The
autolysis loop of activated
protein C (APC) is five residues longer than the
autolysis loop of other
vitamin K-dependent coagulation
proteases. To investigate the role of this loop in the zymogenic and
anticoagulant properties of the molecule, a
protein C mutant was constructed in which the
autolysis loop of the
protein was replaced with the corresponding loop of
factor X. The
protein C mutant was activated by
thrombin with approximately 5-fold higher rate in the presence of Ca2+. Both kinetics and direct binding studies revealed that the Ca2+ affinity of the mutant has been impaired approximately 3-fold. The result of
a factor Va degradation assay revealed that the
anticoagulant function of the mutant has been improved 4-5-fold in the absence but not in the presence of
protein S. The improvement was due to a better recognition of both the P1-Arg506 and P1-Arg306 cleavage sites by the mutant
protease. However, the plasma half-life of the mutant was markedly shortened due to faster inactivation by plasma
serpins. These results suggest that the
autolysis loop of
protein C is critical for the Ca(2+)-dependence of activation by
thrombin. Moreover, a longer
autolysis loop in APC is not optimal for interaction with
factor Va in the absence of
protein S, but it contributes to the lack of
serpin reactivity and longer half-life of the
protease in plasma.