Factor VIIa-induced p44/42 Mitogen-activated Protein Kinase Activation Requires the Proteolytic Activity of Factor VIIa and Is Independent of the Tissue Factor Cytoplasmic Domain

Abstract

The extrinsic pathway of blood coagulation is initiated when factor VIIa (FVIIa) 1 circulating in plasma binds to the integral membrane protein, tissue factor (TF), exposed to the blood upon injury of the vessel wall. TF consists of a 219-residue (1-219) extracellular domain, a 23-residue (220 -242) transmembrane domain, and a 21-residue (243-263) cytoplasmic domain. The extracellular part of TF is structured in two fibronectin type III-like domains, which shows structural and sequence homology to the cytokine receptor superfamily. The important role of TF in hemostasis and thrombotic disorders such as atherogenesis is well established (see (1-5)). Recent findings suggest that TF may participate in biological processes other than hemostasis such as angiogenesis (6), embryo vascularization (7), and tumor metastasis (8, 9). Furthermore, it has been reported that binding of FVIIa to cell surface TF-induced intracellular Ca 2ϩ oscillations in a number of TFexpressing cells (10, 11), transient phosphorylation of tyrosine in monocytes (12), alteration in gene expression in fibroblasts (13), and enhanced expression of urokinase receptor in pancreatic cancer cells (14). Additional information about FVIIa/TFinduced signal transduction comes from our previous report showing that binding of FVIIa to cell surface TF resulted in phosphorylation of p44/42 MAPK (15).A potential role for the TF cytoplasmic domain in signal transduction was indicated by studies showing that TF expression markedly increased the metastatic potential of melanoma cells (16) and that the pro-metastatic property was critically dependent on this domain (8, 9). This supposition was further substantiated by experiments showing that the cytoplasmic domain of TF can be phosphorylated by a protein kinase C-dependent mechanism (17) and that a synthetic peptide based on the cytoplasmic domain can work as a substrate for cell lysate protein kinase activity (18). Furthermore, cysteine 245 in the TF cytoplasmic domain was shown to be acylated with long chain fatty acids (19).Although a number of studies, as referred above, suggest that TF is involved in induction of an intracellular activity, it is not clear how signal transduction is mediated across the membrane as a result of binding of FVIIa to TF, just as the role of the cytoplasmic domain of TF in this process is still unclear. The present study explores the effect of removing the TF cytoplasmic domain on p44/42 MAPK signaling and further examines the importance of FVIIa catalytic activity in mediating the activation of the p44/42 MAPK pathway. Our data show that the cytoplasmic TF domain with its putative sites for regulatory modifications is not required for FVIIa-induced p44/42 MAPK phosphorylation. Furthermore, the data provide evidence that specific FVIIa catalytic activity is required and that other serine proteases fail to mimic the response, suggesting that TF/FVIIa may be activating a novel receptor on the cell surface. EXPERIMENTAL PROCEDURESCell Culture-The baby hamster kidney cell line BHK-21 tk...

Tissue factor–mediated endocytosis, recycling, and degradation of factor VIIa by a clathrin-independent mechanism not requiring the cytoplasmic domain of tissue factor

“…The resulting cell lines, BHK(TF ⌬cyto ) and BHK(TF C245S ), were used to elucidate the role of the cytoplasmic domain in TF-dependent internalization of VIIa. As previously described by Sorensen et al, 25 the Xa generation activity of the BHK(TF ⌬cyto ) and BHK(TF C245S ) cell lines was comparable to that of the BHK cell line transfected with full-length TF, BHK(TF), whereas the VIIa binding capacity of the BHK(TF C245S ) cell line was approximately 50% of that observed with the 2 other TFexpressing cell lines. Figure 4 shows VIIa cell surface association, internalization, and degradation observed with BHK(TF), BHK(TF ⌬cyto ), and BHK(TF C245S ).…”

“…Generation of BHK cells that were stably transfected with full-length TF (BHK(TF)), a cytoplasmic domain-deleted construct of TF (BHK(TF ⌬cyto )), or a substitution of serine for cysteine at amino acid residue 245 (C245S) construct of TF (BHK(TF C245S )) was previously described. 25 …”

Gene induction by coagulation factor Xa is mediated by activation of protease-activated receptor 1

“…15,17 Although it is controversial whether a cell surface receptor for Xa, termed EPR-1, 18 is involved in the Xa-dependent gene induction, 9,15,17,19 all recent studies concur that blocking the proteolytic function of Xa with specific inhibitors abolishes Xa signaling. Consistent with a requirement for proteolytic function, a recent study 20 indicated that the activation of murine fibroblasts by Xa and VIIa can be mediated by PAR-2, though other studies 21,22 concluded that PAR-2 is not the receptor activated by VIIa in different cell types.…”

Protease activated receptors: theme and variations

“…This basic phenomenon was obnserved in several mammalian cell lines, umbilical vein endothelial cells and injected Xenopus laevis ocytes (see references below, although not in baby hamster kidney (BHK) cells (Peterson et al, 2000). It does not require the cytoplasmic domain of tissue factor (Sorensen et al, 1999;Camerer et al, 2000). Tryptase is able to cleave and activate PAR2, but with a potency that is much less than trypsin.…”