Functional analysis of mutant variants of thrombin-activatable fibrinolysis inhibitor resistant to activation by thrombin or plasmin.

BACKGROUND: Thrombin-activatable fibrinolysis inhibitor (TAFI) defines a pathway that functionally links the coagulation and fibrinolytic cascades. TAFI is activated by proteolytic cleavage, a reaction that can be performed by thrombin and plasmin, but most efficiently by thrombin in complex with the endothelial cofactor thrombomodulin (TM). The respective roles of these activators in regulating the TAFI pathway are largely unknown. OBJECTIVE AND METHODS: In the present study, we constructed and expressed mutant variants of TAFI that have key substitutions in the amino acids surrounding the scissile Arg92-Ala93 bond. RESULTS AND
CONCLUSIONS: We identified variants that showed patterns of resistance to specific activators. For example, the P91S, R92K and S90P variants exhibited specific impairment of activation by thrombin or thrombin-TM, thrombin alone, and thrombin alone or plasmin, respectively. The variants that we tested also showed antifibrinolytic potentials that can be rationalized in terms of which enzymes are capable of activating them. On the other hand, certain predictions from peptide studies of mutations that would be expected to interfere with plasmin cleavage were not satisfied by our data, indicating that protein context, as well as the identity of amino acids at protease cleavage sites, dictates protease specificity.