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.
