Protective effects of (−)-epicatechin against nitrative modifications of fibrinogen

Abstract

Fibrinogen appears to be particularly sensitive to toxic action of peroxynitrite; a potent oxidizing and nitrating species. An increased nitration of fibrinogen has been reported in cardiovascular diseases. The defense mechanisms against PN are crucial for complex hemostasis process. Flavonoids have antioxidative properties and could protect biomolecules against action of peroxynitrite. The aim of our studies was to establish, if (−)-epicatechin may in vitro protect fibrinogen molecule against peroxynitrite-induced nitration of tyrosines and change its thrombin-catalyzed polymerization. The exposure of purified fibrinogen (6μM) to peroxynitrite (1–100μM) resulted in both structural modifications and clotting ability of this glycoprotein. Peroxynitrite at the concentration of 1μM increased maximum velocity of Fg polymerization, whereas exposure to 100μM PN resulted in a significant decrease of Vmax. (−)-Epicatechin (1–100μM) caused a dose-dependent inhibition of 3-nitrotyrosine formation in fibrinogen treated with peroxynitrite (100μM) in both Western blot assays and C-ELISA assays. At the highest concentration of (−)-epicatechin (100μM) the level of 3-NT in fibrinogen reached the control values. At lower doses (−)-epicatechin reduced tyrosine nitration by approx. 23% and 40% at the concentration of 1μM and 10μM, respectively. (−)-Epicatechin also abolished the pro-thrombotic effect of peroxynitrite on fibrinogen clotting. The presented in vitro results demonstrated for the first time that (−)-epicatechin might have protective effects against the impairment of structure and properties of Fg, caused by action of the strong biologic oxidant/nitration and inflammatory mediators.