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
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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 of TAFIa transiently in response to sublethal or lethal doses of E. coli. Interestingly, baboons generated less TAFIa when infused with thrombin followed by the blocking of endothelial protein C receptor (EPCR) than when the baboons were infused with only thrombin. Human patients that underwent hip replacement surgery showed sustained, elevated levels of TAFIa for up to 42 days after surgery. The third objective was to determine the kinetics of TAFIa-catalyzed release of bound plasminogen from soluble fibrin degradation products. The kinetics conformed to the Michaelis-Menten equation, which allowed the determination of Km, kcat and Vmax values for varying concentrations of TAFIa. In addition, the enzymatic activity of TAFIa appeared to saturate, probably because at a high concentration of TAFIa, the kinetics of plasminogen release were rate limiting.