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J Thromb Haemost. 2008 Nov;6(11):1892-9. doi: 10.1111/j.1538-7836.2008.03130.x. Epub 2008 Aug 22.

Discovery of novel mechanisms and molecular targets for the inhibition of activated thrombin activatable fibrinolysis inhibitor.

Author information

1
Laboratory for Pharmaceutical Biology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Leuven.

Abstract

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.

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