Probing activation‐driven changes in coagulation factor IX by mass spectrometry

Freato, Nadia; Alphen, Floris P.J. van; Boon-Spijker, Mariëtte; Biggelaar, Maartje van den; Meijer, Alexander B.; Mertens, Koen; Ebberink, Eduard H T M

doi:10.1111/jth.15288

Cited by 8 publications

(7 citation statements)

References 54 publications

(138 reference statements)

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“…The overall conversion rate of small peptide‐like substrates by FIXa seems to be relatively low [84] . FIX plasma activity levels can be measured in either an activated partial thromboplastin time (APTT)‐based one‐stage assay or a chromogenic assay [85] .…”

Section: Coagulation and Fibrinolysis – Proteasesmentioning

confidence: 99%

“…The overall conversion rate of small peptide-like substrates by FIXa seems to be relatively low. [84] FIX plasma activity levels can be measured in either an activated partial thromboplastin time (APTT)-based one-stage assay or a chromogenic assay. [85] This assay employs FXIa as a FIX activator, followed by the formation of the tenase complex with FVIIIa and anionic PL detection of generated FXa by a caged substrate.…”

Section: Factor Ixamentioning

confidence: 99%

See 1 more Smart Citation

Activity Sensing of Coagulation and Fibrinolytic Proteases

Sondag¹,

Verhoeven²,

Löwik³

et al. 2023

Chemistry A European J

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The blood coagulation cascade is a complex physiological process involving the action of multiple coupled enzymes, cofactors, and substrates, ultimately leading to clot formation. Serine proteases have a crucial role, and aberrations in their activity can lead to life-threatening bleeding disorders and thrombosis. This review summarizes the essential proteases involved in blood coagulation and fibrinolysis, the endogenous peptide sequences they recognize and hydrolyze, and synthetic peptide probes based on these sequences to measure their activity. The information in this review can contribute to developing novel anticoagulant therapies and specific substrates for point-of-care diagnosis of coagulation pathologies.

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Section: Coagulation and Fibrinolysis – Proteasesmentioning

confidence: 99%

Section: Factor Ixamentioning

confidence: 99%

Activity Sensing of Coagulation and Fibrinolytic Proteases

Sondag¹,

Verhoeven²,

Löwik³

et al. 2023

Chemistry A European J

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show abstract

“…[25][26][27][28] Structural studies on the Xase complex have proposed multiple binding sites between the fVIIIa A2, A3, and C2 domains, and fIXa catalytic and Gla domains. [29][30][31][32][33][34][35] A putative allosteric network in the fIXa catalytic domain between the 99-loop and a cluster of solvent-exposed helices, collectively described as exosite II, has been proposed to modulate the conformation of the 99loop 36,37 and is a potential binding site for fVIIIa. 31,38 The crystal structure of the prothrombinase complex, formed by factors Va/Xa, which are homologous to fVIIIa/fIXa, respectively, suggests the catalytic domain docks onto the A2 and A3 domains.…”

Section: Introductionmentioning

confidence: 99%

SAXS analysis of the intrinsic tenase complex bound to a lipid nanodisc highlights intermolecular contacts between factors VIIIa/IXa

et al. 2022

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The intrinsic tenase (Xase) complex, formed by factors (f)VIIIa and fIXa, forms on activated platelet surfaces and catalyzes the activation of factor X to Xa, stimulating thrombin production in the blood coagulation cascade. The structural organization of the membrane-bound Xase complex remains largely unknown, hindering our understanding of the structural underpinnings that guide Xase complex assembly. Here, we aimed to characterize the Xase complex bound to a lipid nanodisc with biolayer interferometry (BLI), Michaelis-Menten kinetics, and small angle X-ray scattering (SAXS). Using immobilized lipid nanodiscs, we measured binding rates and nanomolar affinities for fVIIIa, fIXa, and the Xase complex. Enzyme kinetic measurements demonstrated the assembly of an active enzyme complex in the presence of lipid nanodiscs. An ab initio molecular envelope of the nanodisc-bound Xase complex allowed us to computationally model fVIIIa and fIXa docked onto a flexible lipid membrane and identify protein-protein interactions. Our results highlight multiple points of contact between fVIIIa and fIXa, including a novel interaction with fIXa at the fVIIIa A1-A3 domain interface. Lastly, we identified hemophilia A/B-related mutations with varying severities at the fVIIIa/fIXa interface that may regulate Xase complex assembly. Together, our results support the use of SAXS as an emergent tool to investigate the membrane-bound Xase complex and illustrate how mutations at the fVIIIa/fIXa dimer interface may disrupt or stabilize the activated enzyme complex.

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“…[23][24][25][26] Structural studies on the Xase complex have proposed multiple binding sites between the fVIIIa A2, A3, and C2 domains, and fIXa catalytic and Gla domains. [27][28][29][30][31][32][33] A putative allosteric network in the fIXa catalytic domain between the 99-loop and a cluster of solvent-exposed helices, collectively described as exosite II, has been proposed to modulate the conformation of the 99loop 34,35 and is a potential binding site for fVIIIa. 29,36 The crystal structure of the prothrombinase complex, formed by factors Va/Xa, which are homologous to fVIIIa/fIXa, respectively, suggests the catalytic domain docks onto the A2 and A3 domains.…”

Section: Introductionmentioning

confidence: 99%

SAXS analysis of intrinsic tenase complex bound to lipid nanodisc highlights intermolecular contacts between factors VIIIa/IXa

Childers

Peters

Lollar

et al. 2021

Preprint

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The intrinsic tenase (Xase) complex, formed by factors (f)VIIIa and fIXa, forms on activated platelet surfaces and catalyzes the activation of factor X to Xa, stimulating thrombin production in the blood coagulation cascade. The structural organization of the membrane-bound Xase complex remains largely unknown, hindering our understanding of the structural underpinnings that guide Xase complex assembly. Here, we aimed to characterize the Xase complex bound to a lipid nanodisc with biolayer interferometry (BLI) and small angle X-ray scattering (SAXS). Using immobilized lipid nanodiscs, we measured binding rates and nanomolar affinities for fVIIIa, fIXa, and the Xase complex. An ab initio molecular envelope of the nanodisc-bound Xase complex allowed us to computationally model fVIIIa and fIXa docked onto a flexible lipid membrane and identify protein-protein interactions. Our results highlight multiple points of contact between fVIIIa and fIXa, including a novel interaction with fIXa at the fVIIIa A1-A3 domain interface. Lastly, we identified hemophilia A/B-related mutations with varying severities at the fVIIIa/fIXa interface that may regulate Xase complex assembly. Together, our results support the use of SAXS as an emergent tool to investigate the membrane-bound Xase complex and illustrate how mutations at the fVIIIa/fIXa dimer interface may disrupt or stabilize the activated enzyme complex.

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Probing activation‐driven changes in coagulation factor IX by mass spectrometry

Abstract: This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Cited by 8 publications

References 54 publications

Activity Sensing of Coagulation and Fibrinolytic Proteases

Activity Sensing of Coagulation and Fibrinolytic Proteases

SAXS analysis of the intrinsic tenase complex bound to a lipid nanodisc highlights intermolecular contacts between factors VIIIa/IXa

SAXS analysis of intrinsic tenase complex bound to lipid nanodisc highlights intermolecular contacts between factors VIIIa/IXa

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