Synergistic induction of tissue factor by coagulation factor Xa and TNF: evidence for involvement of negative regulatory signaling cascades

Abstract

Enzymes of the blood coagulation pathway enhance the inflammatory response leading to endothelial dysfunction, accounting, in part, for the vascular complications occurring in sepsis and cardiovascular disease. The responses of endothelial cell activation include induction of the expression of tissue factor (TF), a membrane glycoprotein that promotes thrombosis, and of E-selectin, a cell adhesion molecule that promotes inflammation. In this report, we demonstrate synergistic interactions between the coagulation factor Xa (fXa) and the proinflammatory cytokines TNF, IL-1beta, and CD40L, leading to enhanced expression of TF and E-selectin in endothelial cells. A detailed analysis of the molecular pathways that could account for this activity of fXa showed that fXa inhibited the cytokine-induced expression of dual specificity phosphatases, MAP kinase phosphatase-L, -4, -5, and -7, blocking a negative regulatory effect on c-Jun N-terminal kinase. The synergistic interaction between fXa and TNF was also involved in the inhibition of A20 and IkappaBalpha expression in the IkappaB kinase-NF-kappaB pathway. The data indicate that inhibition of negative regulatory signaling accounts for the amplification of cytokine-induced endothelial cell activation by fXa.

Fig. 1.

Coagulation protease, fXa, up-regulates endothelial TF expression induced by proinflammatory cytokines. (A) Time course of TF expression as detected by TF immunoblotting in endothelial cells from different origins stimulated by fXa (10 nM), TNF (1 ng/ml), or both. (B) Time course of TF-dependent coagulation in HUVEC stimulated by fXa (10 nM), TNF (1 ng/ml), or both. (C) TF induction by fXa (10 nM) combined with IL-1β (1 ng/ml) or CD40L (10 μg/ml). Expression was measured by anti-TF immunoblotting (Upper) and by TF-dependent coagulation assay (Lower). Representative data from three independent experiments are reported as average ± standard deviation.

Fig. 2.

The expression of E-selectin is up-regulated by fXa. (A) Time course of E-selectin expression as detected by immunoblotting in HUVECs stimulated by fXa (10 nM), TNF (5 ng/ml), or both. (B) fXa-induced up-regulation of E-selectin or TF requires its proteolytic activity. C921-78 (100 nM), a fXa-specific inhibitor, was added to HUVECs during fXa (10 nM) and TNF (1 ng/ml) stimulation. The expression of E-selectin (Upper) or TF(Lower) was detected by immunoblotting. (C) TF-dependent coagulation assay in the presence of C921-78 (100 nM) during the stimulation of HUVECs by fXa (10 nM) and TNF (1 ng/ml). Representative data from three independent experiments are reported as average ± standard deviation.

Fig. 3.

Effect of fXa on TNF-induced JNK signaling pathway. Time course of JNK and c-Jun phosphorylation as detected by immunoblotting with anti-p-JNK and anti-p-c-Jun, respectively (Top) and the control antibodies anti-JNK and anti-c-Jun, respectively (Bottom). Cells were incubated with fXa (10 nM), TNF (5 ng/ml), or both for the indicated times. Representative data from three independent experiments are reported.

Fig. 4.

fXa decreases TNF-induced expression of specific MAPK phosphatases. Time course of relative expression of the phosphatases in HUVECs stimulated by fXa (10 nM), TNF (5 ng/ml), or both, as detected by real-time PCR by using specific primers for (A) PP2A, (B) MKP-L, (C) MKP-4, (D) MKP-5, or (E) MKP-7. (F) Effect of fXa (10 nM) on TNF-induced (5 ng/ml) protein expression of MKP-5 as detected by immunofluorescent staining in HUVECs stimulated for 60 min. a–d represent immunofluorescent staining using anti-MKP-5, whereas e–h represent DAPI staining of the corresponding field. (G) Effect of fXa (10 nM) on TNF-induced (5 ng/ml) protein expression of MKP-7 as detected by immunoblotting with anti-MKP-7 in HUVECs stimulated for 60 min.

Fig. 5.

PAR1 and -2 agonists mimic the inhibitory effects of fXa, decreasing TNF-induced expression of the dual specificity phosphatases. Relative expression of the phosphatases (A) MKP-L, (B) -4, (C) -5, or (D) -7 in HUVECs stimulated for 60 min by TNF (5 ng/ml) in the presence of 100 μM of the indicated PAR peptide agonists.

Fig. 6.

fXa enhances TNF-induced NFκB activation. (A) Time course of IκBα degradation in HUVECs incubated with fXa (10 nM), TNF (5 ng/ml), or both, as detected by anti-IκBα immunoblotting. (B) p65 nuclear localization as detected by p65 immunostaining: HUVECs were incubated for 120 min with fXa (10 nM), TNF (5 ng/ml), or both. a–d represent immunofluorescent staining using anti-p65, whereas e–h are DAPI staining of the corresponding field. Representative data from three independent experiments are reported.

Fig. 7.

Inhibition of TNF-induced expression of IκBα and A20 by fXa or PAR agonists, as detected by real-time-PCR. (A) Time course of IκBα expression in HUVECs stimulated for the indicated length of time with fXa (10 nM), TNF (5 ng/ml), or both. (B) IκBα expression in HUVECs incubated for 60 min with 100 μM peptide agonists with or without the addition of TNF (5 ng/ml). (C) Time course of A20 expression in HUVECs stimulated for the indicated length of time with fXa (10 nM), TNF (5 ng/ml), or both. (D) A20 expression in HUVECs incubated with 100 μM peptide agonists with or without the addition of TNF (5 ng/ml) for 60 min. Representative data from at least three independent experiments are reported.