Increased zymogen activity of thrombin-activatable fibrinolysis inhibitor prolongs clot lysis

Summary.  Background and objectives:  Thrombin-activatable fibrinolysis inhibitor (TAFI) is a zymogen that can be activated by proteolytic cleavage into the active enzyme TAFIa. Hydrolysis of the C-terminal lysines on fibrin by TAFIa results in a down-regulation of fibrinolysis. Recent studies demonstrated that the zymogen also exerts an intrinsic enzymatic activity. Our objective was to identify and characterize zymogen-stimulatory nanobodies. Methods and results:  The screening of 24 nanobodies against TAFI revealed that two nanobodies (i.e. Vhh-TAFI-a51 and Vhh-TAFI-i103) were able to stimulate the zymogen activity 10- to 21-fold compared with the baseline zymogen activity of TAFI. The increase in catalytic efficiency can be attributed mainly to an increased catalytic rate, as no change in the K(M) -value was observed. The stability, the susceptibility towards PTCI and GEMSA and the kinetics of the stimulated zymogen activity differ significantly from those of TAFIa activity. Epitope mapping revealed that both Asp(75) and Thr(301) are major determinants in the binding of these nanobodies to TAFI. Localization of the epitope strongly suggests that this instability is as a result of a disruption of the stabilizing interactions between the activation peptide and the dynamic flap region (residues 296-350). In TAFI-depleted plasma reconstituted with a non-activatable variant of TAFI (TAFI-R92A), clot lysis could be prolonged by nanobody-induced stimulation of its zymogen activity as well as by increasing its concentration. Conclusions:  Increasing the zymogen activity of TAFI results in an antifibrinolytic effect.

Jaar van publicatie:2012