Abstract
Serine protease inhibitors ("serpins") are highly homologous proteins which inhibit selected "target" serine proteases by acting as a pseudo-substrate. Their specificity is primarily determined by the amino acid sequence around the carboxyl-terminally located reactive center (P1-P1'). In addition, the association rate constant between a serpin and a serine protease can be dramatically increased by non-protein cofactors, such as heparin in the case of thrombin inhibition by antithrombin III. In an attempt to alter the specificity of PAI-1 from an inhibitor of the fibrinolytic system to an inhibitor of coagulation, we replaced P1-P1' or P3 through P3' of the reactive center of PAI-1 by the corresponding residues of antithrombin III and assessed whether the mutant proteins, purified from lysates of transformed Escherichia coli cells, had acquired thrombin inhibitory properties. The experiments were performed in the presence and absence of vitronectin, a multifunctional protein which has been shown to bind PAI-1 in plasma and in the matrix of endothelial cells. The second-order rate constants for t-PA inhibition of "wild-type" PAI-1 and PAI P1-P1'ATIII, irrespective of the presence of vitronectin, were similar, whereas replacing P3-P3' resulted in a 40-fold decrease of the second-order rate constant towards t-PA, again independent of vitronectin. In the absence of vitronectin, reactivity of PAI-1 and its "antithrombin III-like" variants towards thrombin was slow; however, PAI-1 P3-P3' ATIII had a 10-fold higher k1 than wild-type PAI-1 (1.3 x 10(4) M-1 s-1 versus 1.1 x 10(3) M-1 s-1). In contrast, in the presence of vitronectin, PAI-1 and even more rapidly PAI-1 P3-P3'ATIII were found to be effective thrombin inhibitors, with k1 values of 2.2 x 10(5) M-1s-1 and 1.8 x 10(6) M-1 s-1, respectively. Thus, in the presence of vitronectin, PAI-1 P3-P3'ATIII displays a 3-fold higher k1 with thrombin than with t-PA. It is shown that vitronectin enhances, in a dose-dependent manner, the formation of sodium dodecyl sulfate-resistant complexes between PAI-1 or mutants thereof and thrombin. Therefore, vitronectin is the first protein described to function as a cofactor for serpin specificity. PAI-1 is proposed to be a versatile inhibitor which, in the presence of vitronectin, can modulate both coagulation and fibrinolysis.
Highlights
The present study was initiated to elucidate determinants of the protease specificity of PAI-1. rPAI-1 was expressed in E. coli and purified to homogeneity using a convenient three-step purification procedure
This material was indistinguishable from reference ECCM-PAI- with respect to: (a) dependence on in vitro activation, (b) specific activity of the activated preparations, and (c) inhibition kinetics towards t-PA
Several factors have been postulated to contribute to the protease specificity of serpins: 1) the amino acid sequence, especially residue Pl, around the reactive site (Travis and Salvesen, 1983); 2) specific domains in the serpins involved in specialized functions, such as a site in a*-antiplasmin which binds to a lysine-binding site in kringle 1 and/or kringle 4 of plasmin (Christensen and Clemmensen, 1977; Wiman and Collen, 1978)
Summary
Their specificity is primarily determined by the amino acid sequence around the carboxyl-terminally located reactive center (Pl-Pl’). The association rate constant between a serpin and a serine protease can be dramatically increased by non-protein cofactors, such as heparin in the case of thrombin inhibition by antithrombin III. In an attempt to alter the specificity of PAI- from an inhibitor of the fibrinolytic system to an inhibitor of coagulation, we replaced Pl-Pl’ or P3 through P3’ of the reactive center of PAI- by the corresponding residues of antithrombin. The experiments were performed in the presence and absence of vitronectin, a multifunctional protein which has been shown to bind PAI- in plasma and in the matrix of endothelial cells. In the presence of vitronectin, PAI- 1 and even more rapidly PAZ- 1 P3-
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