The factor VIII assay problem: neither rhyme nor reason

Lollar, Pete

doi:10.1046/j.1538-7836.2003.00484.x

Cited by 18 publications

(19 citation statements)

References 15 publications

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“…In the in vitro part of this study, 3 different assays were used for determining FVIII levels, specifically, one-stage and chromogenic FVIII:C assays and an ELISA FVIII antigen assay. We observed the previously described discrepancy in FVIII levels for rFVIII concentrates determined with the one-stage vs. the chromogenic activity assay, with an underestimation with the former assay [23][24][25][26][27]. Levels obtained with the immunological antigen assay were very similar to those obtained with the chromogenic activity assay.…”

Section: Discussionsupporting

confidence: 68%

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In vitro and in vivo stability of diluted recombinant factor VIII for continuous infusion use in haemophilia A

et al. 2009

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Factor VIII (FVIII) replacement by continuous infusion (CI) is used postoperatively or after significant bleeding. For young paediatric patients, CI may require FVIII dilution. Variable stabilities of diluted full-length recombinant FVIII Kogenate FS (KG-FS) have been reported under different storage conditions. We investigated the recovery and stability of diluted KG-FS in vitro and in vivo. Kogenate FS was diluted to 50-120 U mL(-1) and its recovery and stability in glass vials or polypropylene syringes was determined. Furthermore, stability of KG-FS diluted to 80 U mL(-1)'administered' via single- and double-pump mock CI systems was tested. Finally, the in vivo stability of KG-FS diluted to approximately 60 U mL(-1) and administered postsurgically by CI with the double-pump to a paediatric patient with severe haemophilia A undergoing implantable venous access device placement was investigated. Initial KG-FS dilution resulted in a 10-20% FVIII loss; a further 25-30% loss occurred over 72 h in vials or syringes. With the double-pump, 1 h recovery was 35%, increasing to 80% by 24 h; the initial losses were because of the Y-infusion of a 10-fold larger volume of saline concomitantly with the FVIII. In vivo, CI resulted in stable FVIII activity levels within the target range. These in vitro results are important for the generation of CI guidelines for diluted KG-FS in the paediatric haemophilic population. That FVIII losses occur upon dilution and with the double-pump does not preclude use of diluted KG-FS. Indeed, stable FVIII levels were maintained when diluted KG-FS was administered by CI with the double-pump to a paediatric patient postsurgically.

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Section: Discussionsupporting

confidence: 68%

“…Notably, the relative FVIII levels over time were similar with the 3 different assays. For the in vivo study discussed below, we followed the suggestion of Lollar [26], and adopted the one‐stage assay to investigate the stability of diluted KG‐FS during CI.…”

Section: Discussionmentioning

confidence: 99%

In vitro and in vivo stability of diluted recombinant factor VIII for continuous infusion use in haemophilia A

et al. 2009

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“…The inconsistent use of the one‐ or two‐stage assays for the quantification of FVIII has been the source of analytical complication 77. Even within the same assay, use of recombinant or plasma standards can lead to different interpretations of data 54.…”

Section: Resultsmentioning

confidence: 99%

Allometry of Factor VIII and Informed Scaling of Next-Generation Therapeutic Proteins

Kosloski

Pisal

Mager

et al. 2013

Journal of Pharmaceutical Sciences

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Allometric scaling has been applied to the pharmacokinetics (PK) of factor VIII (FVIII), but published relationships are based on relatively small subsets of available data. Numerous next generation forms of FVIII are being developed (e.g. Fc fusion, PEGylated, and liposomal formulations) and traditional pharmacokinetic scaling of these products would not incorporate the wealth of existing knowledge for current FVIII therapy in humans. We conducted a meta-analysis and developed allometric relationships of FVIII from over 100 PK studies collected from literature. Normalized Wajima curves were used to relate mean FVIII profiles between species. An ‘informed scaling’ approach was derived for predicting first-in-human PK parameters and demonstrated with a case study for an Fc fusion FVIII. NCA values for FVIII PK were well described by the allometric equations CL=6.59·W0.85 and Vss=65.0·W0.97. A subset of studies characterized by two compartment modeling showed strong linearity in scaling of total clearance and central volume, but more variability in distributional clearance and peripheral volume. Wajima curves for FVIII superimposed across species and the disposition of Fc fusion FVIII in humans was well predicted by ‘informed scaling.’ This approach might be generally applicable for predicting human PK of next generational therapeutics.

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“…In contrast, established fVIII assays have an fVIII time-log linear range of 0.01 to 0.3 units/mL. 44 Thus, a coagulation assay based on the activated platelet membrane rather than PLVs may have potential as the basis of a clinical assay with a broader range. In addition, the sensitivity of the assay to fVIII inhibition by ESH4 and ESH8 differed by .10-fold compared with commercially available 1-stage and chromogenic assays.…”

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confidence: 99%

Platelet binding sites for factor VIII in relation to fibrin and phosphatidylserine

Gilbert

Novakovic

Shi

et al. 2015

Blood

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• Coagulation fVIII binds to a protein complex, including fibrin, on stimulated platelets rather than to membrane PS.• Anti-fVIII antibodies inhibit function on platelets differently than on phospholipid vesicles used in clinical assays.Thrombin-stimulated platelets expose very little phosphatidylserine (PS) but express binding sites for factor VIII (fVIII), casting doubt on the role of exposed PS as the determinant of binding sites. We previously reported that fVIII binding sites are increased three-to sixfold when soluble fibrin (SF) binds the a IIb b 3 integrin. This study focuses on the hypothesis that platelet-bound SF is the major source of fVIII binding sites. Less than 10% of fVIII was displaced from thrombin-stimulated platelets by lactadherin, a PS-binding protein, and an fVIII mutant defective in PS-dependent binding retained platelet affinity. Therefore, PS is not the determinant of most binding sites. FVIII bound immobilized SF and paralleled platelet binding in affinity, dependence on separation from von Willebrand factor, and mediation by the C2 domain. SF also enhanced activity of fVIII in the factor Xase complex by two-to fourfold. Monoclonal antibody (mAb) ESH8, against the fVIII C2 domain, inhibited binding of fVIII to SF and platelets but not to PS-containing vesicles. Similarly, mAb ESH4 against the C2 domain, inhibited >90% of platelet-dependent fVIII activity vs 35% of vesicle-supported activity. These results imply that platelet-bound SF is a component of functional fVIII binding sites. (Blood. 2015;126(10):1237-1244 IntroductionFactor VIII (fVIII) binds to platelet membranes where it serves as a cofactor for the enzyme, factor IXa, in the intrinsic factor Xase complex, 1,2 converting the zymogen factor X to factor Xa. 3,4 The importance of the factor Xase complex is illustrated by the disease hemophilia, in which deficiency of fVIII (hemophilia A) or factor IX (hemophilia B) leads to life-threatening bleeding. In spite of the central importance of the platelet membrane, the platelet fVIII binding sites have been only partially characterized.fVIII circulates in plasma in a noncovalent complex with von Willebrand factor (VWF). Binding to VWF is mediated by the same motifs that bind platelet and phospholipid membranes. 5,6 After dissociation from VWF, fVIII binds specifically to membranes containing phosphatidylserine (PS), which is exposed on the platelet membrane in response to stimulation by several agonists. 1,6 The residual uncertainty about the identity of platelet binding sites relates to the quantity of PS exposed following stimulation by physiologic agonists and the availability of specific reagents to block the exposed PS. Thrombin stimulates platelets to expose limited PS, resulting in an outer membrane composition of 1% to 4% PS. 7,8 This amount of PS may remain below the threshold to support the observed expression of 200 to 1600 binding sites per platelet. 9,10 In contrast, the combination of thrombin and collagen, or higher concentrations of the calcium ionophore, A23187, le...

show abstract

The factor VIII assay problem: neither rhyme nor reason

Cited by 18 publications

References 15 publications

In vitro and in vivo stability of diluted recombinant factor VIII for continuous infusion use in haemophilia A

In vitro and in vivo stability of diluted recombinant factor VIII for continuous infusion use in haemophilia A

Allometry of Factor VIII and Informed Scaling of Next-Generation Therapeutic Proteins

Platelet binding sites for factor VIII in relation to fibrin and phosphatidylserine

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