Plasminogen activator inhibitor 1 (PAI-1) levels are elevated in a number of life-threatening conditions and often correlate with unfavorable outcomes. Spontaneous inactivation due to active to latent transition limits PAI-1 activity in vivo. While endogenous vitronectin (Vn) stabilizes PAI-1 by 1.5-2.0-fold, further stabilization occurs in a "molecular sandwich" complex (MSC) in which a ligand that restricts the exposed reactive center loop is bound to PAI-1/Vn. The effects of S195A two-chain urokinase (tcuPA) and Vn on inactivation of wild-type (wt) glycosylated (Gl-PAI-1), nonglycosylated (rPAI-1), and nonglycosylated Q123K PAI-1 (lacks Vn binding) forms were studied. S195A tcuPA decreased the rate constant (kL) for spontaneous inactivation at 37 °C for rPAI-1, Q123K, and Gl-PAI-1 by 6.7-, 3.4-, and 7.8-fold, respectively, and both S195A tcuPA and Vn by 66.7-, 5.5-, and 103.3-fold, respectively. Analysis of the temperature dependences of kL revealed a synergistic increase in the Gibbs free activation energy for spontaneous inactivation of wt Gl-PAI-1 and rPAI-1 in MSC from 99.8 and 96.1 to 111.3 and 107.0 kJ/mol, respectively, due to an increase in the activation enthalpy and a decrease in the activation entropy. Anti-PAI-1 monoclonal antibodies (mAbs) competing with proteinase also stabilize PAI-1/Vn. The rate of inhibition of target proteinases by MSCs, with a stoichiometry close to unity, was limited by the dissociation (k = 10(-4) to 10(-3) s(-1)) of S195A tcuPA or mAb. The stabilization of PAI-1 in MSCs in vivo may potentiate uncontrolled thrombosis or extravascular fibrin deposition, suggesting a new paradigm for using PAI-1 inhibitors and novel potential targets for therapy.