title: In vitro and in vivo characterisation of the profibrinolytic effect of an inhibitory anti-rat TAFI nanobody.
abstract: One of the main disadvantages of current t-PA thrombolytic treatment is the increased bleeding risk. Upon activation, thrombin activatable fibrinolysis inhibitor (TAFI) is a very powerful antifibrinolytic enzyme. Therefore, co-administration of a TAFI inhibitor during thrombolysis could reduce the required t-PA dose without compromising the thrombolytic efficacy. In this study we generated and characterised a nanobody that is inhibitory towards rat TAFI and evaluated its profibrinolytic property in vitro and in vivo. Nanobody VHH-rTAFI-i81 inhibits (at a 16-fold molar ratio nanobody over TAFI) the thrombin/thrombomodulin (T/TM)-mediated activation of rat TAFI (rTAFI) by 83 ± 1.8% with an IC50 of 0.46 (molar ratio nanobody over TAFI). The affinity (KA) of VHH-rTAFI-i81 for rTAFI, as determined by surface plasmon resonance (Biacore®), is 2.5 ± 0.2 x 10(10) M(-1) and illustrates a very strong binding. In an in vitro clot lysis assay, administration of VHH-rTAFI-i81 strongly enhances the degree of lysis and reduces time to reach full lysis of t-PA-mediated clot lysis. Epitope mapping discloses that Lys392 is of primary importance for the nanobody/rTAFI interaction besides minor contributions of Tyr175 and Glu183. In vivo application of VHH-rTAFI-i81 in a tissue factor-induced mouse thromboembolism model significantly decreases fibrin deposition in the lungs in the absence of exogenous administered t-PA. Nanobody VHH-rTAFI-i81 is a very potent inhibitor of T/TM-mediated TAFI activation. Co-administration of this nanobody and t-PA enhances the fibrinolytic efficacy. In an in vivo mouse thromboembolism model, VHH-rTAFI-i81 reduces fibrin deposition in the lungs.
authors.forename: Maarten L V
authors.initials: ML
authors.lastname: Hendrickx
authors.forename: Monika
authors.initials: M
authors.lastname: Zatloukalova
authors.forename: Gholamreza
authors.initials: G
authors.lastname: Hassanzadeh-Ghassabeh
authors.forename: Serge
authors.initials: S
authors.lastname: Muyldermans
authors.forename: Ann
authors.initials: A
authors.lastname: Gils
authors.affiliation: Paul Declerck, Laboratory for Therapeutic and Diagnostic Antibodies, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Campus Gasthuisberg, O&N2, PB 820, Herestraat 49, B-3000 Leuven, Belgium, Tel.: +32 16 32 34 31, Fax: +32 16 32 34 60, E-mail: paul.declerck@pharm.kuleuven.be.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Declerck
mesh_terms: D000818:Animals; D019138:Antibodies, Blocking / Q000235:genetics / Q000378:metabolism*; D025901:Carboxypeptidase B2 / Q000378:metabolism; D004195:Disease Models, Animal; D004359:Drug Therapy, Combination; D018604:Epitope Mapping; D005260:Female; D005337:Fibrin / Q000378:metabolism; D005343:Fibrinolytic Agents / Q000627:therapeutic use*; D006801:Humans; D066298:In Vitro Techniques; D008168:Lung / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D011485:Protein Binding; D051381:Rats; D061905:Single-Domain Antibodies / Q000008:administration & dosage* / Q000009:adverse effects / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013917:Thrombin / Q000378:metabolism; D013923:Thromboembolism / Q000188:drug therapy*; D018180:Thrombomodulin / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000627:therapeutic use
chemical_list: D019138:Antibodies, Blocking; D005343:Fibrinolytic Agents; D061905:Single-Domain Antibodies; D018180:Thrombomodulin; D005337:Fibrin; D025901:Carboxypeptidase B2; D013917:Thrombin; D010959:Tissue Plasminogen Activator
keywords: TAFI; TAFI inhibition; fibrinolysis; nanobody; profibrinolytic; thromboembolism