Characterization of TAFI Activation by the Thrombin-Thrombomodulin Complex
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Once activated, protein C (PC) and thrombin-activable fibrinolysis inhibitor (TAFI) initiate anticoagulant and anti-fibrinolytic pathways, respectively. PC and TAFI are activated by the thrombin-thrombomodulin (TM) complex. Endothelial PC receptor (EPCR), which binds PC, enhances the activation of PC, but not TAFI. We investigated PC and TAFI activation on cultured human endothelial cells (HUVEC) in the absence or presence of an antibody against EPCR. In the absence of antibody, PC is the favoured substrate of the thrombin-TM complex. In contrast, in the presence of antibody, PC activation is reduced to a level similar to that of TAFI, suggesting that EPCR acts as a molecular switch that influences substrate preference. PC does not compete with TAFI for activation and vice versa, raising the possibility that the two proteins interact with distinct populations of thrombin-TM complexes on endothelial cell surface. Based on our previous work, the TM-dependence of TAFI activation by thrombin is mediated through exosite interactions involving a positively-charged patch surrounding Lys 42, Lys 43, and Lys 44 on TAFI. Using TAFI variants with one, two or three of these Lys residues replaced with Ala, we determined that each individual Lys residue contributes equally to overall TAFI activation by thrombin-TM and that they act in a cooperative fashion to promote activation. This cooperative interaction is evident in lysis assays where threshold-like behaviour is evident; thus, the anti-fibrinolytic effect of TAFIa is only attenuated when its generation falls below this threshold. Capitalizing on this Lys-dependent interaction of TAFI with thrombin-TM, we synthesized 10-amino acid peptide analogs of this region. Analogs with one, two or three Lys residues progressively attenuated TAFI activation by thrombin-TM. This was a specific effect because (a) the peptide analogs had no effect on PC activation, (b) the inhibitory effect was lost if the three Lys residues were replaced with Thr, and (c) epsilon amino-caproic acid, a Lys analog, had no effect. These studies not only suggest that lysine residues at 42/43/44 comprise an exosite that mediates the interaction of TAFI, but not PC, with thrombin-TM, but also identify novel reagents that have potential antithrombotic activity by modulating TAFI activation.