Functional properties and active-site topographies of factor X Gla- and prothrombin Gla-domain chimeras of activated protein C.
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 A for APC and 84-86 A 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 A for wild-type APC. Further studies revealed that neither chimera elicits a protective signaling response in the TNF-alpha-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.
Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, Missouri 63104, USA.
Shabir H
SH
Qureshi
Likui
L
Yang
Chandrashekhara
C
Manithody
Jong-Sup
JS
Bae
Alireza R
AR
Rezaie
D000925:Anticoagulants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D001665:Binding Sites; D002448:Cell Adhesion / Q000187:drug effects; D002460:Cell Line; D015943:Factor Va / Q000378:metabolism; D005170:Factor X / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects; D010539:Permeability / Q000187:drug effects; D010743:Phospholipids / Q000378:metabolism; D011486:Protein C / Q000737:chemistry* / Q000378:metabolism*; D017293:Protein S / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011516:Prothrombin / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology
D000925:Anticoagulants; D010743:Phospholipids; D011486:Protein C; D017293:Protein S; D011993:Recombinant Fusion Proteins; D015943:Factor Va; D011516:Prothrombin; D005170:Factor X