Comparison of chromogenic substrates for tissue plasminogen activator and the effects on the stability of plasminogen activator inhibitor type-1

Abstract

We have studied the effect of low molecular weight compounds on the stability 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 immobilization, the half-life of PAI-1 at pH 7.4 and 25°C increased from 8.2 to 14.7 h. We found that most chromogenic substrates employed to directly measure tissue plasminogen activator (tPA) have a destabilizing effect on PAI-1, resulting in a decrease in half-life. Similar results were obtained with non-glycosylated PAI-1 from E. coli . Therefore, the catalytic constants for the different substrates for tPA were determined for comparison. However, the apparent rate constant for tPA could not be correlated with the observed effect on PAI-1. Moreover, the destabilizing effect of the chromogenic substrates on PAI-1 is separated from the competition in binding to tPA. The best commercial tPA substrates have a terminal -Gly-Arg- p -NA (-Gly-Arg- p -nitroaniline) moiety. All these substrates accelerated the inactivation of both free and immobilized PAI-1 in a concentration-dependent manner. The weak substrate 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 terminal -Pro-Arg- p -NA, only weakly destabilized PAI-1. Two chromogenic plasmin substrates containing a terminal-Lys- p -NA had no destabilizing effect. 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 electrophoresis (SDS-PAGE) and with plasmon resonance using BIAcore. Both techniques showed that PAI-1 is irreversibly converted by these chromogenic substrates into an inactive form, while the cleaved substrates had no such effect.