In vitro oxidative damage to tissue-type plasminogen activator: a selective modification of the biological functions

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.

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.

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.

(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.