FIBRINOLYTIC EFFECT OF ONE-CHAIN TTSSUE-TYPE PLASMINOGEN ACTIVATOR

Abstract

The fibrinolytic properties of authentic one- and two-chain recombinant tPA were compared to those of a plasmin resistant one-chain tPA analogue, tPA-Gly275, which is point mutated in Arg275 of the activation site. The proteins were characterised by reversed phase HPLC, reduced SDS-PAGE, and their concentrations determined by the BCA (modified Lowry) method. When equivalent conc. of these enzymes were tested for fibrinolytic activity by means of clot lysis and fibrin plate lysis methods, the values found for two-chain tPA were consistently 50% higher than one-chain tPA forms. The time course for plasmin catalysed one-chain tPA cleavage during fibrin clot lysis was determined by means of 125I-tPA. The cleavage is not instantaneous, and one-chain tPA may account for a considerable fraction of the total amount of plasmin formed. This is confirmed by similar experiments with 125I-tPA-Gly275, which is essentially intact one-chain tPA at the time of fibrin clot lysis. The effect of tPA activation site cleavage was also studied using plasmin coupled to sepharose beads. Plasmin sepharose was removed at different time intervals, and tPA was tested for amidolytic activity with > Glu-Gly-Arg-pNA and fibrinolytic activity as measured by fibrin clot lysis time as well as by fibrin plate methods. The results indicate that in the presence of fibrin, plasminogen can be activated by one-chain tPA at a considerable rate. On the other hand, the fact that the fibrinolytic activity measured by conventional assays is lower with one-chain tPA than with two-chain tPA should be considered when of these methods are used for standardization.