A Novel TAFIa Assay and its Use to Measure TAFI Activation in vivo in Primate Models and the Determination of the Kinetics of TAFIa-Catalyzed Release of Bound Plasminogen from Soluble Fibrin Degradation Products

Thrombin-activatable fibrinolysis inhibitor (TAFI), also called procarboxypeptidase U (proCPU), is a plasma zymogen that can be activated by thrombin, the thrombin-thrombomodulin complex, or plasmin. The activated form of TAFI (TAFIa, CPU) removes C-terminal lysine residues of plasmin-modified fibrin (FN’) that mediate positive feedback in plasminogen (Pg) activation, thereby attenuating fibrinolysis. The plasma concentration of TAFI is ~75nM. Since the half-maximal effect of TAFIa occurs at 1nM, only about 1.3 % of TAFI needs to be activated to exert an effect on clot lysis. The first objective of this work was to design a novel assay that can measure functional TAFIa levels in plasma. We have successfully developed a reliable TAFIa assay that is sensitive to TAFIa concentrations as low as 12pM. This assay is not confounded by endogenous levels of Pg or by a naturally occurring polymorphism at position 325 that affects the half-life of TAFIa. The second objective was to measure TAFI activation in vivo in primates in response to thrombin generation, E. coli-induced sepsis, or post-surgical stress. It was found that chimpanzees generated substantial amounts of TAFIa in a transient manner which had a half-life of ~8 to 10 minutes in response to thrombin generation. Baboons generated very high levels o