Abstract
Plasminogen activator inhibitor-1 (PAI-1) is a unique member of the serpin superfamily. The alternative behavior of PAI-1 as an inhibitor, a non-inhibitory substrate, or a non-reactive latent form has been shown to be dependent on the initial conformation. In this study, we have evaluated the effect of a substitution outside the reactive site loop (P18) or in the reactive site loop (P6 and P10) on proteinase specificity and conformational transitions in PAI-1. Wild-type PAI-1 (wtPAI-1) revealed the same conformational distribution pattern toward tissue-type plasminogen activator (t-PA) as toward urokinase-type plasminogen activator (u-PA) (i.e. 53 +/- 6. 9% active, 36 +/- 6.8% latent, and 12 +/- 1.9% substrate). Inactivation of wtPAI-1 resulted in the conversion of the labile active form into the latent form while the stable substrate form remained unchanged. PAI-1-P6 (Val --> Pro at P6) revealed a target specificity for t-PA (39 +/- 7% versus 3 +/- 2% of the theoretical maximal value toward t-PA and u-PA, respectively), PAI-1-P10 (Ser --> Pro at P10) was 4-fold more active toward u-PA than toward t-PA, and PAI-1-P18 (Asn --> Pro at P18) exhibited inhibitory properties exclusively toward u-PA (41 +/- 10%). Surprisingly, inactivation of these mutants revealed functional and conformational transitions distinct from those observed for wtPAI-1. Inactivation of PAI-1-P6(Val --> Pro) resulted in a total conversion of the active form into the latent form and in a partial conversion of the substrate form into the latent form. The active forms of both PAI-1-P10(Ser --> Pro) and PAI-1-P18(Asn --> Pro) are also labile but, in contrast to the active form of wtPAI-1, convert into substrate forms. Based on the existence of various conformations of PAI-1, we propose an alternative reaction scheme describing the putative interactions between serpins and their target proteinases. The unusual conformational and functional flexibility of PAI-1 that, according to the current study, appears not to be restricted to the reactive site loop further underlines the importance of potential structural rearrangements (e.g. upon binding to cofactors) in PAI-1 (or serpins in general) for its functional behavior at particular biological sites.
Highlights
Plasminogen activator inhibitor 1 (PAI-1),1 a glycoprotein with an apparent molecular weight of 50,000 [1], is the most important physiological inhibitor of tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator and inhibits both proteinases very rapidly with secondorder rate constants of more than 2 ϫ 107 MϪ1 sϪ1 [2, 3]
Materials and Methods—The chromogenic substrate S-2403 was obtained from Chromogenix (Molndal, Sweden). t-PA was a kind gift from Boehringer Ingelheim (Brussels, Belgium); low molecular weight two-chain urokinase-type plasminogen activator (u-PA) was prepared from single-chain u-PA kindly provided by Dr Lijnen (University of Leuven, Belgium)
Reaction Products Formed after Incubation of Wild-type PAI-1 (wtPAI-1) and PAI-1 Mutants With t-PA and u-PA at 37 °C—Incubation of wtPAI-1 at 37 °C with a 2-fold molar excess of t-PA revealed the formation of t-PA1⁄7PAI-1 complexes (49 Ϯ 6%, mean Ϯ S.D., n ϭ 4), small amounts of cleaved derivative (13 Ϯ 1%), and residual non-reactive material (38 Ϯ 5%) (Fig. 2A and Table I)
Summary
Materials and Methods—The chromogenic substrate S-2403 was obtained from Chromogenix (Molndal, Sweden). t-PA (predominantly single chain) was a kind gift from Boehringer Ingelheim (Brussels, Belgium); low molecular weight two-chain u-PA (tcu-PA) was prepared from single-chain u-PA (scu-PA) kindly provided by Dr Lijnen (University of Leuven, Belgium). Inactivation of PAI-1 Samples—PAI-1 samples (except PAI-1-P18) were inactivated by diluting the samples to a final concentration of 150 –190 g/ml using the appropiate diluent (containing Tween 80 and Na2HPO4) to obtain a buffered solution with 45 mM phosphate, 70 mM NaCl, and 0.01% Tween 80, pH 7.4, followed by incubation at 37 °C for 24 h. Under these conditions, PAI-1-P18 exhibited extensive precipitation. Statistical Analysis—The statistical significance of differences was evaluated using Student’s t-test; p values Ͼ0.05 were considered non-significant
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