Nitration of human plasminogen by RAW 264.7 macrophages reduces streptokinase-induced plasmin activity

Abstract

Despite many years of study, clinical trials of new drugs to prevent thrombosis have often been disappointing. Part of the problem lies in our incomplete understanding of the regulation of plasminogen activation and/or inhibition in vivo. We have previously shown that in vitro nitration of plasminogen in plasma by peroxynitrite resulted in decreased plasmin activity. We hypothesized that macrophages may be agents of plasminogen nitration and designed this study to prove this hypothesis. We first better characterized our previous observations using purified plasminogen instead of whole plasma, studied the time and concentration dependence of these reactions, and co-incubated plasminogen with macrophages, as well as with non-inflammatory cells as controls, to assess nitration and impaired activity. When plasminogen (10 micromol/L) is incubated in the presence of SIN-1 (0.01-2 mmol/L), plasmin activity (generated by streptokinase) is reduced in a time- and concentration-dependent fashion. We performed experiments incubating human plasminogen in the presence of murine RAW264.7 macrophages, allowing for free diffusion of reactive oxygen species, while preventing the action of proteases. In this way we show that incubation of plasminogen with macrophages also decreases plasmin activity, while increasing nitration of the molecule, an effect that is already apparent after 2 h and reaches a plateau of 60% inhibition after 24 h of incubation. This effect appears specific for macrophages, since 31EG4 murine mammary cells used in parallel and under the same conditions failed to produce any deleterious changes in plasminogen. Our data on quick functional inactivation of plasminogen by nitration, mediated by macrophages, adds a new pathophysiological dimension to our previous work showing plasminogen as a target for peroxynitrite damage. Nitrosative stress may be implicated in impaired fibrinolysis. New therapeutic approaches for nitrosative stress in atherosclerosis and diabetes should limit the formation of superoxides and peroxynitrite.

Antithrombin activity is inhibited by acrolein and homocysteine thiolactone: Protection by cysteine

“…This is an essential homeostatic system that controls the balance between coagulation and fibrinolysis (Baruah et al, 2006). We have shown previously that plasminogen is sensitive to inactivation by nitrogen species attacking Tyr residues (Hathuc et al, 2006;Gugliucci and Menini, 2003). Since plasminogen lacks critical Lys or Cys residues, we included it in our experiments to rule out non-specific effects of acrolein or homocysteine thiolactone on AT (denaturing, heavy aggregate formation etc).…”

“…Thrombosis producing myocardial infarction and stroke is the final common pathway of atherosclerosis and also a complication of many other chronic diseases such as diabetes and renal failure (Bick, 1994;Nieuwdorp et al, 2005). Two key protein systems that control the delicate balance between coagulation and fibrinolysis are antithrombin and plasminogen and both have been previously shown to be inhibited by carbonyl or nitrosative stress (Thomas, 2001;Sakurai et al, 1988;Hathuc et al, 2006;Gugliucci and Menini, 2003).…”

Antioxidant action of six Trifolium species in blood platelet experimental system in vitro

“…The first report suggesting the possibility of ROS generation in platelets was published in 1977 [ 30 ]. Since then, it has been found that oxidative stress influence functions of various elements of the haemostatic system, including modulation of platelet activity [ 31 – 33 ], changes in fibrinogen polymerization [ 34 , 35 ] as well as impairment of fibrinolysis [ 36 , 37 ]. Activation of blood platelets initiates conformational changes in platelet membrane receptors and numerous intra-platelet processes such as exocytosis of granules, secretion of vasoactive mediators, and cytoskeleton reorganization.…”

Homocysteine and its thiolactone impair plasmin activity induced by urokinase or streptokinase in vitro

“…oxidative stress), results obtained by Lind et al [3] demonstrated the sensitivity of plasmin for oxidative inactivation. Studies of Hathuc et al [4], with the use of SIN-1, showed an evident correlation between plasmin inhibition and tyrosine nitration. In other experiments of those authors, after 24 h of incubation of plasminogen with macrophages, about 60% decrease of plasmin activity associated with the increased nitration of tyrosine residues in plasminogen molecule, was observed.…”