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

Abstract

Summary. Background: Down-regulation of fibrinolysis due to cleavage of C-terminal lysine residues from partially degraded fibrin is mainly exerted by the carboxypeptidase activity of activated thrombin-activatable fibrinolysis inhibitor (TAFIa). Recently, some intrinsic carboxypeptidase activity (i.e. zymogen activity) was reported for the proenzyme (TAFI); however, there is some discussion about its ability to cleave high molecular weight substrates. Objective: We aimed to identify and characterize nanobodies toward mouse TAFI (mTAFI) that stimulate the zymogen activity and to test their effect in an in vitro clot lysis assay and an in vivo mouse thromboembolism model. Methods and results: Screening of a library of nanobodies toward mTAFI revealed one nanobody (VHH-mTAFI-i49) that significantly stimulates the zymogen activity of mTAFI from undetectable (< 0.35 U mg À1 ) to 4.4 U mg À1 (at a 16-fold molar ratio over mTAFI). The generated carboxypeptidase activity is unstable at 37°C. Incubation of mTAFI with VHHmTAFI-i49 revealed a time-dependent reduced activatability of mTAFI. Epitope mapping revealed that Arg227 and Lys212 are important for the nanobody/mTAFI interaction and suggest destabilization of mTAFI by disrupting the stabilizing interaction between the activation peptide and the dynamic flap region. In vitro clot lysis experiments revealed an enhanced clot lysis due to a reduced activation of mTAFI during clot formation. In vivo application of VHH-mTAFI-i49 in a mouse thromboembolism model decreased dose-dependently the fibrin deposition in the lungs of thromboembolism-induced mice. Conclusion: The novel, nanobody-induced, reduced activatability of mTAFI demonstrates to be a very potent approach to enhance clot lysis.

Measuring fibrinolysis: from research to routine diagnostic assays

“…Markers of ongoing fibrinolysis such as D‐dimer and plasmin‐α 2 AP (PAP) complexes are commonly used. Interest in PAI‐1 and TAFI as drug targets to modulate hemostasis has focused attention on the need for reliable assays .…”

Innovative thrombolytic strategy using a heterodimer diabody against TAFI and PAI-1 in mouse models of thrombosis and stroke

“…In a model of thromboplastin-induced thromboembolism, which is well established for the evaluation of profibrinolytic agents, 17,18,23,32 the diabody was tested to determine an effective dose. Because of the extremely low baseline levels of PAI-1 in mice, endotoxemic mice were used in this type of acute model to obtain a potential contributing effect of PAI-1 inhibition.…”

“…The thromboprophylactic capacity of the diabody was evaluated in a well-established mouse model of venous thromboembolism. 17,18,23,24 In addition, the effect of the diabody on brain ischemia/reperfusion injury was assessed in a mechanical transient middle cerebral artery occlusion (tMCAo) model in which brain lesion and neurologic/motor outcome were measured. Furthermore, the profibrinolytic capacity was evaluated in 2 stroke models in which in situ clots are induced in the vascular lumen of the middle cerebral artery (MCA) via a thrombin-and a ferric chloride (FeCl 3 )-induced MCA occlusion (MCAo) model.…”

Novel Thrombolytic Drug Based on Thrombin Cleavable Microplasminogen Coupled to a Single‐Chain Antibody Specific for Activated GPIIb/IIIa

“…This new thrombolytic agent also presents the advantage to, presumably, be unaffected by circulating TAFI and PAI‐1, which have been identified as major causal factors of fibrinolysis failure . Indeed, both of these fibrinolysis inhibitors inhibit or indirectly reduce the action of plasminogen activators, which we bypassed with our approach.…”