Vascular Endothelial Growth Factor Production by Fibroblasts in Response to Factor VIIa Binding to Tissue Factor Involves Thrombin and Factor Xa

Abstract

Tissue factor (TF) assembled with activated factor VII (FVIIa) initiates the coagulation cascade. We recently showed that TF was essential for FVIIa-induced vascular endothelial growth factor (VEGF) production by human fibroblasts. We investigated whether this production resulted from TF activation by its binding to FVIIa or from the production of clotting factors activated downstream. Incubation of fibroblasts with a plasma-derived FVIIa concentrate induced the generation of activated factor X (FXa) and thrombin and the secretion of VEGF, which was inhibited by hirudin and FXa inhibitors. By contrast, the addition of recombinant FVIIa to fibroblasts did not induce VEGF secretion unless factor X was present. Moreover, thrombin and FXa induced VEGF secretion and VEGF mRNA accumulation, which were blocked by hirudin and FXa inhibitors, respectively. The effect of thrombin was mediated by its specific receptor, protease-activated receptor-1; in contrast, the effect of FXa did not appear to involve effector cell protease receptor-1, because it was not affected by an anti-effector cell protease receptor-1 antibody. An increase in intracellular calcium with the calcium ionophore A23187 or intracellular calcium chelation by BAPTA-AM had no effect on either basal or FXa-induced VEGF secretion, suggesting that the calcium signaling pathway was not sufficient to induce VEGF secretion. Finally, FVIIa, by itself, had no effect on mitogen-activated protein (MAP) kinase activation, contrary to thrombin and FXa, which activate the p44/p42 MAP kinase pathway, as shown by the blocking effect of PD 98059 and by Western blotting of activated MAP kinases. These findings indicate that FVIIa protease induction of VEGF expression is mediated by thrombin and FXa generated in response to FVIIa binding to TF-expressing fibroblasts; they also exclude a direct signaling involving MAP kinase activation via the intracellular domain of TF when expressed by these cells. (Arterioscler Thromb Vasc Biol. 2000;20:1374-1381.) Key Words: vascular endothelial growth factor tissue factor activated factor VII fibroblasts proteases T issue factor (TF), the cell-surface receptor for activated factor VII (FVIIa), is the primary regulator of blood coagulation. The TF-FVIIa complex cleaves and activates factors IX and X into factors IXa and factor Xa (FXa), respectively, which lead to thrombin generation. 1 Beyond its role as a procoagulant activator, TF participates in other cellular processes, including metastasis, 2 tumor-associated angiogenesis, 3 and embryogenesis. 4 Accordingly, several cell functions are modified in response to FVIIa binding to TF, its receptor. These include intracellular signaling, 5 activation of the p44/42 mitogen-activated protein (MAP) kinase pathway , 6 induction of tyrosine phosphorylation in monocytes, 7 upregulation of poly(A) polymerase, 8 cell spreading and phosphorylation of focal adhesion kinase, 9 enhanced expression of urokinase receptor by pancreatic cancer cell lines, 10 and vascular endothelial growth factor (VEGF)...

An engineered muscle flap for reconstruction of large soft tissue defects

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A novel function of tissue factor pathway inhibitor-2 (TFPI-2) in human glioma invasion

“…As noted above, TFPI-2 inhibits the tissue factor VIIa complex (Sprecher et al, 1994;Dvorak, 1987). Tissue factor also upregulated the production of vascular endothelial cell growth factor (Ollivier et al, 1998) and uPAR (Taniguchi et al, 1998) in tumor cells, and its levels perfectly correlated with the degree of tumor angiogenesis and progression in ®brosarcomas, melanomas, breast and pancreatic tumors (Contrino et al, 1996;Mueller et al, 1992;Nakagawa et al, 1998;Mueller and Ruf, 1998). Recently it was reported (Neaud et al, 2000) that human liver myo®broblasts promote in vitro invasion of human hepatocellular carcinoma (HCC) cells through a hepatocyte growth factor (HGF)/urokinase/plasmin-dependent mechanism.…”

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Jak-Stat signal transduction pathway through the eyes of cytokine class II receptor complexes

“…It does not demonstrate any structural similarity to either the IFNs or IL-10. Nevertheless, recent reports implicate induction of certain biological activities in cells after binding of FVIIa to TF, suggesting the existence of signal transduction events (Bromberg et al, 1995;Pendurthi et al, 1997;Ollivier et al, 1998;Poulsen et al, 1998;Cunningham et al, 1999;Camerer et al, 1999). While, there is no data demonstrating direct activation of the Jak-Stat pathway by TF:FVIIa complex, cells transfected with a reporter gene under control of the IFN-ginducible promoter responded to FVIIa treatment by upregulation of reporter gene expression, although the authors suggested that MAP kinase activation was responsible for FVIIa-induced transcription of a reporter gene (Figure 3) (Poulsen et al, 1998).…”