REVIEW ARTICLE The platelet contribution to cancer progression N. M. BAMBACE and C. E. HOLMES Division of Hematology and Oncology, Department of Medicine, University of Vermont, Burlington, VT, USA To cite this article: Bambace NM, Holmes CE. The platelet contribution to cancer progression. J Thromb Haemost 2011; 9: 237–49. Summary. Traditionally viewed as major cellular components in hemostasis and thrombosis, the contribution of platelets to the progression of cancer is an emerging area of research interest. Complex interactions between tumor cells and circu- lating platelets play an important role in cancer growth and dissemination, and a growing body of evidence supports a role for physiologic plate let receptors and platelet agonists in can cer metastases and angiogenesis. Platelets provide a procoagulant surface f acilitating amplification of cancer-related coagulation, and can be recruited to shroud tumor cells, thereby shielding them from immune responses, and facilitate cancer growth and dissemination. Experimental blockade of key platelet receptors, such as GP1b/IX/V, GPIIbIIIa and GPVI, has b een s hown t o attenuate metastases. Platelets are also recognized as dynamic reservoirs of proangiogenic and anti-angiogenic proteins that can be manipulated pharmacologically. A bidirectional rela- tionship between platelets and tumors is also seen, with evidence of Ôtumor conditioningÕ of platelets. The platelet as a reporter of malignancy and a targeted delivery system for antican cer therapy has also been proposed. The development of platelet inhibitors that influence malignancy progression and clinical testing of c urrently available a ntiplatelet drugs represen ts a promising a rea o f t argeted c ancer therapy . Keywords: angiogenesis, cancer, metastases, platelets, TCIPA. Introduction Tumor cells interact with all major components of the hemostatic system, including platelets. Platelets a nd platelet activation have been linked to key steps in cancer progression (summarized in Fig. 1). The contribution of platelets to malignancy progression has been suggested to be a n organized process that underlies the pathobiology of cancer growth and dissemination rather than a simple epiphenomenon of neopla- sia (reviewed in [1]). Here, we highlight current insights into how platelets contribute to cancer growth, maintenance and propagation and identify potential targets and directions for platelet-directed anticancer therapy in the future. Platelet structure and function Often numbering over 3–4 trillion in a n individual patient with cancer, platelets represent the smallest circulating hematopoi- etic cells and are anucleate fragments formed from the cytoplasm o f m egakaryocytes. The platelet membrane consists of phospholipids and is covered with glycoproteins and integrins, which are essential for adhesion, aggregation and activation, the critical s teps in platelet-mediated hemostasis. Important platelet membrane receptors include Glycoprotein Ib-IX-V (GPIb-IX-V), Glycoprotein VI (GPVI) and Glyco- protein IIb-IIIa (GPIIb-IIIa, also as integrin aIIbb3), receptors that are essential for complete adhesion and aggregation [2,3]. Additional important receptors found on platelet membranes include the protease-activated receptors (PAR ), PAR-1 and PAR-4, and the P2 receptors, P2Y 1 and P2Y 12 ,which principally mediate activation a nd aggregation [4]. Platelets also contain three types of granules: (i) dense granules containing platelet agonists such as serotonin and ADP that serve to amplify platelet activation, (ii) a granules containing proteins that enhance the activation process a nd participate i n coagulation; and (iii) lysosomal granules containing glycosid- ases and proteases [5]. Many of the major structural components of platelets and platelet receptors that contribute to hemostasis have a lso b een found to relate to malignancy progression (reviewed in Table 1). For example, in addition to coagulation-related proteins, platelets also store proteins within the alpha granule that can regulate angiogenesis and metastases [2,6]. Further, platelet receptors such as GPIIb/IIIa can mediate platelet angiogenic protein release in addition to their more t raditional role in fibrinogen binding. At least one study has f ound ultrastructural c hanges in platelets from p atients w ith l ung cancer, including an increase in the number of platelet alpha granules [7]. Interestingly, these r esearchers also f ound that the number of alpha granules was associated with survival. Functionally, platelets are complex cells capable of shape change, translational protein production, protein and metab- olite release, cell-cell interactions and paracrine regulation. Most of these functions relate to the processes of platelet activation and aggregation that occur following exposure to Correspondence: Chris E. Holmes, Department of Medicine, Hematology and Oncology, University of Vermont, Burlington, VT 05401, USA. Tel.: +1 802 656 0302; fax: +1 802 656 0390. E-mail: ceholmes@uvm.edu Journal of Thrombosis and Haemostasis, 9: 237–249 DOI: 10.1111/j.1538-7836.2010.04131.x Ó 2011 International Society on Thrombosis and Haemostasis an in vivo stimulus. In thrombosis formation, thrombin and collagen contribute substantially, but not exclusively, to platelet activation in vivo [2]. In malignancy, tumor cells can activate platelets by direct contact, or via release of mediators such as ADP, thrombin, thromboxane A2 or tumor-associ- ated proteinase s [8–11]. The relative importance of each platelet activator in malignancy is unknown and some data suggest the mechanism of platelet activation by tumor cells may be tumor cell specific and, in some cases, mutually exclusive [12]. Several studies have suggested an increase in platelet activation in the blood of patients with cancer [13–19]. Both tumor secretion of activators and direct contact with tumors have been related to t his platelet activation [ 20–22]. To date, no differences in platelet receptor function or composition have been described in patients with a ctive malignancy a s compared with healthy subjects to explain this increase in platelet activation. Early observations on platelets and cancer Gasic et al., in 1968 [23], fi rst described t he association b etween platelet number and metastatic cancer potential. This group found neuraminidase-induced thrombocytopenia was associ- ated with decreased metastasis of T A3 ascites tumor cells. This antimetastatic effect was neutralized by infusion of platelet- rich plasma (PRP). Thrombocytop enia experimentally induced by a variety of mechanisms has also been associated with a reduction in the number of m etastases in tumor transplant models [23,24]. Thrombocytosis is observed i n 10–57% of patients with cancer, with the number varyin g bas ed o n cancer type [1]. The relationship between elevated platelet count and malignant tumors was initially reported b y Reiss et al. in 1872 [25]. Subsequent studies have established this relationship for common cancers, including colorectal, l ung and breast cancer, as well as gastric, renal and most urogenital malignancies [26– 32]. Further, for the majority of malignancies, the extent of platelet count elevation is inversely correlated with survival, making thrombocytosis a marker of poor prognosis [26–32]. Insights into the mechanisms underlying the initial observa- tions of thrombocytosis in malignancy h ave been forthcoming in more recent decades. Sierko & Wojtukiewicz [1] have recently summarized mechanisms underlying the humoral interaction between tumor cells, bone marrow e ndothelial cells (BMEC) and megakaryocytes. An important driver for thrombocytosis in malignancy is the secretion o f t umor-derived cytokines such as IL-1, G M-CSF, G-CSF and IL-6, which stimulate thrombopoiesis through a thrombopoietin-depen - dent mechanism, influencing largely m egakaryopoietic growth and differentiation [33–38]. Megakaryocytes have a similar ability to secrete inflammatory cytokines, which can in turn influence bone marrow endothelial cells to support mega- karyocytopoiesis [39,40]. VEGF and b-FGF are also released by megakaryocytes, a nd influence megakaryocytic m aturation and transendothelial migration via an autocrine loop [41–43]. Although incompletely elucidated, the interactions between tumor c ells, megakaryocytes and bone marrow endothelial cells appear to promote thrombopoiesis, and may influence tumor angiogenesis. Tumor cell-induced platelet aggregation, activation and metastases Platelets contribute t o critical steps in cancer metastasis, including facilitating tumor cell m igration, invasion [44–46] and a rrest within the vasculature [47–49]. In cellular models of both breast cancer and ovarian cancer, invasiveness has increased following exposure to platelets [46,50]. In the latter, both activated platelet membranes and platelet releasate increased invasion. Platelet contents may be released into the peritumoral space following platelet activation and enhance tumor cell e xtravasation and metastases [51–55]. An important step in metastatic dissemination is the breakd own of v essel basement membrane. By releasing proteolytic enzymes such as gelatinase, heparanase and various matrix metalloproteinases (MMPs), activated platelets can directly degrade s tructural components, or alternatively, support this p rocess b y a ctivating other proteinases and/or enabling tumor cells and endothelial cells to do the same [46,56–58]. Moreover, modulation of proteolytic activity is a ccomplished by g rowth factors released Platelet 1 Angio- genesis Tumor microenvironment Tumor cell Tumor thrombus 4 2 3 Fig. 1. Platelets are involved in ke y steps of malignancy progression. In in vitro and in vivo m urine models, a role for platelets has b een d emonstrated in tumor m etastasis, tumor growth a nd angiogenesis. Our w orking unders tan ding of t he ro le of pla te let s in ma lig nan cy in vol ves : (i) tum or ce ll- induced platelet aggregation can occur following tumor cell intravasation into the v asculature, thereby ÔprotectingÕ or ÔcloakingÕ circulating tumor cells from p hysical clearance a nd immune s urveillance, (ii) platelets facil- itate t umor cell arrest within t he vasculature, endothelial cell retraction and subsequent tissue invasion, (iii) platelets induce endothelial cell pro- liferation and new blood vessel formation, which are requisi te for tumor- associated angiogenesis and growth and (iv) platelet-tumor and platelet- stromal interactions in t he tumor microenvironment depend, in part, on platelet activation a nd platelet protein release, w hich contribute to the inflammatory response. Additional platelet-related proteins an d metabo- lites that facilitate proteolysis and tissue remodelling also e nhance tumor growth and metastasis (including bony metastases). 238 N. M. Bambace and C. E. Holmes Ó 2011 International Society on Thrombosis and Haemostasis by platelets, a topic recently reviewed by Sierko & Wojtukiewicz [1]. Tumor cells have the ability t o aggregate platelets, a finding first reported in 1968, and referred to as tumor cell-induced platelet aggregation ( TCIPA) [23]. It is now recognized that this aggregation correlates with the metastatic potential of cancer cells in b oth in vitro and in vivo mod els of experimental metastasis [59,60]. The mechanisms by which tumor cells induce platelet aggregation m ay differ by cancer type, but have in common t he theme o f conferring surviva l advantage. I n turn, platelets can protect tumor cells in at least two ways: by coating them and thereby directly shielding t hem from physical stressors within the vasculature and by p ermitting e vasion from the i mmune system Õs effector cells. For example, platelets h ave been shown to protect tumors from NK cells and T NF-a cytotoxicity [61,62]. Timar et al. [63] have raised the hypothesis that some malignant ce lls can acquire a platelet-like phenotype, with expression of similar adhesion molecules and receptors. This concept of Ôplatelet-mimicry Õ h as been suggested to relate to the perceived lack of tumor-directed immune surveillance. Recently, platelet-derived transforming growth factor-b has been shown to down-regulate the activating immunoreceptor NKG2D on NK cells and impair NK cell antitumor activity [64]. Tumor-platelet aggregates have the a bility to d isseminate and embolize within the pulmonary microvasculature and have been directly observed to do s o in m urine models [65]. A brief discussion of several major mechanisms of TCIPA a nd tumor Table 1 Key p latelet components and their c ontribu tion to hemostasis a nd malignancy Platelet component Principal role in thrombus formation Role in malignancy Reference In vitro models In vivo models GPIIb/IIIa (a IIbb3 ) Activation allows fibrinogen binding and platelet plug reinforcement Tumor cell and platelet interaction (via fibronectin, fibrinogen and VWF) demonstrated in numerous cell lines; inhibition decreases TCIPA and platelet-mediated angiogenic growth factor release Decreased pulmonary metastasis following inhibition of receptor by antibody and receptor antagonists [3,60,71,86–88,90,160, 165,166] GP Ib-IX-V Binding of von Willebrand factor; anchors platelet to subendothelium Limited data to suggest role in TCIPA; conflicting data on tumor cell-platelet interactions Pulmonary metastasis decreased in mice lacking GPIb but increased when GPIb functionally inhibited by monovalent, monoclonal antibodies [71,91,92] GPVI Platelet adhesion to collagen Not studied to date 50% reduction in pulmonary metastases in GPVI-deficient mice [93] P-selectin Mediates platelet- leukocyte tethering; triggers leukocyte activation Facilitated interaction between tumor cells and endothelial cells via sialylated fucosylated carbohydrates Deficiency or blockade of P- selectin inhibits the formation of melanoma metastases [94–100] P2Y receptors ADP-mediated platelet aggregation ADP-mediated VEGF release from platelets; ADP induced TCIPA ADP depletion associated with reduced metastases [67–71,124,133,163,167] PAR receptors Thrombin mediated platelet activation Selective release of angiogenesis influencing proteins; induces TCIPA Promote metastases [11,122,123,168] Alpha granules Storage of proteins that enhance adhesive process: fibrinogen, VWF, MMP-II, P-selectin, factor V, PF-4, platelet activating factor Uptake and storage of angiogenic proteins that are selectively packaged and released: VEGF, b-FGF endostatin, angiostatin, TSP-1; storage and release of proteolytic enzymes and metastasis influencing proteins Maintenance of intra-tumor vascular integrity [6,117,120–122,126,141] Platelet microparticles Enhances thrombosis and secondary hemostasis Increased tumor cell invasiveness, metastasis, MMP-2 up-regulation and angiogenesis; increased leukemia, prostate and breast cancer invasion/ migration Increased chemo- invasiveness and metastases formation in lung cancer models [101–108] The platelet contribution to cancer progression 239 Ó 2011 International Society on Thrombosis and Haemostasis cell-induced platelet activation follows as their understanding is pivotal to the development of s elective agents t argeting the pharmacologic inhibition of these central pathways (see also [66] for extensive review). Adenosine diphosphate (ADP) is contained in platelet dense granules and is considered a secondary mediator of aggrega- tion. The major ADP receptors, P2Y 1 and P2Y 12 , are both involved in platelet aggregation. Stimulation t hrough these receptors a lso leads to shape change a nd thromboxane A 2 generation by platelets [67]. ADP contributes to TCIPA induced by various tumor cell lines, including neuroblastoma, melanoma and breast carcinoma [68,69]. The P2Y 12 receptor plays a c entral role in platelet activation a nd in TCIPA [ 70,71]. For example, by generating ADP, MCF-7 breast carcinoma cells activate and aggregate platelets via the P2Y 12 receptor [71]. Thrombin has a multifaceted role in hemostasis and represents a key link between primary and secondary coagu- lation responses. Thrombin has also been linked to tumori- genesis and angiogenesis, with thrombin signaling being a major contributor to metastatic tumor dissemination [72]. Thrombin has been detected in situ in numerous tumor types, including small c ell lung cancer, r enal cell, melanoma and ovarian cancer [73–75]. Tumor-enhancing effects of thrombin include i nduction of TCIPA, increased tumor-cell adhesiveness, promigratory and c hemotactic effects, an d up-regulation of VEGF expression by tumor cells [76–79]. Importantly, t hrom- bin is also the most potent platelet activator, and exerts its function via the platelet PAR receptors, PAR-1 and PAR-4. Secretion of ADP and thrombin by human tumor cells activates platelets and recruits them to participate in TCIPA [11]. Following thrombin-mediated platelet activation, up to 300 biologically act ive molecules can be released and deposited ad lib at sites of vascular i njury, at the s ite of a wound or within the tumor and tumor vasculature [6]. Cathepsin B, cancer procoagulant factor and the matrix metalloproteinases (MMPs) are contributors to TCIPA. Cathepsin B and cancer procoagulant factor can induce platelet aggregation when released by tumor cells [80,81]. MMPs have demonstrated a similar ability to induce TCIPA in vitro [82]. MMPs can be released by both platelets and cancer cells in vivo ( reviewed in [83]). Jurasz and colleagues have identified enhanced generation of MMP-2 as the potential cause of human platelet aggregability in the setting of metastatic prostate cancer [66]. Thromboxane A2 (TXA2) and its receptor (TX) also play integral roles in platelet-tumor aggregation. It has been shown that TX mediates platelet aggregation induced by murine and tumor cell lines [84]. TXA2 can be generated by platelets as a result of activation induced by other platelet agonists, an observation that h ighlights the complex a nd interrelated nature of platelet functional responses in the tumor. Platelet adhesion receptors also play a critical r ole in t umor- platelet cross-talk and the process o f h ematogeneous metastasis (recently r eviewed i n [ 85]). T he r ole of the GPIIb-IIIa receptor in TCIPA has been established f or decades, and numerous metastatic models have highlighted the importance of this receptor in the t umor-platelet i nteraction model [86–89]. A recent role for the GPIIb/IIIa receptor in the release of proangiogenic proteins and fibrinogen has also been elucidated [87,90]. The i nvolvement of the i ntegrin receptor G PIba in tumor metastasis, on the other hand, has b een more difficult to define [59,86,91,92]. Recently, the GPVI s urface receptor, a member of the immunoglobulin superfamily, which p rincipally binds collagen, has become a subject of active investigation. Importantly, a 50% reduction in experimental pulmonary metastases in GPVI-deficient mice was reported by Jain et al. [93]. Clinically, patients with GPVI deficiency exhibit a mild bleeding tendency, suggesting that this receptor could poten- tially be inhibited without major hemostatic consequence. Finally, P -selectin is expressed on a ctivated platelets a nd endothelial cells and has been identified as an important mediator of the interaction between these cells and the vessel wall [94]. This f acilitated interaction also applies t o tumor cells as P-selectin can bind t o different tumor c ell lines through binding of sialylated fucosylated carbohydrates [95,96]. In a similar manner, P-selectin appears to facilitate interactions between t umor cells and t he surrounding endothelium, at least in the case of melanoma [97]. Deficiency or blockade of P- selectin has inhibited the formation of metastasis in various other e xperimental models [97,98]. This effect is most pro- nounced in mucin-producing cancers [99,100]. Platelet microparticles and malignancy When platelets are activated or exposed to high shear stress, they release particles expressing membrane receptors and cytoplasmic constituents termed platelet microparticles (PMPs). A growing body of literature s upports the d irect involvement of PMPs in malignant cell proliferation and growth. PMPs have the ability to induce chemotaxis of many hematopoietic cells and increase their adhesive affinity to fibrinogen [101]. PMPs express multiple proteins and chemo- kine receptors, which can be transferred to surrounding cell membranes, including m alignant cells, which then b enefit from enhanced invasiveness [102–105]. In vitro , P MPs h ave been shown to induce p roliferation and tube formation of human umbilical vein endothelial cells, to increase trans-matrigel chemoinvasion of lung cancer cell lines, and to increase invasiveness of breast cancer cells [105]. In vivo, angiogenesis can be observed in the heart of ischemic rats when PMPs are injected into myocardium [106]. Injection of murine Lewis lung cancer cells coated with platelet PMPs was associated with significantly more metastatic lung disease [107]. Janowska-Wierczorek et al. [105] have recently demon- strated that PMPs promote adhesion of tumor cells to endothelium, induce chemotaxis and chemoinvasion, and up- regulate MMP production. MMP-2 up-regulation and increased malignant cell invasiveness have also recently been reported in prostate cancer [108]. PMPs appear to represent an important aspect of the functional interaction between tumors and p latelets and may represent a novel treatment approach in the future. 240 N. M. Bambace and C. E. Holmes Ó 2011 International Society on Thrombosis and Haemostasis The role of platelets in angiogenesis Evidence supporting the link between platelets and angiogen- esis has a ccumulated s ince Pined o and F olkman first raise d this hypothesis [109]. The growth of s olid tumors and formation of metastases depend on the generation of neovessels, and it is recognized that tumor cells cannot grow beyond 2–3 mm in size without a new vascular network [110]. These vessels are needed not only to sustain and nourish the developing tumor cells, but also to allow d elivery o f proteases and c ytokines that permit further invasion, extravasation and dissemination. This elaborate delivery and transportation system exists secondary to an altered b alance between angiogenesis stimulators and inhibitors. These proteins are released by many components of the tumor microenvironment, including the tumor itself. This tumor microenvironment is comprised of stromal fibroblasts, resident macrophages and mast cells, mononuclear cells and platelets [111–115]. Platelets contain over 30 important angiogenesis regulating proteins. Platelets are now recognized as the major source of VEGF (a pro-angiogenic protein) in serum a s t he platelet pool comprises > 80% of total circulating VEGF in patients with cancer as well as healthy individuals [116,117]. Of interest is the observation that i n s ome cancers, plate let-derived VEGF better predicts tumor progression than serum levels of VEGF [118]. Platelets also c ontain proteins that i nhibit angiogenesis, including platelet factor-4 (PF-4), TSP-1 and endostatin [119,120]. Under normal physiologic conditions, platelets have been suggested to release angiogenic proteins to promote wound healing. These p ro-angiogenic proteins are later counterbal- anced by the r elease of angiogenic inhibitors from stro mal cells andplatelets,tostopuncontrolledgrowthinlaterstagesof healing in non-malignant wounds [121]. These angiogenic mediators are packaged into d istinct alpha granule popula- tions, and selective release based on selective engagement of platelet receptors has been proposed [122]. Ma and colleagues first introduced the concept of differential release of platelet angiogenic proteins, by demonstrating that PAR-1 activation was associated with VEGF r elease a nd suppression of endost- atin, while PAR-4 activation, conversely, s timulated endostatin release and suppressed release of VEGF [123]. These investi- gators subsequently treated rats with established gastric ulcers with an oral PAR-1 antagonist or vehicle. In this model, significant healing of ulcers did not occur in the rats treated with the PAR-1 antagonist [123]. Subsequently, the ADP receptors, P2Y 1 and P2Y 12 , have been demonstrated to participate in the regulation of angio- genic protein release, though this pathway of platelet activation appears to release less VEGF than thrombin-mediated activa- tion [124]. ADP-mediated platelet ac tivation is associated with a net increase in the release of VEGF in healthy individuals, with no effect on endostatin release. T his VEGF r elease can be abolished by selectively inhibiting the P2Y 12 receptor [124]. The source and mechanism of platelet-derived angiogenesis proteins remain under a ctive investigation in both h ealthy individuals and patients with cancer. Recent s tudies have offered insight. For example, in the circulation, platelets have been shown to uptake and store proteins that regulate angiogenesis [1,125,126]. In addition to protein u ptake, Zaslavsky et al. [120] have recently demonstrated that the platelet source of TSP-1 is megakaryocyte derived, suggesting that enhanced production or endocytosis by marrow precursor cells ma y c ontribute to t he platelet angiogenic p rotein c ontent. Based o n t he findings that VEGF-A was regulated by Il-6 in a megakaryoblastic cell line, Salgado et al. [127] bring forward the h ypothesis that higher VEGF l evels in cancer patients may partly result from an IL-6 mediated up-regulation of the expression of VEGF-A in platelet precursors. In vitro, proangiogenic effects of platelets were observed by Pipili-Synetos et al. [128], who noted that platelets stimulated endothelial cell proliferation and growth of capillary-like structures in Matrigel assays. An additional in vivo model of angiogenesis showed a reduction of retinal neovascularization in mice with induction of thrombocytopenia as well as inhibition of platelet aggregation by a highly specific alpha- IIbbeta3 receptor antagonist or aspirin [129]. This resulted in a 35–50% reduction of retinal neovascularization, further supporting the platelet contribution to angiogenesis [129]. Kisucka et al. also examined the role of platelets in four in vivo animal models of angiogenesis using both a cornea and Matrigel assay. They report t hat platelet-depleted mice experienced a significant reduction in corneal neovasculariza- tion and developed hemorrhage, and postulate that platelets support angiogenesis through release of growth factors and platelet-vessel w all i nteractions [130]. Brill has also demon- strated the role of platelet microparticles in models of angiogenesis [106]. Importantly, a clear understanding of the contribution of platelets specifically to tumor-associated angiogenesis remains under investigation. For example, while platelets enhance angiogenesis as in the examples above, platelet-endothelial interactions in tumor microvessels have been found to be reduced in murine models of tumor angiogenesis [131]. The platelet as a scavenger of VEGF and therefore a potent anti- angiogenic cellular component of the tumor microvasculature couldalsobeconsidered. A complex and bidirectional relationship between tumor cells and platelets There is growing evidence to suggest that the interplay between platelets and tumors is neither passive n or unidirectional (Fig. 2). Complex relationships between host, tumor and platelet within th e cancer patient will need to be carefully delineated and significant research efforts are required if antiplatelet therapy is to be used successfully in the clinical setting. The platelet role in coagulation-mediated cancer progression, the platelet contribution to the tumor-stromal interaction and the contribution of platelets to inflammation and its subsequent role in malignancy progression are just several examples of these relationships [132]). Shared tumor cell The platelet contribution to cancer progression 241 Ó 2011 International Society on Thrombosis and Haemostasis and p latelet agonists and receptors offer both opportu nity and potential obstacles for d rug targeting. For example, drugs that inhibit the P2Y receptors on platelets may also interact with endothelial and cancer cell P2Y receptors and contribute to the overall impact of the drug [133–135]. The well-delineated role of thrombin signaling and activation of PARs found on malignant cells is another example of shared targets between tumor cells and platelets (reviewed in [136,137]. Some evidence suggests that platelets can be conditioned in vivo by tumor cells to deliver anti-angiogenic proteins [121,138]. In a murine model, Kerr et al. [138] have r ecently d emonstrated that platelets preferentially store tumor-derived GM-CSF, TPO, TNF-a,TGF-bı and especially MCP-1 over host-derived proteins. An emerging concept in the literature focuses on the platelet as a reporter of malignancy. For e xample, both platelet associated PF-4 and TSP-1 have been associated with early cancer growth and been proposed as biomarker s of early tumor progression [120,139]. Platelet granule proteins not only promote growth o f tumor vessels, but prevent tumor hemorrhage, presumably by main- taining the integrity of the existing tumor vascular supply [140,141]. Though t he precise mechanism underlying this phenomenon has not been fully elucidated, t his appears t o occur i ndependently from thrombus formation. The prevention of tumor hemorrhage by platelets has more recently b een found to relate, i n part, to their ability to modulate vascular damage by tumor-infiltrating leukocytes [142]; an observation that further illustrates the complex tumor-stromal interaction, including the ability of platelet to influenc e inflammatory responses [140,143–145]. These observations suggest that the mechanism underlying the maintenance of neoplastic v essels by platelets m ay b e distinct from that used fo r maintenance of host vessels, rendering pharmacologic inhibition of the former plausible. Selective platelet storage and release of stimulatory, inhibitory and regulatory proteins represents a novel concep- tual framework to be explored in the understanding of tumor angiogenesis. Antiplatelet therapy in the treatment of cancer In 1989 and 1993, Dr Leo Zacharski and colleagues, writing for the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis Sub- committee on Hemostasis and Malignancy, published an update of clinical trials using antiplatelet therapy and antico- agulants i n c ancer [146,147]. A t the time, over 2 0 studies, most of them pilot studies with 50 patients or fewer, were reported in the literat ure using an ant iplatelet drug in the treatment (not prevention) of cancer. T he majority of thes e studies focused o n the drug dipyridamole in non-randomized studies, which reported variable response rates. An a nalogue of dipyridamole (RA-233, mopidamol) has a lso been studied in prospective randomized studies, with no survival benefit demonstrated in small cell and ovarian cancer but an approxim ate 1 00-day improvement in s urvival in non-small-cell lung cancer patients [148–150]. The r emaining prospective studies using antiplatelet therapy focused on the use of aspirin in renal cell and small cell lung cancer and showed no effect [151]. Aspirin use has been most extensively studied in colorectal and breast cancer, with demonstrated efficacy in the colorectal cancer prevention setting [152]. Aspirin-mediated inhibition of platelet aggrega- tion is well documented, and recently aspirin has also been shown to attenuate platelet protein release [153]. In vivo data suggesting a possible inhibitory role in the formation of metastasis were initially reported by Gasic et al. [154], who observed metastatic inhibition of MCA6 ascites sarcoma cells in mice, in the presence of a spirin. In a more recent publication, aspirin but not indomethaci n suppressed the formation o f lung metastasis in a metastatic hepato-cellular murine model [155]. Antimetastatic effects of aspirin, however, have not been seen consistently in all laboratory models. Fig. 2. A d iagramatic representation of th e multiple bidirectional inter- actions between pla telets and tum or cells. P latelets and tumor cells express many of the s ame receptors, illustrating the concept of Ôplatelet mimicryÕ. These receptors, such as GPIb and GPIIbIIIa, may participate in T CIPA by promoting a rrest of tumor cells i n the vasculature and b y promoting interactions with bridging proteins such as VWF, fibronectin and fibrin- ogen. Tissue factor expression is u p-regulated in tumor cells, leading to thrombin generation. Tumor cells also have the ability t o directly secrete platelet agonists such as ADP and thrombin, which activate platelet s in the tumor microenvironment. In turn, activated platelets secrete g rowth factors and prot einases that can re gulate tumor growth a nd invasion. Activated plate lets shed m icroparticles, which facilitate cell i nvasion and angiogenesis. ADP, a denosine diphosphate; PAR, proteinase-activated receptors; P2Y, P2Y receptors; GPIb, Glycoprotein I b; GPIIbIIIa, Glycoprotein IIbIIIa ; VWF, V on Willebrand f actor; Lpa, lyso phos- phatidic acid; Txa2, thromboxane; PMP, plate let microparticle. 242 N. M. Bambace and C. E. Holmes Ó 2011 International Society on Thrombosis and Haemostasis Clinical data evaluating the impact of aspirin therapy on cancer survival have begun to emerge. Fontaine et al. [156] have rece ntly reported p re liminary d ata suggesting t hat aspirin used in combination w ith the surgical treatment o f non-small- cell lung cancer is assoc iated with increased survival. Similarly, in a prospective observational study of women d iagnosed with breast cancer, as reported in the NursesÕ Health Study, aspirin use w as associated with decreased risk of breast cancer recurrence and death [157]. Add itionally, a spirin use was found to decrease the proangiogenic effects of tamoxifen in patients with breast cancer [116]. Importantly, the clinical benefits of t he drug are likely to also relate to its anti- inflammatory effects. A review of selected published human clinical studies using antiplatelet th erapy in the treatment of c ancer is found in Table 2. T his t able does not contain data related to the role o f antiplatelets (such as aspirin) in cancer prevention or the potential antiplatelet (p-selectin inhibition) e ffect of heparin (the latter recently reviewed in [85]). Additional p ilot studies of antiplatelet drugs alone or in combination with additional chemotherapy have been reviewed by Hejna et al. [158]. The table h ighlights the paucity of clinical trial data using currently available antiplatelet agents. Importantly, while we have recently reported on t he use o f aspirin therapy i n women with breast cancer receiving tamoxifen therapy [116,159], there is a paucity of data to support t he combination of antiplatelet therapy with existing tumor-targeted therapy. Despite the limited number of prospective randomized trials, the laboratory data using antiplatelet therapy continue to accumulate. Early laboratory studies focused on p rosta- cyclin and p rostacyclin analogues, which have been previ- ously reviewed [158]. In addition, blockade of the GPIIb/IIIa receptor using t he monoclonal a nitbody 10E5, an inhibitor of human platelet GPIIb/IIIa, decreased lung colonization of cancer cells [160]. A challenging aspect of the administration of GPIIb/IIIa antagonists in the clinical setting has been the need for intravenous administration of these agents, which are now widely used in high-risk acute coronary syndromes. Recently, however, an oral inhibitor of GPIIb/IIIa, XV454, has h alted experimental metastasis formation in a murine Table 2 Clinical out comes associated with the u se of platelet inhibitors in patients with canc er. Limited clinical da ta are a va ilable on t he impact of plat elet inhibitors on clinical o utcomes in patients diagnosed wit h cancer. Murine model data are r eviewed in the text and in Table 1 Platelet inhibitor or modulator Mechanism of platelet inhibition Type of cancer(s) studied Protocol designs Observations in clinical studies Reference Aspirin Inhibits platelet thromboxane production and platelet aggregation (anti-neoplastic effects of this drug are also anticipated to rely on COX-2 tissue and tumor inhibition) Colon cancer Double blind No difference in overall survival [169] SCLC Unblinded, randomized No effect on survival [170] Renal cell carcinoma Prospective randomized No significant response or effect on survival [151] Breast cancer Prospective observational study Decreased recurrence and mortality from breast cancer [157] NSCLC (early stage) Retrospective analysis Increased survival post-resection [171] Prostate cancer Retrospective analysis Improved PSA control in patients undergoing radiation [172] Benoral (aspirin- acetaminophen conjugate) Breast cancer Double blind No significant response or improved survival [173] Clopidogrel P2Y12 receptor antagonist; inhibits platelet aggregation induced by ADP Prostate cancer Retrospective analysis Improved PSA control in patients undergoing radiation [172] RA-233 (Mopidamole) Dipyridamole derivative; increase in platelet cyclic AMP; decreased platelet aggregation Colon cancer Double blind No significant response [149] NSCLC (early stage) Double blind Improvement in survival in limited stage/resected disease; no effect in disseminated disease [149,150] SCLC Prospective randomized No significant response [150] Ovarian cancer Prospective randomized trial No effect on survival or recurrence [148] Dipyridamole Increase in platelet cyclic AMP; decreased platelet aggregation Colon cancer (advanced) Prospective randomized trial No impact on survival or response [174] NSCLC (advanced stage) Prospective non-randomized No significant response compared with historical controls [175] The platelet contribution to cancer progression 243 Ó 2011 International Society on Thrombosis and Haemostasis model o f lung c ancer [87]. I ntegrilin, a commercially available platelet-specific aIIbb3 integrin antagonist, was administered to mice after establishment of bony metastases in a study by Boucharaba and colleagues, evaluating the role of platelet-derived lysophosphatidic acid. This resulted in thrombocytopenia, decreased circulating Lpa plasma levels and a significant reduction in the number of osteolytic bony metastases [51]. Wenzel et al. have recently reported successful in vivo reduction of pulmonary metastases in a murine model of breast cancer using the platelet aggregation inhibitor cilostazol. By administrating liposomal cilostazol intravenously, they observed decreased ex vivo platelet aggregability and decreased platelet-tumor complex formation [161]. Similar results were obtained using liposomal dipyramidole [162]. Few studies evaluating the common ADP receptor inhibitors, clopidogrel and ticlopidine, h ave been reported a nd they demonstrated limited success [163]. Conclusion Platelets play a multifaceted and important role in cancer biology (Table 3). The existing research suggests a compelling biological rationale for attempting to disrupt tumor-platelet cross-talk, with the goal of down-regulating tumor invasion, angiogenesis and spread. In the laboratory, platelet receptors, both constitutive and activation dependent, such a s G P1b/IX/ V, P-selectin and alphaIIb-beta3 integrin, c an promote the progression and metastases of various tumor types and are obvious targets for further clinical study [164]. Additionally, control of the platelet reservoir of angiogenic proteins, which are both secreted and sequestered in a selective manner, represents an approach to angiogenic control within t he tumor microenvironment. The study of platelet inhibitors in the clinical setting will require a careful consideration of not only cancer type but stage of disease targeted. Importantly, appropriate trial endpoints must be chosen that are not by des ign predicated on dir ect and toxic tumor effects and secondary rapid cell kill and tumor shrinkage. A potential barrier that surrounds chronic admin- istration of antiplatelet agents in the setting of active malig- nancy is directly related to the paramount role that platelets play in maintaining hemostasis. Currently available oral antiplatelet agents irreversibly inhibit their target, making the risk of bleeding more difficult to mitigate. Future work in the development of novel agents would ideally yield a molecule able to inhibit platelet-tumor interaction while maintaining sufficient platelet function to prevent bleeding. Potential new classes of a gents include antibodies against P-selectin, platelet- specific oral integrin i nhibitors, PAR-1 antagon ists and block - ade of platelet-derived LPA. How should we combine antiplatelet therapy with con- ventional cancer cell-directed therapy? Will other host factors that influence platelet activation, such as diabetes, be important in patient selection [135]? Existing antiplatelet drugs, such as aspirin and clopidogrel, remain understudied as ad juvants t o conventional chemotherapeutic and hor- monal therapies, particularly in animal models and the clinical setting. Increasing our translational database on the anticancer biology of a ntiplatelet strategies to i nclude com- bination therapy and studies directed at prevention vs. l ow burden vs. h igh burden disease are imperative f or the successful clinical translation of results. Importantly, we have learned much from the use of antiplatelet therapy in the treatment of cardiovascular d isease, such as t he concept of drug resistance. These considerations might be applied prospectively in oncologic s tudies. F uture c linical trials formally addressing the role of antiplatelet therapy will need rigorous attention to patient selection, combination therapy with existing agents and trial e ndpoints but offer the hematologic community a significant opportunity to poten- tially improve cancer outcomes. Table 3 Overview of important platelet-cancer cell interactions and their po ten tial influence on cancer p rogression. A full discussion of t hese interactions is found in the text. These observa tions reflect in vitro and murine model data Platelet-related mechanism Effect Platelet activation Increased in patients with cancer Facilitated by contact with tumor cells and tumor release/production of platelet agonists such as ADP and thrombin Platelet activation enhances tumor cell-induced platelet aggregation, releases chemotactic cytokines, proteolytic enzymes and platelet microparticles that can support cancer growth and extravasation as well as angiogenesis Platelet activation provides a procoagulant surface to facilitate cancer-related coagulation Inhibition of key platelet activation and aggregation receptors decreases metastases Tumor-cell-induced platelet aggregation (TCIPA) Platelet aggregation correlates with metastatic potential in in vivo and in vitro models Protection of tumor cells from environment Platelets provide mechanical shielding from physical stressors Platelet-derived proteins down-regulate immune cells, thereby impairing their antitumor activity Production of platelet microparticles (PMPs) Transfer of receptors to tumor cell membranes, which may increase invasiveness. 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