L-Homocysteine (Hcys) occurs in human blood in free (i.e. as reduced form—about 100 nM) and protein bound form. Increased concentration of Hcys in the blood is called hyperhomocysteinemia (about [15 lM). Mechanisms involved in the relationship between hyperhomocysteinemia and haemostatic process are still unclear and sometimes controversial. In the literature there are few papers describing studies on the effects of Hcys on proteins that participate in blood coagulation and fibrinolysis in human. The most reactive form of Hcys is its cyclic thioester— homocysteine thiolactone (HTL; formed through editing mechanisms with methionyl t-RNA synthetase), which represents up to 0.29% of plasma total homocysteine, and the Hcysor tiolactone-induced modifications of haemostatic proteins (N-homocysteinylation or S-homocysteinylation) seem to be the main mechanism of biotoxicity of these compounds. Fibrinogen, plasminogen (Plg) and other haemostatic proteins can be also covalently modified by Hcys and HTL [1, 2]. The aim of our study was to establish and compare the effect of a reduced form of L-Hcys (C95%, Sigma Chemical Company, St. Louis, MO, USA) and its cyclic thioester (C99%, Sigma Chemical Company, St. Louis, MO, USA) on the fibrinolytic system (using human plasma or purified plasminogen). Blood samples were taken from 12 healthy volunteers (aged between 26 and 32 years; mean 29.7 ± 2.5) without cardiovascular disorders, allergy and lipid or carbohydrate metabolism disorders, untreated with drugs. Healthy subjects did not use addictive substances and antioxidant supplementation, their diet was balanced (meat and vegetables), lived in similar socio-economic conditions. Subjects with significant medical illness were excluded. They were no smokers. Human blood was collected at 3.2% citrate (used to 12 mM final concentration) and immediately centrifuged (20009g, 15 min) to get plasma. Plg was isolated by affinity chromatography on Lysine–Sepharose [3]. The natural concentration of total Hcys in plasma was 10.6 ± 3.8 lM. The endogenous concentration of reduced form of Hcys and HTL was about 100 ± 11.2 and 0–35 nM, respectively. The classical technique HPLC has been used to analysis Hcys or HTL in human plasma. The HPLC analysis was performed with a Hewlett-Packard 1100 Series system according to Glowacki et al. [4] and Bald et al. [5]. The concentration of Plg in plasma was 2 ± 0.3 lM. Samples of human Plg (2 lM) were exposed to the reduced form of L-homocysteine (at a final concentration between 10 and 100 lM); or to homocysteine thiolactone (at a final concentration between 0.1 and 1 lM) in the presence of 100 mM potassium phosphate buffer, pH 7.4, for 30 min at 37 C. Human plasma was exposed also to the reduced form of L-homocysteine (at a final concentration between 10 and 100 lM); or to homocysteine thiolactone (at a final concentration between 0.1 and 1 lM) for 30 min at 37 C. The tested concentrations of Hcys or HTL correspond to levels found in human plasma during hyperhomocysteinemia in vivo. Plasmin activity and Plg activation were estimated by the hydrolysis of chromogenic substrate by streptokinase (SK) or by tissue plasminogen activator (tPA); assays were performed at room temperature in 96well polystyrene flat-bottom plates. The absorbance measurements were performed in a microplate reader (Bio-Rad Microplate Reader, model 550) at 415 nm. No activity of generated plasmin was detected in the absence of SK or tPA. We also studied changes of proteolytic activities of J. Kolodziejczyk J. Malinowska P. Nowak B. Olas (&) Department of General Biochemistry, Institute of Biochemistry, University of Łodź, Banacha 12/16, 90-237 Lodz, Poland e-mail: olasb@biol.uni.lodz.pl