The conversion of active to latent plasminogen activator inhibitor-1 is an energetically silent event

Abstract

PAI-1 is a proteinase inhibitor, which plays a key role in the regulation of fibrinolysis. It belongs to the serpins, a family of proteins that behave either as proteinase inhibitors or proteinase substrates, both reactions involving limited proteolysis of the reactive center loop and insertion of part of this loop into beta-sheet A. Titration calorimetry shows that the inhibition of tissue-type plasminogen and pancreatic trypsin are exothermic reactions with DeltaH = -20.3, and -22.5 kcal.mol(-1), respectively. The Pseudomonas aeruginosa elastase-catalyzed reactive center loop cleavage and inactivation of the inhibitor is also exothermic (DeltaH = -38.9 kcal.mol(-1)). The bacterial elastase also hydrolyses peptide-bound PAI-1 in which acetyl-TVASSSTA, the octapeptide corresponding to the P(14)-P(7) sequence of the reactive center loop is inserted into beta-sheet A of the serpin with DeltaH = -4.0 kcal.mol(-1). In contrast, DeltaH = 0 for the spontaneous conversion of the metastable active PAI-1 molecule into its thermodynamically stable inactive (latent) conformer although this conversion also involves loop/sheet insertion. We conclude that the active to latent transition of PAI-1 is an entirely entropy-driven phenomenon.

FIGURE 1

(A) Calorimetric data for the active to latent transition of PAI-1; 100 μl (2.7 nmol) of stock solution of serpin was injected into 1.4 ml of 50 mM Hepes, 25mM NaCl, pH 7.4, 37°C, and the thermal power was recorded for 16 h. (B) Calorimetric data obtained from an identical experiment using the stable variant PAI-1-P₁₄ as a control of the stability of the calorimeter. The arrow indicates the time at which the samples of PAI-1 were injected into the calorimeter cell.

FIGURE 2

Thermal power generated during the inhibition of tPA by PAI-1 at pH 7.4 and 25°C. Aliquots of PAI-1 (0.18 nmol in 15 μl) were successively added (in 20 s) to 1.4 ml buffer containing 1.69 nmol of active tPA while recording the thermal power. The inset shows the quantities of heat calculated from the area of each peak as a function of the molar ratio of PAI-1 to tPA.

FIGURE 3

Thermal power generated during the inhibition of trypsin by PAI-1 at pH 7.4 and 25°C. Aliquots of trypsin (0.38 nmol in 6 μl) were successively added (in 10 s) to 1.4 ml buffer containing 1.53 nmol of active PAI-1 while recording the thermal power. The inset shows the quantities of heat calculated from the area of each peak as a function of the molar ratio of trypsin to tPA.

FIGURE 4

Thermal power generated during the PsE-catalyzed inactivation of PAI-1 at pH 7.4 and 24°C. The reaction was initiated by injecting 36 pmol PsE (20 μl in 30 s) into 1.4 ml buffer containing varying amounts of PAI-1: 0.94 nmol (curve A), 1.88 nmol (curve B), 3.76 nmol (curve C), 5.64 nmol (curve D), and 7.52 nmol (curve E). The inset shows the quantity of heat as a function of the quantity of PAI-1 present in the reaction mixtures. The curve is theoretical and has been generated using the best estimate of ΔH (−38.9 kcal.mol⁻¹) calculated by linear regression analysis.

FIGURE 5

Comparison of the three-dimensional structures of (A) active (Nar et al., 2000), (B) latent (Mottonen et al., 1992), and (C) cleaved (Aertgeerts et al., 1995) PAI-1. The RCL is in red, the P₁ and P′₁ residues of the RCL are blue and yellow spheres, respectively, and strand s1C is in green. In latent PAI-1, s1C extracted from sheet C is shown in green as an extension of the RCL. Figure prepared with MOLMOL (Koradi et al., 1996).