title: Identification and characterisation of monoclonal antibodies that impair the activation of human thrombin activatable fibrinolysis inhibitor through different mechanisms.
abstract: Thrombin activatable fibrinolysis inhibitor (TAFI) forms a molecular link between coagulation and fibrinolysis and is a putative target to develop profibrinolytic drugs. Out of a panel of monoclonal antibodies (MA) raised against TAFI-ACIIYQ, we selected MA-TCK11A9, MA-TCK22G2 and MA-TCK27A4, which revealed high affinity towards human TAFI-TI-wt. MA-TCK11A9 was able to inhibit mainly plasmin-mediated TAFI activation, MA-TCK22G2 inhibited plasmin- and thrombin-mediated TAFI activation and MA-TCK27A4 inhibited TAFI activation by plasmin, thrombin and thrombin/thrombomodulin (T/TM) in a dose-dependent manner. These MA did not interfere with TAFIa activity. Using an eight-fold molar excess of MA over TAFI, all three MA were able to reduce clot lysis time significantly, i.e. in the presence of exogenous TM, MA-TCK11A9, MA-TCK22G2 and MA-TCK27A4 reduced clot lysis time by 47 ± 9.1%, 80 ± 8.6% and 92 ± 14%, respectively, compared to PTCI. This effect was even more pronounced in the absence of TM i.e. MA-TCK11A9, MA-TCK22G2 and MA-TCK27A4 reduced clot lysis time by 90 ± 14%, 140 ± 12% and 147 ± 29%, respectively, compared to PTCI. Mutagenesis analysis revealed that residues at position 268, 272 and 276 are involved in the binding of MA-TCK11A9, residues 147 and 148 in the binding of MA-TCK22G2 and residue 113 in the binding of MA-TCK27A4. The present study identified three MA, with distinct epitopes, that impair the activation of human TAFI and demonstrated that MA-TCK11A9 which mainly impairs plasmin-mediated TAFI activation can also reduce significantly clot lysis time in vitro.
authors.affiliation: Laboratory for Pharmaceutical Biology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, O&N II, Leuven, Belgium.
authors.forename: Niraj
authors.initials: N
authors.lastname: Mishra
authors.forename: Ellen
authors.initials: E
authors.lastname: Vercauteren
authors.forename: Jan
authors.initials: J
authors.lastname: Develter
authors.forename: Riet
authors.initials: R
authors.lastname: Bammens
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Declerck
authors.forename: Ann
authors.initials: A
authors.lastname: Gils
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D000915:Antibody Affinity / Q000235:genetics; D025901:Carboxypeptidase B2 / Q000037:antagonists & inhibitors / Q000276:immunology*; D002460:Cell Line; D004789:Enzyme Activation / Q000187:drug effects; D018604:Epitope Mapping; D005341:Fibrinolysin / Q000378:metabolism; D005342:Fibrinolysis / Q000187:drug effects; D005343:Fibrinolytic Agents / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D051381:Rats; D016415:Sequence Alignment; D054556:Venous Thromboembolism / Q000097:blood / Q000188:drug therapy* / Q000201:enzymology*
chemical_list: D000911:Antibodies, Monoclonal; D005343:Fibrinolytic Agents; D025901:Carboxypeptidase B2; D005341:Fibrinolysin