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Tissue factor: a conventional or alternative target in cancer therapy.

BACKGROUND: Tissue factor (TF) is an evolutionary conserved glycoprotein that plays an important role in the pathogenesis of cancer. TF is expressed in 2 naturally occurring protein isoforms, membrane-bound full-length (fl)TF and soluble alternatively spliced (as)TF. Both isoforms have been shown to affect a variety of pathophysiologically relevant functions, such as tumor-associated angiogenesis, thrombogenicity, tumor growth, and metastasis. Therefore, targeting TF either by direct inhibition or indirectly, i.e., on a posttranscriptional level, offers a novel therapeutic option for cancer treatment.

CONTENT: In this review we summarize the latest findings regarding the role of TF and its isoforms in cancer biology. Moreover, we briefly depict and discuss the therapeutic potential of direct and/or indirect inhibition of TF activity and expression for the treatment of cancer.

SUMMARY: asTF and flTF play important and often distinct roles in cancer biology, i.e., in thrombogenicity and angiogenesis, which is mediated by isoform-specific signal transduction pathways. Therefore, both TF isoforms and downstream signaling are promising novel therapeutic targets in malignant diseases.

Cancer is a leading cause of death worldwide (1, 2). Lung cancer and colorectal cancer are among the most common malignant diseases (3, 4). Tissue factor (TF) [3]is an evolutionary highly conserved glycoprotein expressed in humans and several other species (5, 6). Human TF is genetically coded by the TF gene coagulation factor III, tissue factor (F3) [4] and transcribed to TF premature-messenger RNA (pre-mRNA; Fig. 1) (7). Because of alternative splicing TF is expressed in 3 mRNA splice variants: full-length (fl)TF, alternatively spliced (as)TF, and a third variant named TF-A. Translation of flTF and asTF mRNA splice variants leads to the generation of the flTF isoform, a membrane-bound and highly procoagulant protein (8), and soluble asTF with low prothrombogenic potential but strong proangiogenic, cell proliferation-facilitating, and prosurvival activities (2, 9, 10). The TF-A mRNA splice variant is not translated to a protein (Fig. 1) because of termination sequences within alternative exon 1A, leading to an early translation stop (11). TF-A mRNA expression has been detected in several cancer cell lines as well as in human endothelial cells (5, 11). However, the biological function of TF-A is still unknown (5, 11). flTF and asTF are expressed in several types of cancer cells and tumors and play important roles in cancer biology, i.e., in thrombogenicity, survival, tumor growth, angiogenesis, signaling, invasion, and metastasis (2-4, 8, 12, 13). Moreover, both TF isoforms are also involved in other pathologies, such as cardiovascular diseases (14, 15). Therefore, targeting specific TF isoforms offers novel potential therapeutic options for the treatment of cancer patients.

In this review, we summarize the latest findings regarding the role of TF isoforms in cancer biology. Moreover, we briefly discuss the therapeutic potential of direct and indirect inhibition of TF activity and/or isoform expression for cancer treatment.

TF Isoform Expression in Cancer

The expression of TF and its isoforms is induced and highly regulated in several types of cancer, i.e., in lung and breast cancer (3, 4, 16). Several factors are involved in enhanced TF expression in tumor tissues, such as hypoxia or genetic modifications of oncogenes and tumor suppressor genes, e.g. tumor protein p53 (TP53) and Kirsten rat sarcoma viral oncogene homolog (KRAS) (2, 4, 17). These stimuli and events were shown to induce the transcription of the TF gene F3. Yu et al. demonstrated that mutation of the oncogene KRAS led to increased TF expression in colorectal cancer cells (17). In 2009, Regina and colleagues showed that mutation of the oncogene TP53 increased F3 transcription in non-small cell lung cancer (4). Moreover, they found that increased TF expression was associated with reduced survival of patients with non-small cell lung cancer (4). In 2013, Sun et al. showed hypoxia to induce the generation of whole TF mRNA in breast cancer cells (18).

TF isoform expression is also modulated on a posttranscriptional level. Differential TF isoform expression is regulated by several factors, i.e., serine/arginine-rich (SR) proteins and SR protein kinases (19). In 2010, Chandradas et al. showed that the SR proteins SRp40, SC35, ASF/SF2, and SRp55 modulate TF isoform expression in the human monocytic leukemia cell line THP-1. They found that binding of SRp40 and SC35 to regulatory motifs in the TF pre-mRNA sequence increased the generation of asTF (20). Moreover, they depicted binding of ASF/SF2 and SRp55 to regulatory premRNA sites to facilitate flTF expression in THP-1 cells (20). The authors suggested that SR protein-mediated control of asTF and flTF generation could potentially affect cancer-related thrombogenicity and angiogenesis (20). Recently, we demonstrated hypoxia to induce asTF and flTF expression in A549 lung cancer cells (2). In this context, we found the SR protein kinases cdc2-like kinase (Clk)1 and 4 to be involved in posttranscriptional regulation of hypoxia-induced TF isoform expression in A549 cells (2). MicroRNAs (miRNAs) are also involved in posttranscriptional expression control of TF and its isoforms. In 2011, Zhang et al. demonstrated that inhibition of miR-19a led to increased expression of total TF in breast cancer cells (16). They showed that increased TF concentrations were associated with increased breast cancer invasiveness (16). Recently, we demonstrated that inhibition of miR-19a and miR-126 increased flTF and asTF generation in human endothelial cells. This led to increased flTF-mediated procoagulant activity (15). These data show that both transcriptional and posttranscriptional regulation play an important role for cancer-related increase and modulation of the expression and activity of TF and its isoforms.

Impact of TF Isoforms on Signaling in Cancer

Both flTF and asTF differentially modulate cancer cell signaling and other biological functions, such as cell pro liferation and angiogenesis (13, 21). There is genetic evidence indicating that protease-activated receptors (PARs) are crucial for flTF-induced effects in cancer (22). The proposed mechanisms and aspects of potential pharmacological treatment strategies have been reviewed in detail elsewhere (22). flTF has been shown to mediate cell signaling via PAR-2 and downstream signaling proteins, i.e., protein kinase C (PKC) and extracellular signal-regulated kinase 1 and 2 (13, 23). In this context, Hu et al. found that flTF induced cell proliferation and migration of colon cancer cells via PAR-2-mediated signaling (23).

In contrast to flTF, asTF was demonstrated to mediate cell signaling independently of PAR-2 (10, 21, 24). In 2009, van den Berg and colleagues found that asTF directly binds to 33 and (31 integrins. This induced angiogenesis in vitro and in vivo independent of PAR-2 (24). We also showed that asTF induced the proangiogenic potential and proliferation rate of immortalized cardiomyocytes in a PAR2-in-dependent manner (10). Moreover, we found asTF to induce chemotaxis of human monocytic THP-1 leukemia cells as well as cell proliferation of A549 lung cancer cells (2, 10). Finally, Kocaturk and colleagues demonstrated that asTF, but not flTF, increased breast cancer cell proliferation via binding to [beta]1 integrins in vitro (21). Furthermore, they showed that asTF inhibition reduced tumor growth and proliferation in vivo (21). These data indicate that both TF isoforms are able to activate distinct signaling pathways, leading to an isoform-specific modulation of cancer-related biologic processes, such as tumor growth and angiogenesis (13,21).

The Role of TF Isoforms in Cancer-Related Thrombosis

Thrombotic events play a clinically significant role in the pathophysiology of cancer (3, 8). The procoagulant activity of asTF is controversial (7, 8, 25). We found no detectable effect of increased asTF expression on endothelial thrombogenicity (5). Moreover, Yu and Rak demonstrated that flTF, rather than asTF, mediated the prothrombogenic activity of colorectal carcinoma cells (8). In line with this, Hobbs et al. showed that asTF overexpression had no detectable impact on the procoagulant activity of pancreatic MiaPaCa-2 cancer cells (12). In contrast, Bogdanov and colleagues found that asTF exhibited, at a minimum, low procoagulant activity when exposed to phospholipids in conditioned medium (7). Similarly, Unruh et al. showed that overexpression of asTF slightly increased the prothrombotic potential of pancreatic ductal adenocarcinoma cells (25). Davila et al. also found that asTF, which was secreted by pancreatic tumor cells, exhibited low procoagulant activity (26). In both studies, the low procoagulant potential of asTF was dependent on its association with phospholipid surfaces in vitro and in vivo (25, 26). These findings indicate that, if at all, asTF exhibits only low prothrombogenic potential (7, 25). Thus, the influence of asTF on cancer-related thrombosis remains unclear.

In contrast to asTF, flTF was demonstrated to be the major source of procoagulant activity in cancer settings (8). The expression of flTF is increased in many types of cancer, i.e., in lung cancer patients or pancreas carcinomas (3, 8). These increased flTF concentrations are associated with an increased rate of thrombotic events, which significantly contributes to morbidity and mortality of cancer patients (3, 8). In 2009, Zwicker et al. found that the amount of flTF-bearing microparticles was increased in patients with advanced malignancy and pancreatic cancer (27). This was associated with an increased risk of venous thromboembolic events (27). Substantiating this, Yu and Rak demonstrated that shedding of flTF-containing microparticles into the medium increased the prothrombogenic activity of A431 squamous cell carcinoma and HCT116 colorectal carcinoma cells (8). In agreement with this, Davila et al. showed flTF to induce thrombogenicity of breast cancer cells (28). The role of microparticles in cancer-associated thrombosis has been well summarized in a review by Geddings and Mackman (29).

TF Isoforms and Angiogenesis in Cancer

Angiogenesis is crucial for tumor growth (2, 12). Both TF isoforms were demonstrated to induce angiogenesis in cancer (2, 10, 12, 30). Hobbs et al. showed that asTF overexpression increased the microvascular density in a pancreatic cancer tumor model and thus promoted cancer-related angiogenesis in vivo (12). In line with this, we and other groups also found asTF to mediate proangiogenic processes (2, 10, 24). We demonstrated that asTF overexpression in murine HL-1 cells as well as in A549 lung cancer cells increased the proangiogenic potential of these cells (2, 10). In 2009, van den Berg et al. demonstrated in endothelial cells that asTF-induced proangiogenic processes were mediated via integrin ligation, which was independent of PAR-2 signaling (24).

flTF was also found to induce angiogenesis in cancer (13, 30). In 2008, Versteeg and colleagues found that flTF induced angiogenesis via PAR-2 signaling in human MDA-MB-231 breast cancer cells (30). Moreover, they showed that flTF-activated PAR-2 signaling leads to increased angiogenesis in a mammary tumor mouse model in vivo (13). These data demonstrate that flTF and asTF play an important role in cancer-related angiogenesis.

The Influence of asTF and flTF on Cancer Cell Proliferation and Tumor Growth

Both TF isoforms have been shown to enhance cancer cell proliferation and tumor growth (2, 12, 30). In 2008, Versteeg and colleagues demonstrated that inhibition of flTF-mediated PAR-2 signaling reduced tumor growth in an in vivo breast cancer model (30). In vitro, blocking of flTF had no detectable impact on MDA-MD-231 cell proliferation (30). In line with this, Yu et al. found that flTF expression in colorectal cancer had no influence on cell proliferation in vitro but specifically modulates tumor growth in vivo (17).

In contrast to flTF, asTF was shown to promote cell proliferation in vitro as well as tumor growth in vivo (2, 10, 12, 21). Hobbs et al. found asTF overexpression to enhance tumor growth in vivo in a pancreatic cancer model (12). Substantiating this, we demonstrated asTF overexpression to increase cell proliferation of murine HL-1 cells and human A549 lung cancer cells in vitro (2, 10). This was mediated via asTF-induced expression of proliferation-facilitating factors, such as monocyte chemotactic protein-1 (MCP-1) (2, 10,31). In accordance with this, Kocaturk and colleagues demonstrated that increased asTF expression enhanced breast cancer cells proliferation in vitro and tumor growth in vivo via [beta]1 integrin signaling (21).

The Role of TF Isoforms in Cancer Cell Migration, Tumor Metastasis, and Invasiveness

Both TF isoforms are known to modulate cancer cell migration, metastasis, and invasiveness (2, 20, 32). In 2011, we found that increased amounts of asTF in cell culture media induced migration of THP-1 leukemia cells in vitro (10). In line with this, Unruh et al. showed that increased asTF expression in pancreatic ductal adenocarcinomas led to the generation of metastases in distal lymph nodes of mice (25). In human endothelial cells, van den Berg and colleagues demonstrated that asTF-induced cell migration is mediated via integrin [alpha]v]beta]3 interaction (24).

flTF was also found to affect cancer cell migration. The first evidence for the involvement of TF in tumor metastasis, via both the TF-triggered coagulation pathway as well as cellular events mediated via the cytoplasmic domain of TF, came from the work of Palumbo and Degen (33) as well as Ruf and Muller (34). Recently, additional mechanistic insights have come from other groups. Dorfleutner et al. showed flTF to suppress integrin [alpha]3[beta]1-dependent migration of A7 melanoma cells (32). Hu and colleagues reported that flTF promoted colon cancer cell migration through PAR-2 signaling (23). These finding suggest that asTF as well as flTF affect cancer cell migration, metastasis, and tumor invasion.

Available data consistently show that both TF isoforms play an important and often distinct role in cancer-related hemostatic and nonhemostatic pathophysiological processes, i.e., tumor angiogenesis, thrombosis, and metastasis.

Therapeutic Implications of Targeting TF

Both TF isoforms play important roles in the pathophysiology of cancer. flTF, which is highly expressed in several types of cancer, is involved in cancer-related thrombosis, tumor growth, and metastasis (2-4, 8, 12, 13). In contrast to flTF, asTF exhibits low prothrombogenic activity (2, 8, 12). Moreover, asTF does not affect physiological functions of flTF in vessel wall hemostasis and blood coagulation control (19). asTF has been shown to modulate cell survival, proliferation, and angiogenesis in experimental cancer settings (2, 9, 12, 21). Targeting flTF and asTF or their isoform-specific functions may thus offer novel potential options for the treatment of cancer (Table 1). Over the last few years, several molecules and drugs targeting TF, its isoforms, and TF-mediated signaling have been developed and tested for their therapeutic potential for the treatment of cancer and other human diseases. In this section we will describe and discuss the therapeutic potential of the most promising tools.

One potential therapeutic approach is to target flTF-mediated PAR-2 signaling via inhibition of coagulation factors (F) VII and X, because both factors are needed for efficient PAR-2 signaling. As thoroughly reviewed by Schaffner and Ruf, flTF-mediated signaling affects different aspects of cancer biology depending on the tumor type, stage, and receptor types (35). Substantiating this, inhibition of direct flTF:FVIIa signaling by monoclonal anti-flTF antibody 10H10 reduced tumor growth in aggressive breast cancer, whereas inhibition of TF-induced coagulation by monoclonal anti-flTF antibody 5G9 had only a minimal effect (30). In contrast, 5G9-mediated blocking of coagulation inhibited hematogenous metastasis, whereas inhibition of flTF:FVIIa signaling by 10H10 had no influence on metastatic tumor homing (30, 36). The divergent effects of TF isoform-mediated direct and/or indirect signaling on cancer biology were reviewed in detail elsewhere (35).

First studies were performed to test the therapeutic potential of blocking FVII- and FX-mediated effects on TF in cancer. FVII and FX generation can be reduced via vitamin K antagonists, which are already approved drugs and have been used for a long time as anticoagulants (37). Indeed, in a large population-based study, long-term treatment with vitamin K antagonists was associated with reduced risk for cancer development, especially prostate cancer (37). Substantiating this, Nakchbandi et al. reported that low doses of the vitamin K antagonist warfarin increased survival of patients with pancreatic carcinoma (38). However, it is thus far unclear whether these effects depend solely on flTF-mediated processes. Furthermore, treatment with vitamin K antagonists bears the risk of severe bleeding events. This points toward the need for specifically modifying TF:FVIIa-dependent signaling without affecting coagulation pathways when targeting TF as a therapeutic option in cancer treatment.

Another promising opportunity is inhibition of flTF/FVIIa via PCI-27483 or the nematode anticoagulant protein rNAPc2. In vivo, rNAPc2 reduced tumor growth, angiogenesis, and metastasis in a Lewis lung carcinoma model in mice (39). However, the therapeutic value of these compounds is unclear because of the lack of adequate human studies. In 2006, Love et al. started a first safety study of rNAPc2 to prevent tumor progression and metastases in colon cancer (registered in the National Institutes of Health database clinicalTrials.gov; ID: NCT00443573). In 2009, Hedrick et al. initiated a study of safety and tolerability of the selective FVIIa inhibitor PCI-27483 in patients with pancreatic cancer receiving gemcitabine (ClinicalTrials.gov, ID: NCT01020006). However, for both studies no results were published at the time this review was written.

In 1999, Hu and colleagues generated immunoconjugates Of inactivated FVII and the Fc effector domain of human IgG1 (40). The TF-targeting FVII domain mediated highly specific delivery of this molecule to TF. Specific binding of this immunoconjugate to TF effectively inhibited tumor growth and caused regression of human melanoma in a mouse xenograft model (40).

In 2014, Breij and colleagues analyzed the cytotoxic potential of a TF antibody--drug conjugate on different human tumors (41). They demonstrated that a conjugate of a TF-specific antibody and the cytotoxic agent monomethyl auristatin E exhibited potent and TF expression--dependent cytotoxicity in vitro (41). Moreover, this antibody-drug conjugate showed excellent antitumor activity in patient-derived xenograft models of 7 different flTF-expressing solid cancers in vivo with only marginal effects on coagulation (41). In another study, the therapeutic potential of the flTF-specific antibody 10H10 was determined in cancer (30). Versteeg et al. showed that inhibition of flTF by 10H10 effectively suppressed breast cancer tumor growth and metastasis in vitro and in vivo (30). Recently, Wong et al. initiated a study dealing with the effect of ALT-836, a chimeric anti--human TF monoclonal antibody, in combination with gemcitabine on locally advanced or metastatic solid tumors (clinicalTrials.gov, ID: NCT01325558). At the time of this writing, no direct results for this study have been published. However, these novel tools need further investigation to define potential benefits as well as undesirable adverse effects.

The therapeutic value of TF inhibition was also tested in other human diseases, such as in atherosclerosis and coronary artery disease (42, 43). In vivo studies showed that overexpression of TF pathway inhibitor (TFPI), an endogenous inhibitor of TF activity, in balloon-injured atherosclerotic arteries reduced TF-mediated thrombogenicity and vascular remodeling in a hyperlipidemic Watanabe rabbit model (44). Badimon et al. showed that treatment of human atherosclerotic plaques with a polyclonal anti-TF antibody significantly reduced plaque thrombogenicity ex vivo (42). In 2005, the results of the PROXIMATE-TIMI 27 [PROXimal Inhibition of coagulation using a Monclonal Antibody to Tissue factor (SunolcH36)-Thrombolysis in Myocardial Infarction 27] trial revealed that treatment of coronary artery disease patients with a chimeric mouse/human monoclonal TF antibody (Sunol-cH36) significantly reduced thrombin generation in a dose-dependent manner (43). Concomitantly, a dose-related incidence of mucosal bleeding was observed which was supposed to result from effects on platelets (43). This might impair the safety of the investigated antibody.

In 2001, Abraham et al. published results from a prospective, randomized, placebo-controlled, and multinational phase II clinical trial assessing the safety of recombinant (r)TFPI administration, which indicated that treatment of sepsis patients with rTFPI reduced 28-day all-cause mortality compared to controls (45).

Taken together, recent antitumor strategies targeting flTF or flTF-mediated PAR-2 signaling show promising results but need further evaluation, especially in clinical studies, to assess potential benefits as well as to detect undesirable side effects.

Because asTF has an isoform-specific unique C terminus (7), specific antibodies directed against this unique C terminus could be used to inhibit asTF and its pathophysiologically relevant functions without affecting other factors and/or coagulation. Bogdanov and colleagues developed an asTF-specific antibody (7). This antibody was shown to specifically bind asTF in experimental cancer settings (2, 10) and to affect breast cancer cell proliferation in vitro and tumor growth and angiogenesis in vivo (21), therefore offering the opportunity to directly target asTF. However, a potential inhibitory effect of asTF antibodies on asTF-mediated functions needs to be investigated and mechanistically analyzed in further studies.

In contrast to flTF, asTF was found to mediate its proangiogenic activity independently of PAR-2 via integrins, such as integrin [alpha]v[beta]3 (10, 24). Thus, asTF-specific signaling via integrins seems to be another potential target for antiangiogenetic therapy in cancer. This option was tested in vitro and in vivo (2, 10, 24). In 2009, van den Berg et al. found that blocking of [beta]1 and [beta]3 integrins as well as treatment with a TF antibody that disrupts asTF-integrin interaction reduced aortic sprouting ex vivo in a mouse aortic ring model (24). Substantiating this, we demonstrated that pharmacologic inhibition of integrin [alpha]v[beta]3 via cyclic RGD peptides reduced proliferation and the proangiogenic potential of asTF-overexpressing human lung cancer cells in vitro (2). Further studies are needed to definitively assess the safety and efficacy of targeting asTF-induced integrin signaling as a therapeutic option for cancer treatment.

Another therapeutic strategy might be the application of silencing oligos directed against the asTF mRNA-specific exon 4-6 transition sequence. However, this option has not been studied so far. Therefore, it is necessary to focus on the development of such asTF-specific tools. Moreover, asTF-directed tools and anticancer strategies deserve to be further studied, especially in adequate in vivo cancer models. It is essential to determine their potential therapeutic value as well as possible negative and undesirable side effects.

Manipulation of TF isoform expression on the posttranscriptional level via affecting miRNA-regulated mechanisms or alternative splicing may offer another possibility for cancer treatment in the future (Table 1). Posttranscriptional effects of miRNAs can be modulated by specific synthetic miRNAs (mimics) or miRNA inhibitors (antagomirs) (15, 16). Moreover, several pharmacological tools have already been developed to modulate alternative splicing, i.e., inhibitors of SR protein kinases, such as the Clk inhibitor KH-CB19 and the DNA topoisomerase I inhibitor camptothecin (2, 5, 14). However, the biological effects of these substances have only been tested in vitro so far. Therefore, little is known about their pathophysiological relevance, therapeutic potential, and toxicity. Thus, further studies are needed to gain further insights into posttranscriptional regulation mechanisms in cancer as well as the pathophysiological impact of such posttranscriptional manipulations in vivo.

Conclusions

Both TF isoforms play essential and often isoform-specific roles in cancer biology and pathophysiology, i.e., in cancer-related thrombogenicity, metastasis, and angiogenesis (2,8,21,23,30). Differential TF isoform functions are suggested to be mediated by isoform-specific signal transduction via PAR-2 or integrins [alpha]v[beta]3, respectively (13, 23, 24).

Several studies indicate that flTF, asTF, and TF isoform--specific signaling are promising new targets for the treatment of cancer and other human diseases (13, 21, 23, 42, 45). Moreover, preliminary human studies and clinical trials were done to characterize the therapeutic value of drugs and molecules interacting with TF, its isoforms, and associated signaling pathways, such as a study indicating that indirect inhibition of TF activity by vitamin K antagonists was associated with a reduced risk for cancer development (37). Other clinical trials have revealed beneficial effects of direct antibody--or rTFPI-mediated blocking of TF activity in different human pathologies (43, 45). However, the number of adequate TF isoform-specific tools, such as drugs, antibodies, or silencing oligos, is limited so far. Thus, future studies should focus on the development of novel therapeutic tools for asTF- and/or flTF-targeting treatment approaches as well as on the characterization of their therapeutic potential and possible risks. Therefore, such TF isoform--specific anticancer strategies need to be further studied, especially in adequate in vivo models as well as in clinical trials to assess their potential benefits as well as undesirable side effects in cancer settings.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contribution to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest.

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(42.) Badimon JJ, Lettino M, Toschi V, Fuster V, Berrozpe M, Chesebro JH, Badimon L. Local inhibition of tissue factor reduces the thrombogenicity of disrupted human atherosclerotic plaques: effects of tissue factor pathway inhibitoron plaque thrombogenicity under flow conditions. Circulation 1999;99:1780-7.

(43.) Morrow DA, Murphy SA, McCabe CH, Mackman N, Wong HC, Antman EM. Potent inhibition of thrombin with a monoclonal antibody against tissue factor (Sunol-cH36): results of the PROXIMATE-TIMI 27 trial. Eur Heart J 2005;26:682-8.

(44.) Zoldhelyi P, Chen ZQ, Shelat HS, McNatt JM, Willerson JT. Local genetransfer of tissue factor pathway inhibitor regulates intimal hyperplasia in atherosclerotic arteries. Proc Natl Acad Sci USA 2001;98:4078-83.

(45.) E, Reinhart K, Svoboda P, SeibertA, Olthoff D, Dal NA, et al. Assessment of the safety of recombinant tissue factor pathway inhibitor in patients with severe sepsis: a multicenter, randomized, placebo-controlled, single-blind, dose escalation study. Crit Care Med 2001;29:2081-9.

Andreas Eisenreich, [1] * Juliane Bolbrinker, [1] and Ulrike Leppert [2]

[1] Charite-Universitatsmedizin Berlin, CC04, Institut fiir Klinische Pharmakologie und Toxikologie, Berlin, Germany; [2] Charite-Universitatsmedizin Berlin, CCO2, Institut fur Physiologie, Berlin, Germany.

* Address correspondence to this author at: Charite-Universitatsmedizin Berlin, CC04, Institut fur Klinische Pharmakologie und Toxikologie, Chariteplatz 1, 10117 Berlin, Germany. Fax +49-30-4507525112; e-mail andreas.eisenreich@charite.de.

Received May 24, 2015; accepted January 14, 2016.

Previously published online at DOI: 10.1373/clinchem.2015.241521

[3] Nonstandard abbreviations: TF, tissue factor; pre-mRNA, premature messenger RNA; fl, full-length; as, alternatively spliced; SR, serine/arginine rich; Clk, cdc2-like kinase; miRNA, microRNA; PAR, protease-activated receptor; PKC, protein kinase C; MCP-1, monocyte chemotactic protein-1; F, coagulation factor; TFPI, TF pathway inhibitor; r, recombinant.

[4] Human genes: F3, coagulation factor III, tissue factor; TP53, tumor protein p53; KRAS, Kirsten rat sarcoma viral oncogene homolog.

Caption: Fig. 1. Scheme of the TF isoform expression. The TF premature (pre)mRNA is generated by transcription of the human TF gene F3. The arrow depicts the transcription start. Because of constitutive or alternative splicing, 3 TF mRNA splice variants are generated on the post transcriptional level. Removal of all introns (constitutive splicing) leads to the generation of the fITF variant. Additional removal of exon 5 by alternative splicing leads to the production of asTF. Retention of a part of intron 1 as alternative exon 1A in the mature transcript (alternative splicing) leads to the generation of the third splice variant, TF-A. The mRNA variants fITF and asTF are translated in membrane-bound fITF or soluble asTF protein, respectively. Because of termination sequences within the alternative exon 1A no protein is generated from the TF-A mRNA variant.
Table 1. Therapeutic implications: strategies directed against TF
and its isoforms.

Substance/       Directed            Effects              Reference
compound         against

Antibody      flTF             Cytotoxic effect on    Breijet al. (41)
drug                           cancer cells (in
conjugate                      vitro, human);
of TF and                      Antitumor activity
monomethyl                     in patient-derived
auristatin                     xenograft models
E                              (in vivo, mouse,
                               human)

Anti-flTF     flTF             Reduced breast         Versteeg et al.
antibody                       cancer tumor growth    (30)
10H10                          and metastasis (in
                               vitro + in vivo,
                               human, mouse)

Anti-TF       TF               Reduced thrombus       Morrow (43)
antibody                       formation in
Sunol-cH36                     coronary artery
                               disease patients
                               (in vivo, human)

FVII/Fc       TF               Reduced tumor          Hu et al. (40)
immuno-                        growth of human
conjugates                     melanoma in a
                               mouse xenograft
                               model (in vivo)

rTFPI         TF               Reduced                Zoldhelyi et al.
                               thrombogenicity and    (44),
                               vascular remodeling    Abraham et al.
                               (in vivo, rabbit);     -45
                               Reduced mortality
                               in sepsis (in vivo,
                               human)

              FVII FX/PAR-2    Long-term use          Pengo et al.
Vitamin K     signaling        reduces risk of        (37),
antagon-                       cancer (in vivo,       Nakchbandi et al.
ists                           human); Low-dose       (38)
                               warfarin improved
                               survival of
                               patients with
                               pancreatic
                               carcinoma

              asTF/integrin    Reduced cell           Eisenreich et al.
Cyclic RGD    signaling        proliferation and      (2,10)
peptides                       angiogenesis in
                               lung cancer cells
                               (in vitro, mouse,
                               human)

Anti-[beta]/  asTF/integrin    Reduced                van den Berg et
anti-[beta]1  signaling        angiogenesis,          al. (24)
antibodies                     endothelium (in
                               vitro + in vivo,
                               mouse, human)

rNAPc2        flTF/FVII        Reduces tumor          Hembrough et al.
              complex          angiogenesis,          (39)
                               growth, and
                               metastasis in a
                               Lewis lung cancer
                               model (in vivo,
                               mouse)

miR/19,       flTF/asTF        miR-19 mimic:          Eisenreich and
miR19a,                        reduced total TF       Rauch (15), Zhang
miR/126                        expression in          et al. (16)
(mimics/                       breast cancer cells    (16)
antago-                        (in vitro, human);
mirs)                          miR- 19a and miR-
                               126 Inhibition of
                               Clk1 and 4 (KH-
                               CB19, siRNAs):
                               reduced expression
                               of flTF and asTF as
                               well as decreased
                               proangiogenic
                               potential of lung
                               cancer cells (in
                               vitro, human)

KH-CB19       flTF/asTF        Inhibition:            Eisenreich et
(Clk                           increased              al. (2),
inhibitor)                     expression (flTF       Zhang et al. (16)
                               and asTF) and
                               endothelial
                               thrombogenicity (in
                               vitro, human)
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Author:Eisenreich, Andreas; Bolbrinker, Juliane; Leppert, Ulrike
Publication:Clinical Chemistry
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Date:Apr 1, 2016
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