title: Engineering a disulfide bond to stabilize the calcium-binding loop of activated protein C eliminates its anticoagulant but not its protective signaling properties.
abstract: In addition to an anticoagulant activity, activated protein C (APC) also exhibits anti-inflammatory and cytoprotective properties. These properties may contribute to the beneficial effect of APC in treating severe sepsis patients. A higher incidence of bleeding because of its anticoagulant function has been found to be a major drawback of APC as an effective anti-inflammatory drug. In this study, we have prepared a protein C variant in which an engineered disulfide bond between two beta-sheets stabilized the functionally critical Ca2+-binding 70-80 loop of the molecule. The 70-80 loop of this mutant no longer bound Ca2+, and the activation of the mutant by thrombin was enhanced 60-80-fold independently of thrombomodulin. The anticoagulant activity of the activated protein C mutant was nearly eliminated as determined by a plasma-based clotting assay. However, the endothelial protein C receptor- and protease-activated receptor-1-dependent protective signaling properties of the mutant were minimally altered as determined by staurosporine-induced endothelial cell apoptosis, thrombin-induced endothelial cell permeability, and tumor necrosis-alpha-mediated neutrophil adhesion and migration assays. These results suggest that the mutant lost its ability to interact with the procoagulant cofactors but not with the protective signaling molecules; thus this mutant provides an important tool for in vivo studies to examine the role of anticoagulant versus anti-inflammatory function of activated protein C.
authors.affiliation: Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri 63104, USA.
authors.forename: Jong-Sup
authors.initials: JS
authors.lastname: Bae
authors.forename: Likui
authors.initials: L
authors.lastname: Yang
authors.forename: Chandrashekhara
authors.initials: C
authors.lastname: Manithody
authors.forename: Alireza R
authors.initials: AR
authors.lastname: Rezaie
mesh_terms: D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D000925:Anticoagulants / Q000737:chemistry* / Q000378:metabolism; D017209:Apoptosis; D002118:Calcium / Q000737:chemistry*; D002461:Cell Line, Transformed; D002465:Cell Movement; D004220:Disulfides; D006801:Humans; D009154:Mutation; D011485:Protein Binding; D011486:Protein C / Q000737:chemistry*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D018180:Thrombomodulin / Q000737:chemistry
chemical_list: D000893:Anti-Inflammatory Agents; D000925:Anticoagulants; D004220:Disulfides; D011486:Protein C; D018180:Thrombomodulin; D002118:Calcium