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

Abstract

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 KM‐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 Asp75 and Thr301 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.

HypE-specific Nanobodies as Tools to Modulate HypE-mediated Target AMPylation

“…A recent study characterized small molecule inhibitors of VopS; however, these molecules failed to rescue VopS-induced cell rounding and subsequent cell death in tissue culture assays (22). There are other examples of inhibiting or neutralizing V H Hs (35), but only a few V H Hs have shown stimulatory activity, such as a pair of V H Hs capable of increasing the zymogen activity of the carboxypeptidase TAFI (38). The HypE-specific V H Hs remain functional when expressed in HeLa cells, as indicated by co-localization with HypE.…”

Identification of a novel, nanobody‐induced, mechanism of TAFI inactivation and its in vivo application

“…Furthermore, TAFI zymogen activity can be induced by binding of nanobodies . Based on the current observations and our previous study , it is tempting to speculate that it should be feasible to raise nanobodies to other proenzymes (plasminogen, protein C, factors of the coagulation cascade, etc. ), resulting in similar effects (i.e.…”

“…The stability of the VHH-mTAFI-i49-induced zymogen activity was investigated through incubation of the mTAFI with VHH-mTAFI-i49 for 10 min at 25°C prior to incubation for different time periods (5,10,20,40,60, and 120 min) at 37°C. The residual activity was determined as described previously [15].…”

“…In addition to the intrinsic zymogen activity, Mishra et al . demonstrated that it is possible to enhance the zymogen activity of TAFI by nanobodies. Addition of these nanobodies to TAFI‐depleted plasma, which was reconstituted with a nonactivatable TAFI mutant, TAFI‐TI‐R92A, resulted in a significant prolongation of clot lysis time.…”

“…Foley et al [14] demonstrated that TAFI is effective in cleaving a small substrate, but due to its inability to cleave plasmin-modified fibrin, the authors concluded that it had no effect on the attenuation of fibrinolysis. In addition to the intrinsic zymogen activity, Mishra et al [15] demonstrated that it is possible to enhance the zymogen activity of TAFI by nanobodies. Addition of these nanobodies to TAFI-depleted plasma, which was reconstituted with a nonactivatable TAFI mutant, TAFI-TI-R92A, resulted in a significant prolongation of clot lysis time.…”