Functional properties and active-site topographies of factor X Gla- and prothrombin Gla-domain chimeras of activated protein C

Abstract

Substitution of the Gla-domain of activated protein C (APC) with the Gla-domain of prothrombin (APC-PTGla) improves the anticoagulant activity of APC independent of protein S. Previous FRET studies showed that this substitution alters the active-site topography of this mutant, rendering it identical to the active site of the APC–protein S complex. In this study, we characterized the functional properties and the active-site topography of another APC chimera containing the Gla-domain of factor X (APC-FXGla). We discovered that the anticoagulant activity of this mutant was similarly improved independent of protein S. The average distance of the closest approach ( L) between the donor dye fluorescein attached to the active site of APC derivatives and the acceptor dye octadecylrhodamine incorporated into PC/PS vesicles was determined to be 99 Å for APC and 84–86 Å for both APC-PTGla and APC-FXGla. Protein S minimally influenced the L values of the APC chimeras, however, it lowered this value to 87 Å for wild-type APC. Further studies revealed that neither chimera elicits a protective signaling response in the TNF-α-activated endothelial cells. These results suggest that unique structural features within the Gla-domain of APC enable the protease to interact with endothelial protein C receptor in the antiinflammatory pathway, while the same features also cause an inherently lower specific activity for APC in the anticoagulant pathway. This adaptation has made APC a cofactor-dependent protease, requiring the cofactor function of protein S for its optimal anticoagulant function, which appears to involve the alteration of the active-site topography of APC above the membrane surface.