Development of sandwich‐type ELISAs for the quantification of rat and murine thrombin activatable fibrinolysis inhibitor in plasma

Hillmayer, Kerstin; Brouwers, Els; León-Tamaríz, Fabián; Meijers, Joost C. M.; Marx, Pauline F.; Declerck, Paul; Gils, Ann

doi:10.1111/j.1538-7836.2007.02789.x

Cited by 10 publications

(11 citation statements)

References 27 publications

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“…Antibody-dependent assays suffer from affinity differences between the TAFI-325-Thr and Ile form, and activity-based assays from a difference in half-life. Nevertheless progress in this area over the past few years resulted in a polymorphism-independent activity-based assay [78,79], an assay for the direct measurement of functional TAFIa in plasma [80], the development of various new ELISAs specific for the various TAFI fragments (zymogen, activation peptide, TAFIa/TAFIai) [81] and TAFI from different species (human, mouse, rat) [82][83][84], and (global) fibrinolytic assays [85][86][87]. Also, testing of novel substrates resulted in more selective TAFIa substrates that distinguish between TAFIa and CPN [88].…”

Section: The Tafi Gene Polymorphisms and Tafi Expressionmentioning

confidence: 99%

Recent Developments in Thrombin-Activatable Fibrinolysis Inhibitor Research

Marx

Verkleij²,

Serón³

et al. 2009

MRMC

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Section: The Tafi Gene Polymorphisms and Tafi Expressionmentioning

confidence: 99%

Recent Developments in Thrombin-Activatable Fibrinolysis Inhibitor Research

Marx

Verkleij²,

Serón³

et al. 2009

MRMC

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“…Plasma was prepared by centrifugation of blood at 2000 × g for 20 min. Mouse plasma levels of TAFI and PAI‐1 were determined with previously described ELISAs [16,17]. An ELISA specific for the detection of free α 2 ‐antiplasmin was developed with mAbs directed towards human α 2 ‐antiplasmin that cross‐react with mouse α 2 ‐antiplasmin, i.e.…”

Section: Methodsmentioning

confidence: 99%

The hyperfibrinolytic state of mice with combined thrombin‐activatable fibrinolysis inhibitor (TAFI) and plasminogen activator inhibitor‐1 gene deficiency is critically dependent on TAFI deficiency

Vercauteren

Peeters

Hoylaerts

et al. 2012

Journal of Thrombosis and Haemostasis

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To cite this article: Vercauteren E, Peeters M, Hoylaerts MF, Lijnen HR, Meijers JCM, Declerck PJ, Gils A. The hyperfibrinolytic state of mice with combined thrombin-activatable fibrinolysis inhibitor (TAFI) and plasminogen activator inhibitor-1 gene deficiency is critically dependent on TAFI deficiency. J Thromb Haemost 2012; 10: 2555-62.Summary. Background: Mice with single gene deficiency of thrombin-activatable fibrinolysis inhibitor (TAFI) or plasminogen activator inhibitor-1 (PAI-1) have an enhanced fibrinolytic capacity. Objectives: To unravel the function and relevance of both antifibrinolytic proteins through the generation and characterization of mice with combined TAFI and PAI-1 gene deficiency. Results: Mating of TAFI knockout (KO) mice with PAI-1 KO mice resulted in the production of TAFI/PAI-1 double-KO mice that were viable, were fertile, and developed normally. In a tail vein bleeding model, the bleeding time and hemoglobin content of the TAFI/PAI-1 double-KO mice did not deviate significantly from those of the single-KO mice or of the wild-type (WT) counterparts. Interestingly, in ex vivo rotational thromboelastometry measurements with whole blood samples, TAFI KO mice and TAFI/PAI-1 double-KO mice were more sensitive to fibrinolytic activation with tissuetype plasminogen activator than WT or PAI-1 KO mice. This enhanced fibrinolytic capacity was confirmed in vivo in a mouse thromboembolism model, as shown by decreased fibrin deposition in the lungs of TAFI KO mice and TAFI/PAI-1 double-KO mice as compared with WT or PAI-1 KO mice. Conclusions: TAFI gene inactivation predominantly contributes to the increased fibrinolytic capacity of TAFI and PAI-1 double-gene-deficient mice, as observed in some basic thrombosis models.

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“…MA‐RT13B2, MA‐RT30D8, MA‐RT36A3F5, and MA‐RT36B2) or recombinant activated rat TAFI‐6His (i.e. MA‐RT82F12), as described previously [13,26].…”

Section: Methodsmentioning

confidence: 99%

“…Recombinant rat TAFI wild‐type (rat TAFI‐wt) and human TAFI (Thr 147 ‐Ile 325 ‐isoform) were produced and purified as described previously [13,26].…”

Section: Methodsmentioning

confidence: 99%

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

Hillmayer

Vancraenenbroeck

Maeyer

et al. 2008

Journal of Thrombosis and Haemostasis

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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. , 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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Development of sandwich‐type ELISAs for the quantification of rat and murine thrombin activatable fibrinolysis inhibitor in plasma

Abstract: We developed three highly specific and extremely sensitive sandwich-type ELISAs for the quantification of rat and murine TAFI in plasma. The described ELISAs will facilitate in vivo investigation on the pathophysiological role of TAFI.

Cited by 10 publications

References 27 publications

Recent Developments in Thrombin-Activatable Fibrinolysis Inhibitor Research

Recent Developments in Thrombin-Activatable Fibrinolysis Inhibitor Research

The hyperfibrinolytic state of mice with combined thrombin‐activatable fibrinolysis inhibitor (TAFI) and plasminogen activator inhibitor‐1 gene deficiency is critically dependent on TAFI deficiency

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

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