Design and synthesis of a multivalent catch-and-release assay to measure circulating FXIa.

Autor: van der Beelen SHE; Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, Maastricht, the Netherlands., Agten SM; Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, Maastricht, the Netherlands., Suylen DPL; Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, Maastricht, the Netherlands., Wichapong K; Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, Maastricht, the Netherlands., Hrdinova J; Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, Maastricht, the Netherlands., Mees BME; Vascular Surgery, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, Maastricht, the Netherlands., Spronk HMH; Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, Maastricht, the Netherlands; Internal Medicine, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, Maastricht, the Netherlands., Hackeng TM; Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, Maastricht, the Netherlands. Electronic address: t.hackeng@maastrichtuniversity.nl.
Jazyk: angličtina
Zdroj: Thrombosis research [Thromb Res] 2021 Apr; Vol. 200, pp. 16-22. Date of Electronic Publication: 2021 Jan 18.
DOI: 10.1016/j.thromres.2021.01.002
Abstrakt: Background: Decreased blood coagulation factor (F)XIa levels have been shown to protect from thrombosis without bleeding side effects, but less is known on effects of increased FXIa levels. Studies are hampered by lack of a reliable and robust method for FXIa quantification in blood. We aim to develop a new assay employing a unique multivalent catch-and-release system. The system selectively isolates and protects homodimeric FXIa from plasma and releases free FXIa allowing subsequent quantification.
Methods: A dynamic multivalent construct was synthesized by complexing four identical FXIa inhibitors from the snake Bungarus Fasxiatus to avidin through desthiobiotin-PEG-linkers, allowing dissociation of FXIa by excess biotin. PEG-linker lengths were optimised for FXIa inhibitory activity and analysed by Michaelis-Menten kinetics. Finally, the catch-and-release assay was validated in buffer and plasma model systems.
Results: Monovalent and multivalent inhibitor constructs were successfully obtained by total chemical synthesis. Multimerisation of Fasxiator resulted in a 30-fold increase in affinity for FXIa from 1.6 nM to 0.05 nM. With use of this system, FXIa could be quantified down to a concentration of 7 pM in buffer and 20 pM in plasma.
Conclusion: In this proof-of-concept study, we have shown that the catch-and-release approach is a promising technique to quantify FXIa in plasma or buffer. By binding FXIa to the multivalent construct directly after blood drawing, FXIa is hypothesized to be inaccessible for serpin inhibition or auto inactivation. This results in a close reflection of actual circulating FXIa levels at the moment of blood drawing.
(Copyright © 2021 Elsevier Ltd. All rights reserved.)
Databáze: MEDLINE
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