In vitro oxidative damage to tissue-type plasminogen activator: a selective modification of the biological functions.
OBJECTIVE
Tissue-type plasminogen activator (t-PA) is an important component of the blood fibrinolytic system responsible for thrombus dissolution. It is often required to function under oxidative stress, and exogenous t-PA is used clinically in the treatment of acute myocardial infarction (AMI). The aim of this study was to examine alterations in the residual activity of t-PA pre-treated with certain oxidants.


METHODS
Recombinant t-PA (rt-PA) and native t-PA (nt-PA) were pre-treated with freshly generated hypochlorous acid (HOCl) and chloramine T at varying concentrations. The amidolytic activity, the plasminogenolytic activity and the fibrin-binding affinity were then examined using chromogenic assays based on S-2288 and S-2251.


RESULTS
The amidolytic activity of t-PA was surprisingly found to be rather sensitive (IC50 1 and 12 mumol/1, respectively), and the plasminogenolytic activity rather resistant to pre-treatment with HOCl and chloramine T. The fibrin binding study of treated t-PA revealed substantial loss of binding to CNBr-digested fibrinogen (FDP-CNBr). The velocity of t-PA in reaction with plasminogen remained the same as non-treated t-PA. The possible mechanisms of this asymmetrical oxidative modification of the biological functions are also discussed.


CONCLUSIONS
(1) The catalytic activity of t-PA and the binding affinity for its large-molecule substrate plasminogen, rather than the small-molecule substrate S-2288, are highly resistant to oxidative damage; (2) the fibrin-binding affinity of t-PA can be selectively and asymmetrically damaged by exposure to these oxidants. Thus it is possible that the characteristic advantage of thrombus selectivity of t-PA in both spontaneous thrombolysis and thrombolytic therapy may be diluted in circumstances where toxic and reactive oxidants exist.
Feng
Y H
YH
Department of Cardiovascular Medicine, John Radcliffe Hospital, University of Oxford, UK.
Hart
G
G
D000595:Amino Acid Sequence; D002700:Chloramines / Q000494:pharmacology*; D002863:Chromogenic Compounds; D005342:Fibrinolysis; D006801:Humans; D006868:Hydrolysis; D006997:Hypochlorous Acid / Q000494:pharmacology*; D019272:Leukocyte Elastase; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D018384:Oxidative Stress; D010196:Pancreatic Elastase / Q000235:genetics / Q000378:metabolism; D010958:Plasminogen / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D010959:Tissue Plasminogen Activator / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D014105:Tosyl Compounds / Q000494:pharmacology*; D000515:alpha 1-Antitrypsin / Q000378:metabolism
D002700:Chloramines; D002863:Chromogenic Compounds; D009842:Oligopeptides; D014105:Tosyl Compounds; D000515:alpha 1-Antitrypsin; C037741:alpha 1-antitrypsin-leukocyte elastase complex; C016300:chloramine-T; D006997:Hypochlorous Acid; C041319:isoleucyl-prolyl-arginine-4-nitroanilide; D010958:Plasminogen; D010196:Pancreatic Elastase; D019272:Leukocyte Elastase; D010959:Tissue Plasminogen Activator