We recently demonstrated that the substitution of the
autolysis loop (residues 143 to 154 in the
chymotrypsin numbering system) of activated
protein C (APC) with the corresponding loop of
factor Xa (fXa) renders the APC mutant (APC/fX143-154) susceptible to inhibition by
antithrombin (AT) in the presence of pentasaccharide. Our recent results further indicated, that in addition to an improvement in the reactivity of APC/fX143-154 with AT, both the amidolytic and anti-
factor Va activities of the mutant APC have also been significantly increased. Since the
autolysis loop of APC is five residues longer than the
autolysis loop of fXa, it could not be ascertained whether this loop in the mutant APC specifically interacts with the activated conformation of AT or if a shorter
autolysis loop is responsible for a global improvement in the catalytic activity of the mutant
protease. To answer this question, we prepared another APC mutant in which the
autolysis loop of the
protease was replaced with the corresponding loop of
trypsin (APC/Tryp143-154). Unlike an approximately 500-fold improvement in the reactivity of APC/fX143-154 with AT in the presence of pentasaccharide, the reactivity of APC/Tryp143-154 with the
serpin was improved approximately 10-fold. These results suggest that both the length and structure of residues of the
autolysis loop are critical for the specificity of the coagulation
protease interaction with AT. Further
factor Va inactivation studies with the APC mutants revealed a similar role for the
autolysis loop of APC in the interaction with its natural substrate.