Development of a novel recombinant serpin with potential antithrombotic properties.

Recombinant alpha 1-antitrypsin with a P1 arginine residue (Arg-alpha 1-antitrypsin) is a rapid inhibitor of both thrombin and activated protein C (APC). A series of mutants were made in an attempt to increase the specificity of this serpin for thrombin over APC. Initially, P2 and P'1 residues of Arg-alpha 1-antitrypsin were replaced in single and double mutations by the corresponding residues in antithrombin and C1 inhibitor which are very poor inhibitors of APC. No improvement in selectivity was achieved by these mutations. In fact, all P2/P'1 substitutions led to a decrease in selectivity for thrombin over APC. For example, replacement of the P2 proline of Arg-alpha 1-antitrypsin by glycine decreased the association rate constant (kass) with thrombin by 37-fold while the kass value with APC was reduced by only 16-fold. Cooperative effects were observed with the double P2 and P'1 substitutions; the mutational effects were not additive. The decrease in the kass for thrombin caused by the mutation of the P2 proline to alanine or glycine was 3-fold greater when threonine was present in the P'1 position instead of the normal serine. In contrast to the disappointing results with the P2/P'1 mutations, replacement of the P7 to P'3 residues of alpha 1-antitrypsin by those of antithrombin led to a dramatic increase in selectivity. Although this substitution only affected the kass value with thrombin by 10-fold, a 12,500-fold decrease in this value with APC was observed. Substitution of proline for the P2 glycine of this chimeric serpin increased the kass values with thrombin and APC by 7- and 90-fold, respectively. The effect of the P2 substitution was again found to depend on the sequence surrounding the residue; the change in the kass for APC caused by the P2 Pro-->Gly replacement was 6-fold larger in the chimeric serpin. Evaluation of the kass values of the chimeric serpin with a P2 proline in light of the likely rates of inhibition of thrombin and APC during antithrombotic therapy with heparin suggested that this serpin may have kinetic parameters suitable for an antithrombotic agent.