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Thromb Haemost 2000; 84(05): 871-875
DOI: 10.1055/s-0037-1614130
Review Article
Schattauer GmbH

Effect of Stabilizing versus Destabilizing Interactions on Plasminogen Activator Inhibitor-1

Nele Vleugels
1   From the Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Belgium
,
John Leys
1   From the Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Belgium
,
Isabelle Knockaert
1   From the Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Belgium
,
Paul J. Declerck
1   From the Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Belgium
› Author AffiliationsThis work was supported in part by a grant from the Fund for Scientific Research (F.W.O.-Vlaanderen, project G.0266.97) and by a grant from the Research Fund K.U. Leuven (OT/98/37). N.V. is a research assistant of the Fund for Scientific Research (F.W.O.-Vlaanderen).
We are grateful to Dr. A. Rabijns (Laboratory of Analytical Chemistry and Medical Physicochemistry, KULeuven, Belgium) for assistance in the preparation of Fig. 1. and to Mrs I. Christ and Dr. J. Stassen (Cardiovascular Department, Boehringer Ingelheim Pharma KG, Biberach, Germany) for the measurement of the affinity constants.
Further Information

Publication History

Received 24 November 1999

Accepted after resubmission 26 June 2000

Publication Date:
13 December 2017 (online)

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Summary

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 as well as reversal or removal of charges on the s3C-s4C turn results 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. Whereas PAI-1-stab has a half-life of 150 ± 66 h, PAI-1-stab-R186ER187E, 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) of 175 ± 48 min, 75 ± 34 min, 68 ± 38 min and 79 ± 16 min, respectively. Wild-type PAI-1 (wtPAI-1) had a half-life of 55 ± 19 min. These data indicate that the stabilization induced by the mutated residues in PAI-1-stab is counteracted by the additional mutations, resulting in half-lives similar to that of wtPAI-1, thereby suggesting that the stabilizing and destabilizing forces act mainly independently in these mutants. Extrapolation of these data to other (stable) serpins leads to the hypothesis that the s3C-s4C turn and the distal hinge region of the reactive site loop plays a role for the stability of serpins in general.

Key words

Serpins - PAI-1 - mutagenesis - stability
 

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