Journal of Thrombosis and Haemostasis, 6: 1514–1516
DOI: 10.1111/j.1538-7836.2008.03069.x
COMMENTARY
Cellular origin of microparticles exposing tissue factor in cancer: a mixed double? R. NIEUWLAND Academic Medical Center, Department of Clinical Chemistry, Meibergdreef 9, 1105 AZ Amsterdam, the Netherlands
To cite this article: Nieuwland R. Cellular origin of microparticles exposing tissue factor in cancer: a mixed double? J Thromb Haemost 2008; 6: 1514–6. See also Davila M, Amirkhosravi A, Coll E, Desai H, Robles L, Colon J, Baker CH, Francis JL. Tissue factor-bearing microparticles derived from tumor cells: impact on coagulation activation. This issue, pp 1517–24.
In 1995, Kakkar and coworkers showed that there is a strong association between malignant disease, circulating tissue factor (TF) and extrinsic coagulation activation. Patients suffering from malignant diseases such as gastrointestinal or gynecological cancers were shown to have, compared with healthy controls, elevated plasma concentrations of non-cell bound tissue factor, activated factor VII (FVIIa), thrombin–antithrombin complexes and prothrombin fragment F1+2 [1]. For a long time, however, the precise cellular origin and nature of this plasma TF in cancer patients has remained elusive and puzzled oncologists as well as hematologists. Then what precisely is the cellular origin of this TF? Cancer cells release tissue factor-exposing vesicles Normal cells and cancer cells release microparticles and exosomes into their environment. Microparticles are budded off from the cell surface and are best known for their ability to support coagulation. Exosomes, which are stored in intracellular multivesicular bodies and are released when the membrane of the multivesicular body fuses with the cellsÕ plasma membrane, efficiently modulate the immune response. Already in 1982, Dvorak and coworkers demonstrated that tumor-derived procoagulant activity (PCA) Ôis associated with sedimentable, ultramiscroscopic plasma membrane-derived vesiclesÕ in vitro (cancer cell-conditioned culture medium) as well as in vivo (Ôascitis tumor fluidÕ from animals). These vesicles, isolated by centrifugation at 100 000 · g, ranged in size from 15 and 800 nm (median 60 nm) [2]. In 1983, these investigators showed that cancer cell-derived vesicles support coagulation via various mechanisms, i.e. Ôone procoagulant activity associated with shed tumor vesicles behaved as tissue factorÕ, and Ôshed tumor Correspondence: Rienk Nieuwland, Academic Medical Center, Department of Clinical Chemistry, Meibergdreef 9, 1105 AZ Amsterdam, the Netherlands. Tel.: +31 20 5664851; fax: +31 20 6091222. E-mail
[email protected]
vesicles also acted at a second step late in the clotting cascade at the level of prothrombinase generation, presumably by providing a phospholipid surfaceÕ [3]. A decade later, in 1993, from four cases of TrousseauÕs syndrome, i.e. cancer patients who have Ôspontaneous recurrent or migratory episodes of venous thrombosis, arterial emboli due to nonbacterial thrombotic endocarditis, or bothÕ, it was concluded that Ôtwo properties of a tumor can account for the pathogenesis of TrousseauÕs syndrome: The first is that the malignant cell expresses tissue factor on its external surface. The second is that the tumor cells are anatomically positioned so that cells or vesicles shed from them are exposed to the circulating blood, either directly or by their entrance into the circulatory system from the lymphatic systemÕ [4]. Concurrently, other investigators concluded that Ôa continuing entrance into the circulation of tissue factor from malignant cells is responsible for the manifestations of TrousseauÕs syndrome in most patientsÕ [5]. Taken together, these studies demonstrate that the strong association between malignant disease and coagulation activation may – at least partially – be explained by the release of TFexposing vesicles from cancer cells into the blood or other body fluids, which in turn may contribute to the low grade disseminated intravascular coagulation and thrombotic episodes which are characteristic of TrousseauÕs syndrome. Other potential sources of TF-exposing vesicles The true cellular origin of microparticle-associated TF in cancer patients, however, has proven surprisingly difficult to establish. Patients with disseminated breast and pancreatic cancer have increased levels of microparticle-associated TF in plasma compared with controls, and the patients with a low likelihood of survival have (in plasma) both a high microparticle-associated TF activity and increased numbers of epithelial mucin (MUC1) exposing microparticles. [6] Whether or not MUC1-exposing microparticles, for example microparticles originating from tumor cells, expose TF, however, was not investigated. Surprisingly, a low number 2008 International Society on Thrombosis and Haemostasis
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of microparticles was present that stained positive for both MUC1 and glycoprotein IIIa (CD61; integrin b3). As glycoprotein IIIa is abundantly exposed on platelets and platelet-derived microparticles, they concluded that Ôa small part of circulating microparticles seemed to result from fusion of cellular vesicles originating from malignant epithelial cells and plateletsÕ. Whether or not these particular microparticles expose TF, however, was not investigated. Membrane fusion of cancer cell-derived vesicles with other cells (and possibly vesicles) was initially reported in the early 1980s, and this process makes it very difficult to establish the true cellular origin of microparticle-associated TF in plasma of cancer patients [7]. Membrane fusion, however, is not limited to cancer cellderived vesicles. Tissue factor-exposing microparticles from monocytes also fuse with activated platelets, thereby delivering their tissue factor to the platelet surface [8]. A recent case report of a 55-year-old patient with a giant-cell lung carcinoma showed that a minor fraction of the TF-exposing microparticles in the patientÕs plasma expose CD14 (LPS receptor), indicating that these microparticles originate from monocytes. The cellular origin of the majority of the TFexposing microparticles, however, was not established [9]. In a recent study on 20 patients with advanced colorectal cancer, the numbers of circulating TF-exposing microparticles was found to be 2-fold increased compared with controls. On average, 52% of these (TF-exposing) microparticles exposed CD41a (glycoprotein IX; a platelet marker), 24% exposed CD45 (LCA or leukocyte-common antigen; a general marker of leukocytes) and 32% exposed CD14 [10]. From this study, it was concluded that most of the TF-exposing microparticles are of platelet origin. As no double labeling experiments were performed to study possible co-localization of CD45 or CD14 with CD61, it can not be excluded that TF is exposed on microparticles showing characteristics of both leukocytes and platelets, thus reflecting membrane fusion. Alternatively, although still controversial, given the fact that platelets themselves are important for storage of tissue factor, then also the CD61and TF-exposing microparticles may originate from platelets directly without prior membrane fusion. Taken together, leukocytes, in particular monocytes, and platelets may contribute to the pool of TF-exposing microparticles in cancer patients. Microparticles in cancer patients: a mixed double? To make the cellular origin of TF-exposing microparticles in cancer patients even more complex, the cellular origin of CD61-exposing microparticles may be questioned for two reasons. First, many cancer cells in blood are coated with platelets, presumably to escape from immune surveillance [11]. Membrane fusion may then transfer tissue factor and other membrane proteins, including glycoprotein IIb–IIIa, from cancer cells to platelets and vice versa. Second, many cancer cells express integrins, including glycoprotein IIb–IIIa, to 2008 International Society on Thrombosis and Haemostasis
mimick non-transformed cells such as platelets, and to promote cell adhesion [12]. Thus, staining solely with for example CD61 does not exclude the possibility that these microparticles in fact originate from cancer cells. In sum, the true cellular origin of microparticle-exposed TF in cancer patients is still not fully established. Evidence that cancer cells contribute to the circulating pool of coagulant TF in vivo In this issue of the Journal of Thrombosis and Haemostasis, a study of Davila and coworkers may shed more light on the cellular origin of plasma TF in cancer patients [13]. Human pancreatic cancer cells (L3.6pl) were injected in the pancreas of immune incompetent (nude) mice. At various time intervals, mouse blood was collected and human TF antigen (ELISA) and activity (thrombin generation assay, with and without antihuman tissue factor) were determined in the cell-free plasma. Three weeks after injection, TF antigen was detectable, and the amount of TF antigen was related to the tumor weight. In addition, the mouse plasma samples were also shown to be capable of initiating (human) TF-dependent thrombin generation. This study demonstrates in a set of elegant experiments that plasma TF originates, in an animal model of cancer, at least in part from tumor cells. The authors conclude that Ôcirculating tumor-derived tissue factor exhibited PCA ex vivoÕ, but strictly speaking they did not demonstrate this, as they did not isolate microparticles from the cell-free mouse plasma samples to study their procoagulant activity. It is striking that also in this study the levels of TF antigen and activity are somewhat discrepant. As outlined by the authors, ÔTF activity was detected in the plasmas of tumor-bearing mice only when circulating tissue factor was present beyond certain concentrationÕ. Therefore, they conclude that Ôcirculating active tissue factor has to reach concentrations sufficient to overcome the physiological threshold of the in vivo anticoagulant systems and trigger a detectable activation of the coagulation cascadeÕ. In fact, this is precisely what we showed earlier when studying the procoagulant activity of human microparticles, isolated from pericardial wound blood of patients undergoing cardiac surgery or healthy individuals, in a rat venous stasis model [14]. With regard to the cellular origin and function of TFexposing microparticles in cancer, many questions remain unanswered. The study of Davila shows directly that TF does originate from cancer cells, but it does not exclude that concurrently TF originates from other cell types. It also remains obscure to which extent exosomes expose procoagulant TF. Davila et al. describe that part of the TF activity is associated with exosomes using a 0.1-lm filter, but additional evidence is essential to support this initial and important observation. Perhaps, the two most important questions, however, are (i) the biological benefits a tumor may have to release TF-exposing microparticles, and (ii) whether membrane fusion between (TF-exposing) microparticles and cells not only transfers a procoagulant phenotype, but also transmits other TF-mediated functions to such cells, including the ability to
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support angiogenesis, to protect cells from apoptosis and transmembrane signaling. Taken together, by showing that cancer cells contribute to the pool of circulating TF-exposing microparticles in vivo is a further step in our understanding of the complex relationship between tumors, TF and the prothrombotic tendency in cancer patients. Disclosure of Conflict of Interests The author states that he has no conflict of interest. References 1 Kakkar AK, DeRuvo N, Chinswangwatanakul V, Tebbutt S, Williamson RCN. Extrinsic-pathway coagulation activity in cancer with high factor VIIa and tissue factor. Lancet 1995; 346: 1004–5. 2 Dvorak HF, Quay SC, Orenstein NS, Dvorak AM, Hahn P, Bitzer AM, Carvalho AC. Tumor shedding and coagulation. Science 1981; 212: 923–4. 3 Dvorak HF, van de Water L, Bitzer AM, Dvorak AM, Anderson D, Harvey VS, Bach R, Davis GL, deWolf W, Carvalho AC. Procoagulant activity associated with plasma membrane vesicles shed by cultured tumor cells. Cancer Res 1983; 43: 4434–42. 4 Callander N, Rapaport SI. TrousseauÕs syndrome. West J Med 1993; 158: 364–71. 5 Rapaport SI. Blood coagulation and its alterations in hemorrhagic and thrombotic disorders. West J Med 1993; 158: 153–61.
6 Tesselaar ME, Romijn FP, van der Linden K, Prinas FA, Bertina RM, Osanto S. Microparticle-associated tissue factor activity: a link between cancer and thrombosis? J Thromb Haemost 2006; 5: 520–7. 7 Poste G, Nicolson GL. Arrest and metastasis of blood-borne tumor cells are modified by fusion of plasma membrane vesicles from highly metastatic cells. Proc Natl Acad Sci USA 1980; 77: 399–403. 8 del Conde I, Shrimpton CN, Thiagarajan P, Lo´pez JA. Tissue factorbearing microvesicles arise from lipid rafts and fuse with activated platelets to initiate coagulation. Blood 2005; 106: 1604–11. 9 del Conde I, Bharwani LD, Dietzen DJ, Pendurthi U, Thiagarajan P, Lo´pez JA. Microovesicle-associated tissue factor and TrousseauÕs syndrome. J Thromb Haemost 2007; 5: 70–4. 10 Hron G, Kollars M, Weber H, Sagaster V, Quehenberger P, Eichinger S, Kyrle PA, Weltermann A. Tissue factor-positive microparticles: cellular origin and association with coagulation activation in patients with colorectal cancer. Thromb Haemost 2007; 97: 119–23. 11 Betz SA, Foucar K, Head DR, Chen IM, Willman CL. False-positive flow cytometric platelet glycoprotein IIb/IIIa expression in myeloid leukemias secondary to platelet adherence to blasts. Blood 1992; 79: 2399–403. 12 Merono A, Lucena C, Lopez A, Garrido JJ, Perez de LL, Llanes D. Immunohistochemical analysis of ß3 integrin (CD61): expression in pig tissues and human tumors. Histol Histopathol 2002; 17: 347–52. 13 Davila M, Amirkhosravi A, Coll E, Desai H, Robles L, Colon J, Baker CH, Francis JL. Tissue factor-bearing microparticles derived from tumor cells: impact on coagulation activation. J Thromb Haemost 2008; 6: 1517–24. 14 Biro´ E´, Sturk-Maquelin KN, Vogel GM, Meuleman DG, Smit MJ, Hack CE, Sturk A, Nieuwland R. Human cell-derived microparticles promote thrombus formation in vivo in a tissue factor-dependent manner. J Thromb Haemost 2003; 1: 2561–8.
2008 International Society on Thrombosis and Haemostasis
