Structural determinants of protease-activated receptor 1 cleavage by activated protein C

Activated protein C (APC) performs cytoprotective functions mediated by cleavage of the protease-activated receptor 1 (PAR1) in the presence of the endothelial protein C receptor and signaling through β-arrestin-2. APC cleaves PAR1 at R41 and R46, but the specificity of the reaction is low. In contrast, thrombin cleaves PAR1 at R41 only in a reaction that is independent of endothelial protein C receptor, producing a proinflammatory response mediated by signaling through G-protein intermediates and features high specificity. The molecular basis of this difference between APC and thrombin remains unknown.

To identify the structural determinants of APC that influence PAR1 specificity.

Using available structural information, we engineered thrombin determinants of PAR1 recognition into APC. Specifically, we replaced T99 with Leu and swapped the entire 37- and 60-loops of APC with those of thrombin (eg, APC_60/T99L and APC_37/60/T99L).

The engineered APC variants featured up to 80-fold enhanced specificity toward PAR1 mediated by increased cleavage at R41 and decreased cleavage at R46. Notably, the variants APC_60/T99L and APC_37/60/T99L also showed significantly reduced activity toward factor Va.

The 37, 60, and 99 segments of APC determine the cytoprotective and anticoagulant properties of the enzyme.