J Pharm Belg. 2000 Mar-Apr; 55(2): 57-8.

Effect of stabilizing versus destabilizing interactions on plasminogen activator inhibitor-1.

Vleugels N, Leys J, Knockaert I, Declerck PJ.

Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Belgium.

Plasminogen activator inhibitor-1 (PAI-1) is a unique member of the serpin family, as it spontaneously converts into a latent conformation. However, the exact mechanism of this conversion is not known. Previous studies reported that neutralizing monoclonal antibodies (MAs) as well as reversal or removal of charges on the s3C-s4C turn ('gate-region') result in a destabilization of PAI-1 leading to an accelerated conversion to its latent form. In this study the effect of the reversal or removal of charges in this 'gate region' (R186E/R187E, H190E/K191E, H190L/K191L and R356E) on a stable PAI-1-variant (PAI-1-stab) was investigated. PAI-1-stab-R186E-R187E, PAI-1-stab-H190E-K191E, PAI-1-stab-H190L-K191L and PAI-1-stab-R356E have a strongly decreased half-life (p < 0.005 versus PAI-1-stab) resulting in half-lives similar or slightly increased to that of wild-type PAI-1 (wtPAI-1). Reversal of the positively charged residues at position 186/187 or at position 356 and removal at position 190/191 do not only have a destabilizing effect on the active conformation, but also on the substrate conformation. However, reversal of the charge at position 190/191 does not affect the stability of the substrate conformation. Moreover this study is to the best of our knowledge the first to report that the distal hinge region of the reactive site loop (Arg 356) may be involved in the substrate behaviour of PAI-1. In addition our results suggest an important role for the gate-region for the inactivation of PAI-1 through the conversion to latency both for the destabilizing mutations and for the neutralizing MAs.