Methodology of the One‐stage Assay of Factor Viii(viii:c)

Over, J

doi:10.1111/j.1600-0609.1984.tb02764.x

Cited by 72 publications

(34 citation statements)

References 36 publications

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“…One-stage clotting assays were performed using substrate plasma chemically depleted of factor VIII 25 and assayed using a Diagnostica Stago clotting instrument. Plasma was incubated with activated partial thromboplastin time reagent (bioMérieux, Durham, NC) for 6 minutes at 37°C after which a dilution of factor VIII was added to the cuvette.…”

Section: One-stage Clotting Assaymentioning

confidence: 99%

Generation of enhanced stability factor VIII variants by replacement of charged residues at the A2 domain interface

Wakabayashi

Varfaj

DeAngelis

et al. 2008

Blood

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Factor VIII consists of a heavy chain (A1A2B domains) and light chain (A3C1C2 domains), whereas the contiguous A1A2 domains are separate subunits in the cofactor, factor VIIIa. The intrinsic instability of the cofactor results from weak affinity interactions of the A2 subunit within factor VIIIa. The charged residues Glu272, Asp519, Glu665, and Glu1984 appear buried at the interface of the A2 domain with either the A1 or A3 domain, and thus may impact protein stability. To determine the effects of these residues on procofactor/ cofactor stability, these residues were individually replaced with either Ala or Val, and stable BHK cell lines expressing the B-domainless proteins were prepared. Specific activity and thrombin generation parameters for 7 of the 8 variants were more than 80% the wild-type value. Factor VIII activity at 52°C to 60°C and the decay of factor VIIIa activity after thrombin activation were monitored. Six of the 7 variants showing wild-type-like activity demonstrated enhanced stability, with the Glu1984Val variant showing a 2-fold increase in thermostability and an approximately 4-to 8-fold increase in stability of factor VIIIa. These results indicate that replacement of buried charged residues is an effective alternative to covalent modification in increasing factor VIII (VIIIa) stability. (Blood. 2008;112:2761-2769) IntroductionFactor VIII, a plasma protein that is decreased or defective in patients with hemophilia A, circulates as a noncovalent, metal ion-dependent heterodimer. This procofactor form of the protein consists of a heavy chain (HC) composed of A1(a1)A2(a2)B domains and a light chain (LC) composed of (a3)A3C1C2 domains, with the lower case "a" representing short (ϳ 30-40 residue) segments rich in acidic residues (for review, see Fay 1 ). Factor VIII is activated by proteolytic cleavages at the a1A2, a2B, and a3A3 junctions catalyzed by thrombin or factor Xa. The product of this reaction, factor VIIIa, is a heterotrimer composed of subunits designated A1, A2, and A3C1C2 that functions as a cofactor for the serine protease factor IXa in the membrane-dependent conversion of zymogen factor X to the serine protease, factor Xa (for review, see Fay 1 ).Reconstitution studies have shown that the factor VIII heterodimeric structure is supported by both electrostatic and hydrophobic interactions, 2,3 and the interchain affinity is further strengthened by factor VIII binding von Willebrand factor. 2,4 Metal ions also contribute to the interchain affinity and activity parameters. 5 Calcium is required to yield the active factor VIII conformation. Mutagenesis studies mapped a calcium-binding site to a segment rich in acidic residues within the A1 domain (residues 110-126) and identified specific residues within this region prominent in the coordination of the ion. 6 A recent intermediate resolution X-ray structure 7 confirmed this calcium-binding site as well as suggested a second potential site within the A2 domain. This structure also showed occupancy of the 2 type 1 copper ion sites within...

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Section: One-stage Clotting Assaymentioning

confidence: 99%

Generation of enhanced stability factor VIII variants by replacement of charged residues at the A2 domain interface

Wakabayashi

Varfaj

DeAngelis

et al. 2008

Blood

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“…The test was first described by Langell in 1953 [18]. Nowadays, it is performed identically to the original method and has been extensively reviewed by Over [19] and more recently by Mannucci [20]. The test is based upon the ability of a highly diluted patient sample to shorten the clotting time in a medium that additionally consists of FVIII deficient plasma, an aPTT reagent (phospholipids and a negatively charged surface or liquid contact activator) and calcium.…”

Section: One-stage Fviii:c Activity Assaymentioning

confidence: 99%

Diagnosis of factor VIII deficiency

Verbruggen

Meijer²,

Nováková

2008

Haemophilia

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Summary. The correct diagnosis of factor VIII deficiency and the assessment of severity of the disease are essential for a patient-tailored treatment strategy. An optimal diagnostic procedure comprises sensitive and specific screening methods and factor VIII activity assays. Different screening reagents show variable characteristics and receiver operator characteristic curves are presented showing the relation between sensitivity and specificity of eleven activated partial thromboplastin time reagents. The details of the three methods for factor VIII activity assay, onestage and two-stage assay and chromogenic assays, are discussed. The chromogenic assay seems to be more sensitive than the one-stage assay with regard to the detection of severe haemophilia. Discrepant results obtained with one-stage and two-stage assays are reviewed and discussed.

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“…Biological activity of FVIII glycoforms was determined using a one-stage aPTT assay (28). Briefly, the samples (full-length or deglycosylated rFVIII) were mixed with FVIII-deficient plasma as substrate.…”

Section: Activity Of Factor VIII and Its Glycoformsmentioning

confidence: 99%

Role of Glycosylation in Conformational Stability, Activity, Macromolecular Interaction and Immunogenicity of Recombinant Human Factor VIII

Kosloski

Miclea

Balu‐Iyer

2009

AAPS J

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Abstract. Factor VIII (FVIII) is a multi-domain glycoprotein that is an essential cofactor in the blood coagulation cascade. Its deficiency or dysfunction causes hemophilia A, a bleeding disorder. Replacement using exogenous recombinant human factor VIII (rFVIII) is the first line of therapy for hemophilia A. The role of glycosylation on the activity, stability, protein-lipid interaction, and immunogenicity of FVIII is not known. In order to investigate the role of glycosylation, a deglycosylated form of FVIII was generated by enzymatic cleavage of carbohydrate chains. The biochemical properties of fully glycosylated and completely deglycosylated forms of rFVIII (degly rFVIII) were compared using enzyme-linked immunosorbent assay, size exclusion chromatography, and clotting activity studies. The biological activity of degly FVIII decreased in comparison to the fully glycosylated protein. The ability of degly rFVIII to interact with phosphatidylserine containing membranes was partly impaired. Data suggested that glycosylation significantly influences the stability and the biologically relevant macromolecular interactions of FVIII. The effect of glycosylation on immunogenicity was investigated in a murine model of hemophilia A. Studies showed that deletion of glycosylation did not increase immunogenicity.

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Methodology of the One‐stage Assay of Factor Viii(viii:c)

Cited by 72 publications

References 36 publications

Generation of enhanced stability factor VIII variants by replacement of charged residues at the A2 domain interface

Generation of enhanced stability factor VIII variants by replacement of charged residues at the A2 domain interface

Diagnosis of factor VIII deficiency

Role of Glycosylation in Conformational Stability, Activity, Macromolecular Interaction and Immunogenicity of Recombinant Human Factor VIII

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