A functional assay for measuring activated thrombin-activatable fibrinolysis inhibitor in plasma.
Thrombin-activatable fibrinolysis inhibitor (TAFI) is a procarboxypeptidase found in plasma that is activated by thrombin, the thrombin-thrombomodulin complex, or plasmin. The active carboxypeptidase, TAFIa, attenuates fibrinolysis by removing newly exposed carboxy-terminal lysine residues on fibrin. The half-maximal effect of TAFIa on clot lysis occurs at 1 nM and the maximal effect occurs at 20 nM. Since the circulating concentration of the procarboxypeptidase is approximately 75 nM, only a small portion needs to be activated to have a significant effect on clot lysis. Several assays to measure total plasma TAFI levels and plasma TAFIa levels after it is fully activated exist. However, no currently available assay is sufficiently sensitive and specific to measure endogenous TAFIa in plasma. We have devised a new sensitive and specific assay for TAFIa in plasma that is based on physiologic function. This assay is based on the fact that TAFIa decreases the cofactor activity of high-molecular-weight fibrin degradation products in the stimulation of plasminogen cleavage in a concentration-dependent fashion. With this assay, we can measure TAFIa concentrations as low as 10 pM in plasma and it is not affected by variability in other hemostatic factors. This assay is reliable and repeatable with intra- and interassay variabilities of 6.5 and 6.1%, respectively.
Neill
Erin K T
EK
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada K7L 3N6.
Stewart
Ronald J
RJ
Schneider
Mark M
MM
Nesheim
Michael E
ME
D025901:Carboxypeptidase B2 / Q000097:blood*; D002850:Chromatography, Gel; D005338:Fibrin Fibrinogen Degradation Products / Q000378:metabolism; D006801:Humans; D010958:Plasminogen / Q000378:metabolism; D015203:Reproducibility of Results
D005338:Fibrin Fibrinogen Degradation Products; D010958:Plasminogen; D025901:Carboxypeptidase B2