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Discovery of novel mechanisms and molecular targets for the inhibition of activated thrombin activatable fibrinolysis inhibitor
Author(s) -
HILLMAYER K.,
VANCRAENENBROECK R.,
DE MAEYER M.,
COMPERNOLLE G.,
DECLERCK P. J.,
GILS A.
Publication year - 2008
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/j.1538-7836.2008.03130.x
Subject(s) - fibrinolysis , thrombin , chemistry , lysis , epitope , in vitro , ic50 , tissue plasminogen activator , monoclonal antibody , plasminogen activator , microbiology and biotechnology , plasminogen activator inhibitor 1 , biochemistry , pharmacology , antibody , platelet , medicine , biology , immunology
Summary. Background : Thrombin activatable fibrinolysis inhibitor (TAFI) is an important regulator of fibrinolysis and an attractive target to develop profibrinolytic drugs. Objective : To analyze the (inhibitory) properties of five monoclonal antibodies (mAbs) directed towards rat TAFI (i.e. MA‐RT13B2, MA‐RT30D8, MA‐RT36A3F5, MA‐RT36B2 and MA‐RT82F12). Methods and results : Direct interference of the mAb with rat activated TAFI (TAFIa) activity was assayed using a chromogenic activity assay. This revealed reductions of 79% ± 1%, 54% ± 4%, and 19% ± 2% in activity in the presence of a 16‐fold molar excess of MA‐RT13B2, MA‐RT36A3F5, and MA‐RT82F12, respectively whereas MA‐RT30D8 and MA‐RT36B2 had no direct inhibitory effect. Additionally, MA‐RT13B2 and MA‐RT36A3F5 reduced rat TAFIa half‐life by 56% ± 2% and 61% ± 3%. Tissue‐type plasminogen activator mediated in vitro clot lysis was determined using rat plasma. Compared to potato tuber carboxypeptidase inhibitor, MA‐RT13B2, MA‐RT30D8, MA‐RT36A3F5, and MA‐RT82F12 reduced clot lysis times by 86% ± 14%, 100% ± 5%, 100% ± 10%, and 100% ± 11%, respectively. During epitope mapping, Arg 227 and Ser 251 were identified as major residues interacting with MA‐RT13B2. Arg 188 and His 192 contribute to the interaction with MA‐RT36A3F5. Arg 227, Ser 249 , Ser 251 , and Tyr 260 are involved in the binding of MA‐RT30D8 and MA‐RT82F12 with rat TAFI(a). The following mechanisms of inhibition have been deduced: MA‐RT13B2 and MA‐RT36A3F5 have a destabilizing effect on rat TAFIa whereas MA‐RT30D8 and MA‐RT82F12 partially block the access to the active site of TAFIa or interact with the binding of TAFIa to the blood clot. Conclusions : The described inhibitory mAb towards rat TAFIa will facilitate TAFI research in murine models. Additionally, we reveal novel molecular targets for the direct inhibition of TAFIa through different mechanisms.