Abstract 38: Identification of Thrombomodulin Binding Sites on Thrombin Activatable Fibrinolysis Inhibitor that Mediate Accelerated Activation by Thrombin

Thrombin activatable fibrinolysis inhibitor (TAFI) is a human plasma zymogen that provides a molecular connection between coagulation and fibrinolysis. TAFI is activated through proteolytic cleavage by thrombin, thrombin in complex with the endothelial cell cofactor thrombomodulin (TM), or plasmin to generate the enzyme activated TAFI (TAFIa). TAFIa possesses basic carboxypeptidase activity which down-regulates fibrin clot lysis by removing carboxyl-terminal lysine residues from partially degraded fibrin thereby attenuating the positive feedback in the fibrinolytic cascade. Accumulating evidence from several studies suggests that TM and TAFI make direct contacts such that the enhancement of TAFI activation by TM is through binding at sites remote from the activation site (Arg92-Ala93). The elements of TAFI structure that allow accelerated activation of thrombin by TM are largely unknown. Therefore, alanine scanning mutagenesis of surface-exposed charged residues on TAFI was used to identify sites that mediate acceleration of activation by TM and which thus may indicate sites where TM binds to TAFI directly. The rates of activation by thrombin of the variants was measured in the presence or absence of TM. The variants R12A, E28A and R15A exhibited the lowest enhancement of catalytic efficiency in the presence of TM with decreases of 3.0-fold, 3.2-fold and 2.6-fold, respectively, compared to wild-type. The variants D75A/E77A/D78A, E106A, E112A/E116A, R12A/R15A and D54A/D56A showed about half the extent of rate enhancement of wild-type. On the other hand, the variants E99A and E116G exhibited approximately 2.0 to 2.5- fold increases in rate enhancement which may indicate increased binding between TAFI and TM. We determined the antifibrinolytic potential of each variant using an in vitro plasma clot lysis assay. The variants that showed reduced activation by thrombin/TM correspondingly showed impaired stimulation of antifibrinolytic potential by TM. Overall, the data indicate that the variants that showed the strongest TM dependence were focused around the activation peptide which would be near where the C-loop of EGF-3 of TM would contact TAFI. These residues contribute to the increased efficiency of TAFI activation by thrombin.