Identification of a binding site for blood coagulation factor IXa on the light chain of human factor VIII.

Lenting, Peter J.; Donath, M.-J. S. H.; Mourik, Jan A. van; Mertens, Koen

doi:10.1016/s0021-9258(17)37260-5

Cited by 161 publications

(19 citation statements)

References 43 publications

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“…In contrast, the presence of FVIII mediated a greater TH than emicizumab at 4 min perfusion time. After activation, the affinities of FVIII to FIXa and FX ( K d 3.6–300 nM, 32‐35 0.165–3 µM, 36‐39 respectively) have been reported to be much higher than those of emicizumab ( K d 1.52 μM, 1.85 μM, respectively), 31 and consequently, these differences could be reflected in the final thrombus size.…”

Section: Discussionmentioning

confidence: 99%

Emicizumab improves thrombus formation of type 2A von willebrand disease under high shear condition

Yaoi

Shida

Kitazawa³

et al. 2021

Haemophilia

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factor (VWF) plays a crucial role in both physiological haemostasis and pathological arterial thrombosis. 1 At the high shear rates, VWF mediates the initial tethering of platelets to the injured vessel surface by bridging platelet receptor glycoprotein (GP) Ib and subendothelial collagen. 1 This initial platelet adhesion on the subendothelial surface leads to platelet activation and conformational

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Section: Discussionmentioning

confidence: 99%

Emicizumab improves thrombus formation of type 2A von willebrand disease under high shear condition

Yaoi

Shida

Kitazawa³

et al. 2021

Haemophilia

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“…Several groups have provided a wealth of biochemical data on the intermolecular contacts between fVIIIa and fIXa, enhancing our understanding of how the Xase complex is formed and regulated. [27][28][29][30][31][32][33] Calculating an ab initio molecular envelope of the Xase:ND complex allowed us to computationally dock fVIIIa to fIXa (Figure 3F) and investigate the feasibility of previously proposed protein-protein interactions as well as speculate on novel points of contact (Figure 4). Formation of the Xase complex is not predicted to alter the redox states of the disulfide bridges in fVIIIa or fIXa, 78 limiting any conformational rearrangements to flexible linkers between domains.…”

Section: Fviiia Binds To Fixa Via the A2 A3 And C2 Domainsmentioning

confidence: 99%

“…[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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“…Plasma-derived FIX was purified as described elsewhere [16]. Factor VIII (FVIII) was purified as outlined previously [17]. FX was purified as described in [18].…”

Section: Proteinsmentioning

confidence: 99%

“…The monoclonal anti-FIX antibody CLB-FIX 14 has been described in [19]. Polyclonal antibodies against FIX were obtained as described in [17].…”

Section: Proteinsmentioning

confidence: 99%

Surface-loop residue Lys316 in blood coagulation Factor IX is a major determinant for Factor X but not antithrombin recognition

KOLKMAN¹,

MERTENS²

2000

Biochem. J.

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The active site of activated Factor IX (FIXa) and related blood-coagulation enzymes is surrounded by a number of highly variable surface loops, which contribute to the characteristic substrate specificity of each individual enzyme. FIX residue Lys(316) is located in one of these loops and mutation of this residue to Glu is associated with haemophilia B. In the present study we investigated the functional role of Lys(316) in human FIXa by analysing the purified and activated FIX mutants FIXa-K316E and FIXa-K316A. FIXa-K316E was indistinguishable from normal FIXa in binding the competitive active-site inhibitor p-aminobenzamidine. In addition, substitution of Glu for Lys(316) had no significant effect on the reactivity towards various synthetic tripeptide substrates. Inhibition by the macromolecular inhibitor antithrombin was only slightly reduced for both FIXa mutants (less than 2-fold). In contrast, proteolytic activity of FIXa-K316E towards the natural substrate Factor X (FX) was virtually lacking, while the Lys(316) to Ala mutation resulted in a more than 10-fold reduction in FX activation. Thus residue Lys(316) plays a key role in FIXa activity towards FX. The requirement for Lys at position 316 for FX activation was also evident in the presence of the cofactor activated Factor VIII, although to a lesser extent than in its absence. These data demonstrate that Lys(316) specifically determines the reactivity of FIXa towards its natural substrate FX, but not to synthetic peptide substrates or antithrombin.

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Identification of a binding site for blood coagulation factor IXa on the light chain of human factor VIII.

Cited by 161 publications

References 43 publications

Emicizumab improves thrombus formation of type 2A von willebrand disease under high shear condition

Emicizumab improves thrombus formation of type 2A von willebrand disease under high shear condition

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

Surface-loop residue Lys316 in blood coagulation Factor IX is a major determinant for Factor X but not antithrombin recognition

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