Factor Xa Induces Tissue Factor Expression in Endothelial Cells by P44/42 MAPK and NF-κB-Dependent Pathways

Background

Tissue factor (TF) is an initiator of coagulation. The serine protease factor Xa (FXa) is the convergence point of the extrinsic and intrinsic components of the coagulation cascade. In addition to its hemostatic function, FXa elicits inflammatory responses in endothelial cells that may be important in surgical procedures in which inflammation is triggered. This study tested the hypothesis that FXa can up-regulate TF on vascular endothelial cells by a mitogen-activated protein kinase (MAPK)- and NF-κB-dependent pathway.

Methods and Results

Incubation of cultured human umbilical vein endothelial cells (HUVECs) with FXa increased TF protein expression and activity in a dose-dependent manner. Pre-incubation of HUVECs with the serine protease inhibitor antithrombin, which targets not only thrombin but also FXa and FIXa, inhibited FXa-induced TF expression, but the selective thrombin inhibitor hirudin did not inhibit FXa-induced TF expression, ruling out a thrombin-mediated pathway. After 10 min incubation with HUVECs, FXa rapidly induced P44/42 MAPK activation (immunoblotting of phosphorylated P44/42 MAPK) with a peak at 30 min. The MEK 1/2 inhibitor PD98059 partially reduced FXa-induced TF expression and activity (3.82 ± 0.11 vs 6.54 ± 0.08 fmol/min/cm², P < 0.05). NF-κB was activated by FXa, confirmed by cytoplasmic IkBα degradation and increased NF-κB P65 nuclear translocation. Interruption of the NF-κB pathway by the IkBα phosphorylation inhibitor Bay 11-7802 abrogated FXa-induced TF protein expression and activity (1.93 ± 0.02 versus 6.54 ± 0.08 fmol/min/cm², P < 0.05). However, inhibition of PI3 kinase by LY 294002 did not attenuate FXa-induced TF protein expression and activity.

Conclusions

(1) FXa up-regulates TF protein expression and activity in HUVECs, (2) FXa-induced up-regulation of TF is independent of the thrombin-PAR1 pathway, and (3) the MAPK and NF-κB pathways, but not PI3 kinase pathway, are involved in FXa-induced TF expression on human umbilical endothelial cells. FXa may be a feed-forward alternative mechanism of activating TF expression and activity, thereby increasing a procoagulant state or inflammation. This mechanism may be important in the pro-inflammatory state initiated by cardiac surgical procedures.

Key Words: endothelial cell, factor Xa, MAP kinase, NF-κB, tissue factor