Reactive oxygen species induce a procoagulant state in endothelial cells by inhibiting tissue factor pathway inhibitor.

ABSTRACT

Tissue factor pathway inhibitor (TFPI) is a serine-protease inhibitor, which modulates coagulation tissue factor-dependent (TF). It binds directly and inhibits the TF-FVII/FVIIa complex as well as FXa. Time to reperfusion of acute ischemic myocardium is essential for tissue salvage. However, reperfusion also results in a unique form of myocardial damage, such as contractile dysfunction, decreased coronary flow and altered vascular reactivity. Oxidants and reactive oxygen species (ROS) formation is increased in the post-ischemic heart and is responsible of post-ischemic injury. It has been reported that ROS promote a procoagulant state via TF expression while no data are available on the effect on TFPI. Endothelial cells were incubated with two different ROS generating systems, xanthine (X)/xanthine oxidase (XO) for 5 min, or H2O2 (500 µM) for 24 h. TFPI activity was measured in supernatants by chromogenic assay and TFPI-mRNA analyzed by RT-PCR 2 h after ROS exposure. Unstimulated cells and cells exposed to either X or XO served as controls. Western blot and ligand dot blot was performed to evaluate ROS effect on TFPI structure and binding to FXa. ROS generated by X/XO as well as H2O2 system resulted in decreased TFPI activity compared to unstimulated cells while X or XO alone had no effect. No differences in TFPI mRNA levels versus controls was observed. A significant degradation of TFPI was induced by ROS exposure, resulting in a decreased ability to bind FXa. ROS induce a procoagulant state in endothelial cells by altering TFPI structure, resulting in inhibition of TFPI binding to Factor Xa and loss of activity. This phenomenon might have important consequences during reperfusion of post-ischemic myocardium.