Summary
Plasminogen activator inhibitor-1 (PAI-1), the primary physiological inhibitor of
both tissue-type plasminogen activator and urokinasetype plasminogen activator in
plasma, is a well established risk factor in thrombotic diseases. Reduction of active
PAI-1 levels may lead to a decreased tendency of thrombosis. Compounds that can suppress
pharmacologically active PAI-1 levels are therefore considered as putative drugs.
In the present study, we describe the PAI-1 neutralizing properties and mechanism
of a newly selected compound (i. e. fendosal, HP129) in comparison to four previously
reported compounds (i. e. AR-H029953XX, XR1853, XR5118 and the peptide TVASS) using
different assays. The inhibitory effect of these compounds on active PAI-1 was analyzed
by a plasmin-coupled chromogenic assay (Coaset® t-PA), direct chromogenic assays (t-PA,
u-PA) and quantification of complex formation by ELISA, SDS-PAGE and surface plasmon
resonance. Comparative evaluation of the obtained IC50 values reveals large differences
[i. e. IC50 of 15 µM (HP129) vs. >1000 µM (XR5118) determined at 37° C using SDS-PAGE] between the compounds studied.
Importantly, the relative potency of the various compounds is also dependent on the
method used (10 to 170-fold differences in IC50 values). Characterization of the PAI-1
forms (i. e. active, non-reactive and substrate) generated upon inactivation reveals
that the newly described compound HP129 induces a unique pathway (i. e. active to
non-reactive conversion via a substrate-behaving intermediate) of inactivation compared
to the other compounds.
Taken together, these data strongly suggest that the various compounds act through
different mechanisms. In addition, the results stress the necessity for a careful
selection of the method used for the evaluation of PAI-1 inhibitors, preferably requiring
a panel of screening methods.
Keywords
PAI-1 - PAI-1 inhibitor - serpin - thrombosis
