Engineering a disulfide bond to stabilize the calcium-binding loop of activated protein C eliminates its anticoagulant but not its protective signaling properties

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...

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Vydáno v:The Journal of biological chemistry Ročník 282; číslo 12; s. 9251
Hlavní autoři: Bae, Jong-Sup, Yang, Likui, Manithody, Chandrashekhara, Rezaie, Alireza R
Médium: Journal Article
Jazyk:angličtina
Vydáno: United States 23.03.2007
Témata:
Anti-Inflammatory Agents - pharmacology
Anticoagulants - chemistry
Anticoagulants - metabolism
Apoptosis
Calcium - chemistry
Cell Line, Transformed
Cell Movement
Disulfides
Humans
Mutation
Protein Binding
Protein C - chemistry
Protein Structure, Secondary
Protein Structure, Tertiary
Signal Transduction
Thrombomodulin - chemistry
ISSN:0021-9258
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Shrnutí: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.
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ISSN:0021-9258
DOI:10.1074/jbc.M610547200