The fraction of recombinant factor VIII:Ag unable to bind von Willebrand factor has no FVIII coagulant activity: studies in vitro (original) (raw)
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Potency and mass of factor VIII in FVIII products
Haemophilia, 2009
Introduction-Several factor (F) VIII products of different origin and structure are being used for hemophilia A treatment worldwide. The assessment of FVIII concentration in these products is done using activity assays, which are dependent upon the assay and its modifications.
Thrombosis Research, 1996
Factor VII (FVI1)and tissue factor (TF) form a binary complex which initiatesthe extrinsicpathway of the blood coagulationcascade. The infrequenttripeptide motif Trp-Lys-Scr(WKS) is foundthree timesin TF, It has been suggestedthat the motif is involved in bindingof TF to FVIl(a). Also, Lysl65 and Lys'66 of TF have been reportedto be importantfor factorX activation.To clucidatcthe molecularinteractions between TF and FVIIa, and the interactionsbetweenthe binarycomplexand FX, we examinedthe inhibitoryeffectof syntheticTF and FVII peptideanalogs.One-and twostage chromogenicassayswere employed,as WC1l as one-stagecoagulationassay. The pcptidc analogsof TF possessedthe WKS motif, the double lysineresiduesor other regionsof TF. Syntheticpeptidesof FVH Encompassing sequencesof the FV[12g5.305 region were included for comparative purposes. TF154.167 and FVI13~o-305 significantlyinhibitedboth FX activationand plasma coagulation.FVI12~5-294 acted synergistically,increasing the effect observed by FVI1300-305on FX activation. However, TF]63-175 possessing the double Iysine residues did not inhibit FX activation,indicatingthat inhibitionof FXa formationand coagulationby TF154-167is due to the region 154-162of TF. None of the peptides,includingthe WKS tripeptide, interferedwith the FVIIa activityof the TF/FVHacomplex.Thus, the results do not suggestthatthe WKSmotifsare necessa~for bindingof TF to FVIIa but that the third WKS motifmay be of importanceforthe activationof FX. Copyright@1996ElsevierScienceLtd
Activation, activity and inactivation of factor VIII in factor VIII products
Haemophilia, 2016
Introduction: Factor VIII (FVIII) products used in haemophilia A treatment show inter-and intra-product and inter-assay differences in specific activity. The mechanistic basis of these differences remains unclear. Aim: The aim of this study was to mechanistically compare the functional properties of an in-house excipient-free full-length FVIII standard and pharmacologic recombinant products containing full-length (products A and B) or B-domainless (C and D) FVIII. Methods: Factor VIII protein concentration was quantitated by ELISA. Product potency determinations (APTT, intrinsic tenase assays) and kinetic analyses detailing these products' activations by thrombin and FXa, their spontaneous and activated protein C (APC) catalysed inactivation and their performances in coagulation proteome reconstructions were studied +/À von Willebrand factor (VWF). Computational models were developed to facilitate interpretation of empirical data. Results: Factor VIII protein content per manufacturer activity unit was highest for product C with the other three products similar to the standard. Potency estimates, done five different ways, varied 20-30% in inter-and intra-assay comparisons, with product B consistently showing lower specific activity. Kinetic analyses showed the five FVIII species to differ somewhat in maximum rate of activation, the maximum level of activity achieved, the rate of spontaneous or APC catalysed inactivation and the magnitude of the effect of VWF on these parameters. When evaluated both computationally and empirically in the context of tissue factor initiated thrombin generation, product C appears the most dissimilar. Conclusion: Assessments of FVIII activation/inactivation dynamics report larger differences between FVIII products than standard functional assays. However, all FVIII products promote a 'normal' thrombin generation response to TF.
Vox Sanguinis, 2012
Background and Objectives We studied the structural and functional properties of von Willebrand factor (VWF) molecules present in a very high-purity plasmaderived factor VIII concentrate (VHP pdFVIII-Factane Ò) because several observations suggest that the presence of VWF in factor VIII (FVIII) preparations may decrease their immunogenicity. Materials and Methods Ten marketed batches of VHP pdFVIII (Factane Ò) with levels of VWF ranging from 15 to 39 IU ⁄ 100 IU FVIII were analysed. The VWF multimeric pattern was studied by agarose gel electrophoresis. The binding of VWF to FVIII was studied by gel filtration and ELISA. The binding of VWF to GPIb was analysed by ELISA. Results The results showed that high-molecular-weight multimers of VWF were present in VHP pdFVIII (Factane Ò). VWF subunits maintain a triplet structure similar to that of normal plasma. Regardless of the VWF content, all FVIII molecules of each batch were co-eluted with VWF, and no free FVIII was detectable. By immunoassays, VWF was found to be able to bind to FVIII and platelet GPIb in a similar manner to that of VWF in normal plasma. Conclusions In all the VHP pdFVIII (Factane Ò) batches studied, regardless of the level of VWF, the structure and capacity of VWF binding to FVIII and to platelet GPIb were fully preserved.
sue factor:factor VIIa–catalyzed factor IX and factor X activation by TFPI and TFPI constructs. J Thromb Haemost 2014; 12: 1826–37. Summary. Background: TFPI is a Kunitz-type protease inhibitor that downregulates the extrinsic coagulation pathway by inhibiting factor Xa (FXa) and FVIIa. All three Kunitz domains (KD1, KD2, and KD3) and protein S are required for optimal inhibition of FXa and FVIIa. There is limited information on Kunitz domain requirements of the inhibition of TF:FVIIa–catalyzed FIX and FX activation by TFPI. Aim: To investigate the role of the Kunitz domains of TFPI and protein S in the inhibition of FX and FIX activation. Methods: Inhibition of TF:FVIIa– catalyzed FX and FIX activation by full-length TFPI (TFPI FL) and TFPI constructs was quantified from progress curves of FXa and FIXa generation measured with chromogenic substrates. Results and conclusions: TFPI FL inhibited TF:FVIIa–catalyzed FIX activation with a K i of 16.7 nmol L –1. Protein S reduced the K i to 1.0 nmol L –1. TFPI 1-150 and KD1-KD2 had 10-fold higher K i values and were not stimulated by protein S. Single Kunitz domains were poor inhibitors of TF:FVIIa-catalyzed FIX activation (K i >800 nM). FX activation was measured at limiting FVIIa and excess TF or vice versa. At both conditions, TFPI FL , TFPI 1-150 , and KD1-KD2 showed similar inhibition of FX activation. However, at low phospholipid concentrations , TFPI FL was ~ 15-fold more active than TFPI 1-150 or KD1-KD2. Apparently, excess phospholipids act as a kind of sink for TFPI FL , limiting its availability for TF: FVIIa inhibition. Preformed FXa:TFPI FL/1-150 complexes rapidly and stoichiometrically inhibited FIX and FX activation by TF:FVIIa, indicating that binary TFPI:FXa complex formation is the limiting step in TF:FVIIa inhibition. Protein S also enhanced inhibition of TF:FVIIa–cata-lyzed FX activation by TFPI.
PLoS ONE, 2014
Recombinant factor VIII Fc (rFVIIIFc) is a fusion protein consisting of a single Bdomain-deleted (BDD) FVIII linked recombinantly to the Fc domain of human IgG1 to extend half-life. To determine if rFVIIIFc could be further improved by maintaining the heavy and light chains within a contiguous single chain (SC), we evaluated the activity and function of SC rFVIIIFc, an isoform that is not processed at residue R1648. SC rFVIIIFc showed equivalent activity in a chromogenic assay compared to rFVIIIFc, but approximately 40% activity by the one-stage clotting assay in the presence of von Willebrand Factor (VWF), with full activity in the absence of VWF. Moreover, SC rFVIIIFc demonstrated markedly delayed thrombin-mediated release from VWF, but an activity similar to that of rFVIIIFc upon activation in FXa generation assays. Therefore, the apparent reduction in specific activity in the aPTT assay appears to be primarily due to delayed release of FVIII from VWF. To assess whether stability and activity of SC rFVIIIFc were affected in vivo, a tail vein transection model in Hemophilia A mice was utilized. The results demonstrated similar pharmacokinetic profiles and comparable efficacy for SC rFVIIIFc and rFVIIIFc. Thus, while the single chain configuration did not promote enhanced half-life, it reduced the rate of release of FVIII from VWF required for activation. This impaired release may underlie the observed reduction in the one-stage clotting assay, but does not appear to affect the physiological activity of SC rFVIIIFc.
Management of bleeding disorders: basic science
Haemophilia, 2012
A fraction of FVIII:Ag in commercial recombinant FVIII (rFVIII) cannot bind VWF whereas all the FVIII:Ag in plasma-derived FVIII (pd-FVIII) concentrates does. To compare the FVIII:C activities of the fractions of rFVIII:Ag that can and cannot bind VWF. The FVIII:Ag contents of the rFVIII Kogenate, and Advate and a pd-FVIII-pd-VWF (Fanhdi) were measured by ELISA. The FX activation was initiated by adding 1.0 IU of FVIII:C of each FVIII-containing product to a coagulant phospholipids suspension containing 1.0 nM FIXa, 100 nM FX, 1 lM hirudin and 2 mM calcium chloride and measured after 1, 5 and 10 min. The same approach was followed after adding 2.0 IU of pd-VWF to1.0 IU of FVIII:C of Kogenate or Advate. The FVIII:Ag content/IU of FVIII:C of Kogenate, Advate and Fanhdi were 1.80 ± 0.05, 1.31 ± 0.9 and 0.84 ± 1.5 IU respectively.