Concerted action of coagulation factors on cell survival

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Tissue factor (TF) together with factor (F)VIIa not only induces coagulation, but is also involved in pathological processes such as tumor metastasis [1, 2]. Recently, it has been shown by Sørensen et al. [3] that 100 nm of FVIIa inhibits apoptosis in TF-expressing cells. The authors conclude that the antiapoptotic function of TF:FVIIa could, in concert with TF:FVIIa-induced gene upregulation and cell migration, facilitate tumor growth and metastasis. Many TF-dependent physiological processes could well be mediated by intracellular signaling. After initial reports on the induction of signal transduction by individual coagulation factors, it has recently been shown that the combined action of coagulation factors might be more relevant [4]. TF:FVIIa-induced signal transduction may also be mediated by factor (F)Xa or thrombin generation, since both FXa and thrombin are potent activators of signal transduction [5]. As in vivo TF:FVIIa complexation directly leads to the production of FXa and thrombin, the concerted action of these coagulation factors might well be highly relevant. To examine whether TF effects in tumor growth and metastasis rely on FVIIa, FXa, thrombin, or the combined action of these proteases, we compared the relative effects of FVIIa and those of TF:FVIIa-induced FXa and thrombin generation on cell survival of baby hamster kidney cells stably transfected with TF (BHKTF) [6]. Figure 1A shows that FVIIa alone indeed induces cell survival. However, the presence of both FVIIa and FX (thus resulting in FXa generation) provides a much better cell survival stimulus. Moreover, when prothrombin was added in combination with FVIIa and FX (subsequently resulting in the generation of FXa and thrombin), cell survival was even more prominent. Furthermore, the concerted action of these factors was potently inhibited by the FXa inhibitor tick anticoagulant protein (TAP), and, to a lesser extent, the thrombin inhibitor hirudin (Fig. 1B). Thus, whereas FVIIa alone might have a small effect on tumor growth and metastasis, it is conceivable that, if coagulation factor-induced signaling indeed plays a role in tumor growth and metastasis, it will be the combined action of FVIIa, FXa and thrombin that is physiologically relevant. Concerted action of coagulation factors on survival. (A) BHKTF cells were serum starved for 16 h and subsequently incubated with either 10 nm factor (F)VIIa (Novo Nordisk, Bagsvaerd, Denmark), 10 nm FVIIa/130 nm factor (F)X (Enzyme Laboratories, South Bend, IN, USA) or 10 nm FVIIa/130 nm FX/1.3 µm prothrombin (Enzyme Laboratories). After 2 days, survival was assessed using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Results are expressed as mean ± SEM of three separate experiments. (B) BHKTF cells were serum starved for 16 h and subsequently incubated with 10 nm FVIIa/130 nm FX/1.3 µm prothrombin directly or after preincubation for 30 min with 1 µm tick anticoagulant protein (kind gift from G. Vlasuk, Corvas International, Inc., San Diego, CA, USA) or 50 U mL−1 hirudin (Calbiochem, La Jolla CA, USA). After 2 days, survival was assessed using a MTT reduction assay. Results are expressed as mean ± SEM of three separate experiments. Sørensen and colleagues exploited a model system in which serum-starved cells were used to induce apoptosis. One could argue that such a system is not physiologically relevant. Indeed, tumor cells that are not in contact with serum will most likely not be in contact with coagulation factors either. Therefore one might question the relevance of FVIIa-induced cell survival for tumor growth and metastasis. However, recently we showed that FVIIa, either alone or in combination with FX, also induces survival in cells that have been detached from the extracellular matrix [7]. As loss of adhesion represents one of the key processes in metastasis, this observation supports the hypothesis that coagulation factor-induced cell survival might be of physiological relevance. In conclusion, it appears that the survival effects mediated by the interaction of FVIIa with TF are mediated, at least in a physiological context, by the concerted action of coagulation factors FVIIa, FXa and thrombin.