A3 Domain Region 1803–1818 Contributes to the Stability of Activated Factor VIII and Includes a Binding Site for Activated Factor IX

Bloem, Esther; Meems, Henriet; Biggelaar, Maartje van den; Mertens, Koen; Meijer, Alexander B.

doi:10.1074/jbc.m113.500884

Cited by 13 publications

(14 citation statements)

References 37 publications

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“…The K m values (1.50 ± 0.20 and 1.29 ± 0.18 nmol L −1 , respectively) for FIXa with WT and P1809L mutant obtained were similar (data not shown). The finding suggested that P1809L mutation appeared not to affect the FVIII and FIXa association, likely supporting the recent report on the A3 domain (residues 1811–1818, particular Phe1816)–FIXa interaction , and was unlikely to have disturbed the FIXa‐binding sites on the A3 and C2 molecules.…”

Section: Resultssupporting

confidence: 83%

Mild hemophilia A patient with novel Pro1809Leu mutation develops an anti‐C2 antibody inhibiting allogeneic but not autologous factor VIII activity

Yada

Nogami

Takeyama

et al. 2015

Journal of Thrombosis and Haemostasis

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M. Mild hemophilia A patient with novel Pro1809Leu mutation develops an anti-C2 antibody inhibiting allogeneic but not autologous factor VIII activity. J Thromb Haemost 2015; 13: 1843-53.Summary. Background: In mild hemophilia A (MHA) patients, the risk of inhibitor development is generally low, but some factor VIII (FVIII) gene missense mutations are associated with a higher inhibitor incidence. Objective: To investigate the mechanism(s) of inhibitor development in MHA. Methods and results: A patient, HA78, with MHA with a novel P1809L missense mutation in the A3 domain, exhibited significant residual FVIII activity (FVIII:C 10 IU dL À1 ), despite the development of an inhibitor (5.6 BU mL À1). Purified HA78-IgG significantly depressed FVIII:C from normal plasma but not from patient's plasma without inhibitor, indicating that this IgG inhibited allogeneic but not autologous FVIII. The HA78-IgG blocked thrombin and FXa-catalyzed FVIII cleavage but had little effect on FVIII binding to von Willebrand factor and phospholipid. The IgG recognized a C2 epitope close or overlapping the previously described anti-C2 ESH8 epitope. Similarly, a recombinant FVIII-P1809L mutant was little inactivated by HA78-IgG. This mutant demonstrated 3-fold lower binding affinities to von Willebrand factor and phospholipid compared with wild-type, while reactions with thrombin or FXa were not impaired. Reaction of FVIII-P1809L with the alternative anti-C2 ESH4 showed only an~20% inhibition compared with wild-type FVIII but was similar to wild-type after incubation with ESH8. A surface plasmon resonance-based assay demonstrated that anti-C2 ESH4 bound to FVIII-P1809L with~10 2 -fold lower affinity compared with ESH8. Conclusion: These results indicated that the P1809L mutation in A3 induced the conformational change in the FVIII molecule that hampered antigenic determinant(s) located in the C2 domain and might result in the inhibitor development.

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Section: Resultssupporting

confidence: 83%

Mild hemophilia A patient with novel Pro1809Leu mutation develops an anti‐C2 antibody inhibiting allogeneic but not autologous factor VIII activity

Yada

Nogami

Takeyama

et al. 2015

Journal of Thrombosis and Haemostasis

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“…A number of site-specific mutagenesis and domain-swapping studies have been reported in order to understand the functional role of various domains of FVIIIa and FIXa involved in the tenase complex formation [7,17,18]. The A2 and A3 domains have been largely implicated to possess the binding interface with SP and EGF domains of FIXa respectively.…”

Section: Resultsmentioning

confidence: 99%

“…The EGF2 domain region surrounding Asn91 residue and the 1811-1818 sequence of A3 domain were suggested to be involved in FVIIIa.FIXa interactions [17,22]. As shown in Table 1 , the side-chain of Lys91 of EGF2 domain makes hydrogen-bonding interactions with the side-chains of Thr1695, Lys1693 and Asn1770.…”

Section: Resultsmentioning

confidence: 99%

Structural insights into the interaction of blood coagulation co-factor VIIIa with factor IXa: A computational protein–protein docking and molecular dynamics refinement study

Venkateswarlu

2014

Biochemical and Biophysical Research Communications

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Coagulation factor X (FX) zymogen activation by factor IXa (FIXa) enzyme plays a critical role in the middle-phase of coagulation cascade. The activation process is catalytically inert and requires FIXa binding and complex formation with co-factor VIIIa (FVIIIa). In order to understand the structural details of the FVIIIa:FIXa complex, we employed knowledge-driven protein-protein docking and aqueous-phase MD refinement methods to develop a stable structural complex between FVIIIa and FIXa. The model shows that all four domains of FIXa wrap across FVIIIa that spans the co-factor binding surface of A2, A3 and C1 domains. The region surrounding the 558-helix of the A2-domain of FVIIIa is predicted to be the key interaction site with the helical segments of Lys293-Lys301 and Asp332-Arg338 residues of the serine-protease domain of FIXa. The hydrophobic helical stack between the GLA and EGF1 domains of FIXa is predicted to be primary interacting region with the A3-C2 domain interface of FVIIIa.

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“…Because of the high structural similarity between FVIII and factor V (FV), chimeric variants of FVIII and FV have been utilized in a number of studies to gain insight into the structure and function of FVIII and FV . Factor V (FV) is the cofactor for FXa in the coagulation cascade and can form together with FXa and procoagulant phospholipid membranes the prothrombinase complex that efficiently converts prothrombin into thrombin .…”

Section: Introductionmentioning

confidence: 99%

Unique surface‐exposed hydrophobic residues in the C1 domain of factor VIII contribute to cofactor function and von Willebrand factor binding

Przeradzka

Freato

Boon-Spijker

et al. 2020

Journal of Thrombosis and Haemostasis

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Background:The identity of the amino acid regions of factor VIII (FVIII) that contribute to factor IXa (FIXa) and von Willebrand factor (VWF) binding has not been fully resolved. Previously, we observed that replacing the FVIII C1 domain for the one of factor V (FV) markedly reduces VWF binding and cofactor function. Compared to the FV C1 domain, this implies that the FVIII C1 domain comprises unique surfaceexposed elements involved in VWF and FIXa interaction.Objective: The aim of this study is to identify residues in the FVIII C1 domain that contribute to VWF and FIXa binding. Methods:Structures and primary sequences of FVIII and FV were compared to identify surface-exposed residues unique to the FVIII C1 domain. The identified residues were replaced with alanine residues to identify their role in FIXa and VWF interaction. This role was assessed employing surface plasmon resonance analysis studies and enzyme kinetic assays.Results: Five surface-exposed hydrophobic residues unique to the FVIII C1 domain, ie, F2035, F2068, F2127, V2130, I2139 were identified. Functional analysis indicated that residues F2068, V2130, and especially F2127 contribute to VWF and/or FIXa interaction. Substitution into alanine of the also surface-exposed V2125, which is spatially next to F2127, affected only VWF binding. Conclusion:The surface-exposed hydrophobic residues in C1 domain contribute to cofactor function and VWF binding. These findings provide novel information on the fundamental role of the C1 domain in FVIII life cycle. K E Y W O R D Sfactor VIII, factor IXa, kinetics, surface plasmon resonance, Von Willebrand factor | 365 PRZERADZKA Et Al.

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A3 Domain Region 1803–1818 Contributes to the Stability of Activated Factor VIII and Includes a Binding Site for Activated Factor IX

Cited by 13 publications

References 37 publications

Mild hemophilia A patient with novel Pro1809Leu mutation develops an anti‐C2 antibody inhibiting allogeneic but not autologous factor VIII activity

Mild hemophilia A patient with novel Pro1809Leu mutation develops an anti‐C2 antibody inhibiting allogeneic but not autologous factor VIII activity

Structural insights into the interaction of blood coagulation co-factor VIIIa with factor IXa: A computational protein–protein docking and molecular dynamics refinement study

Unique surface‐exposed hydrophobic residues in the C1 domain of factor VIII contribute to cofactor function and von Willebrand factor binding

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