Thrombin Activatable Fibrinolysis Inhibitor (TAFI). Focus on Activated TAFI
Trombine Activeerbare Fibrinolyse Inhibitor (TAF). Focus op geactiveerd TAFI
Ceresa, Erik; M9716739
Activated Thrombin Activatable Fibrinolysis Inhibitor (TAFIa), formed upon activation of TAFI by thrombin/thrombomodulin, removes C-terminal lysines from partially degraded fibrin, thereby removing the cofactor function for t-PA mediated plasminogen activation. As a decreased rate of TAFI activation might contribute to bleeding disorders, an increased rate of TAFI activation might lead to thrombosis. This makes TAFI an interesting target for therapeutic intervention.In the past years, different TAFI inhibitors have been identified. However, these compounds often lack potency, specificity and consequently safety. In this context, monoclonal antibodies (MA) are better tools for therapeutic interaction with TAFI. We generated 19 MA with an inhibition up to 92%. Characterization of the inhibition mechanism revealed that 14 MA blocked the activation of TAFI whereas 5 MA inhibited the enzymatic activity. The activation inhibiting MA could reduce clot lysis timewhereas the activity inhibiting MA could not.Since 2000, several epidemiological studies have been investigating therole of TAFI in cardiovascular diseases such as thrombosis, infarction,stroke etc . but the outcome of these studies was inconclusive. ThreeELISAs with distinct reactivities were developed: recognizing either exclusively non-activated TAFI, the released activation peptide or exclusively activated TAFI. These ELISAs constitute more sensitive markers to study the relationship between TAFI and cardiovascular diseases.There are no known physiologic inhibitors of TAFIa. In contrast, TAFIa is down-regulated by a temperature dependent instability which is causedby a conformational change. This results in the loss of TAFIa activity within a few minutes (t1/2, 37°C = 6 min). This instability has compromised not only the studies on the structural-functional relationships of TAFI but has also prevented crystallization and consequently the development of structure based TAFI inhibitors. We used an ad random as well as a site-directed mutagenesis approach in order to stabilize the TAFIa enzyme. Combination of five mutations, Ser305Cys, Thr325Ile, Thr329Ile, His333Tyr and His335Gln, resulted in an 180-fold increased stability (t1/2,37°C = 19u) and in an 18-fold increased antifibrinolytic potential. In agreement with a conformational change, a quenching of the intrinsic fluorescence was found upon inactivation of TAFIa. This revealed that the conformational change is characterized by a fast and a slow transition. The availability of a more stable TAFIa variant will facilitate the search for inhibitors and allow further structural analysis to elucidate the mechanisms of the instability of TAFIa.