COMPARISON OF CHROMOGENIC SUBSTRATES FOR TISSUE-PLASMINOGEN ACTIVATORAND THE EFFECTS ON THE STABILITY OF PLASMINOGEN-ACTIVATOR INHIBITOR TYPE-1

We have studied the effect of low molecular weight compounds on the st ability of glycosylated human plasminogen activator inhibitor type-1 ( PAI-1), both in solution and immobilized by amine coupling to dextran. PAI-1 in buffer spontaneously loses its active conformation. By immob ilization, the half-life of PAI-I at pH 7.4 and 25 degrees C increased from 8.2 to 14.7 h. We found that most chromogenic substrates employe d to directly measure tissue plasminogen activator (tPA) have a destab ilizing effect on PAI-I, resulting in a decrease in half-life. Similar results were obtained with non-glycosylated PAI-1 from E. coli Theref ore, the catalytic constants for the different substrates for tPA were determined for comparison. However, the apparent rate constant for tP A could not be correlated with the observed effect on PAI-1. Moreover, the destabilizing effect of the chromogenic substrates on PAI-I is se parated from the competition in binding to tPA. The best commercial tP A substrates have a terminal -Gly-Arg-p-NA (-Gly-Arg-p-nitroaniline) m oiety. All these substrates accelerated the inactivation of both free and immobilized PAI-1 in a concentration-dependent manner. The weak su bstrate with a fluorogenic leaving group, instead of para nitroaniline (p-NA), had a strong destabilizing effect, In contrast, the products after hydrolysis of these substrates, the peptides and the chromophore no longer had an effect on PAI-1. The weak tPA substrate, with a term inal -Pro-Arg-p-NA, only weakly destabilized PAI-1. Two chromogenic pl asmin substrates containing a terminal-Lys-p-NA had no destabilizing e ffect. The best general PAI-1 assay may thus be to use plasminogen and fibrin, monitoring tPA activity by measuring plasmin activity with a chromogenic plasmin substrate. The destabilization effect was further studied with sodium dodecyl sulphate polyacrylamidle gel electrophores is (SDS-PAGE) and with plasmon resonance using BIAcore. Both technique s showed that PAI-1 is irreversibly converted by these chromogenic sub strates into an inactive form, while the cleaved substrates had no suc h effect.