100,000),2 a review3 of the Medicare database suggests that for those tumors with a high association with VTE (eg, pancreas, brain, ovary, etc), the risk may be as much as 10 times that of patients without cancer (ie, approximately 100/10,000). The best estimates of overall incidence, however, are determined by extraction of data on symptomatic VTE from large, prospective cohort studies of patients with breast cancer, where incidence rates vary from as low as 0.1% in stage I disease in untreated patients to as high as 18% in stage III/IV disease in patients treated with chemotherapy and hormones.4

Risk for symptomatic VTE in surgical patients, determined by retrospective analysis of the data from control groups in large, randomized, controlled trials of anticoagulant prophylaxis, also appears to be higher in those with cancer than in those without cancer (approximately 2.2 hazard ratio).5 The rates of recurrence of VTE have recently been studied in a large, prospective 11-year trial,6 and shown to be significantly increased in patients with cancer vs patients without cancer (hazard ratio, 3.2; 95% confidence interval, 1.9 to 5.4). The reverse is also true: patients with true idiopathic VTE have a significantly higher risk (fourfold to ninefold) of having an underlying carcinoma diagnosed within 6 months to 1 year of the presentation of symptomatic VTE.7 The risk rate for an occult neoplasm is particularly high for those patients with a second episode of VTE for which no other predisposition can be found.7

The pathogenesis of VTE in cancer is complex but relates principally to the procoagulant properties of tumor cells themselves, tumor-associated endothelial cells, and host inflammatory cells. In addition, an unfortunate concatenation of abnormalities of the normal defense mechanisms (eg, stasis, vascular defects, reduction in circulating inhibitors, and cell-associated anticoagulants and fibrinolytic activators) predisposes patients with cancer to hypercoagulability.8 In particular, the cell surface receptor protein TF plays a key role.


   TF
TOP
 Abstract
 Introduction
 Cancer and Thrombosis;...
 TF
Thrombin
 Fibrin
 Therapeutic Applications
 Concluding Remarks
 References
 
As is discussed extensively elsewhere in this monograph, thrombin, generated in response to TF availability, induces clot formation, activates platelets, and mediates feedback amplification of various earlier steps in clotting, ultimately leading to the deposition of fibrin. Thrombin also mediates a number of additional cellular responses, including angiogenesis, by cleaving protease-activated receptors (PARs) that are linked to various signaling cascades (vide infra). Thus, in addition to its role in promoting hypercoagulability in patients with cancer, it is now believed that thrombin is central to the pathobiology of tumor growth and metastasis.9 It has been postulated that aberrant TF expression, plus deregulation of mechanisms controlling TF procoagulant activity, contribute to the systemic hypercoagulability inherent to many patients with cancer, which is ultimately mediated by thrombin generation.

Aberrant TF Expression in Tumors
Up-regulation of TF gene expression appears to be characteristic of malignant cells and normal host cells responding to inflammatory or remodeling signals (eg, tumor-associated endothelial cells, monocytes, macrophages, and fibroblasts). TF shares homology with members of the cytokine receptor superfamily.10 Therefore, it is not surprising that cytokines and growth factors generated by inflammatory and malignant cells induce TF expression.11 Inappropriate expression of TF alters the behavior of cells. Cancer cells transfected with TF complementary DNA exhibit a more malignant phenotype both in vitro and in vivo compared to parent cell lines.12 13 Aberrant TF expression has been detected in a variety of human tumors,8 including glioma,14 15 breast cancer,16 17 non-small cell lung cancer,18 19 leukemia,20 colon cancer,21 22 23 24 and pancreatic cancer,25 26 but generally is not found in corresponding normal tissues. Elevated TF expression in tumors has been correlated with unfavorable prognostic indicators, such as increased angiogenesis, advanced stages of disease, and the multidrug resistant phenotype,27 that contribute to poorer survival rates in cancer patients. In colorectal cancer, for example, increased TF positivity in higher grade tumors has been correlated directly with increased vascular density and vascular endothelial growth factor (VEGF) expression, as well as clinical stage of colorectal cancer, Duke classification, and angiogenesis.21 Similar correlations between TF expression, VEGF expression, and microvessel density have also been found in non-small cell lung cancers19 and breast cancer.17

TF and Angiogenesis
TF appears to play a critical role in both physiologic and pathologic angiogenesis. It is well established that TF deficiency in the transgenic mouse (TF0/0) causes embryonic lethality by day 10.5 due to impaired vascular integrity and abnormal development of the yolk sac.28 29 30 Similar histopathology associated with lethality occurs with VEGF-deficient embryos,31 32 suggesting that TF and VEGF regulate similar functions. The switch to an angiogenic phenotype requires a shift in balance between endogenous proangiogenic and antiangiogenic factors that regulate vessel growth and development (Fig 1) . Aberrant expression of TF in tumors contributes to the angiogenic phenotype in part by up-regulating the expression of the proangiogenic protein VEGF and down-regulating the expression of the antiangiogenic protein thrombospondin.33 34 TF-positive tumors have higher levels of microvessel density and VEGF expression than TF-negative colorectal tumors.21 Tissue factor and VEGF have also been found to be colocalized in tumor cells of human lung and breast cancer specimens.35 Analysis of several human breast cancer35 and melanoma36 cell lines revealed a significant correlation between the level of synthesis of VEGF and TF in vitro. Subcutaneous inoculation of a high TF- and VEGF-producing melanoma cell line (RPMI-7951) into mice with severe combined immunodeficiency yielded highly vascular tumors in vivo.36 Similar experiments with a low TF- and VEGF-producing cell line (WM-115) produced relatively avascular tumors in vivo. However, when a low TF- and VEGF-producing melanoma cell line (HT144) that had been transfected with full-length TF complementary DNA was used in these experiments, vascular tumors grew that expressed high levels of both TF and VEGF. These studies support the hypothesis that TF regulates VEGF synthesis and contributes to tumor angiogenesis.

TF and VEGF participate in a vicious cycle of clot formation and tumor growth. Not only does TF induce VEGF, but the converse also holds true since VEGF in turn up-regulates the expression of TF on endothelial cells by activating the early growth response-1 gene.37 Using specific low-molecular-weight inhibitors, Blum and colleagues38 investigated signaling cascades that regulate TF expression in human umbilical vein endothelial cells; these investigators described a mechanism that triggers differential activation of TF on endothelial cells during physiologic vs pathologic conditions. Under physiologic conditions, Blum and colleagues38 postulate that plasma components, such as growth factors, continuously activate the phosphatidyl 3-kinase (PI3-K)-Akt signaling pathway that causes suppression of TF production in normal endothelial cells. Within tumors, PI3-K activity is diminished due to poor perfusion conditions that limit access to plasma components. Decreased PI3-K activity concurrent with increased p38 and Erk-1/2 mitogen-activated protein kinase (MAPK) activity induce positive regulation of TF expression by VEGF in tumor-related endothelial cells.38 Another recent study lends support to this novel mechanism; Kim and colleagues39 reported that angiopoietin-1, an activator of intracellular PI3-K-Akt signaling, inhibits tumor necrosis factor-{alpha} and VEGF-induced expression of TF in endothelial cells.

Clotting-Dependent and -Independent Pathways of TF-Induced Angiogenesis
We believe that TF contributes to tumor angiogenesis via both clotting-dependent and -independent mechanisms.40 41 Clotting-dependent pathways likely involve activation of the TF receptor via ligand binding followed by downstream production of thrombin and ensuing clot formation. Clotting-independent pathways appear to involve phosphorylation of the cytoplasmic domain of the TF receptor and subsequent downstream signaling events that occur independent of thrombin production or clot formation, and possibly even independent of ligand activation (Fig 2 ).34 Both pathways can directly and/or indirectly contribute to angiogenesis and tumor progression.



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  		Figure 2. TF, thrombin, and tumor angiogenesis. Top: Clotting-independent mechanisms. TF can induce angiogenesis via mechanisms that are independent of thrombin generation and fibrin deposition. These mechanisms are primarily mediated by the constitutive and aberrant expression of TF observed in many tumor cells and associated vascular endothelial cells (VECs). The serine residues on the cytoplasmic tail of the TF receptor can be phosphorylated by PKC, independent of ligand binding. Phosphorylation of the receptor initiates downstream signaling cascades that result in the transcriptional activation or inactivation of different genes. VEGF, a proangiogenic factor, is up-regulated by TF activation, while thrombospondin (TSP)-1, an antiangiogenic factor, is down-regulated by TF activation. VEGF, in turn, up-regulates TF to continue the vicious cycle of tumor growth and clot formation. VEGF also increases vascular permeability that leads to plasma protein leakage and the deposition of a fibrin-rich proangiogenic matrix around tumor cells and vascular endothelial cells. Increased VEGF and decreased thrombospondin-1 induce the proliferation of endothelial cells that contributes to increased tumor angiogenesis. The binding of factor VII (FVII) to its cognate ligand TF results in the activation of factor VII (FVIIa) and an increase in intracellular calcium (Ca2+). Intracellular Ca2+ activates PKC that, in turn, phosphorylates the cytoplasmic tail of the TF receptor. Ligand binding also promotes the attachment of the ABP-280 to the cytoplasmic tail, resulting in the assembly of actin filaments. This association regulates MAPK signaling and phosphorylation of focal adhesion kinases (FAK) and downstream signaling cascades that promote increased endothelial cell adhesion and migration essential for tumor angiogenesis. Reproduced with permission.9 Center: Thrombin and angiogenesis: clotting-independent mechanism. TF can induce angiogenesis via thrombin generation, independent of fibrin deposition and clot formation. Activation of the TF receptor occurs via binding of its cognate ligand, factor VII. Factor VII is activated (FVIIa) and the TF/activated factor VII complex in turn activates factor X (FXa). If the tissue factor pathway inhibitor (TFPI) does not bind and inactivate the TF/activated factor VII/activated factor X ternary complex, activated factor X dissociates from the complex and associates with another phospholipid membrane in the presence of Ca2+ and activated factor V to form the prothrombinase complex that proteolytically converts prothrombin to thrombin. Thrombin can induce angiogenesis independent of clot formation by cleaving the cell membrane-bound PARs. Thrombin-generated cleavage of part of the N-terminal domain of the PARs exposes a neo-N-terminus that functions as a tethered ligand. This tethered ligand binds intramolecularly to the second transmembrane domain of this seven-transmembrane G-protein–coupled receptor. Thrombin cleaves PAR-1, PAR-3, and PAR-4, but not PAR-2. Other proteases, such as trypsin, tryptase, the TF/activated factor VII complex, or activated factor X, can activate PAR-2. Activation of the PARs causes a conformational change that results in the exchange of bound GDP for GTP on associated G proteins. The G proteins are comprised of an {alpha} subunit that contains the nucleotide binding site and a ß and {gamma} heterodimer. Tissue-specific expression of various G-protein subunits confers differential responses to thrombin. The specific signal transduction cascade induced by PAR activation depends on the type of G-protein subunit that is attached to the PAR. Signal transduction cascades, such as the MAPKs, can lead to the transcriptional activation of a number of genes that are involved in angiogenesis. Thrombin activation of PARs leads to the up-regulation of many angiogenesis-related genes, including VEGF, VEGF receptors, TF, bFGF, and MMP-2. These genes can lead to a number of pleiotropic responses, such as change in endothelial cell shape, increased vascular permeability, increased endothelial cell proliferation, and increased proteolysis, which all contribute to increased tumor angiogenesis. Reproduced with permission.9 Bottom: Fibrin and angiogenesis: clotting-dependent mechanisms. TF can also induce angiogenesis by clotting-dependent mechanisms via thrombin generation and fibrin deposition. Generation of thrombin from the prothrombinase complex results in an active serine protease that cleaves fibrinopeptide A (FPA) and fibrinopeptide B (FPB) from the fibrinogen molecule, resulting in the eventual conversion of soluble fibrinogen to XLF. Elevated levels of plasma FPA have been correlated with tumor growth and progression. Thrombin also activates platelets. Deposition of activated platelets with XLF forms the clot. Clot formation and dissolution contributes to tumor growth and angiogenesis. Activated platelets release a number of proangiogenic factors from their {alpha} granules, including VEGF, bFGF, and platelet-derived growth factor (PDGF), that contribute to increased tumor and endothelial cell proliferation and migration. VEGF induces plasma protein leakage that results in an extravascular XLF matrix around tumor cells. This matrix serves as a supportive scaffold that facilitates endothelial cell migration and tubule formation. The fibrinolytic degradation of the fibrin matrix also contributes to angiogenesis since degradation results in the exposure of proangiogenic cryptic sites that facilitate cell adhesion and migration, and the release of small proangiogenic fragments and sequestered growth factors. Reproduced with permission from Fernandez et al.9

 
TF is classified as an immediate early gene. Following cell activation, transcription factors bind to corresponding binding sites located on the promoter region of the TF gene and rapidly induce its expression independent of clotting. Some transcription factors, including specificity protein-1 (SP-1), activator protein-1 (AP-1), and nuclear factor-{kappa}B, are involved in the regulation of both TF and VEGF, offering a potential mechanism for their co-localization and co-regulation in many tumors. Since VEGF is characterized as one of the most potent regulators of angiogenesis, the direct association of TF with VEGF offers strong support for a link between TF and tumor angiogenesis. Differential signaling pathways may control TF-induced regulation of VEGF during physiologic and pathologic angiogenesis. Using human fibroblasts, Ollivier and colleagues42 43 reported that TF-induced production of VEGF required the binding of activated factor VII to TF and subsequent generation of activated factor X and thrombin. However, in some (but not all) malignant melanoma cell lines,44 TF-mediated regulation of VEGF is regulated independent of clotting via activation of the cytoplasmic tail of TF, rather than via the ligand-binding extracellular domain.36 The serine residues on the cytoplasmic tail of the TF receptor can be phosphorylated by protein kinase C (PKC)-mediated mechanisms.34 45

The cytoplasmic tail of TF appears to regulate nonclotting-dependent mechanisms, including cytoskeletal reorganization, vascular remodeling, angiogenesis, and cellular metastasis. Following extracellular binding of the TF receptor, the actin-binding protein 280 (ABP-280) is recruited to the cytoplasmic tail, where it participates in the assembly of actin filaments (Fig 2 , top).46 The carboxyl terminus of ABP-280 associates with the cytoplasmic domain of TF, while its amino terminus interacts with the actin filaments. This association regulates MAPK signaling and phosphorylation of focal adhesion kinases that promote cell adhesion and migration. Mechanisms mediated by the cytoplasmic tail of TF are important for embryonic vessel development, tumor angiogenesis, and metastasis. In an in vivo model of metastasis, Bromberg et al47 demonstrated that the cytoplasmic tail of TF was essential for metastasis. Mueller and Ruf,48 in studies of transfected Chinese hamster ovary cells, concluded that the extracellular proteolytic activity of the TF receptor was also required for TF-dependent metastasis. This mechanism may be mediated via ensuing PAR activation that leads to the phosphorylation of the Ser residues in the cytoplasmic tail of the TF receptor.


   Thrombin
TOP
 Abstract
 Introduction
 Cancer and Thrombosis;...
 TF
 Thrombin
Fibrin
 Therapeutic Applications
 Concluding Remarks
 References
 
Thrombin and Angiogenesis: Clotting-Independent Mechanism
Although thrombin is best known for its direct role in clot formation via platelet activation and fibrin deposition, it also regulates cellular behavior independent of clotting by activating G-protein–coupled PARs that orchestrate a network of signaling cascades (see the article by Dr. Brass in this Supplement) [Fig 2 , center]. Many of the effects of thrombin in cancer may be mediated by promoting angiogenesis in vivo, independent of its clotting activity,49 50 inducing microvessel infiltration and in vitro promoting PKC-dependent morphologic differentiation of human umbilical vein endothelial cells.51 Under most physiologic conditions, endothelial cells reside in a quiescent state. In response to vascular injury, thrombin activates the proliferation and migration of endothelial cells for promotion of wound repair. In the context of a tumor microenvironment, these actions invoke the recruitment of new blood vessels into the developing tumor. Tumor cells secrete the proangiogenic factor VEGF to attract new vessels.

Thrombin stimulates the release of VEGF (and other growth factors) from the {alpha} granules of platelets.52 53 Thrombin indirectly up-regulates the transcription of VEGF by inducing the production of reactive oxygen species and the expression of the hypoxia-inducible factor-1{alpha} transcription factor. The hypoxia-inducible factor-1 complex binds to the VEGF gene and induces its transcription.54 Thrombin directly up-regulates VEGF expression, via PAR activation55 56 and angiopoietin-2, the proangiogenic factor that antagonizes the blood vessel-stabilizing effects of angiopoietin-1.57 58 Thrombin promotes reversible rounding of endothelial cells and increases vascular permeability,59 resulting in plasma protein leakage and the development of a provisional proangiogenic matrix.60

Angiogenesis involves activation of endothelial cells, invasion of the endothelial cells through their basement membrane, and migration to distal sites. In vitro studies51 61 have demonstrated that thrombin contributes to each of these events. Thrombin decreases adhesion of endothelial cells to basement membrane proteins via cyclic adenosine monophosphate, making them more mobile. Thrombin also mobilizes adhesion molecules (eg, P-selectin) to the endothelial surface that facilitate platelet and tumor cell adhesion. Immobilized thrombin functions as a chemoattractant to endothelial cells by inducing their migration and invasion toward high concentrations of the serine protease, as might exist at a site of injury or within a tumor microenvironment. Endothelial cells attach to thrombin via the angiogenic {alpha}vß3 integrin, which is up-regulated by thrombin. This attachment provides endothelial cells with survival signals during their anchorage-independent migration.51 Thrombin also facilitates invasion through the basement membrane by activating the collagen type IV degrading enzyme, gelatinase A, also known as matrix metalloproteinase (MMP)-2.61 Interestingly, both {alpha}vß3 and MMP-2 functionally coexist on the surface of angiogenic capillaries.62

Thrombin dramatically increases the growth and metastatic potential of tumor cells, although these effects may be attributed in part to its proangiogenic effects.58 63 By mobilizing adhesion molecules, such as the {alpha}IIbß3 integrin,64 65 66 P-selectin,67 68 and CD40 ligand,69 to the cell surface, thrombin enhances adhesion between tumor cells, platelets, endothelial cells, and the extracellular matrix, and contributes to tumor progression. Thrombin also triggers the release of growth factors,70 chemokines, and extracellular proteins71 that promote the proliferation and migration of tumor cells. The prometastatic activity of thrombin has been demonstrated in vivo with experimental pulmonary metastasis models that showed dramatic fold induction of lung metastases with thrombin-treated tumor cells compared to untreated tumor cells.65 72 73 The principal thrombin receptor, PAR-1, has been implicated in the promotion of these effects.74 Most of the cellular effects elicited by thrombin are mediated through the activation and subsequent signal transduction cascades of members of the PAR family, suggesting that proteolytic activity of thrombin is essential for the mediation of these events.

Thrombin PAR Receptors in Cancer
The expression of PAR-1, the predominant thrombin receptor, has been correlated with the malignant phenotype. Using both breast cancer cell lines and breast cancer specimens, Even-Ram and colleagues63 found a direct correlation between elevated PAR expression and invasive potential. Whereas normal or premalignant breast specimens lack detectable PAR-1 expression, infiltrating ductal carcinomas express very high levels. Transfection of the metastatic MDA-435 breast cancer cell line with PAR-1 antisense complementary DNA significantly reduces its invasive potential.63

Both thrombin and trypsin are common proteases secreted by malignant cells that are thought to contribute to their metastatic potential. Expression of PAR-1 and PAR-2 has been identified in tumor cells, endothelial cells, vascular smooth-muscle cells, smooth-muscle actin-positive stromal fibroblasts, mast cells, and macrophages within the metastatic tumor microenvironment.75 Although PAR-1 and PAR-2 expression has been observed in normal endothelial cells, vascular smooth-muscle cells, and mast cells, these receptors have not been detected in normal (benign) epithelial cells or normal stromal fibroblasts. These results suggest that the tumor microenvironment might be receptive to PAR-mediated gene induction, offering potential mechanisms for metastasis promoted by thrombin.


   Fibrin
TOP
 Abstract
 Introduction
 Cancer and Thrombosis;...
 TF
 Thrombin
 Fibrin
Therapeutic Applications
 Concluding Remarks
 References
 
Fibrin and Angiogenesis: Clotting-Dependent Mechanisms
Thrombin is an effective activator of angiogenesis via both clotting-independent and -dependent mechanisms. Whereas clotting-independent mechanisms are believed to be mediated by PAR activation and ensuing signal transduction cascades, clotting-dependent mechanisms involve platelet activation and fibrin deposition (Fig 2 , bottom). Tumor vessels are inherently leaky as a consequence of the hyperpermeable effects of VEGF/vascular permeability factor. Despite its rather large molecular weight, the plasma protein fibrinogen is capable of leaking into the extravascular tissue. Fibrinogen then binds to specific receptors on inflammatory cells and tumor cells and is cleaved by thrombin generated in the local tumor microenvironment. This fibrin can be found within the vascular endothelium of neoangiogenic vessels in the tumor,17 bound to inflammatory cells or tumor cells, or deposited around tumor cells as a provisional scaffold that facilitates further angiogenesis.76

Plasma levels of fibrinopeptide A, the first thrombin cleavage product of fibrinogen, varies in proportion to tumor growth and regression in patients with cancer,77 78 79 80 81 consistent with the reported increased turnover rate of plasma fibrinogen in such patients.82 Deposition of cross-linked fibrin (XLF) characterizes a variety of human malignant tumors,83 including breast,17 lung,35 brain,84 and prostate.85 In histologic specimens from patients with breast cancer or benign breast tumors, XLF was found within the endothelium of angiogenic vessels of invasive cancer specimens but not within the vessels of benign tumors.17

Fibrinogen/Fibrin Degradation Products
Several biologically active plasmin and/or thrombin cleavage fragments of fibrinogen and/or XLF elicit proangiogenic or antiangiogenic effects that may contribute to wound healing and tumorigenesis. Fibrinogen or fibrin fragment E, for example, can stimulate angiogenesis in some in vitro86 and in vivo assay systems,87 while in other systems quantifiable inhibition of angiogenesis has been demonstrated comparable to endostatin, the endogenous peptide now in clinical cancer trials.86 Elevated plasma levels of fibrin d-dimer may be of prognostic significance in patients with advanced stages of colorectal,88 lung,89 and breast90 cancer. A recent study91 reported a significant correlation between elevated plasma d-dimer levels and fibrinogen levels, numbers of metastatic sites, tumor load, progression kinetics, and survival in patients with breast cancer. Of interest, serum levels of VEGF, serum interleukin-6, and calculated VEGF load in platelets were also positively correlated with plasma d-dimer levels in this study.91

Fibrin(ogen) and Adhesion Molecules
Cleavage and degradation of fibrinogen and fibrin expose cryptic sites in the molecules that facilitate adhesion to cell-surface receptors.92 Fibrin bridges cell-matrix interactions essential for physiologic and pathologic events, including inflammation, hemostasis, wound healing, and tumor angiogenesis,93 which is accomplished via multifunctional domains within fibrin(ogen). For example, binding of endothelial cells to fibrin via the adhesion molecule vascular endothelial cadherin may be necessary for capillary tube formation, a critical step in angiogenesis.94 The fibrin matrix that develops around tumors provides a provisional proangiogenic scaffold that supports vessel formation and stimulates endothelial cell proliferation and migration.95

Endothelial cells express different adhesion molecules on their surface based on the extracellular matrices they encounter. During wound healing, the fibrin matrix provokes an angiogenic response by up-regulating the expression of {alpha}vß3 receptors that facilitate endothelial invasion96 and capillary tube formation.97 The {alpha}vß3 integrins provide survival signals to endothelial cells during their interaction with fibrin. In vitro studies98 have shown that {alpha}vß3 messenger RNA of human dermal microvascular cells is more stable in fibrin gels compared to collagen gels. When the collagen-rich matrix from mature granulation tissue replaces the fibrin clot in a wound, the integrin expression changes on the endothelial cells, signaling completion of repair and the end of the angiogenic state. Tumors have been characterized as "wounds that do not heal" because the continuous deposition of fibrin around tumors evoked by VEGF-induced vessel leakiness creates a persistent proangiogenic environment.78 99 100

Fibrin Matrix and Growth Factors
The fibrin matrix functions as a multifunctional scaffold (promoting matrix-cell interactions essential for neovascularization) and as a "storehouse" for proangiogenic growth factors, such as basic fibroblast growth factor (bFGF), VEGF, and insulin-like growth factor-1. Within the fibrin matrix, sequestered growth factors are protected from proteolytic degradation.101 Degradation of the matrix by proteolytic enzymes, generated during invasion by endothelial and tumor cells, releases sequestered growth factors to bind cognate receptors on the invading cells, promoting cell proliferation and migration for tumor angiogenesis.102 103 Fibrin also stimulates the synthesis and secretion of pro-angiogenic factors, such as interleukin-8, from tumor cells,104 and promotes an "autocrine" procoagulant loop by inducing TF expression in endothelial cells.105

Fibrinogen-Deficient Mice
Although fibrin(ogen) plays a pivotal role in tumor angiogenesis and progression, studies in fibrinogen-deficient mice suggest the role is permissive but not requisite. Palumbo and colleagues106 demonstrated comparable tumor growth of experimental tumors in fibrinogen-deficient mice and control mice. Although lack of fibrinogen did not impair growth and angiogenesis of the primary tumor, significantly reduced lung metastases were noted, perhaps due to decreasing adhesion and stability of the metastatic cells.107 The thrombin inhibitor hirudin further decreased the metastatic potential in fibrinogen-deficient mice, suggesting that nonclotting properties of thrombin (eg, PAR activation) may contribute importantly to the metastatic process.


   Therapeutic Applications
TOP
 Abstract
 Introduction
 Cancer and Thrombosis;...
 TF
 Thrombin
 Fibrin
 Therapeutic Applications
Concluding Remarks
 References
 
TF- and Thrombin-Targeted Cancer Therapy
Selective expression of TF on vascular endothelial cells and tumor cells of malignant but not benign tumors makes TF an ideal target for directed cancer therapeutics. Using a number of different approaches, several groups108 109 110 111 112 have demonstrated that TF-targeted cancer therapies in vivo can be efficacious. Hu and Garen110 designed an immunoconjugate (icon) molecule that consists of a mutated mouse factor VII and the Fc effector domain of an IgG1 Ig. After encoding the icon in a replication-competent vector, it was injected into human prostate cancer and human melanoma xenograft tumors grown in mice with severe combined immunodeficiency. The icon selectively localized to TF-expressing tumor cells and caused a cytolytic immune response against the Fc domain of the icon that resulted in significant tumor debulking. Nilsson and colleagues108 used antibody-mediated targeting of TF to the oncofetal extradomain B of fibronectin, expressed by blood vessels of malignant but not benign tissues, to selectively induce thrombosis at the tumor site. This approach also produced significant tumor debulking, although residual tissue grew back in some of the mice. Since this latter treatment produced low toxicity, higher doses may improve efficacy. Although TF-targeted therapeutics have only been evaluated in animal models to date, phase I clinical studies are in progress; these trials represent a novel strategy that holds significant promise for treating patients with cancer.

Thrombin-targeted, anticoagulant strategies designed to affect both the prothrombotic properties of tumors and their growth and metastatic potential have been evaluated in a number of preclinical and clinical studies over many years.8 113 114 115 116 To date, however, none of these studies have provided convincing evidence that this approach can predictably improve survival in cancer. Recent retrospective analyses of large VTE prophylaxis and treatment trials have once again stimulated interest in this concept that antithrombotic agents might increase survival in patients with cancer (see below).

Differences have been reported in survival benefits of patients with cancer treated short term with low-molecular-weight heparin (LMWH) for proximal deep vein thrombosis, as compared with unfractionated heparin (UFH). For example, an overall mortality rate of 9.6% in hospitalized patients with cancer receiving standard UFH compared to 4.7% in patients with cancer treated with LMWH (p = 0.05).117 118 Prandoni and colleagues119 reported an overall mortality rate in LMWH-treated patients with cancer of 7%, compared to 12% in the UFH-treated patients (not statistically significant). Subgroup analysis results such as these have prompted others to conduct large meta-analyses that address the question of a perceived 3-month survival advantage for LMWH-treated patients with cancer enrolled in VTE trials. Lensing and coworkers,120 for example, reported an overall mortality rate of 3.9% for the patients treated with LMWH, compared to 7.1% for those treated with standard UFH, a relative risk reduction of 47% (p = 0.04). In the 195 patients with cancer in the studies subjected to meta-analysis, the mortality risk reduction was 64% in favor of LMWH (p < 0.01). Several subsequent meta-analyses,121 122 123 encompassing > 3,000 patients, came to the same conclusion, one that could not be drawn for the comparison analysis of patients without cancer in the same studies. However, in the absence of data from prospective, randomized controlled trials, it is difficult to interpret this intriguing retrospective data.

Two recent prospective studies of LMWH therapy of patients with cancer either with124 or without125 underlying thrombosis were designed with survival either as a primary125 or secondary124 end point. In neither study was overall survival significantly affected by LMWH,124 125 although subgroup analysis of patients with more limited disease demonstrated a significant survival advantage for the LMWH-treated patients.125 126 Studies in progress should provide additional insights into this important question. At this time, however, routine use of antithrombotic agents cannot be recommended for patients with cancer without active VTE or a history of VTE.

Thrombotic Complications of Antiangiogenic Agents
Since it is believed that all tumors require angiogenesis to grow and metastasize, targeting tumor vasculature with antiangiogenic agents has developed into a novel strategy for treating many cancers. In contrast to standard chemotherapeutic agents, antiangiogenic agents generally elicit few toxic side effects. However, an unexpected high incidence of both arterial and venous thrombosis has recently been reported in a number of clinical trials127 128 129 130 131 in which patients were treated with both antiangiogenic agents and standard chemotherapy. This serious complication has been observed with several promising antiangiogenic agents, including SU5416,130 131 which is a potent inhibitor of the VEGF receptor 2, and thalidomide.127 128 131 132 133 134 135 Although a prospective evaluation of VTE in patients with cancer treated with these agents has not been published, VTE rates as high as 40% have been reported in some case series.

No pathophysiologic study that explains this high rate of VTE has been published. However, it is plausible that synergistic vascular toxicity occurs between antiangiogenic agents and chemotherapy drugs. Indeed, virtually all chemotherapeutic agents injected IV stimulate increased thrombin generation, an increase that can be prevented by immediate pretreatment with IV UFH.81 Therefore, adding anticoagulants to combination drug regimens including agents that interact with the endothelium may help to prevent some of these thrombotic complications by blocking thrombin generation.81 128

Further stimulation for the addition of anticoagulants to cancer treatment regimens comes from recent experimental studies136 137 138 139 140 in which nonanticoagulant properties of anticoagulant drugs have been exploited to reduce tumor growth, angiogenesis, and metastasis. If the results of current prospective, randomized controlled trials of anticoagulant drugs in cancer support the findings of these earlier studies, combination regimens of standard chemotherapeutics with anticoagulants may provide added benefit for control of tumor growth, while reducing the risks for serious thrombotic complications.


   Concluding Remarks
TOP
 Abstract
 Introduction
 Cancer and Thrombosis;...
 TF
 Thrombin
 Fibrin
 Therapeutic Applications
 Concluding Remarks
References
 
Angiogenesis may be the central link between the coagulation cascade and tumorigenesis. Key players of the coagulation cascade, including TF, thrombin, and fibrin induce angiogenesis via clotting-independent and/or clotting-dependent mechanisms. Targeting one or more of these proteins may add to the armamentarium of novel approaches to controlling cancer, reducing the incidence of VTE and, thereby, improving overall patient survival and quality of life.


   Footnotes
 
Abbreviations: ABP-280 = actin-binding protein 280; bFGF = basic fibroblast growth factor; LMWH = low-molecular-weight heparin; MAPK = mitogen-activated protein kinase; MMP = matrix metalloproteinase; PAR = protease-activated receptor; PI3-K = phosphatidylinositol 3-kinase; PKC = protein kinase C; TF = tissue factor; UFH = unfractionated heparin; VEGF = vascular endothelial growth factor; VTE = venous thromboembolism; XLF = cross-linked fibrin


   References
TOP
 Abstract
 Introduction
 Cancer and Thrombosis;...
 TF
 Thrombin
 Fibrin
 Therapeutic Applications
 Concluding Remarks
 References
 

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