An assay for measuring functional activated thrombin-activatable fibrinolysis inhibitor in plasma.
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 mediates a positive feedback mechanism in plasminogen (Pg) activation, thereby attenuating fibrinolysis. The plasma concentration of TAFI is approximately 75 nM. Because the half-maximal effect of TAFIa occurs at 1 nM, only approximately 1.3% of TAFI needs to be activated to exert an effect on clot lysis. The assay is performed by mixing soluble FN' covalently attached to a quencher and fluorescein-labeled Pg. The sample containing TAFIa is then added, and the rate of fluorescence increase due to removal of C-terminal lysine from FN' and loss of Pg binding is measured with a fluorescence plate reader. The assay was shown to be sensitive for TAFIa at a concentration as low as 12 pM. The intraassay variability and interassay variability of the assay were 6.3 and 8.3%, respectively. This assay was not confounded by the naturally occurring TAFI Thr325Leu polymorphism that affects the thermal stability of TAFIa or endogenous plasminogen in plasma.
Kim
Paula Y G
PY
Department of Biochemistry, Queen's University, Kingston, ON, K7L 3N6, Canada.
Foley
Jonathan
J
Hsu
Grace
G
Kim
Paul Y
PY
Nesheim
Michael E
ME
D025901:Carboxypeptidase B2 / Q000097:blood*; D019793:Fluorescein; D006801:Humans; D015203:Reproducibility of Results
D025901:Carboxypeptidase B2; D019793:Fluorescein