Structures of importance for the stability of antiplasmin as studied by site-directed mutagenesis.

Human antiplasmin, a fast-acting inhibitor of plasmin in plasma, belongs to the serpin super-family of proteins. Like other members of this family, antiplasmin has a scissile peptide bond exposed within a reactive centre loop, typically present at the surface of the molecule. Antiplasmin is stable at neutral pH, but at acidic pH or at elevated temperatures it rapidly becomes inactivated. Data regarding "native" antiplasmin have demonstrated that both polymerization processes and formation of latent molecules are important in this respect. In this work we used site-directed mutagenesis to produce 11 single-site mutants (mainly within Abeta-sheet, Bbeta-sheet and reactive centre loop), which were expressed in Drosophila S2 cells, purified and characterized. Five of the 11 mutants were found to have a deviating stability at decreased pH. Glu346Thr was the only mutant with a lesser stability as compared to wt-antiplasmin, but the other 4 were more stable. The most stable mutant, His341Thr, was 7-fold more stable at pH 4.9 as compared to wt-antiplasmin. The wt-antiplasmin had a much more pronounced tendency to polymerize at decreased pH, as compared to "native" antiplasmin. However, many of the mutants clearly rather formed latent molecules, as judged both from PAGE-analysis at non-denaturing condition and reactivation experiments.

Inactivation of antiplasmin at low pH: evidence for the formation of latent molecules.

Several serine proteinase inhibitors (serpins) are metastable proteins which under certain conditions may undergo conformational changes resulting in the insertion of the reactive centre loop into the so-called Abeta-sheet and hence forming latent molecules. Here we have studied the inactivation of antiplasmin as a function of pH and temperature with time. At decreased pH (4.9-5.8) and at room temperature, antiplasmin activity decreased following first-order kinetics. Analysis by polyacrylamide gel electrophoresis under non-denaturing conditions demonstrated that only minor amounts of polymerized material formed after extensive incubation (4 days) at room temperature. However, on incubation at elevated temperatures (45 or 55 degrees C), a rapid formation of polymerized material was observed. We also demonstrated that antiplasmin inactivated by treatment at pH approximately 5 at room temperature spontaneously slowly regained some activity if incubated in a buffer of neutral pH. Furthermore, by treatment with 4 M guanidinium chloride for about 30 min, followed by dialysis against a neutral phosphate buffer, considerable activity (almost 40%) was regained. Thus, we conclude that antiplasmin, at least partially, at lower temperatures is transformed into a latent form, which could be reactivated, in a similar manner as PAI-1. At increased temperature, however, polymerization seems to be the predominant reason for inactivation.

Identification of amino acids in antiplasmin involved in its noncovalent 'lysine-binding-site'-dependent interaction with plasmin.

The lysine-binding-site-mediated interaction between plasmin and antiplasmin is of great importance for the fast rate of this reaction. It also plays an important part in regulating the fibrinolytic enzyme system. To identify structures important for its noncovalent interaction with plasmin, we constructed seven single-site mutants of antiplasmin by modifying charged amino acids in the C-terminal part of the molecule. All the variants were expressed in the Drosophila S2 cell system, purified, and shown to form stable complexes with plasmin. A kinetic evaluation revealed that two mutants of the C-terminal lysine (K452E or K452T) did not differ significantly from wild-type antiplasmin in their reactions with plasmin, in either the presence or absence of 6-aminohexanoic acid, suggesting that this C-terminal lysine is not important for this reaction. On the other hand, modification of Lys436 to Glu decreased the reaction rate about fivefold compared with wild-type. In addition, in the presence of 6-aminohexanoic acid, only a small decrease in the reaction rate was observed, suggesting that Lys436 is important for the lysine-binding-site-mediated interaction between plasmin and antiplasmin. Results from computerized molecular modelling of the C-terminal 40 amino acids support our experimental data.