Site‐directed mutagenesis of alanine‐382 of human antithrombin III

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Abstract
Antithrombin III Hamilton is a structural variant of antithrombin III (AT-III) with normal heparin affinity but impaired scrine protcase inhibitory activity. The molecular defect of AT-III-Hamilton is a substitution of threonine for alanine at amino acid residue 382. Recently it has been shown that both plasma-derived and cell-free-derived AT-III-Hamilton polypeptides act as substrates rather than inhibitors of thrombin and factor Xa. In the present study, the cell-free expression phagemid vector pGEM-32f(+)-AT-III1–433 was mutated at amino acid residue 382 of AT-III to generate 7 cell-free-derived variants. All these cell-free-derived AT-III variants were able to bind heparin as effectively as cell-free-derived normal AT-III. In terms of α-thrombin inhibitory activity each variant reacted differently. Variants could be grouped into 3 categories with respect to thrombin-AT-III complex formation: (1) near normal activity (glycine, isolcucine, leucine, valine): (2) low activity (threonine, glutamine): (3) no detectable activity (lysine). These data suggest that mutations at position 382 of AT-III may have a variable effect on protease inhibitory activity, depending on either the stability of the P12-P9 region of the exposed loop of AT-III, or the inability of the amino acid residue at position 382 to interact with a conserved hydrophobic pocket consisting of phenylalanine (at positions 77,221 and 422) and isolcucine (position 412) residues.