Tissue factor and cellular signalling

The ability of the TF: F Vila complex to initiate the extrinsic pathway of coagulation (section 1.1) was established at the beginning of last century. More recently novel functions of the TF: F Vila complex in cellular signalling have emerged. The initial data supporting a role for TF in cellular signalling was circumstantial in nature. The recognition of TF as a member of the cytokine receptor superfamily (Bazan J.F., 1990) suggested that TF might function in cellular signalling. In addition, the observation that phosphorylation of the serine residues occurs in the intracellular domain of TF (Zioncheck T.F. 1992 and Mody R.S., 1997) alludes to the existence of a TF-dependent signal transduction pathway. Further observations, demonstrating the involvement of TF in the alteration of cellular phenotype, confirmed a signalling function for TF.

The initial biological evidence that indicated a role for TF in cellular signalling was derived from studies in vivo that demonstrated the involvement of TF in angiogenesis. The first evidence to emerge that implicated a role for TF in angiogenesis was derived from studies of tumour genesis. The malignancy of tumours is synonymous with the ability of the tumour to metastasise and tumour metastasis is dependent upon angiogenesis at two stages of the pathogenic pathway (Folkman J., 1995). Firstly, metastatic cells are not shed from a primary tumour until after it has become vascularised. Second, upon arrival at the target organ metastatic cells must undergo neovascularisation to enable the metastasis to establish itself and grow.

A study in the early eighties that demonstrated the deposition of fibrin at tiie leading edge of breast tumours alluded to the involvement of TF in malignancy (Dvorak H.F., 1981). However, not until the next decade was a correlation between TF expression and metastatic capacity conclusively demonstrated (Mueller B.M., 1992). A number of subsequent studies confirm the association of TF with metastatic capacity in both humans (Contrino J., 1996, Vrana J.A, 1996, Pasqualini M.E. 1997, Koomagi R. 1998, Sawada M., 1999, Wojtukiewicz M.Z., 1999, Sawasda Y., 1999) and mice (Zhang Y., 1994 and Bromberg M.E., 1995) and also in cellular model systems (Kakkar A.K., 1999). A close correlation between the levels of VEGF and TF has been reported in neoplasia (Contrino J., 1996) furthermore, TF and VEGF co-localise in lung and breast cancer (Shoji M., 1998). VEGF is a cytokine that acts upon endothelial cells to promote vascular permeability, endothelial cell growth and angiogenesis (Connolly D.T., 1989 and Ferrara N., 1997). A significant relationship between TF expression, VEGF expression and microvessel density in human non-small cell lung

carcinoma has been reported (Koomagi M., 1998). The TF: FVIIa complex is capable of inducing VEGF (Ollivier V., 1998) and the existence of a positive feedback loop is suggested by the ability of VEGF to induce TF (Clauss M., 1996 and Camera M., 1999).

Increased TF activity accompanying angiogenesis has been also been observed in diabetic patients with retinopathic complications arising from neovascularisation of the retina (Zumbach M., 1997). The involvement of TF in the metastasis of tumours appears to result from the involvement of TF in angiogenesis. Metastasis of tumours and microvessel density are both linked to poor patient prognosis (Weidner N, 1991), thus TF activity may be indicative of the gravity of the disease. In addition, patients with malignant disease are at increased risk of thrombotic complications (Baron J.A., 1998), due to raised circulating levels of TF and excess thrombin generation (Kakkar A.K., 1995).

The ability of TF to support angiogenesis has been directly demonstrated. Enhancement of angiogenesis mediated by TF has been demonstrated in a diffusion chamber assay in rats, in an in vitro assay of bovine aortic endothelial cells in collagen gels (Watanabe T., 1999) and in tumours and in wounds (Nakagawa K., 1998). The role of TF in vasculogenesis during embryo development in mice has been recently recognised (Carmeliet P., 1996). Inactivation of the TF gene in mice resulted in abnormal circulation from yolk sac to embryo beyond embryonic day 8.5. TF deficiency compromises vascular development at a critical time when vessels develop a muscular wall in response to the physiological challenge of blood pressure load. This dysfunction seems to be related to a paucity of mesenchymal pericytes. This study demonstrates an intrinsic capacity for TF to function in de novo vascular modelling (vasculogenesis). The involvement of TF in vasculogenesis alludes to a potential for TF to be involved in the mechanisms of angiogenesis, where the circulatory network is extended from pre-existing vessels rather than occurring de novo.

More recently a number of studies have reported the activation of signalling pathways and the induction of genes by TF signalling. A list of genes induced by the TF: FVIIa complex is presented in figure 3.1. TF up-regulates several growth factors that have been reported to promote angiogenesis including, vascular endothelial growth factor (VEGF, Ollivier V.,

1998), FGF-5, and CTGF (Camerer E., 2000a). CTGF and FGF-5 promote both the growth of connective tissue and angiogenesis and CTGF itself has been associated with atherosclerosis (Camerer E., 2000a). The binding o f FVIIa to TF also induces expression of the poIy(A) polymerase gene (PAP, Pendurthi U.R., 1997), a gene that is up-regulated in many human cancer tissues, particularly in colon carcinoma.

Not only does TF induce the expression of angiogenic proteins but it is itself up-regulated in response to recognised angiogenic stimuli including hypoxia (Lawson C.A., 1995 and

Amirkhosravi A., 1998), VEGF (Ollivier V., 1998), TGF-P (Ranganathan G., 1991) TNF- a (Martin N.B., 1993 and Camera B., 1999), PDGF-PP (Xeureb, 1997), oestrogen (Quirk S.M., 1998) and bFGF (Camerer E., 2000). The mechanism of the involvement of TF in angiogenesis is explored further in chapter 5.

Figure 3.1: Activation of cellular responses by the tissue factor: FVIIa complex

• Transcriptional regulators

p38 MAP kinase^, p44/42 MAP kinase (erkl/2) c-jun N-terminal kinase^, c-fos®, e g r - r ^ \ ETRlOl®, BTEB2^ c-myc®, fra-l®,NFKB"

• Protein kinases

Phospholipase C PKC, G-proteins • Growth factors

Amphiregulin ^ hbEGF *, FGF-5 VEGF '' • Proinflammatory mediators

IL -lp IL-8 LIF MIP2a®, prostaglandin E2 receptor® • Proteins involved in cellular reorganisation/ migration

GTPases; RhoE®, cdc42^, Rac^, urokinase receptor (uPAR)*’’ ®, PAI-2 ®, collagenases 1 and 3 ®, CTGF ®, Cyr61 PDGF-BB-stimulated chemotaxis^, Src-like kinases (c-src, lyn and yes)^, c-Akt IgE RI y-chain', phosphatidylinositol 3-kinase ^

• Intercellular communication Jagged1®

• RNA processing

Tristetraproline®, cyclophilin ®, poly(A) polymerase® • Stress/damage inducible gene

GADD45®

a; Camerer E., 1999, b; Taniguchi T., 1998, c: Siegbahn A., 2000, d: Versteeg H., 2000, e: Pendurthi U., 1997, f: Pendurthi U., 2000, g: Camerer E., 2000, h: Mechtcheriakova D,, 1999, i: MasudaM., 1996, j: Sorensen B.B., 1999, k: Poulsen L.K., 1998