Engineering a Disulfide Bond To Stabilize the Calcium Binding Loop of Activated Protein C Eliminates Its Anticoagulant but Not Protective Signaling Properties.

In addition to its potent anticoagulant activity, activated protein C (APC) also exhibits anti-inflammatory and cytoprotective properties. The latter properties of APC are thought to be responsible for its beneficial effect in treating severe sepsis patients. A higher incidence of bleeding due to its anticoagulant function has been found to be a major drawback of APC as an effective anti-inflammatory drug. Thus, there is intense interest in developing APC variants that have no anticoagulant activity, but have normal protective properties. In this study, we have prepared a protein C variant in which an engineered disulfide bond between two β-sheets has stabilized the functionally critical Ca²⁺-binding 70–80 loop of the molecule. The 70–80 loop of this mutant no longer binds Ca²⁺ and the activation of the mutant by thrombin has been enhanced 60–80-fold independent of thrombomodulin. The anticoagulant activity of the APC variant has been essentially eliminated as determined by both factor Va degradation and plasma-based clotting assays. However, the endothelial protein C receptor- and protease-activated receptor-1-dependent protective signaling properties of the APC variant have remained intact as determined by staurosporine-induced endothelial cell apoptosis, thrombin-induced endothelial cell permeability and TNF-α-mediated neutrophil adhesion and migration assays. These results suggest that modulation of the structure and/or activity of APC by Ca²⁺ is required for its anticoagulant, but not for its anti-inflammatory activity, thus the protein C variant may potentially be a safer drug for treating severe sepsis patients in both activated and zymogenic forms.