To cite this article: Buelens K, Hassanzadeh-Ghassabeh G, Muyldermans S, Gils A, Declerck PJ. Generation and characterization of inhibitory nanobodies towards thrombin activatable fibrinolysis inhibitor. J Thromb Haemost 2010; 8: 1302-12.Summary. Background and objective: As activated thrombinactivatable fibrinolysis inhibitor (TAFIa) is a potent antifibrinolytic enzyme, the development of TAFI inhibitors is a new promising approach in the development of profibrinolytic drugs. We, therefore, aimed to generate nanobodies, camelidderived single-domain antibodies towards TAFI. Methods and results: This study reports the generation and characterization of a panel of 22 inhibitory nanobodies. This panel represents a wide diversity in mechanisms for interference with the functional properties of TAFI as the nanobodies interfere with various modes of TAFI activation, TAFIa activity and/or TAFI zymogen activity. Nanobodies inhibiting TAFIa activity and thrombin/thrombomodulin-mediated TAFI activation revealed profibrinolytic properties in a clot lysis experiment with exogenously added thrombomodulin (TM), whereas nanobodies inhibiting plasmin-mediated TAFI activation only revealed profibrinolytic properties in a clot lysis experiment without TM. The results of in vitro clot lysis experiments provided evidence that inhibitory nanobodies penetrate the clot better compared with inhibitory monoclonal antibodies. Conclusions: These data suggest that the generated nanobodies are potent TAFI inhibitors and are a step forward in the development of a profibrinolytic drug. They might also be an excellent tool to unravel the role of the physiological activators of TAFI in various pathophysiological processes.

Abstract-Activated Thrombin Activatable Fibrinolysis Inhibitor (TAFIa) exerts an antifibrinolytic effect by removing C-terminal lysines from partially degraded fibrin. These lysines are essential for a rapid conversion of plasminogen to plasmin by tissue type plasminogen activator. TAFI is heavily glycosylated at Asn 22 , Asn 51 , Asn 63 , and Asn 86 . Although the glycans occurring at the glycosylation sites have previously been identified, the biochemical role of these glycans is not known yet. Therefore, we have determined the biochemical importance of the glycosylation in TAFI. Four single, 6 double, 4 triple, and 1 quadruple mutant, in which asparagine was replaced by glutamine, were constructed and transfected into HEK293T cells. Based on the determination of antigen and activity levels on conditioned medium, 4 single and 1 triple mutant were purified and their biochemical properties were determined. The glycosylation knockout mutants did neither reveal an altered fragmentation pattern nor differences in TAFIa stability, but TAFI-N51Q, TAFI-N63Q, and TAFI-N22Q-N51Q-N63Q revealed a decreased TAFIa activity, an increased intrinsic catalytic activity of the zymogen, and a decreased antifibrinolytic potential compared with TAFI-wild-type, whereas TAFI-N22Q and TAFI-N86Q revealed an increased antifibrinolytic potential probably because of an increased catalytic efficiency toward the physiological substrate. From these data it can be concluded that mainly the glycosylation at Asn 86 contributes to the biochemical characteristics of TAFI. Furthermore we provide evidence that the activation peptide stays in close proximity to the TAFIa moiety after activation.

Summary. Background: Because activated thrombin activatable fibrinolysis inhibitor (TAFIa) has very powerful antifibrinolytic properties, co-administration of t-PA and a TAFIa inhibitor enhances t-PA treatment. Objective: We aimed to generate nanobodies specifically inhibiting the TAFIa activity and to test their effect on t-PA induced clot lysis. Results: Five nanobodies, raised towards an activated more stable TAFIa mutant (TAFIa A ), are described. These nanobodies inhibit specifically TAFIa activity, resulting in an inhibition of up to 99% at a 16-fold molar excess of nanobody over TAFIa, IC 50 Õs range between 0.38-and > 16-fold molar excess. In vitro clot lysis experiments in the absence of thrombomodulin (TM) demonstrate that the nanobodies exhibit profibrinolytic effects. However, in the presence of TM, one nanobody exhibits an antifibrinolytic effect whereas the other nanobodies show a slight antifibrinolytic effect at low concentrations and a pronounced profibrinolytic effect at higher concentrations. This biphasic pattern was highly dependent on TM and t-PA concentration. The nanobodies were found to bind in the active-site region of TAFIa and their timedependent differential binding behavior during TAFIa inactivation revealed the occurrence of a yet unknown intermediate conformational transition. Conclusion: These nanobodies are very potent TAFIa inhibitors and constitute useful tools to accelerate fibrinolysis. Our data also demonstrate that the profibrinolytic effect of TAFIa inhibition may be reversed by the presence of TM. The identification of a new conformational transition provides new insights into the conformational inactivation of the unstable TAFIa.

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.

The formation of thiadiazolidines 3a‐c and dithiazolidines 4a‐c and 5a‐b from the title reactions has been studied in detail under a variety of conditions. On the basis of kinetic measurements, isomerization studies and cross experiments a mechanism is proposed involving path (b) (Scheme 1) as the first step, followed by a series of isomerizations as shown in Scheme 3.