IMPACT OF MUTATIONS AT THE P4 AND P5 POSITIONS ON THE REACTION OF ANTITHROMBIN WITH THROMBIN AND ELASTASE

Antithrombin(AT) is a serpin capable of trapping thrombin (IIa) in a s table and covalent complex Complex formation is prevented by leukocyte elastase (LE) cleavage near the AT reactive centre. We mutated the kn own LE cleavage sites of AT to explore the possibility of producing an LE-resistant AT molecule. Initially, six rabbit AT variants differing only at residue 390 (P4) were generated in a cell-free system, and ge l-based assays were used to assess IIa-mediated complex formation and LE-mediated cleavage of the variants. Substitution of charged residues (Glu or Arg) reduced complex formation by 50-60%, while the Ser varia nt was incapable of inhibiting thrombin; LE reactivity was less affect ed. The least (Trp) and most (Ser) affected variants were expressed in COS-1 cells. Again, the Ser variant was incapable of detectably reduc ing the rate of thrombin-mediated amidolysis while the Trp variant inh ibited thrombin at a slightly reduced rate (-28%). LE inactivated the Trp variant and the wild-type AT to a similar extent. Recreation of th e Trp mutation in COS-derived human AT showed similar results. Since r etention of LE-sensitivity could have arisen due to cleavage at Val389 (P5), we produced and characterized a human AT substitution mutant wi th Trp at both P4 and P5. This variant showed a slight reduction in th rombin inhibitory activity (-22%), but remained susceptible to LE inac tivation. These results suggest either that LE cleaves at secondary si tes if its primary cleavage sites are blocked, or that the substrate s pecificity of LE differs in polypeptides as compared to peptide substr ates. (C) 1997 Elsevier Science Ltd.