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DOI: 10.1055/s-0040-1721422
Hydrogen–Deuterium Exchange Mass Spectrometry Identifies Activated Factor IX-Induced molecular Changes in Activated Factor VIII
Funding This study has been funded by Landsteiner Stichting voor Bloedtransfusie Research (LSBR 1417).Abstract
Hydrogen–deuterium exchange mass spectrometry (HDX-MS) was employed to gain insight into the changes in factor VIII (FVIII) that occur upon its activation and assembly with activated factor IX (FIXa) on phospholipid membranes. HDX-MS analysis of thrombin-activated FVIII (FVIIIa) revealed a marked increase in deuterium incorporation of amino acid residues along the A1–A2 and A2–A3 interface. Rapid dissociation of the A2 domain from FVIIIa can explain this observation. In the presence of FIXa, enhanced deuterium incorporation at the interface of FVIIIa was similar to that of FVIII. This is compatible with the previous finding that FIXa contributes to A2 domain retention in FVIIIa. A2 domain region Leu631-Tyr637, which is not part of the interface between the A domains, also showed a marked increase in deuterium incorporation in FVIIIa compared with FVIII. Deuterium uptake of this region was decreased in the presence of FIXa beyond that observed in FVIII. This implies that FIXa alters the conformation or directly interacts with this region in FVIIIa. Replacement of Val634 in FVIII by alanine using site-directed mutagenesis almost completely impaired the ability of the activated cofactor to enhance the activity of FIXa. Surface plasmon resonance analysis revealed that the rates of A2 domain dissociation from FVIIIa and FVIIIa-Val634Ala were indistinguishable. HDX-MS analysis showed, however, that FIXa was unable to retain the A2 domain in FVIIIa-Val634Ala. The combined results of this study suggest that the local structure of Leu631-Tyr637 is altered by FIXa and that this region contributes to the cofactor function of FVIII.
Keywords
hydrogen–deuterium exchange - factor VIII - factor IXa - A2 domain retention - interactive regionsAuthors' Contributions
J.v.G., N.F., M.B.-S, and M.A.P. performed the experiments; C.v.d.Z. provided technical assistance; J.v.G., N.F., M.v.d.B., and A.B.M. designed the experiments; E.E. assisted with setting up of HDX-MS experiments; J.v.G., N.F., M.A.P., and M.v.d.B. analyzed the results; N.F. and J.v.G. made the figures; and J.v.G., N.F., M.v.d.B., and A.B.M. wrote the article.
* Both authors contributed equally to this work.
Publication History
Received: 24 June 2020
Accepted: 21 October 2020
Article published online:
10 December 2020
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