Factor VIII-driven changes in activated factor IX explored by hydrogen-deuterium exchange mass spectrometry.

Autor:	Freato N; Department of Molecular and Cellular Hemostasis, Sanquin Research, Amsterdam, The Netherlands; and., Ebberink EHTM; Department of Molecular and Cellular Hemostasis, Sanquin Research, Amsterdam, The Netherlands; and., van Galen J; Department of Molecular and Cellular Hemostasis, Sanquin Research, Amsterdam, The Netherlands; and., Fribourg C; Department of Molecular and Cellular Hemostasis, Sanquin Research, Amsterdam, The Netherlands; and., Boon-Spijker M; Department of Molecular and Cellular Hemostasis, Sanquin Research, Amsterdam, The Netherlands; and., van Alphen FPJ; Department of Molecular and Cellular Hemostasis, Sanquin Research, Amsterdam, The Netherlands; and., Meijer AB; Department of Molecular and Cellular Hemostasis, Sanquin Research, Amsterdam, The Netherlands; and.; Department of Biomolecular Mass Spectrometry and Proteomics, and., van den Biggelaar M; Department of Molecular and Cellular Hemostasis, Sanquin Research, Amsterdam, The Netherlands; and., Mertens K; Department of Molecular and Cellular Hemostasis, Sanquin Research, Amsterdam, The Netherlands; and.; Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, Utrecht, The Netherlands.
Jazyk:	angličtina
Zdroj:	Blood [Blood] 2020 Dec 03; Vol. 136 (23), pp. 2703-2714.
DOI:	10.1182/blood.2020005593
Abstrakt:	The assembly of the enzyme-activated factor IX (FIXa) with its cofactor, activated factor VIII (FVIIIa) is a crucial event in the coagulation cascade. The absence or dysfunction of either enzyme or cofactor severely compromises hemostasis and causes hemophilia. FIXa is a notoriously inefficient enzyme that needs FVIIIa to drive its hemostatic potential, by a mechanism that has remained largely elusive to date. In this study, we employed hydrogen-deuterium exchange-mass spectrometry (HDX-MS) to investigate how FIXa responds to assembly with FVIIIa in the presence of phospholipids. This revealed a complex pattern of changes that partially overlaps with those changes that occur upon occupation of the substrate-binding site by an active site-directed inhibitor. Among the changes driven by both cofactor and substrate, HDX-MS highlighted several surface loops that have been implicated in allosteric networks in related coagulation enzymes. Inspection of FVIIIa-specific changes indicated that 3 helices are involved in FIXa-FVIIIa assembly. These are part of a basic interface that is also known as exosite II. Mutagenesis of basic residues herein, followed by functional studies, identified this interface as an extended FVIIIa-interactive patch. HDX-MS was also applied to recombinant FIXa variants that are associated with severe hemophilia B. This revealed that single amino acid substitutions can silence the extended network of FVIIIa-driven allosteric changes. We conclude that HDX-MS has the potential to visualize the functional impact of disease-associated mutations on enzyme-cofactor complexes in the hemostatic system. (© 2020 by The American Society of Hematology.)
Databáze:	MEDLINE
Externí odkaz:	Zobrazit plný text záznamu