A NEW FUNCTIONAL ASSAY FOR MEASURING PLASMA TAFIA AND ITS USE TO DEMONSTRATE TAFI ACTIVATION IN VIVO IN A PRIMATE MODEL
Abstract number: P-S-371
Kim1 P.Y., Nesheim2 M.E.
11Biochemistry 22Biochemistry and Medicine, Queen's University, Kingston, Canada
How-to-cite Kim PY, Nesheim ME. A NEW FUNCTIONAL ASSAY FOR MEASURING PLASMA TAFIA AND ITS USE TO DEMONSTRATE TAFI ACTIVATION IN VIVO IN A PRIMATE MODEL. J Thromb Haemost 2007; 5 Supplement 2: P-S-371
Introduction: Thrombin-activatable fibrinolysis inhibitor (TAFI) is a plasma zymogen that can be activated by the thrombin-thrombomodulin complex to form the enzyme, TAFIa, which attenuates fibrinolysis. We have developed a new assay for functional TAFIa activity in plasma and used it to measure TAFI activation in vivo in a primate model.
Methods: TAFIa activity was measured by quantifying the increase in fluorescence over time that occurs when fluorescein-labeled plasminogen is released from soluble fibrin degradation products (FDPs) covalently labeled with the fluorescence quencher QSY through the action of TAFIa on the FDPs. The measurement was performed in a fluorescence microtitre plate reader. The assay was standardized with purified TAFIa added to TAFI-deficient plasma. Chimpanzees were infused over a period of 30 seconds with a bolus of factor Xa and PCPS vesicles at various doses. Samples were obtained before infusion and at 2, 5, 10, 15, 30, 60, and 90 minutes post infusion. Samples were collected into PPAck and citrate. They were chilled and processed to plasma immediately and stored at -80°C.
Results: The assay is sensitive to functional plasma TAFIa over the concentration range of 12.5-200pM. Once the components are mixed, the time to complete the measurement is about 10-20 minutes. Prior to infusion of factor Xa and PCPS, the plasma level of TAFIa was 10237pM. Following infusion of factor Xa and PCPS, the TAFIa level rose to a peak by 10 minutes with levels varying from 700-6800pM, depending on dose of the procoagulant. It then declined with an average half life of 10.41.7 minutes. The TAFIa level was 558pM by 60-90 minutes.
Conclusions: The new assay is relatively easy to use and is very sensitive. The data with the chimpanzees demonstrates a profound and transient activation of TAFI in a primate model in response to a procoagulant stimulus.
To cite this abstract use the following format:
Journal of Thrombosis and Haemostasis 2007; Volume 5, Supplement 2: abstract number
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