Peroxynitrite and fibrinolytic system-The effects of peroxynitrite on t-PA-induced plasmin activity

The aim of the present study was the investigation of peroxynitrite (ONOO−) effects on fibrinolysis in vitro and in silico. The exposure of human plasminogen to ONOO− (10-1000 μM) resulted in a decrease of t-PA-induced amidolytic activity of plasmin; the inhibitory effect was associated with the increasing level of 3-nitrotyrosine in plasminogen/plasmin molecule. Furthermore, ONOO− displayed both the ability to impair the t-PA-induced activation of plasminogen to plasmin, and to reduce the rate of fibrin lysis by plasmin. The susceptibility of plasminogen in blood plasma to nitrative action of ONOO− was revealed by the immunoprecipitation technique. To confirm the hypothesis that 3-nitrotyrosine generation is crucial for the impairment of plasmin activity, (−)-epicatechin, a polyphenolic antioxidant that selectively prevents tyrosine nitration, was used both for in vitro experiments as well as for in silico studies on ONOO−, ONOOH and (−)-epicatechin binding and plasminogen nitration. (−)-Epicatechin effectively protected plasminogen against ONOO−-induced inactivation and significantly reduced the level of 3-nitrotyrosine. The obtained results revealed tyrosine nitration as the most likely mechanism of the inhibitory effect of ONOO− on plasmin(ogen) functions. The possible role of tyrosine modifications was additionally confirmed by bioinformatics calculations with indication of nitration susceptible tyrosine residues.