Antiplasmin the main plasmin inhibitor in blood plasma : studies on structure-function relationships

Abstract

Antiplasmin is an important regulator of the fibrinolytic system. It inactivates plasmin very rapidly. The reaction between plasmin and antiplasmin occurs in several steps: first a lysine-binding site in plasmin interacts with a complementary site in antiplasmin. Then, an interaction occurs between the substrate-binding pocket in the plasmin active site and the scissile peptide bond in the RCL of antiplasmin. Subsequently, peptide bond cleavage occurs and a stable acyl-enzyme complex is formed. It has been accepted that the COOH-terminal lysine residue in antiplasmin is responsible for its interaction with the plasmin lysine-binding sites. In order to identify these structures, we constructed single-site mutants of charged amino acids in the COOH-terminal portion of antiplasmin. We found that modification of the COOH-terminal residue, Lys452, did not change the activity or the kinetic properties significantly, suggesting that Lys452 is not involved in the lysine-binding site mediated interaction between plasmin and antiplasmin.

On the other hand, modification of Lys436 to Glu decreased the reaction rate significantly, suggesting this residue to have a key function in this interaction. Results from computerised molecular modelling indeed supported our experimental data. The interaction between immobilized plasminogen or an elastase degradation product from plasminogen, constituting kringles 1-3 and different purified variants of antiplasmin was then studied by surface plasmon resonance. Again, the data demonstrated that Lys452 is not involved in the lysine-binding site mediated interaction. On the other hand, solid evidence was produced, proving that Lys436 is indeed very important for this interaction. Some evidence was found that Glu443 may also be involved in this interaction.

Molecular modelling experiments suggest that the negatively charged Glu443 is within the expected range from the positively charged Lys436 to form a complementary site to a lysine-binding site. Serine protease inhibitors may under certain conditions undergo conformational changes resulting in the insertion of RCL into the Aß-sheet during formation of latent molecules or polymers. Antiplasmin is stable at neutral pH, but at acidic pH or at elevated temperatures it rapidly becomes inactivated. At decreased pH, antiplasmin activity declined following first-order kinetics. Analysis by PAGE under non-denaturing conditions demonstrated that only minor amounts of polymerized material had formed. However, on incubation at elevated temperatures a rapid formation of polymerized material was observed. Antiplasmin inactivated by treatment at pH ~5 could spontaneously regain activity if incubated at neutral pH.

Furthermore, by treatment of such material with guanidinium chloride followed by dialysis, considerable activity was regained, in contrast to antiplasmin that had been inactivated by polymerization. To better understand these processes, site-directed mutagenesis was employed to produce some interesting variants of antiplasmin, which were purified and characterized. Five of the 11 mutants were found to have a deviating stability at decreased pH. One mutant was less stable as compared to wt-antiplasmin, but the other 4 were more stable. 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 were rather transformed to latent molecules, as judged both from PAGE-analysis at non-denaturing condition and reactivation experiments.