Determination of TAFI in Plasma Using a New Chromogenic Method.

Abstract

TAFI (thrombin-activatable fibrinolysis inhibitor) is a proenzyme similar to carboxypeptidase B and is activated to TAFIa by thrombin and probably plasmin also. Thrombomodulin accelerates TAFI activation by thrombin about 1250-fold. TAFIa is an important inhibitor of fibrinolysis by cleaving C-terminal lysine and arginin residues in the fibrin clot. By this the carboxy-terminal plasminogen binding sites in the fibrin clot are degraded, plasminogen activation by t-PA and in consequence fibrinolysis is most effectively inhibited. Whether elevated TAFI levels correlate with the risk of thrombosis is currently under study. To clarify the role of TAFI in various diseases a simple and rapid assay for determination of TAFI concentration in plasma is desirable. With Pefakit® TAFI we developed a new chromogenic method for determination of TAFI in plasma samples.Method: TAFI is activated by thrombin/thrombomodulin complex in the presence of calcium ions. The activated TAFI cleaves a 3-thia-arginine peptide substrate whose cleavage products subsequently react with Ellman's reagent. Color development is followed up by measuring absorption at 405 nm. The method is the result of synthetic work and substrate screening. The assay was optimized to determine TAFI in human plasma. The concentration of TAFI in plasma sample corresponds to the initial reaction rate of substrate cleavage. TAFI level in each sample can be calculated from a linear standard curve.Results TAFI was measured in plasma of about 50 patients. The plasma levels of TAFI determined with Pefakit® TAFI correlate well with a commercially available chromogenic assay (Actichrome TAFI).Conclusion: Pefakit® TAFI is a fast and precise method to determine TAFI concentration in plasma. There is a linear correlation between TAFI concentration and measuring signal. Because of its simple practicability the assay can be adapted to most of the common automated analyzers for routine application.