We previously demonstrated that the substitution of the autolysis loop (residues 143-154 in chymotrypsin numbering) of APC with the corresponding loop of trypsin (APC-Tryp 143-154) has no influence on the proteolytic activity of the protease toward fVa, however, this substitution increases the reactivity of APC with plasma inhibitors so that the mutant exhibits no anticoagulant activity in plasma. To further investigate the role of the autolysis loop in APC and determine whether this loop is a target for modulation by protein S, we evaluated the activity of APC-Tryp 143-154 toward fVa and several plasma inhibitors both in the absence and presence of protein S. Furthermore, we evaluated the active-site topography of APC-Tryp 143-154 by determining the average distance of the closest approach (L) between a fluorescein dye tethered to a tripeptide inhibitor, attached to the active-site of APC-Tryp 143-154, and octadecylrhodamine dyes incorporated into PCPS vesicles both in the absence and presence of protein S. The activity of APC-Tryp 143-154 toward fVa was identical to that of wild-type APC both in the presence and absence of protein S. However, the reactivity of APC-Tryp 143-154 with plasma inhibitors was preferentially improved independent of protein S. The FRET analysis revealed a dramatic change in the active-site topography of APC both in the absence and presence of protein S. Anisotropy measurements revealed that the fluorescein dye has a remarkable degree of rotational freedom in the active-site of APC-Tryp 143-154. These results suggest that the autolysis loop of APC may not be a target for modulation by protein S. This loop, however, plays a critical role in restricting both the specificity and spatial environment of the active-site groove of APC.