Unexpected Role of PACE/Furin Cleavage Site in FVIII Biology: Implications for Hemophilia a Therapy

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 105-105
Author(s):  
Joshua I Siner ◽  
Julie M Crudele ◽  
Courtney T Connolly ◽  
Shangzhen Zhou ◽  
Elizabeth P. Merricks ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Amino Acid ◽  
Cleavage Site ◽  
Manufacturing Systems ◽  
Hemophilia A ◽  
Wild Type ◽  
Furin Cleavage ◽  
Intracellular Processing ◽  
Liver Gene ◽  
Increased Activity

Abstract The paired basic amino acid cleaving enzyme (PACE)/Furin is a protein convertase system that plays a vital role in several biological processes, including coagulation. The propeptide processing of human FIX by PACE/Furin is a critical posttranslational modification, so cells co-expressing PACE/Furin and FIX are used for production of clinical recombinant protein. In the development of recombinant B-domain deleted (BDD) FVIII for hemophilia A (HA), a 14 amino acid B-domain sequence containing a putative cleavage site for PACE/Furin was retained because it was believed to be critical for intracellular processing and secretion. In contrast to FIX, we report here a surprising detrimental effect of PACE/Furin in FVIII activity and intracellular processing and secretion. We engineered a human FVIII variant where the PACE/Furin site at residues 1645-1648 was deleted from FVIII-BDD (FVIII-ΔP/F). Notably, FVIII-ΔP/F exhibits a 3-fold increased activity over FVIII-BDD (p=0.0004) in a 2-stage APTT assay. Moreover, the A2-domain dissociation of activated FVIII-ΔP/F was also 3-fold longer compared to FVIII-BDD, suggesting a more stable activated FVIII molecule. The amount of FVIII secreted from stably transduced BHK cells was about 3-fold higher for FVIII-ΔP/F than for FVIII-BDD. Conversely, the amount of intracellular FVIII antigen was lower for FVIII-ΔP/F than for FVIII-BDD. To confirm that PACE/Furin was implicated in the underlying mechanisms for the observed differences in FVIII secretion, we inhibited PACE/Furin in FVIII-BDD producing BHKs by transducing them with vectors expressing an engineered α1-antitrypsin variant (haat-PDX) that specifically inhibits PACE/Furin. This resulted in a 40% increase in FVIII secretion (p=0.017) and a decrease in intracellular FVIII-BDD, whereas transduction with the haat-wild type control, which does not inhibit PACE/Furin, did not significantly change the amount of FVIII secreted (p=0.32). Importantly, the secretion and intracellular levels of FVIII-ΔP/F were not affected by the inhibition of PACE/Furin by haat-PDX, indicating that the secretion of this FVIII variant does not benefit from further inhibition of PACE/Furin cleavage. Together these data suggest that the increased secretion of FVIII-ΔP/F compared to FVIII-BDD is due to the former circumventing PACE/Furin. Furin is ubiquitously expressed in mammal tissues. In a stringent cellular model, we used LoVo, a unique human cell line that lacks functional Furin to determine whether expression of FVIII-ΔP/F and FVIII-BDD would differ, as we have observed in cells expressing Furin. Interestingly, the secretion of FVIII-ΔP/F and FVIII-BDD were comparable. This result confirms that FVIII-BDD is secreted better in the absence of Furin. In summary, our novel variant FVIII-ΔP/F exhibits enhanced secretion primarily by bypassing PACE/Furin cleavage; inhibiting this cellular process also enhances the secretion of FVIII. Futhermore in vivo experiments also demonstrated a beneficial effect of FVIII-ΔP/F: HA mice (n=4-7/dose) given adeno-associated viral 8 (AAV8) vectors for liver gene expression of FVIII-ΔP/F resulted in a 3-fold higher circulating FVIII levels than FVIII-BDD-expressing mice (p=0.025). These exciting results from human FVIII-ΔP/F prompt us to test this variant HA canine model. First we found that recombinant canine FVIII with the entire PACE/Furin site deleted (cFVIII-ΔP/F) had increased activity in a 2-stage aPTT assay compared to wild-type cFVIII-BDD. Injection of cFVIII-ΔP/F effectively corrects the hemophilia coagulopathy in two HA dogs. Further, AAV8 liver gene therapy with cFVIII-ΔP/F in additional two HA dogs at doses of ~6 x 1012 vg/kg, a log lower than previously used for canine FVIII-BDD AAV8 gene therapy, resulted in therapeutic levels of cFVIII and shortening of clotting times. Preliminary data on injection of cFVIII-BDD protein was well tolerated in cFVIII-ΔP/F-expressing dogs. In conclusion, these data suggest that PACE/Furin cleavage of FVIII hampers protein biological activity. FVIII variants lacking PACE/Furin recognition sequences are secreted more efficiently and exhibit improved hemostatic effects in both protein- and gene-based strategies. Inhibition of PACE/Furin in manufacturing systems for recombinant human FVIII may increase the yields of protein production. Thus these strategies have a strong rationale for translation to HA therapy. Disclosures No relevant conflicts of interest to declare.

scholarly journals Novel factor VIII variants with a modified furin cleavage site improve the efficacy of gene therapy for hemophilia A

2016 ◽  
Vol 15 (1) ◽  
pp. 110-121 ◽  
Author(s):  
G. N. Nguyen ◽  
L. A. George ◽  
J. I. Siner ◽  
R. J. Davidson ◽  
C. B. Zander ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Cleavage Site ◽  
Hemophilia A ◽  
Furin Cleavage ◽  
Furin Cleavage Site

scholarly journals Novel Human Factor VIII Variant with Impaired Intracellular Processing Exhibits Enhanced In Vivo Efficacy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 256-256
Author(s):  
Giang N. Nguyen ◽  
Christian M. Wood ◽  
Tanner Dirstine ◽  
Robert J. Davidson ◽  
Denise E. Sabatino
Keyword(s):  
Gene Therapy ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Factor Viii ◽  
Cleavage Site ◽  
Human Factor ◽  
Fold Increase ◽  
Intracellular Processing ◽  
Human Factor Viii

Abstract During intracellular processing, B-domain deleted human factor VIII (hFVIII-BDD) undergoes proteolysis at multiple sites with the most predominant cleavage at the furin recognition site (1645-RHQR-1648) at the carboxy-terminus of the B-domain. This cleavage gives rise to two polypeptide chains, the 90 kDa heavy chain (HC) and the 80 kDa light chain (LC), that form a heterodimer which becomes the major secreted form of the protein. In contrast, B-domain deleted canine factor VIII (cFVIII-BDD) is secreted primarily in the uncleaved single chain (SC) form, exhibits higher activity and shows increased expression in the gene therapy setting. To investigate the difference in the amino acid sequence at the furin recognition site between cFVIII-BDD (1645-HHQR-1648) and hFVIII-BDD, our previous work generated and characterized a series of hFVIII-BDD furin site deletion variants. Our studies demonstrated that hFVIII- BDD-del1645-1647 (Δ3) variant is secreted predominantly as SC, with a two-fold increase in activity and improved secretion over hFVIII-BDD. N-terminal sequencing of the hFVIII LC revealed that while hFVIII-BDD is cleaved after R1648 and S1657, cleavage of Δ3 occurs exclusively after S1657. Amino acid sequence analysis revealed differences at this site between hFVIII (S1657/D1658) and cFVIII (P1657/E1658). We hypothesized that modifications to this downstream cleavage site in hFVIII-BDD may further decrease cleavage efficiency, leading to a higher portion of SC, and further increase protein expression after gene therapy. We generated a new variant where this downstream cleavage sequence was modified to that of cFVIII, S1657P/D1658E, in combination with our previously described Δ3 variant. hFVIII-BDD, hFVIII-Δ3, and hFVIII-Δ3-S1657P/D1658E were purified using ion exchange chromatography. Interestingly, optical densitometry analysis of all purified proteins on a reducing SDS-PAGE gel revealed that hFVIII-Δ3-S1657P/D1658E was expressed almost entirely as a SC (91.7% ± 5.1%), similar to what was observed with cFVIII (86.8% ± 1.0%). hFVIII-Δ3 and hFVIII-BDD were 56.5% ± 5.7% and 24.3% ± 0.6% SC, respectively. Once activated by thrombin, hFVIII-Δ3-S1657P/D1658E yielded the same expected species as hFVIII-Δ3 and hFVIII-BDD. In a one-stage aPTT assay, hFVIII-Δ3-S1657P/D1658E had activity comparable to hFVIII-Δ3 and hFVIII-BDD. In the two-stage aPTT assay, hFVIII-Δ3 and hFVIII-Δ3-S1657P/D1658E exhibited a two-fold increase in activity over hFVIII-BDD. Thus, hFVIII-Δ3-S1657P/D1658E was secreted primarily in the SC form and had higher activity similar to the hFVIII-Δ3 variant in vitro. In the setting of gene-based therapeutics, strategies to increase hFVIII expression provide a platform for reducing the vector dose required to achieve therapeutically relevant FVIII levels. To investigate the efficacy of this variant in vivo, the hFVIII-Δ3-S1657P/D1658E variant was introduced into an adeno-associated viral vector serotype 8 expressing the wild type hFVIII-BDD cDNA sequence (AAV8-hAAT-hFVIII-BDD) for liver-targeted delivery. The vector was delivered to immune deficient hemophilia A mice (5x1011 vector genomes/mouse) (n=4/group). At 12 weeks post-vector administration, the hFVIII-Δ3-S1657P/D1658E had a 4.5-fold increase in protein expression over hFVIII-BDD, a significant improvement over hFVIII-Δ3, which was two-fold higher than hFVIII-BDD. Altogether, the data supports that the modifications at the downstream cleavage site at residue S1657 and D1658, in combination with a furin evading variant in hFVIII-BDD, further hinders cleavage during intracellular processing. This unexpectedly leads to additional enhancement of hFVIII-BDD expression in the gene therapy setting. Not only might the studies of these variants improve our understanding of the intracellular processing of hFVIII-BDD, but they also provide a novel approach to increase hFVIII expression that will allow the use of a lower vector dose, improving the safety of gene-based therapeutics. Disclosures Sabatino: Spark Therapeutics: Research Funding.

Codon optimization of human factor VIII cDNAs leads to high-level expression

Blood ◽  
2011 ◽  
Vol 117 (3) ◽  
pp. 798-807 ◽  
Author(s):  
Natalie J. Ward ◽  
Suzanne M. K. Buckley ◽  
Simon N. Waddington ◽  
Thierry VandenDriessche ◽  
Marinee K. L. Chuah ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Viral Vectors ◽  
Hemophilia A ◽  
Lentiviral Vector ◽  
Wild Type ◽  
Fviii Activity ◽  
Human Factor Viii

Abstract Gene therapy for hemophilia A would be facilitated by development of smaller expression cassettes encoding factor VIII (FVIII), which demonstrate improved biosynthesis and/or enhanced biologic properties. B domain deleted (BDD) FVIII retains full procoagulant function and is expressed at higher levels than wild-type FVIII. However, a partial BDD FVIII, leaving an N-terminal 226 amino acid stretch (N6), increases in vitro secretion of FVIII tenfold compared with BDD-FVIII. In this study, we tested various BDD constructs in the context of either wild-type or codon-optimized cDNA sequences expressed under control of the strong, ubiquitous Spleen Focus Forming Virus promoter within a self-inactivating HIV-based lentiviral vector. Transduced 293T cells in vitro demonstrated detectable FVIII activity. Hemophilic mice treated with lentiviral vectors showed expression of FVIII activity and phenotypic correction sustained over 250 days. Importantly, codon-optimized constructs achieved an unprecedented 29- to 44-fold increase in expression, yielding more than 200% normal human FVIII levels. Addition of B domain sequences to BDD-FVIII did not significantly increase in vivo expression. These significant findings demonstrate that shorter FVIII constructs that can be more easily accommodated in viral vectors can result in increased therapeutic efficacy and may deliver effective gene therapy for hemophilia A.

Direct Comparison of Bioengineered Factor VIII Expression Constructs: Towards an Optimal Transgene for Gene Therapy of Hemophilia A.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5477-5477
Author(s):  
Kerry L. Titus ◽  
Paul Lee ◽  
H. Trent Spencer ◽  
Christopher Doering
Keyword(s):  
Gene Therapy ◽  
Amino Acid ◽  
Factor Viii ◽  
Hemophilia A ◽  
Double Mutation ◽  
Specific Amino Acid ◽  
Stable Transfectants ◽  
Glycosylation Sites ◽  
Fviii Activity ◽  
A2 Domain

Abstract A major obstacle for gene therapy of hemophilia A is the achievement of adequate factor VIII (fVIII) expression. Bioengineering strategies have targeted specific sequences within human fVIII that are thought to be responsible for its generally poor expression. Specific amino acid substitutions, L303E/F309S herein referred to as double mutation (DM), function to decrease fVIII binding to BiP, a resident ER chaperone, which results in increased fVIII secretion (Swaroop, Moussalli et al. 1997). Furthermore, addition of 6 N-linked glycosylation sites, designated 226/N6, located within the human B domain also increases human fVIII expression (Miao, Sirachainan et al. 2004). We previously demonstrated that porcine and certain hybrid human/porcine fVIII constructs are expressed at 10 – 14-fold greater levels than human fVIII (Doering, Healey et al. 2002; Doering, Healey et al. 2004). The aim of the current study was to directly compare various fVIII expression constructs in order to determine an optimal transgene for gene therapy applications. The following fVIII constructs were generated: human B-domain-deleted fVIII (HBDD-fVIII), HBDD-fVIII with a 14 amino acid linker between the A2 domain and the activation peptide (HSQ-fVIII), porcine fVIII containing a 24 amino acid linker (HEP-fVIII), hybrid human/porcine-fVIII which has porcine A1 and A3 domains (HP47), and modified HBDD, HSQ and HEP-fVIII constructs containing DM and/or 226/N6. Each construct was transiently transfected into BHK-M cells, and fVIII production between 48 – 72 hrs post-transfection was measured using a one-stage clotting assay. Under these conditions, the addition of the DM and 226/N6 significantly increased fVIII expression for HBDD (P = 0.003), though not for HSQ. Addition of DM or 226/N6 alone did not significantly increase the expression of either human fVIII construct, and furthermore, the addition of DM to HEP-fVIII decreased its expression 98%. HEP-fVIII was expressed at 8-fold or greater levels than any of the other human constructs. Next, ~25 stably transfected BHK-M clones were isolated following transfection with each of the fVIII expression constructs and the rate of fVIII production for each clone was determined. Several clones did not express detectable fVIII activity (<0.01 units/mL) and were excluded from the analysis. Approximately 14% of the total number of clones were excluded, ranging from 0 – 42% for the different constructs. HEP-DM-fVIII was the exception, where 82% of the clones had activity <0.01 units/mL. Mean HEP-fVIII expression was 3.93 ± 3.22 units/mL/24 hr (n = 19) (Figure 1), and HP47 was similarly expressed at 3.21 ± 2.31 units/mL/24 hr (n = 19). All of the HSQ-based constructs and HBDD-DM/226/N6 showed similar mean expression levels (0.28 ± 0.03 units/mL/24 hr) and were significantly higher than HBDD and HBDD-DM, which had a mean of 0.12 ± 0.01 units/mL. In the current study, we provide experimental evidence that the expression of HEP-fVIII and HP47 is superior to other bioengineered fVIII expression constructs, which should eliminate the expression barrier that has hampered the clinical translation of gene therapy for hemophilia A. Figure 1: Stable Transfectants Figure 1:. Stable Transfectants

scholarly journals Efficacy and Safety of Long-term Prophylaxis in Severe Hemophilia A Dogs Following Liver Gene Therapy Using AAV Vectors

2011 ◽  
Vol 19 (3) ◽  
pp. 442-449 ◽  
Author(s):  
Denise E Sabatino ◽  
Amy M Lange ◽  
Ekaterina S Altynova ◽  
Rita Sarkar ◽  
Shangzhen Zhou ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Hemophilia A ◽  
Severe Hemophilia ◽  
Efficacy And Safety ◽  
Liver Gene

scholarly journals Eradication of neutralizing antibodies to factor VIII in canine hemophilia A after liver gene therapy

Blood ◽  
2010 ◽  
Vol 116 (26) ◽  
pp. 5842-5848 ◽  
Author(s):  
Jonathan D. Finn ◽  
Margareth C. Ozelo ◽  
Denise E. Sabatino ◽  
Helen W. G. Franck ◽  
Elizabeth P. Merricks ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Neutralizing Antibodies ◽  
Viral Vectors ◽  
Hemophilia A ◽  
Major Complication ◽  
Complete Disappearance ◽  
Anamnestic Response ◽  
Endogenous Expression ◽  
Liver Gene

Abstract Inhibitory antibodies to factor VIII (FVIII) are a major complication in the treatment of hemophilia A, affecting approximately 20% to 30% of patients. Current treatment for inhibitors is based on long-term, daily injections of large amounts of FVIII protein. Liver-directed gene therapy has been used to induce antigen-specific tolerance, but there are no data in hemophilic animals with pre-existing inhibitors. To determine whether sustained endogenous expression of FVIII could eradicate inhibitors, we injected adeno-associated viral vectors encoding canine FVIII (cFVIII) in 2 strains of inhibitor hemophilia A dogs. In 3 dogs, a transient increase in inhibitor titers (up to 7 Bethesda Units [BU]) at 2 weeks was followed by continuous decline to complete disappearance within 4-5 weeks. Subsequently, an increase in cFVIII levels (1.5%-8%), a shortening of clotting times, and a reduction (> 90%) of bleeding episodes were observed. Immune tolerance was confirmed by lack of antibody formation after repeated challenges with cFVIII protein and normal protein half-life. A fourth dog exhibited a strong early anamnestic response (216 BU), with slow decline to 0.8 BU and cFVIII antigen detection by 18 months after vector delivery. These data suggest that liver gene therapy has the potential to eradicate inhibitors and could improve the outcomes of hemophilia A patients.

scholarly journals Sequence Analysis Indicates that 2019-nCoV Virus Contains a Putative Furin Cleavage Site at the Boundary of S1 and S2 Domains of Spike Protein

2020 ◽  
Author(s):  
Z. Galen WO
Keyword(s):  
Amino Acid ◽  
Cleavage Site ◽  
Spike Protein ◽  
Sequence Motif ◽  
Furin Cleavage ◽  
Host Cell Membrane ◽  
Virus Family ◽  
Prediction Program ◽  
Furin Cleavage Site ◽  
Novel Coronavirus

The infectious 2019-nCoV virus, which caused the current novel coronavirus pneumonia epidemic outbreak, possesses a unique 4-Amino Acid insert at the boundary of the two subdomains (S1 and S2) of Spike protein based on multiple protein sequence alignment with the large SARS and SARS-related virus family. Using Bat CoV_RaTG13 Spike protein as reference (sharing 97% aa identity) the 4-amino acid insert can be identified as PRRA (AA position 681-684). The effect of the 4-AA insertion is the presence of a furin signature sequence motif (PRRARSV) at the boundary of S1 and S2 domains of spike protein. This sequence motif consists the required Arg residue for P1 and P4 position of Furin site. In addition, it contains Arg at P3 site as well as Ser at P1’ site of furin motif. This sequence motif matches Aerolysin furin site in FurinDB and was predicted to be moderately strong (score 0.62) by ProP, a protease cleavage site prediction program. This finding suggests that the infectious 2019-nCoV virus, unlike SARS viruses, may be processed via cellular furin recognition and cleavage of the spike protein before host cell membrane fusion and entry. This putative furin site in spike protein of 2019-nCoV virus, if proven to be functional, suggests the potential of looking into agents inhibiting furin as therapeutic mean for the treatment of the novel coronavirus pneumonia.

scholarly journals Identification of a F.VIII epitope recognized by a human hemophilic inhibitor

Blood ◽  
1989 ◽  
Vol 73 (2) ◽  
pp. 497-499 ◽  
Author(s):  
BC Lubahn ◽  
J Ware ◽  
DW Stafford ◽  
HM Reisner
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Factor Viii ◽  
Fusion Proteins ◽  
Cleavage Site ◽  
Hemophilia A ◽  
Antibody Binding ◽  
Bleeding Disorders ◽  
Enzymatic Cleavage ◽  
Coagulant Activity

Abstract Hemophilia A, one of the most common of the inherited bleeding disorders, results from a deficiency or abnormality of factor VIII (F.VIII). In approximately 15% of persons with hemophilia, treatment with exogenous F.VIII is complicated by the development of anti-F.VIII antibodies which block F.VIII coagulant activity. These antibodies have been termed inhibitors. To localize epitopes recognized by inhibitors, we used a lambda gt11 library which expresses small random fragments of F.VIII as fusion proteins. One epitope has been mapped to the 25-amino acid sequence lys-338 through asp-362 of F.VIII (E338–362). Immunoaffinity-purified antibodies that react with this epitope neutralize F.VIII:C activity. E338–362 is adjacent to an enzymatic cleavage site at arg-372 which is important in F.VIII activation. Hence, an antibody binding to E338–362 would probably block this cleavage and thereby block activation of F.VIII.

scholarly journals Mutations of factor VIII cleavage sites in hemophilia A

Blood ◽  
1988 ◽  
Vol 72 (3) ◽  
pp. 1022-1028 ◽  
Author(s):  
J Gitschier ◽  
S Kogan ◽  
B Levinson ◽  
EG Tuddenham
Keyword(s):  
Amino Acid ◽  
Factor Viii ◽  
Cleavage Site ◽  
Hemophilia A ◽  
Activated Protein C ◽  
Gene Mutations ◽  
Coagulation Factor ◽  
Cleavage Sites ◽  
Severe Hemophilia ◽  
Thrombin Activation

Abstract Hemophilia A is caused by a defect in coagulation factor VIII, a protein that undergoes extensive proteolysis during its activation and inactivation. To determine whether some cases of hemophilia are caused by mutations in important cleavage sites, we screened patient DNA samples for mutations in these sites by a two-step process. Regions of interest were amplified from genomic DNA by repeated rounds of primer- directed DNA synthesis. The amplified DNAs were then screened for mutations by discriminant hybridization using oligonucleotide probes. Two cleavage site mutations were found in a survey of 215 patients. A nonsense mutation in the activated protein C cleavage site at amino acid 336 was discovered in a patient with severe hemophilia. In another severely affected patient, a mis-sense mutation results in a substitution of cysteine for arginine in the thrombin activation site at amino acid 1689. This defect is associated with no detectable factor VIII activity, but with normal levels of factor VIII antigen. The severe hemophilia in this patient was sporadic; analysis of the mother suggested that the mutation originated in her gametes or during her embryogenesis. The results demonstrate that this approach can be used to identify factor VIII gene mutations in regions of the molecule known to be important for function.

scholarly journals Identification of a F.VIII epitope recognized by a human hemophilic inhibitor

Blood ◽  
1989 ◽  
Vol 73 (2) ◽  
pp. 497-499
Author(s):  
BC Lubahn ◽  
J Ware ◽  
DW Stafford ◽  
HM Reisner
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Factor Viii ◽  
Fusion Proteins ◽  
Cleavage Site ◽  
Hemophilia A ◽  
Antibody Binding ◽  
Bleeding Disorders ◽  
Enzymatic Cleavage ◽  
Coagulant Activity

Hemophilia A, one of the most common of the inherited bleeding disorders, results from a deficiency or abnormality of factor VIII (F.VIII). In approximately 15% of persons with hemophilia, treatment with exogenous F.VIII is complicated by the development of anti-F.VIII antibodies which block F.VIII coagulant activity. These antibodies have been termed inhibitors. To localize epitopes recognized by inhibitors, we used a lambda gt11 library which expresses small random fragments of F.VIII as fusion proteins. One epitope has been mapped to the 25-amino acid sequence lys-338 through asp-362 of F.VIII (E338–362). Immunoaffinity-purified antibodies that react with this epitope neutralize F.VIII:C activity. E338–362 is adjacent to an enzymatic cleavage site at arg-372 which is important in F.VIII activation. Hence, an antibody binding to E338–362 would probably block this cleavage and thereby block activation of F.VIII.

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