Abstract Number: PB0261
Meeting: ISTH 2020 Congress
Background: Reduced in vivo levels of FXI have been associated with a lower thrombosis risk in absence of an increased bleeding risk. However, it is not known if increased FXIa levels lead to a higher risk on thrombosis. Previous studies have described methods for indirect measurement of FXIa either by quantification of FXIa-inhibitor complexes or through thrombin generation based assays. However, complex ELISA’s can be influenced by contact activation during blood drawing, while thrombin generation assays are hindered by natural inhibitors of FXIa, resulting in a need for an improved assay to quantify FXIa in the circulation.
Aims: To developed an assay to measure the amount of FXIa present in blood, aiming at isolation of circulating FXIa homodimers through complexation with a multimeric form of the natural FXIa inhibitor Fasxiator.
Methods: Fasxiator (Bugarus Fasciatus: 62 amino acids; 3 disulfide bonds) was synthesized using tBoc-solid phase peptide synthesis protocols in two parts, subsequently ligated using native chemical ligation (NCL) and folded to obtain the native protein containing 3 disulfide bonds. The inhibitor was modified with a desthiobiotin tag to enable tetramer formation of Fasxiator with Avidin. In a proof of concept study desthiobiotin-Fasxiator was coupled to avidin and subsequently used to inhibit and isolate FXIa from plasma. To release FXIa, the multivalent enzyme-inhibitor complex was disrupted by incubation with biotin. Lineweaver Burk plots were made and Biacore assays were performed to study the kinetic parameters of FXIa inhibition.
Results: Multimerization of Fasxiator increased the affinity for FXIa 10-fold, which makes a tetrameric Fasxiator a suitable candidate for FXIa isolation. Using this assay set up We were able to measure FXIa down to 50 pM in blood plasma.
Conclusions: The so designed “catch and release” FXIa assay is a promising approach to isolate and measure FXIa in circulation.
To cite this abstract in AMA style:van der Beelen S, Suylen D, Mees B, Spronk H, Hackeng T. Quantification of Free FXIa by an Innovative “Catch and Release” Assay [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/quantification-of-free-fxia-by-an-innovative-catch-and-release-assay/. Accessed May 6, 2021.
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