Modulation of plasminogen activation and plasmin activity by methylglyoxal modi¢cation of the zymogen Istva¤n Le¤ra¤nt, Krasimir Kolev, Judit Gomba¤s, Raymund Machovich * Department of Medical Biochemistry at Semmelweis University, Budapest, Hungary Received 29 September 1999; received in revised form 14 March 2000; accepted 13 April 2000 Abstract The effect of methylglyoxal on the plasminogen^plasmin system is studied. Treatment of plasminogen with methylglyoxal at a 20-fold molar excess results in covalent modification of the molecule as evidenced by the decreased number of NH 2 side chains, arginine side chain residues and the new band in the non-tryptophan dependent fluorescent spectrum. This structural modification is associated with profound functional alterations : the rate of activation by streptokinase, tissue-type plasminogen activator, urokinase-type plasminogen activator and trypsin decreases and the amidolytic activity of the generated plasmin is impaired. Plasmin treatment with methylglyoxal on the other hand does not alter its steady-state kinetic parameters on a peptidyl-anilide synthetic substrate, indicating that modification susceptible side chains are sensitive to methylglyoxal only in the zymogen. Our data suggest that in vivo fibrinolysis could be impaired under pathological conditions, e.g. increased methylglyoxal formation in diabetes mellitus. ß 2000 Elsevier Science B.V. All rights reserved. Keywords : Plasmin(ogen) ; Methylglyoxal ; Fibrinolysis ; Diabetes mellitus 1. Introduction The long-term complications of diabetes mellitus (diabetic glomerulopathy, retinopathy and neuro- pathy) are attributed, at least in part, to posttransla- tional modi¢cations of proteins [1]. In this regard until recently the attention was mostly concentrated on the e¡ects of glucose. From the experiments undertaken with methylglyoxal, however, it turned out that this aldehyde is more e/cient as a modi¢er of proteins than glucose [2]. The plasma level of methylglyoxal in diabetic patients is elevated [3]. Methylglyoxal is an K-oxoaldehyde, which can inter- act with sulfhydryl, amino or guanidine groups in the side chain of proteins (cysteine, lysine, arginine) (for review see [4]). In diabetes mellitus the isoenzymes of the cyto- chrome P450 IIE1 gene subfamily, which metabolize acetone to methylglyoxal, are induced (for a review see [5]). The induction of these isoenzymes together with the increased availability of acetone can easily result in an elevated rate of methylglyoxal produc- tion. Furthermore, the generation of methylglyoxal from triose-phosphate intermediates is also possible, and both enzymatic and non-enzymatic reactions are involved in this conversion [6], which can also be a factor that contributes to the increased rate of meth- ylglyoxal production in diabetes mellitus [7]. Hemostasis is based on the balance of blood coag- ulation and ¢brinolysis. Plasmin is the activated form 0167-4838 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII:S0167-4838(00)00083-2 * Corresponding author. Fax: +36-1-2667480; E-mail : mr@puskin.sote.hu Biochimica et Biophysica Acta 1480 (2000) 311^320 www.elsevier.com/locate/bba