Background: Thrombin-activatable fibrinolysis inhibitor (TAFI) is a validated target for thrombotic diseases. TAFI is converted in vivo to activated TAFI (TAFIa) by removal of its pro-domain. Whereas TAFI is stable and persists in the circulation, possibly in complex with plasminogen, TAFIa is unstable and poorly soluble, with a half-life of minutes.Objectives: In order to study the molecular determinants of this instability, we studied the influence of protein inhibitors on human TAFIa. Results: We found that protein inhibitors significantly reduced the instability and insolubility of TAFIa. In addition, we solved the 2.5-Å resolution crystal structure of human TAFIa in complex with a potent protein inhibitor, tick-derived carboxypeptidase inhibitor, which gives rise to a stable and soluble TAFIa species. The structure revealed a significant reduction in the flexibility of dynamic segments when compared with the structures of bovine and human TAFI. We also identified two latent hotspots, loop Lβ2β3 and segment α5-Lα5β7-β7, where conformational destabilization may begin. These hotspots are also present in TAFI, but the pro-domain may provide sufficient stabilization and solubility to guarantee protein persistence in vivo. When the pro-domain is removed, the free TAFIa moiety becomes unstable, its activity is suppressed, and the molecule becomes insoluble. Conclusions: The present study corroborates the function of protein inhibitors in stabilizing human TAFIa and it provides a rigid and high-resolution mold for the design of small molecule inhibitors of this enzyme, thus paving the way for novel therapy for thrombotic disorders. © 2010 International Society on Thrombosis and Haemostasis.
|Journal||Journal of Thrombosis and Haemostasis|
|Publication status||Published - 1 Jan 2010|
- Blood coagulation
- Zymogen activation