Mutations associated with hemophilia A in the 558-565 loop of the factor VIIIa A2 subunit alter the catalytic activity of the factor Xase complex

Jenkins, P. Vincent; Freas, Jan; Schmidt, Kyla; Zhou, Qian; Fay, Philip J.

doi:10.1182/blood-2001-12-0361

Cited by 62 publications

(86 citation statements)

References 48 publications

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“…Factor Xase reconstituted using bA2 and A2 derived from thrombin-activated, BHK-expressed factor VIII exhibited similar K d values thus validating our experimental design. Furthermore, the values for K d for A2 binding and k cat for Xa generation using these reagents were similar to the parameter values obtained in the above study (20) using factor Xase formed with thrombin-activated WT factor VIIIa in the presence of phospholipid vesicles.…”

Section: Table 2 Inhibition Parameters For Reconstituted Factor Xase supporting

confidence: 66%

“…However, little experimental evidence is available regarding specific factor VIIIa residues that contribute to this effect. In an earlier report from our laboratory, Jenkins et al (20) examined several point mutations in the 558-loop follow- …”

Section: Discussionmentioning

confidence: 99%

“…According to this model, electrostatic, hydrophobic interactions and hydrogen bonds contribute to forming this interface. In an attempt to determine the role of the 558-loop toward cofactor activity of factor VIIIa, Jenkins et al (20) expressed and purified selected B-domainless factor VIII variants with point mutations in this loop as defined from the hemophilia A data base. Functional assays showed that the mutations affected cofactor potential by altering the catalytic rate constant of factor IXa, however, affinity values of the factor VIII variants for factor IXa remained largely unchanged, likely due to the primary factor IXa-binding interactions involving the A3C1C2 subunit of the cofactor.…”

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confidence: 99%

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Identification of Residues in the 558-Loop of Factor VIIIa A2 Subunit That Interact with Factor IXa

Jagannathan

Ichikawa

Kruger

et al. 2009

Journal of Biological Chemistry

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Factor VIIIa is comprised of A1, A2, and A3C1C2 subunits. Several lines of evidence have identified the A2 558-loop as interacting with factor IXa. The contributions of individual residues within this region to inter-protein affinity and cofactor activity were assessed following alanine scanning mutagenesis of residues 555-571 that border or are contained within the loop. Variants were expressed as isolated A2 domains in Sf9 cells using a baculovirus construct and purified to >90%. Two reconstitution assays were employed to determine affinity and activity parameters. The first assay reconstituted factor Xase using varying concentrations of A2 mutant and fixed levels of A1/A3C1C2 dimer purified from wild type (WT), baby hamster kidney cellexpressed factor VIII, factor IXa, and phospholipid vesicles to determine the inter-molecular K d for A2. The second assay determined the K d for A2 in factor VIIIa by reconstituting various A2 and fixed levels of A1/A3C1C2. Parameter values were determined by factor Xa generation assays. WT A2 expressed in insect cells yielded similar K d and k cat values following reconstitution as WT A2 purified from baby hamster kidney cell-expressed factor VIII. All A2 variants exhibited modest if any increases in K d values for factor VIIIa assembly. However, variants S558A, V559A, D560A, G563A, and I566A showed >9-fold increases in K d for factor Xase assembly, implicating these residues in stabilizing A2 association with factor IXa. Furthermore, variants Y555A, V559A, D560A, G563A, I566A, and D569A showed >80% reduction in k cat for factor Xa generation. These results identify residues in the 558-loop critical to interaction with factor IXa in Xase.Factor VIIIa is an essential blood coagulation protein that acts as a cofactor for the serine protease factor IXa in the conversion of factor X to Xa during the propagation phase of coagulation. Defects or deficiencies in factors VIII and IX result in hemophilia A and B, respectively. Factor VIII is synthesized as a multidomain, single chain precursor with a molecular mass of ϳ300 kDa and domain structure A1-A2-B-A3-C1-C2. It circulates primarily as a heterodimer resulting from proteolysis at the B-A3 junction wherein the heavy chain (A1A2B) and light chain (A3C1C2) are associated by metal ion-dependent and independent linkages (see Ref. 1 for review). Factor VIII is activated by limited proteolysis catalyzed by thrombin or factor Xa to the active cofactor, factor VIIIa. These cleavages convert the heterodimer or single chain precursor to the heterotrimer (2, 3) with the heavy chain-derived A1 subunit and light chain-derived A3C1C2 subunit maintaining stable association, whereas the A2 subunit is weakly associated with the A1/A3C1C2 dimer through electrostatic interactions (4, 5). Factor VIIIa can be reconstituted from the isolated A2 subunit and A1/A3C1C2 dimer to regenerate high levels of cofactor activity (3, 5).Association of factors VIIIa and IXa on a phospholipid surface forms the intrinsic factor Xase complex, which increases the cataly...

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Section: Table 2 Inhibition Parameters For Reconstituted Factor Xase supporting

confidence: 66%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Identification of Residues in the 558-Loop of Factor VIIIa A2 Subunit That Interact with Factor IXa

Jagannathan

Ichikawa

Kruger

et al. 2009

Journal of Biological Chemistry

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“…The selection, subcloning, and cloning of stable transfectants were done by standard methods, and the cloned cells were cultured in roller bottles (22). The conditioned medium was collected daily, and the expressed proteins were purified following chromatography on a SP-Sepharose as described previously (23).…”

Section: Methodsmentioning

confidence: 99%

“…Mutations were introduced into the shuttle constructs using the Stratagene QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) as described previously (22). Factor VIII molecules bearing cluster mutation of 341-345Ala, 347-349Ala, and 361-363Ala were constructed.…”

Section: Methodsmentioning

confidence: 99%

Identification of a Factor Xa-interactive Site within Residues 337–372 of the Factor VIII Heavy Chain

Nogami

Lapan

Zhou

et al. 2004

Journal of Biological Chemistry

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We recently demonstrated that the residues 337-372, comprising the acidic C-terminal region in A1 subunit, interact with factor Xa during the proteolytic inactivation of factor VIIIa (Nogami, K., Wakabayashi, H., and Fay, P. J. (2003) J. Biol. Chem. 278, 16502-16509). We now show this sequence is important for factor Xa-catalyzed activation of factor VIII. Peptide 337-372 markedly inhibited cofactor activation, consistent with a delay in the rate of cleavage at the A1-A2 junction. Studies using the isolated factor VIII heavy chain indicated that the peptide completely blocked cleavage at the A1-A2 junction (IC 50 ‫؍‬ 11 M) and partially blocked cleavage at the A2-B junction (IC 50 ‫؍‬ 100 M). Covalent cross-linking was observed between the 337-372 peptide and factor Xa following reaction with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, and the peptide quenched the fluorescence of dansyl-Glu-Gly-Arg active site-modified factor Xa, suggesting that residues 337-372 directly interact with factor Xa. Studies using a monoclonal antibody recognizing residues 351-365 as well as the peptide to this sequence further restricted the interactive region. Mutant factor VIII molecules in which clustered acidic residues in the 337-372 segment were converted to alanine were evaluated for activation by factor Xa. Of the mutants tested, only factor Xa-catalyzed activation of the D361A/D362A/D363A mutant was inhibited with peak activity of ϳ50% and an activation rate constant of ϳ30% of the wild type values. These results indicate that the 337-372 acidic region separating A1 and A2 domains and, in particular, a cluster of acidic residues at position 361-363 contribute to a unique factor Xa-interactive site within the factor VIII heavy chain that promotes factor Xa docking during cofactor activation.Factor VIII, a plasma protein deficient of defective in individuals with hemophilia A, functions as a cofactor for the serine protease, factor IXa, in the anionic phospholipid surface-dependent conversion of factor X to Xa (1). Factor VIII is synthesized as a multi-domain single chain molecule (A1-A2-B-A3-C1-C2) consisting of 2332 amino acid residues with a molecular mass of ϳ300 kDa (2, 3). Factor VIII is processed to a series of metal ion-dependent heterodimers by cleavage at the B-A3 junction, generating a heavy chain consisting of the A1 and A2 domains, plus heterogeneous fragments of a partially proteolyzed B-domain linked to a light chain consisting of the A3, C1, and C2 domains (2-4).Factor VIII is converted into an active form, factor VIIIa, following limited proteolysis catalyzed by either thrombin or factor Xa (5). Cleavages at Arg 372 and Arg 740 of the heavy chain produce the 50-kDa A1 and 40-kDa A2 subunits. Cleavage of the 80-kDa light chain at Arg 1689 produces a 72-kDa A3-C1-C2 subunit. Additionally, cleavage by factor Xa at Arg 1721 produces a 67-kDa A3-C1-C2 subunit. Proteolysis at Arg 372 and Arg 1689 is essential for generating factor VIIIa cofactor activity (see for review see Ref. 6). Cleavage at the former site ...

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Detection of 95 novel mutations in coagulation factor VIII geneF8responsible for hemophilia A: results from a single institution

et al. 2006

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Hemophilia A (HA) is an X-linked hereditary bleeding disorder defined by a qualitative and/or quantitative factor VIII (FVIII) deficiency. The molecular diagnosis of HA is challenging because of the high number of different causative mutations that are distributed throughout the large F8 gene. The putative role of the novel mutations, especially missense mutations, may be difficult to interpret as causing HA. We identified 95 novel mutations out of 180 different mutations responsible for HA in 515 patients from 406 unrelated families followed up at a single hemophilia treatment center of the Bicêtre university hospital (Assistance Publique-Hôpitaux de Paris [AP-HP], Le Kremlin-Bicêtre). These 95 novel mutations comprised 55 missense mutations, 12 nonsense mutations, 11 splice site mutations, and 17 small insertions/deletions. We therefore developed a mutation analysis based on a body of proof that combines the familial segregation of the mutation, the resulting biological and clinical HA phenotype, and the molecular consequences of the amino acid (AA) substitution. For the latter, we studied the putative biochemical modifications: its conservation status with cross-species FVIII and homologous proteins, its putative location in known FVIII functional regions, and its spatial position in the available FVIII 3D structures. The usefulness of such a strategy in interpreting the causality of novel F8 mutations is emphasized.

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Mutations associated with hemophilia A in the 558-565 loop of the factor VIIIa A2 subunit alter the catalytic activity of the factor Xase complex

Cited by 62 publications

References 48 publications

Identification of Residues in the 558-Loop of Factor VIIIa A2 Subunit That Interact with Factor IXa

Identification of Residues in the 558-Loop of Factor VIIIa A2 Subunit That Interact with Factor IXa

Identification of a Factor Xa-interactive Site within Residues 337–372 of the Factor VIII Heavy Chain

Detection of 95 novel mutations in coagulation factor VIII geneF8responsible for hemophilia A: results from a single institution

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