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Sample records for factor viii fviii

  1. Clotting factor VIII (FVIII) and thrombin generation in camel plasma: A comparative study with humans

    PubMed Central

    Abdel Gader, Abdel Galil M.; Al Momen, Abdul Karim M.; Alhaider, Abdulqader; Brooks, Marjory B.; Catalfamo, James L.; Al Haidary, Ahmed A.; Hussain, Mansour F.

    2013-01-01

    The objective of this study was to characterize the highly elevated levels of clotting factor VIII (FVIII) in camel plasma. Whole blood was collected from healthy camels and factor VIII clotting activity (FVIII:C) assays were conducted using both the clotting and the chromogenic techniques. The anticoagulant citrate phosphate dextrose adenine (CPDA) produced the highest harvest of FVIII:C, the level of plasma factor VIII, compared to heparin:saline and heparin:CPDA anticoagulants. Camel FVIII can be concentrated 2 to 3 times in cryoprecipitate. There was a significant loss of camel FVIII when comparing levels of FVIII in camel plasma after 1 h of incubation at 37°C (533%), 40°C (364%), and 50°C (223%). Thrombin generation of camel plasma is comparable to that of human plasma. It was concluded that camel plasma contains very elevated levels of FVIII:C, approaching 8 times the levels in human plasma, and that these elevated levels could not be attributed to excessive thrombin generation. Unlike human FVIII:C, camel FVIII:C is remarkably heat stable. Taken together, these unique features of camel FVIII could be part of the physiological adaptation of hemostasis of the Arabian camel in order to survive in the hot desert environment. PMID:24082408

  2. A sequence variation scan of the coagulation factor VIII (FVIII) structural gene and associations with plasma FVIII activity levels.

    PubMed

    Viel, Kevin R; Machiah, Deepa K; Warren, Diane M; Khachidze, Manana; Buil, Alfonso; Fernstrom, Karl; Souto, Juan C; Peralta, Juan M; Smith, Todd; Blangero, John; Porter, Sandra; Warren, Stephen T; Fontcuberta, Jordi; Soria, Jose M; Flanders, W Dana; Almasy, Laura; Howard, Tom E

    2007-05-01

    Plasma factor VIII coagulant activity (FVIII:C) level is a highly heritable quantitative trait that is strongly correlated with thrombosis risk. Polymorphisms within only 1 gene, the ABO blood-group locus, have been unequivocally demonstrated to contribute to the broad population variability observed for this trait. Because less than 2.5% of the structural FVIII gene (F8) has been examined previously, we resequenced all known functional regions in 222 potentially distinct alleles from 137 unrelated nonhemophilic individuals representing 7 racial groups. Eighteen of the 47 variants identified, including 17 single-nucleotide polymorphisms (SNPs), were previously unknown. As the degree of linkage disequilibrium across F8 was weak overall, we used measured-genotype association analysis to evaluate the influence of each polymorphism on the FVIII:C levels in 398 subjects from 21 pedigrees known as the Genetic Analysis of Idiopathic Thrombophilia project (GAIT). Our results suggested that 92714C>G, a nonsynonymous SNP encoding the B-domain substitution D1241E, was significantly associated with FVIII:C level. After accounting for important covariates, including age and ABO genotype, the association persisted with each C-allele additively increasing the FVIII:C level by 14.3 IU dL(-1) (P = .016). Nevertheless, because the alleles of 56010G>A, a SNP within the 3' splice junction of intron 7, are strongly associated with 92714C>G in GAIT, additional studies are required to determine whether D1241E is itself a functional variant.

  3. A sequence variation scan of the coagulation factor VIII (FVIII) structural gene and associations with plasma FVIII activity levels

    PubMed Central

    Viel, Kevin R.; Machiah, Deepa K.; Warren, Diane M.; Khachidze, Manana; Buil, Alfonso; Fernstrom, Karl; Souto, Juan C.; Peralta, Juan M.; Smith, Todd; Blangero, John; Porter, Sandra; Warren, Stephen T.; Fontcuberta, Jordi; Soria, Jose M.; Dana Flanders, W.; Almasy, Laura

    2007-01-01

    Plasma factor VIII coagulant activity (FVIII:C) level is a highly heritable quantitative trait that is strongly correlated with thrombosis risk. Polymorphisms within only 1 gene, the ABO blood-group locus, have been unequivocally demonstrated to contribute to the broad population variability observed for this trait. Because less than 2.5% of the structural FVIII gene (F8) has been examined previously, we resequenced all known functional regions in 222 potentially distinct alleles from 137 unrelated nonhemophilic individuals representing 7 racial groups. Eighteen of the 47 variants identified, including 17 single-nucleotide polymorphisms (SNPs), were previously unknown. As the degree of linkage disequilibrium across F8 was weak overall, we used measured-genotype association analysis to evaluate the influence of each polymorphism on the FVIII:C levels in 398 subjects from 21 pedigrees known as the Genetic Analysis of Idiopathic Thrombophilia project (GAIT). Our results suggested that 92714C>G, a nonsynonymous SNP encoding the B-domain substitution D1241E, was significantly associated with FVIII:C level. After accounting for important covariates, including age and ABO genotype, the association persisted with each C-allele additively increasing the FVIII:C level by 14.3 IU dL−1 (P = .016). Nevertheless, because the alleles of 56010G>A, a SNP within the 3′ splice junction of intron 7, are strongly associated with 92714C>G in GAIT, additional studies are required to determine whether D1241E is itself a functional variant. PMID:17209060

  4. The relation between soluble endothelial protein C receptor and factor VIII levels and FVIII/sEPCR index in healthy infants.

    PubMed

    Şimşek Orhon, Filiz; Eğin, Yonca; Ulukol, Betül; Başkan, Sevgi; Akar, Nejat

    2011-03-05

    AMAÇ: Çözünür endotelyal protein C reseptörü (sEPCR) ve faktör VIII (FVIII) trombotik ve inflamatuvar durumlarda potansiyel arabileşenler olarak görülmektedir. Bu çalışmanın amacı; bir grup sağlıklı süt çocuğunda plazma sEPCR ve FVIII düzeyleri arasındaki ilişkiyi tanımlamaktır. YÖNTEMLER: Çalışma grubunu herhangi bir akut ya da kronik hastalığı ve/veya enfeksiyonu olmayan, sağlıklı 6 aylık (Grup 1, n=23) ve 12 aylık (Grup 2, n=27) çocuklar oluşturmaktadır. sEPCR düzeyleri ve FVIII düzeyleri sırasıyla; ELISA ve one stage factor metodu ile çalışılmıştır.

  5. Engineering Factor Viii for Hemophilia Gene Therapy

    PubMed Central

    Roberts, Sean A.; Dong, Biao; Firrman, Jenni A.; Moore, Andrea R.; Sang, Nianli; Xiao, Weidong

    2012-01-01

    Current treatment of hemophilia A by intravenous infusion of factor VIII (fVIII) concentrates is very costly and has a potential adverse effect of developing inhibitors. Gene therapy, on the other hand, can potentially overcome these limitations associated with fVIII replacement therapy. Although hemophilia B gene therapy has achieved promising outcomes in human clinical trials, hemophilia A gene therapy lags far behind. Compared to factor IX, fVIII is a large protein which is difficult to express at sustaining therapeutic levels when delivered by either viral or non-viral vectors. To improve fVIII gene delivery, numerous strategies have been exploited to engineer the fVIII molecule and overcome the hurdles preventing long term and high level expression. Here we reviewed these strategies, and discussed their pros and cons in human gene therapy of hemophilia A. PMID:23565342

  6. Factor VIII therapy for hemophilia A: current and future issues.

    PubMed

    Aledort, Louis; Ljung, Rolf; Mann, Kenneth; Pipe, Steven

    2014-06-01

    Hemophilia A is a congenital, recessive, X-linked bleeding disorder that is managed with infusions of plasma-derived or recombinant factor (F) VIII. The primary considerations in FVIII replacement therapy today are the: 1) immunogenicity of FVIII concentrates, 2) role of longer-acting FVIII products, 3) prophylactic use of FVIII in children and adults with severe hemophilia A, and 4) affordability and availability of FVIII products. Improving patient outcomes by increasing the use of FVIII prophylaxis, preventing or eliminating FVIII inhibitors, and expanding access to FVIII concentrates in developing countries are the major challenges confronting clinicians who care for patients with hemophilia A.

  7. Comparison of factor VIII transgenes bioengineered for improved expression in gene therapy of hemophilia A.

    PubMed

    Dooriss, Kerry L; Denning, Gabriela; Gangadharan, Bagirath; Javazon, Elisabeth H; McCarty, David A; Spencer, H Trent; Doering, Christopher B

    2009-05-01

    Successful gene therapy of hemophilia A depends on the sustained expression of therapeutic levels of factor VIII (fVIII). Because of mRNA instability, interactions with resident endoplasmic reticulum (ER) chaperones, and the requirement for carbohydrate-facilitated transport from the ER to the Golgi apparatus, fVIII is expressed at much lower levels from mammalian cells than other proteins of similar size and complexity. A number of bioengineered forms of B domain-deleted (BDD) human fVIII have been generated and shown to have enhanced expression. Previously, we demonstrated that recombinant BDD porcine fVIII exhibits high-level expression due to specific sequence elements that increase biosynthesis via enhanced posttranslational transit through the secretory pathway. In the current study, high-expression recombinant fVIII constructs were compared directly in order to determine the relative expression of the various bioengineered fVIII transgenes. The data demonstrate that BDD porcine fVIII expression is superior to that of any of the human fVIII variant constructs tested. Mean fVIII expression of 18 units/10(6) cells/24 hr was observed from HEK-293 cells expressing a single copy of the porcine fVIII transgene, which was 36- to 225-fold greater than that of any human fVIII transgene tested. Furthermore, greater than 10-fold higher expression was observed in human cells transduced with BDD porcine fVIII versus BDD human fVIII-encoding lentiviral vectors, even at low proviral copy numbers, supporting its use over other human fVIII variants in future hemophilia A gene therapy clinical trials.

  8. Differential proteolytic activation of factor VIII-von Willebrand factor complex by thrombin

    SciTech Connect

    Hill-Eubanks, D.C.; Parker, C.G.; Lollar, P. )

    1989-09-01

    Blood coagulation factor VIII (fVIII) is a plasma protein that is decreased or absent in hemophilia A. It is isolated as a mixture of heterodimers that contain a variably sized heavy chain and a common light chain. Thrombin catalyzes the activation of fVIII in a reaction that is associated with cleavages in both types of chain. The authors isolated a serine protease from Bothrops jararacussu snake venom that catalyzes thrombin-like heavy-chain cleavage but not light-chain cleavage in porcine fVIII as judged by NaDodSO{sub 4}/PAGE and N-terminal sequence analysis. Using a plasma-free assay of the ability of activated {sup 125}I-fVIII to function as a cofactor in the activation of factor X by factor IXa, they found that fVIII is activated by the venom enzyme. The venom enzyme-activated fVIII was isolated in stable form by cation-exchange HPLC. von Willebrand factor inhibited venom enzyme-activated fVIII but not thrombin-activated fVIII. These results suggest that the binding of fVIII to von Willebrand factor depends on the presence of an intact light chain and that activated fVIII must dissociate from von Willebrand factor to exert its cofactor effect. Thus, proteolytic activation of fVIII-von Willebrand factor complex appears to be differentially regulated by light-chain cleavage to dissociate the complex and heavy-chain cleavage to activate the cofactor function.

  9. Reduction of Factor VIII Inhibitor Titers During Immune Tolerance Induction With Recombinant Factor VIII-Fc Fusion Protein.

    PubMed

    Groomes, Charles L; Gianferante, David M; Crouch, Gary D; Parekh, Dina S; Scott, David W; Lieuw, Kenneth

    2016-05-01

    The development of inhibitors toward factor VIII (FVIII) is a common and serious complication of hemophilia A (HA) therapy. Patients with hemophilia who develop inhibitors often undergo time- and resource-intensive immune tolerance induction (ITI) protocols. We report a 15-month-old male with severe HA and a high-titer inhibitor that occurred while receiving prophylactic treatment with recombinant FVIII (rFVIII), in whom significant inhibitor titer reduction was achieved with thrice weekly infusions of a new, prolonged half-life rFVIII-Fc fusion protein product (trade name Eloctate). Further studies are warranted to explore the potential of Eloctate in ITI protocols.

  10. Development and characterization of lipidic cochleate containing recombinant factor VIII

    PubMed Central

    Miclea, Razvan D.; Varma, Prashant R.; Peng, Aaron; Balu-Iyer, Sathy V.

    2007-01-01

    Hemophilia A, a life threatening bleeding disorder is caused by deficiency of Factor VIII (FVIII). Replacement therapy using rFVIII is the first line therapy for hemophilia A. However, 15-30% of patients develop neutralizing antibody, mainly against the C2, A3 and A2 domains. It has been reported that PS-FVIII complex reduced total and neutralizing anti-rFVIII antibody titers in hemophilia A murine models. Here, we developed FVIII – containing cochleate cylinders, utilizing PS-Ca2+ interactions and characterized these particles for optimal in vivo properties using biophysical and biochemical techniques. Approximately 75% of the protein was associated with cochleate cylinders. Sandwich ELISA, acrylamide quenching and enzymatic digestion studies established that rFVIII was shielded from the bulk aqueous phase by the lipidic structures, possibly leading to improved in vivo stability. Freeze – thawing and rate limiting diffusion studies revealed that small cochleate cylinders with a particles size of 500 nm or less could be generated. The release kinetics and in vivo experiments suggested that there is slow and sustained release of FVIII from the complex upon systemic exposure. In vivo studies using tail clip method indicated that FVIII-cochleate complex is effective and protects hemophilic mice from bleeding. Based on these studies, we speculate that the molecular interaction between FVIII and PS may provide a basis for the design of novel FVIII lipidic structures for delivery applications. PMID:17936245

  11. Factor Activity Assays for Monitoring Extended Half-Life FVIII and Factor IX Replacement Therapies.

    PubMed

    Kitchen, Steve; Tiefenbacher, Stefan; Gosselin, Robert

    2017-04-01

    The advent of modified factor VIII (FVIII) and factor IX (FIX) molecules with extended half-lives (EHLs) compared with native FVIII and FIX represents a major advance in the field of hemophilia care, with the potential to reduce the frequency of prophylactic injections and/or to increase the trough level prior to subsequent injections. Monitoring treatment through laboratory assays will be an important part of ensuring patient safety, including any tailoring of prophylaxis. Several approaches have been used to extend half-lives, including PEGylation, and fusion to albumin or immunoglobulin. Some of these modifications affect factor assays as routinely performed in hemophilia centers; so, laboratories will need to use FVIII and FIX assays which have been shown to be suitable on a product-by-product basis. For some products, there are marked differences between results obtained using one-stage or chromogenic assays and results obtained using different reagents in the one-stage assay. The laboratory should use an assay in which the recovery of the product closely aligns with the assay used by the pharmaceutical company to assign potency to the product, so that the units reported by the laboratory agree with those used to demonstrate efficacy of the product during clinical trials. Reported assay differences in relation to several of the EHL FVIII and FIX molecules will be reviewed in this article.

  12. Circumventing furin enhances factor VIII biological activity and ameliorates bleeding phenotypes in hemophilia models

    PubMed Central

    Siner, Joshua I.; Samelson-Jones, Benjamin J.; Crudele, Julie M.; French, Robert A.; Lee, Benjamin J.; Zhou, Shanzhen; Merricks, Elizabeth; Raymer, Robin; Camire, Rodney M.; Arruda, Valder R.

    2016-01-01

    Processing by the proprotein convertase furin is believed to be critical for the biological activity of multiple proteins involved in hemostasis, including coagulation factor VIII (FVIII). This belief prompted the retention of the furin recognition motif (amino acids 1645–1648) in the design of B-domain–deleted FVIII (FVIII-BDD) products in current clinical use and in the drug development pipeline, as well as in experimental FVIII gene therapy strategies. Here, we report that processing by furin is in fact deleterious to FVIII-BDD secretion and procoagulant activity. Inhibition of furin increases the secretion and decreases the intracellular retention of FVIII-BDD protein in mammalian cells. Our new variant (FVIII-ΔF), in which this recognition motif is removed, efficiently circumvents furin. FVIII-ΔF demonstrates increased recombinant protein yields, enhanced clotting activity, and higher circulating FVIII levels after adeno-associated viral vector–based liver gene therapy in a murine model of severe hemophilia A (HA) compared with FVIII-BDD. Moreover, we observed an amelioration of the bleeding phenotype in severe HA dogs with sustained therapeutic FVIII levels after FVIII-ΔF gene therapy at a lower vector dose than previously employed in this model. The immunogenicity of FVIII-ΔF did not differ from that of FVIII-BDD as a protein or a gene therapeutic. Thus, contrary to previous suppositions, FVIII variants that can avoid furin processing are likely to have enhanced translational potential for HA therapy. PMID:27734034

  13. Targeting factor VIII expression to platelets for hemophilia A gene therapy does not induce an apparent thrombotic risk in mice.

    PubMed

    Baumgartner, C K; Mattson, J G; Weiler, H; Shi, Q; Montgomery, R R

    2017-01-01

    Essentials Platelet-Factor (F) VIII gene therapy is a promising treatment in hemophilia A. This study aims to evaluate if platelet-FVIII expression would increase the risk for thrombosis. Targeting FVIII expression to platelets does not induce or elevate thrombosis risk. Platelets expressing FVIII are neither hyper-activated nor hyper-responsive.

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

    PubMed

    Nguyen, G N; George, L A; Siner, J I; Davidson, R J; Zander, C B; Zheng, X L; Arruda, V R; Camire, R M; Sabatino, D E

    2017-01-01

    Essentials Factor (F) VIII is an inefficiently expressed protein. Furin deletion FVIII variants were purified and characterized using in vitro and in vivo assays. These minimally modified novel FVIII variants have enhanced function. These variants provide a strategy for increasing FVIII expression in hemophilia A gene therapy.

  15. O-phospho-L-serine, multi-functional excipient for B domain deleted recombinant factor VIII.

    PubMed

    Miclea, Razvan D; Purohit, Vivek S; Balu-Iyer, Sathy V

    2007-06-29

    Factor VIII (FVIII) is an important cofactor in the blood coagulation cascade. A deficiency or dysfunction of FVIII causes hemophilia A, a life-threatening bleeding disorder. FVIII circulates in plasma as a heterodimer comprising 6 domains (heavy chain, A1-A2-B and light chain, A3-C1-C2). Replacement therapy using FVIII is the leading therapy in the management of hemophilia A. However, approximately 15% to 30% of patients develop inhibitory antibodies that neutralize the activity of the protein. Neutralizing antibodies to epitopes in the lipid binding region of FVIII are commonly identified in patients' plasma. In this report, we investigated the effect of O-phospho-L-serine (OPLS), which binds to the lipid binding region, on the immunogenicity of B domain deleted recombinant factor VIII (BDDrFVIII). Sandwich enzyme-linked immunosorbent assay (ELISA) studies showed that OPLS specifically bind to the lipid binding region, localized in the C2 domain of the coagulation factor. Size exclusion chromatography and fluorescence anisotropy studies showed that OPLS interfered with the aggregation of BDDrFVIII. Immunogenicity of free- vs BDDrFVIII-OPLS complex was evaluated in a murine model of hemophilia A. Animals administered subcutaneous (sc) injections of BDDrFVIII-OPLS had lower neutralizing titers compared with animals treated with BDDrFVIII alone. Based on these studies, we hypothesize that specific molecular interactions between OPLS and BDDrFVIII may improve the stability and reduce the immunogenicity of BDDrFVIII formulations.

  16. Optimisation of the Factor VIII yield in mammalian cell cultures by reducing the membrane bound fraction.

    PubMed

    Kolind, Mille Petersen; Nørby, Peder Lisby; Berchtold, Martin Werner; Johnsen, Laust Bruun

    2011-02-20

    In vivo, clotting Factor VIII (FVIII) circulates in plasma bound to von Willebrand factor (vWF), and the vWF:FVIII complex prevents binding of FVIII to phosphatidylserine (PS). Activation of FVIII by thrombin releases FVIII from vWF, and subsequently FVIII binds to PS exposed on activated platelets and forms the tenase complex together with clotting Factor IX. In vitro, during serum free production of recombinant FVIII (rFVIII), production cells also expose PS, and since vWF is not present to hinder interaction of secreted rFVIII with PS, rFVIII is partly associated with the cell membrane of the production cells. Recently, we showed that as much as 90% of secreted rFVIII is bound to transiently transfected production cells during serum free conditions. In this study, we investigated the effect of including vWF in the serum free medium, and demonstrate that addition of vWF results in release of active membrane bound rFVIII to the culture medium. Moreover, the attachment of rFVIII to cell membranes of un-transfected HEK293 cells was studied in the presence of compounds that competes for interactions between rFVIII and PS. Competitive assays between iodinated rFVIII (¹²⁵I-rFVIII) and annexin V or ortho-phospho-L-serine (OPLS) demonstrated that annexin V and OPLS were able to reduce the membrane bound fraction of rFVIII by 70% and 30%, respectively. Finally, adding OPLS to CHO cells stably expressing FVIII increased the yield by 50%. Using this new knowledge, the recovery of rFVIII could be increased considerably during serum free production of this therapeutic protein.

  17. Intracellular trafficking of factor VIII to von Willebrand factor storage granules.

    PubMed Central

    Rosenberg, J B; Foster, P A; Kaufman, R J; Vokac, E A; Moussalli, M; Kroner, P A; Montgomery, R R

    1998-01-01

    In plasma, von Willebrand factor (vWf) associates with Factor VIII (FVIII); however, the site at which these proteins first interact has not been defined. Administration of 1-desamino-8-D-arginine vasopressin (DDAVP) causes a rapid, concomitant elevation in plasma levels of both vWf and FVIII, suggesting the existence of a DDAVP-releasable storage pool for both proteins. To determine whether vWf and FVIII can associate intracellularly and colocalize to storage vesicles, we transfected AtT-20 cells with vWf and FVIII expression plasmids. FVIII alone was not detectable within storage granules; however, transfection of vWf cDNA into the same cell caused FVIII to alter its intracellular trafficking and to undergo granular storage, colocalizing to the vWf-containing granules. In contrast, colocalization of FVIII was not observed when these cells were transfected with plasmids encoding defective FVIII-binding vWf mutants. Transfection of bovine endothelial cells with FVIII further demonstrated vesicular storage of FVIII with vWf in Weibel-Palade bodies. Since gene therapy of hemophilia A may ultimately target endothelium or hematopoietic stem cells, the interaction between vWf and FVIII within a secretory cell is important. Thus, vWf can alter the intracellular trafficking of FVIII from a constitutive to a regulated secretory pathway, thereby producing an intracellular storage pool of both proteins. PMID:9449695

  18. Diagnostic accuracy study of a factor VIII ELISA for detection of factor VIII antibodies in congenital and acquired haemophilia A.

    PubMed

    Batty, Paul; Moore, Gary W; Platton, Sean; Maloney, James C; Palmer, Ben; Bowles, Louise; Pasi, K John; Rangarajan, Savita; Hart, Daniel P

    2015-10-01

    Antibody formation to factor VIII (FVIII) remains the greatest clinical and diagnostic challenge to the haemophilia-treating physician. Current guidance for testing for inhibitory FVIII antibodies (inhibitors) recommends the functional Nijmegen-Bethesda assay (NBA). A FVIII ELISA offers a complementary, immunological approach for FVIII antibody testing. It was the aim of this study to retrospectively evaluate the performance of a FVIII ELISA (index) for detection of FVIII antibodies, compared with the NBA (reference). All samples sent for routine FVIII antibody testing at two haemophilia Comprehensive Care Centres, were tested in parallel using the NBA and a solid-phase, indirect FVIII ELISA kit (Immucor). A total of 497 samples from 239 patients (severe haemophilia A=140, non-severe haemophilia A=85, acquired haemophilia A=14) were available for analysis. Sixty-three samples tested positive by the NBA (prevalence 12.7%, 95% confidence interval [CI], 9.9-15.9 %), with a median inhibitor titre of 1.2 BU/ml (range 0.7-978.0). The FVIII ELISA demonstrated a specificity of 94.0% (95%CI, 91.3-96.0), sensitivity of 77.8% (95%CI, 65.5-87.3), negative predictive value of 96.7% (95%CI, 94.5-98.2), positive predictive value 65.3% (95%CI, 53.5-76.0), negative likelihood ratio 0.2 (95%CI, 0.1-0.4), positive likelihood ratio 13.0 (95%CI, 8.7-19.3) and a diagnostic odds ratio of 54.9 (95%CI, 27.0-112.0). Strong positive correlation (r=0.77, p<0.001) was seen between the results of the NBA (log adjusted) and FVIII ELISA optical density. In conclusion, FVIII ELISA offers a simple, specific, surveillance method enabling batch testing of non-urgent samples for the presence of FVIII antibodies.

  19. Analysis of the spatial and temporal characteristics of platelet-delivered factor VIII-based clots.

    PubMed

    Neyman, Michael; Gewirtz, Jamie; Poncz, Mortimer

    2008-08-15

    Normally factor (F) VIII is not expressed in megakaryocytes, but when human FVIII was transgenically expressed in murine megakaryocytes, it was stored in platelet alpha-granules and released at sites of injury. This platelet FVIII (pFVIII) is effective in correcting hemostasis, even in the presence of circulating inhibitors, so it offers a potential gene therapy strategy for hemophilia A. To understand clot development by pFVIII, we have examined clot response to laser injury in both cremaster arterioles and venules in FVIII(null) mice either infused with FVIII or transgenic for pFVIII. In both sets of vessels, pFVIII is at least as effective as infused FVIII. However, there are temporal and spatial differences in fibrin and platelet accumulation within clots depending on how FVIII is delivered. These differences may be related to the temporal and spatial distribution of the alpha-granular-released FVIII within the developing clot, and may explain the increased frequency and size of embolic events seen with pFVIII. These observations may not only have implications for the use of pFVIII in gene therapy for hemophilia A, but may also have physiologic consequences, explaining why many procoagulant factors are delivered both in the plasma and in platelet alpha-granules.

  20. Rituximab in the treatment of acquired factor VIII inhibitors.

    PubMed

    Wiestner, Adrian; Cho, Hearn J; Asch, Adam S; Michelis, Mary Ann; Zeller, Jack A; Peerschke, Ellinor I B; Weksler, Babette B; Schechter, Geraldine P

    2002-11-01

    Autoantibodies against factor VIII (FVIII) are rare but can cause life-threatening bleeding requiring costly factor replacement and prolonged immunosuppression. We report 4 consecutively treated patients whose acquired FVIII inhibitors responded rapidly to immunosuppressive regimens that included rituximab, a monoclonal antibody against CD20(+) B cells. Three patients had spontaneously occurring inhibitors. The fourth, a patient with mild hemophilia A, developed both an autoantibody and an alloantibody following recombinant FVIII treatment. Pretreatment FVIII activities ranged from less than 1% to 4% and inhibitor titers from 5 to 60 Bethesda units (BU). One patient with polymyalgia rheumatica who developed the inhibitor while receiving prednisone responded to single agent rituximab. The hemophilia patient had rapid resolution of the autoantibody, whereas the alloantibody persisted for months. Responses continue off treatment from more than 7 to more than 12 months. This report adds to the growing evidence that rituximab has efficacy in immune disorders resulting from autoantibody formation.

  1. Idiopathic Acquired Hemophilia A with Undetectable Factor VIII Inhibitor

    PubMed Central

    Abt, Nicholas B.; Streiff, Michael B.; Gocke, Christian B.; Kickler, Thomas S.; Lanzkron, Sophie M.

    2014-01-01

    Objective. We present the case of a 73-year-old female, with no family or personal history of a bleeding disorder, who had a classic presentation for acquired hemophilia A. Factor VIII activity was low but detectable and a factor VIII inhibitor was undetectable. Methods. The patient's plasma was comprehensively studied to determine the cause of the acquired coagulopathy. Using the Nijmegen modification of the Bethesda assay, no factor VIII autoantibody was measureable despite varying the incubation time from 1 to 3 hours. Results. The aPTT was prolonged at 46.8 seconds, which did not correct in the 4 : 1 mix but did with 1 : 1 mix. Using a one stage factor VIII activity assay, the FVIII activity was 16% and chromogenic FVIII activity was also 16%. The patient was treated with recombinant FVII and transfusion, significantly reducing bleeding. Long-term therapy was initiated with cyclophosphamide and prednisone with normalization of FVIII activity. Conclusions. Physicians can be presented with the challenging clinical picture of an acquired factor VIII inhibitor without a detectable inhibitor by the Bethesda assay. Standard therapy for an acquired hemophilia A should be considered. PMID:24955264

  2. The intron-22–inverted F8 locus permits factor VIII synthesis: explanation for low inhibitor risk and a role for pharmacogenomics

    PubMed Central

    Lozier, Jay N.; Kasper, Carol K.; Yanover, Chen; Nichols, Timothy

    2015-01-01

    Intron-22-inversion patients express the entire Factor VIII (FVIII)-amino-acid sequence intracellularly as 2 non-secreted polypeptides and have a positive “intracellular (I)-FVIII-CRM” status. Mutations conferring a positive I-FVIII-CRM status are associated with low inhibitor risk and are pharmacogenetically relevant because inhibitor risk may be affected by the nature of the therapeutic FVIII-protein (tFVIII), the affinity of any tFVIII-derived foreign peptide (tFVIII-fp) for any HLA class-II isomer (HLA-II) comprising individual major histocompatibility complex (MHC) repertoires, and the stability of any tFVIII-fp/HLA-II complex. We hypothesize that mutations conferring a completely or substantially negative I-FVIII-CRM status are pharmacogenetically irrelevant because inhibitor risk is high with any tFVIII and individual MHC repertoire. PMID:25406352

  3. Chemical Chaperones Improve Protein Secretion and Rescue Mutant Factor VIII in Mice with Hemophilia A

    PubMed Central

    Milanov, Peter; Abriss, Daniela; Ungerer, Christopher; Quade-Lyssy, Patricia; Simpson, Jeremy C.; Pepperkok, Rainer; Seifried, Erhard; Tonn, Torsten

    2012-01-01

    Inefficient intracellular protein trafficking is a critical issue in the pathogenesis of a variety of diseases and in recombinant protein production. Here we investigated the trafficking of factor VIII (FVIII), which is affected in the coagulation disorder hemophilia A. We hypothesized that chemical chaperones may be useful to enhance folding and processing of FVIII in recombinant protein production, and as a therapeutic approach in patients with impaired FVIII secretion. A tagged B-domain-deleted version of human FVIII was expressed in cultured Chinese Hamster Ovary cells to mimic the industrial production of this important protein. Of several chemical chaperones tested, the addition of betaine resulted in increased secretion of FVIII, by increasing solubility of intracellular FVIII aggregates and improving transport from endoplasmic reticulum to Golgi. Similar results were obtained in experiments monitoring recombinant full-length FVIII. Oral betaine administration also increased FVIII and factor IX (FIX) plasma levels in FVIII or FIX knockout mice following gene transfer. Moreover, in vitro and in vivo applications of betaine were also able to rescue a trafficking-defective FVIII mutant (FVIIIQ305P). We conclude that chemical chaperones such as betaine might represent a useful treatment concept for hemophilia and other diseases caused by deficient intracellular protein trafficking. PMID:22973456

  4. Efficacy and safety of recombinant factor VIII products in patients with hemophilia A.

    PubMed

    Musso, Robert

    2008-10-01

    The introduction of recombinant factor VIII (rFVIII) clotting factor concentrates nearly 20 years ago represented a significant advance in the treatment of hemophilia A. The major advantage of rFVIII products compared with plasma-derived FVIII products is related to product safety, with rFVIII products virtually eliminating bloodborne pathogen transmission. The most challenging aspect of hemophilia A management today is the development of FVIII inhibitors; previously untreated patients are at the highest risk for inhibitor formation. Presented in this article are results of clinical trials in previously treated and untreated patients and postmarketing surveillance studies for the four commercially available rFVIII products (Recombinate, ReFacto, Kogenate FS/Kogenate Bayer and Advate). Recombinant FVIII therapies are highly efficacious when used ondemand and prophylactically, and they have excellent safety profiles; there have been no reports of viral- or prion-based disease transmission associated with rFVIII administration. The incidence rate of inhibitors in previously untreated patients ranges from 15% to approximately 30%. Because rFVIII concentrates have proven efficacy and safety profiles, a number of hemophilia treatment groups recommend rFVIII products as first-line therapy in the management of hemophilia A.

  5. Immunogenicity and pharmacokinetic studies of recombinant Factor VIII containing lipid cochleates

    PubMed Central

    Kosloski, Matthew P.; Peng, Aaron; Varma, Prashant R.; Fathallah, Anas M.; Miclea, Razvan D.; Mager, Donald E.; Balu-Iyer, Sathy V.

    2010-01-01

    Replacement therapy using recombinant factor VIII (rFVIII) is currently the most common therapy for hemophilia A, a bleeding disorder caused by the deficiency of FVIII. However, 15–30% of patients develop inhibitory antibodies against administered rFVIII which complicates the therapy. Encapsulation or association of protein with lipidic structures can reduce this immune response. Previously, we developed and characterized rFVIII-containing phosphatidylserine (PS) cochleate cylinders using biophysical techniques. We hypothesized that these structures may provide a reduction in immunogenicity while avoiding the rapid clearance by the reticuloendothelial system (RES) previously observed with liposomal vesicles of similar composition. We investigated in vivo behavior of the cochleates containing rFVIII including immunogenicity and pharmacokinetics in hemophilia A mice. The rFVIII-cochleate complex significantly reduced the level of inhibitory antibody developed against rFVIII following intravenous (i.v.) administration. Pharmacokinetic modeling allowed assessment of in vivo release kinetics. Cochleates acted as delayed release delivery vehicle with an input peak of rFVIII observed around 2 hrs post-injection. rFVIII associated with cochleates showed limited RES uptake and a similar disposition to the free protein upon release from the structure. Incomplete disassociation from the complex limits systemic availability of the protein. Further formulation efforts are warranted to regulate the rate and extent of release of rFVIII from cochleate complexes. PMID:21114461

  6. Female haemophiliac homozygous for the factor VIII intron 22 inversion mutation, with transcriptional inactivation of one of the factor VIII alleles.

    PubMed

    David, D; Morais, S; Ventura, C; Campos, M

    2003-01-01

    Phenotypic expression of X-linked recessive disorders, including haemophilia A, is rare in females. This report describes a female with sporadic severe haemophilia A. The female patient and her family members were evaluated by coagulation assays. Visible detectable disturbance of X chromosome structure or number, as well as 2N von Willebrand disease, were excluded as possible explanations of the haemophilia A phenotype. Molecular studies, factor VIII (FVIII) intron 22 inversion mutation analysis showed that the severe haemophilia A phenotype is the result of a maternally inherited, distal, FVIII gene inversion and a paternally inherited de novo, also distal, FVIII gene inversion. Furthermore, comparative single-stranded conformation polymorphism analysis revealed the absence of detectable maternally inherited abnormal FVIII gene transcript in the patient's peripheral blood lymphocytes. X chromosome methylation analysis indicates that this could be explained by preferential inactivation of the maternally inherited X chromosome carrying the distal FVIII gene inversion.

  7. Factor VIII alters tubular organization and functional properties of von Willebrand factor stored in Weibel-Palade bodies.

    PubMed

    Bouwens, Eveline A M; Mourik, Marjon J; van den Biggelaar, Maartje; Eikenboom, Jeroen C J; Voorberg, Jan; Valentijn, Karine M; Mertens, Koen

    2011-11-24

    In endothelial cells, von Willebrand factor (VWF) multimers are packaged into tubules that direct biogenesis of elongated Weibel-Palade bodies (WPBs). WPB release results in unfurling of VWF tubules and assembly into strings that serve to recruit platelets. By confocal microscopy, we have previously observed a rounded morphology of WPBs in blood outgrowth endothelial cells transduced to express factor VIII (FVIII). Using correlative light-electron microscopy and tomography, we now demonstrate that FVIII-containing WPBs have disorganized, short VWF tubules. Whereas normal FVIII and FVIII Y1680F interfered with formation of ultra-large VWF multimers, release of the WPBs resulted in VWF strings of equal length as those from nontransduced blood outgrowth endothelial cells. After release, both WPB-derived FVIII and FVIII Y1680F remained bound to VWF strings, which however had largely lost their ability to recruit platelets. Strings from nontransduced cells, however, were capable of simultaneously recruiting exogenous FVIII and platelets. These findings suggest that the interaction of FVIII with VWF during WPB formation is independent of Y1680, is maintained after WPB release in FVIII-covered VWF strings, and impairs recruitment of platelets. Apparently, intra-cellular and extracellular assembly of FVIII-VWF complex involves distinct mechanisms, which differ with regard to their implications for platelet binding to released VWF strings.

  8. Detection of Intracellular Factor VIII Protein in Peripheral Blood Mononuclear Cells by Flow Cytometry

    PubMed Central

    Pandey, Gouri Shankar; Tseng, Sandra C.; Howard, Tom E.; Sauna, Zuben E.

    2013-01-01

    Flow cytometry is widely used in cancer research for diagnosis, detection of minimal residual disease, as well as immune monitoring and profiling following immunotherapy. Detection of specific host proteins for diagnosis predominantly uses quantitative PCR and western blotting assays. In this study, we optimized a flow cytometry-based detection assay for Factor VIII protein in peripheral blood mononuclear cells (PBMCs). An indirect intracellular staining (ICS) method was standardized using monoclonal antibodies to different domains of human Factor VIII protein. The FVIII protein expression level was estimated by calculating the mean and median fluorescence intensities (MFI) values for each monoclonal antibody. ICS staining of transiently transfected cell lines supported the method's specificity. Intracellular FVIII protein expression was also detected by the monoclonal antibodies used in the study in PBMCs of five blood donors. In summary, our data suggest that intracellular FVIII detection in PBMCs of hemophilia A patients can be a rapid and reliable method to detect intracellular FVIII levels. PMID:23555096

  9. Intravenous administration of Factor VIII-O-Phospho-L-Serine (OPLS) complex reduces immunogenicity and preserves pharmacokinetics of the therapeutic protein.

    PubMed

    Gaitonde, Puneet; Purohit, Vivek S; Balu-Iyer, Sathy V

    2015-01-23

    Hemophilia A is a bleeding disorder caused by the deficiency of an important coagulation factor; Factor VIII (FVIII). Replacement therapy using exogenously administered recombinant FVIII is the most commonly used method of treatment. However, approximately 30% of Hemophilia A patients develop neutralizing antibodies (Nabs) against the recombinant protein. Nabs abolish FVIII activity and drastically influence efficacy of the protein. The immunogenic epitopes of FVIII reside predominantly in the C2 domain of FVIII. However, the C2 domain also contains a lipid binding region. O-Phospho-L-Serine (OPLS) which is the head-group moiety of phosphatidylserine, interacts with the lipid binding region of FVIII. Previous studies have shown that FVIII complexed with OPLS lowered Nab development against FVIII following subcutaneous administration. In dendritic cell-T-cell co-culture studies, OPLS treatment increased the secretion of immunosuppressive cytokines (Transforming Growth Factor-β and Interleukin-10), and simultaneously decreased pro-inflammatory IL-17 cytokine. Here, we investigated FVIII immune response and pharmacokinetics upon intravenous administration of FVIII-OPLS complex. We studied the effect of FVIII-OPLS complex on the interaction between a professional antigen presenting cell; dendritic cell and T-cell, and T-cell clonal expansion. Pharmacokinetics parameters were estimated following intravenous administration of FVIII and FVIII-OPLS. The results suggest that OPLS lowers FVIII immune response following intravenous administration. OPLS also hinders FVIII-specific T-cell clonal proliferation and preserves FVIII PK profile. Thus, the ease of protein-lipid complexation, preservation of FVIII activity and in vivo behavior, and improved in vitro FVIII stability, makes OPLS an attractive excipient in the preparation of next generation or biosimilar FVIII products with improved safety profile.

  10. Intravenous administration of Factor VIII - O-Phospho-L-Serine (OPLS) complex reduces immunogenicity and preserves pharmacokinetics of the therapeutic protein

    PubMed Central

    Gaitonde, Puneet; Purohit, Vivek S.

    2014-01-01

    Hemophilia A is a bleeding disorder caused by the deficiency of an important coagulation factor; Factor VIII (FVIII). Replacement therapy using exogenously administered recombinant FVIII is the most commonly used method of treatment. However, approximately 30% of Hemophilia A patients develop neutralizing antibodies (Nabs) against the recombinant protein. Nabs abolish FVIII activity and drastically influence efficacy of the protein. The immunogenic epitopes of FVIII reside predominantly in the C2 domain of FVIII. However, the C2 domain also contains a lipid binding region. O-Phospho-L-Serine (OPLS) which is the head-group moiety of phosphatidylserine, interacts with the lipid binding region of FVIII. Previous studies have shown that FVIII complexed with OPLS lowered Nab development against FVIII following subcutaneous administration. In dendritic cell – T-cell co-culture studies, OPLS treatment increased the secretion of immunosuppressive cytokines (Transforming Growth Factor-β and Interleukin-10), and simultaneously decreased pro-inflammatory IL-17 cytokine. Here, we investigated FVIII immune response and pharmacokinetics upon intravenous administration of FVIII-OPLS complex. We studied the effect of FVIII-OPLS complex on the interaction between a professional antigen presenting cell; dendritic cell and T-cell, and T-cell clonal expansion. Pharmacokinetics parameters were estimated following intravenous administration of FVIII and FVIII-OPLS. The results suggest that OPLS lowers FVIII immune response following intravenous administration. OPLS also hinders FVIII-specific T-cell clonal proliferation and preserves FVIII PK profile. Thus, the ease of protein-lipid complexation, preservation of FVIII activity and in vivo behavior, and improved in vitro FVIII stability, makes OPLS an attractive excipient in the preparation of next generation or biosimilar FVIII products with improved safety profile. PMID:25459532

  11. Hemophilia A gene therapy via intraosseous delivery of factor VIII-lentiviral vectors.

    PubMed

    Miao, Carol H

    2016-01-01

    Current treatment of hemophilia A (HemA) patients with repeated infusions of factor VIII (FVIII; abbreviated as F8 in constructs) is costly, inconvenient, and incompletely effective. In addition, approximately 25 % of treated patients develop anti-factor VIII immune responses. Gene therapy that can achieve long-term phenotypic correction without the complication of anti-factor VIII antibody formation is highly desired. Lentiviral vector (LV)-mediated gene transfer into hematopoietic stem cells (HSCs) results in stable integration of FVIII gene into the host genome, leading to persistent therapeutic effect. However, ex vivo HSC gene therapy requires pre-conditioning which is highly undesirable for hemophilia patients. The recently developed novel methodology of direct intraosseous (IO) delivery of LVs can efficiently transduce bone marrow cells, generating high levels of transgene expression in HSCs. IO delivery of E-F8-LV utilizing a ubiquitous EF1α promoter generated initially therapeutic levels of FVIII, however, robust anti-FVIII antibody responses ensued neutralized functional FVIII activity in the circulation. In contrast, a single IO delivery of G-FVIII-LV utilizing a megakaryocytic-specific GP1bα promoter achieved platelet-specific FVIII expression, leading to persistent, partial correction of HemA in treated animals. Most interestingly, comparable therapeutic benefit with G-F8-LV was obtained in HemA mice with pre-existing anti-FVIII inhibitors. Platelets is an ideal IO delivery vehicle since FVIII stored in α-granules of platelets is protected from high-titer anti-FVIII antibodies; and that even relatively small numbers of activated platelets that locally excrete FVIII may be sufficient to promote efficient clot formation during bleeding. Additionally, combination of pharmacological agents improved transduction of LVs and persistence of transduced cells and transgene expression. Overall, a single IO infusion of G-F8-LV can generate long-term stable

  12. Immunogenicity and pharmacokinetic studies of recombinant factor VIII containing lipid cochleates.

    PubMed

    Kosloski, Matthew P; Peng, Aaron; Varma, Prashant R; Fathallah, Anas M; Miclea, Razvan D; Mager, Donald E; Balu-iyer, Sathy V

    2011-05-01

    Replacement therapy using recombinant factor VIII (rFVIII) is currently the most common therapy for hemophilia A, a bleeding disorder caused by the deficiency of FVIII. However, 15-30% of patients develop inhibitory antibodies against administered rFVIII, which complicates the therapy. Encapsulation or association of protein with lipidic structures can reduce this immune response. Previous studies developed and characterized rFVIII-containing phosphatidylserine (PS) cochleate cylinders using biophysical techniques. It was hypothesized that these structures may provide a reduction in immunogenicity while avoiding the rapid clearance by the reticuloendothelial system (RES) previously observed with liposomal vesicles of similar composition. This study investigated in vivo behavior of the cochleates containing rFVIII including immunogenicity and pharmacokinetics in hemophilia A mice. The rFVIII-cochleate complex significantly reduced the level of inhibitory antibody developed against rFVIII following intravenous (i.v.) administration. Pharmacokinetic modeling allowed assessment of in vivo release kinetics. Cochleates acted as a delayed release delivery vehicle with an input peak of cochleates showed limited RES uptake and associated rFVIII displayed a similar disposition to the free protein upon release from the structure. Incomplete disassociation from the complex limits systemic availability of the protein. Further formulation efforts are warranted to regulate the rate and extent of release of rFVIII from cochleate complexes.

  13. Induction of megakaryocytes to synthesize and store a releasable pool of human factor VIII.

    PubMed

    Wilcox, D A; Shi, Q; Nurden, P; Haberichter, S L; Rosenberg, J B; Johnson, B D; Nurden, A T; White, G C; Montgomery, R R

    2003-12-01

    von Willebrand factor (VWF) is a complex plasma glycoprotein that modulates platelet adhesion at the site of a vascular injury, and it also serves as a carrier protein for factor (F)VIII. As megakaryocytes are the only hematopoietic lineage to naturally synthesize and store VWF within alpha-granules, this study was performed to determine if expression of a FVIII transgene in megakaryocytes could lead to trafficking and storage of FVIII with VWF in platelet alpha-granules. Isolex selected CD34+ cells from human G-CSF mobilized peripheral blood cells (PBC) and murine bone marrow were transduced with a retrovirus encoding the B-domain deleted form of human FVIII (BDD-FVIII). Cells were then induced with cytokines to form a population of multiple lineages including megakaryocytes. Chromogenic analysis of culture supernatant from FVIII-transduced human cells demonstrated synthesis of functional FVIII. Treatment of cells with agonists of platelet activation (ADP, epinephrine, and thrombin receptor-activating peptide) resulted in the release of VWF antigen and active FVIII into the supernatant from transduced cells. Immunofluorescence analysis of cultured human and murine megakaryocytes revealed a punctate pattern of staining for FVIII that was consistent with staining for VWF. Electron microscopy of transduced megakaryocytes using immunogold-conjugated antibodies colocalized FVIII and VWF within the alpha-granules. FVIII retained its association with VWF in human platelets isolated from the peripheral blood of NOD/SCID mice at 2-6 weeks post-transplant of transduced human PBC. These results suggest feasibility for the development of a locally inducible secretory pool of FVIII in platelets of patients with hemophilia A.

  14. Enhanced Proteolytic Processing of Recombinant Human Coagulation Factor VIII B-Domain Variants by Recombinant Furins.

    PubMed

    Demasi, Marcos A; de S Molina, Erika; Bowman-Colin, Christian; Lojudice, Fernando H; Muras, Angelita; Sogayar, Mari C

    2016-06-01

    Recombinant human factor VIII (rFVIII) is used in replacement therapy for hemophilia A. Current research efforts are focused on bioengineering rFVIII molecules to improve its secretion efficiency and stability, limiting factors for its efficient production. However, high expression yield in mammalian cells of these rFVIII variants is generally associated with limited proteolytic processing. Non-processed single-chain polypeptides constitute non-natural FVIII molecule configurations with unpredictable toxicity and/or antigenicity. Our main objective was to demonstrate the feasibility of promoting full-proteolytic processing of an rFVIII variant retaining a portion of the B-domain, converting it into the smallest natural activatable form of rFVIII, while keeping its main advantage, i.e., improved secretion efficiency. We generated and employed a CHO-DG44 cell clone producing an rFVIII variant retaining a portion of the B-domain and the FVIII native cleavage site between Arg(1648) and Glu(1649). By bioengineering CHO-DG44 cells to express stably the recombinant human endoproteases PACE, PACE-SOL, PCSK5, PCSK6, or PCKS7, we were able to achieve complete intra- or extracellular proteolytic processing of this rFVIII variant. Additionally, our quantitative data indicated that removal of the B-domain segment by intracellular proteolytic processing does not interfere with this rFVIII variant secretion efficiency. This work also provides the first direct evidence of (1) intracellular cleavage at the Arg(1648) FVIII processing site promoted by wild-type PACE and PCSK7 and (2) proteolytic processing at the Arg(1648) FVIII processing site by PCSK6.

  15. Quantitative Influence of ABO Blood Groups on Factor VIII and Its Ratio to von Willebrand Factor, Novel Observations from an ARIC Study of 11,673 Subjects.

    PubMed

    Song, Jaewoo; Chen, Fengju; Campos, Marco; Bolgiano, Doug; Houck, Katie; Chambless, Lloyd E; Wu, Kenneth K; Folsom, Aaron R; Couper, David; Boerwinkle, Eric; Dong, Jing-fei

    2015-01-01

    ABO blood groups are known to influence the plasma level of von Willebrand factor (VWF), but little is known about the relationship between ABO and coagulation factor VIII (FVIII). We analyzed the influence of ABO genotypes on VWF antigen, FVIII activity, and their quantitative relationship in 11,673 participants in the Atherosclerosis Risk in Communities (ARIC) study. VWF, FVIII, and FVIII/VWF levels varied significantly among O, A (A1 and A2), B and AB subjects, and the extent of which varied between Americans of European (EA) and African (AA) descent. We validated a strong influence of ABO blood type on VWF levels (15.2%), but also detected a direct ABO influence on FVIII activity (0.6%) and FVIII/VWF ratio (3.8%) after adjustment for VWF. We determined that FVIII activity changed 0.54% for every 1% change in VWF antigen level. This VWF-FVIII relationship differed between subjects with O and B blood types in EA, AA, and in male, but not female subjects. Variations in FVIII activity were primarily detected at low VWF levels. These new quantitative influences on VWF, FVIII and the FVIII/VWF ratio help understand how ABO genotypes differentially influence VWF, FVIII and their ratio, particularly in racial and gender specific manners.

  16. Recombinant factor VIII Fc (rFVIIIFc) fusion protein reduces immunogenicity and induces tolerance in hemophilia A mice

    PubMed Central

    Krishnamoorthy, Sriram; Liu, Tongyao; Drager, Douglas; Patarroyo-White, Susannah; Chhabra, Ekta Seth; Peters, Robert; Josephson, Neil; Lillicrap, David; Blumberg, Richard S.; Pierce, Glenn F.; Jiang, Haiyan

    2016-01-01

    Anti-factor VIII (FVIII) antibodies is a major complication of FVIII replacement therapy for hemophilia A. We investigated the immune response to recombinant human factor VIII Fc (rFVIIIFc) in comparison to BDD-rFVIII and full-length rFVIII (FL-rFVIII) in hemophilia A mice. Repeated administration of therapeutically relevant doses of rFVIIIFc in these mice resulted in significantly lower antibody responses to rFVIII compared to BDD-rFVIII and FL-rFVIII and reduced antibody production upon subsequent challenge with high doses of rFVIIIFc. The induction of a tolerogenic response by rFVIIIFc was associated with higher percentage of regulatory T-cells, a lower percentage of pro-inflammatory splenic T-cells, and up-regulation of tolerogenic cytokines and markers. Disruption of Fc interactions with either FcRn or Fcγ receptors diminished tolerance induction, suggesting the involvement of these pathways. These results indicate that rFVIIIFc reduces immunogenicity and imparts tolerance to rFVIII demonstrating that recombinant therapeutic proteins may be modified to influence immunogenicity and facilitate tolerance. PMID:26775174

  17. Spotlight on the human factor: building a foundation for the future of haemophilia A management: report from a symposium on human recombinant FVIII at the World Federation of Hemophilia World Congress, Melbourne, Australia on 12 May 2014.

    PubMed

    Kessler, C; Oldenburg, J; Ettingshausen, C Escuriola; Tiede, A; Khair, K; Négrier, C; Klamroth, R

    2015-01-01

    Inhibitor development is the most serious and challenging complication in the treatment of severe haemophilia A. Up to 38% of such patients develop inhibitors with current recombinant factor VIII (rFVIII) products produced in hamster cell lines. Human-cl rhFVIII is a new generation fully sulfated B-domain-deleted FVIII coagulant glycoprotein, which is generated from a human cell line. Thus, there are no non-human epitopes which would be potentially immunogenic. This molecule has significantly higher VWF-binding affinity compared with existing full-length rFVIII produced in hamster cell lines. The development aim of Human-cl rhFVIII is to address the challenges of FVIII inhibitors and frequent infusions during prophylaxis. Human-cl rhFVIII's mean half-life is very comparable to some of the newer products which involve modification of the FVIII molecule to extend the circulating half-life. There are promising data concerning the use of a personalized prophylaxis regimen with Human-cl rhFVIII. Preliminary data indicate a median dosing interval of 3.5 days with 66.7% of the patients on a twice per week or fewer infusions schedule combined with a low bleeding rate and no increased FVIII consumption when compared to standard prophylaxis. No product-specific laboratory assay is required to monitor the coagulation activity for Human-cl rhFVIII. The results of registration clinical trials with Human-cl rhFVIII as well as the ongoing studies in previously untreated patients (NuProtect) and personalized prophylaxis study in previously treated patients (NuPreviq), will be discussed. The manufacturer has received marketing authorization for Human-cl rhFVIII in Europe and Canada under the name Nuwiq(®) and plans to launch it in the USA and globally in 2015.

  18. Continuous infusion of porcine factor VIII: stability, microbiological safety and clinical experience.

    PubMed

    DiMichele, D M; Gorman, P O; Kasper, C K; Mannucci, P M; Santagostino, E; Hay, C R M

    2002-01-01

    Porcine factor VIII (pFVIII) is an effective haemostatic treatment for bleeding in selected patients with FVIII inhibitors. Its use is sometimes associated with a transient fall in platelet count and transfusion reactions, the risk of which may be related to the rate of administration. Theoretical considerations suggest that the administration of pFVIII by continuous infusion should be effective, and could have pharmacokinetic advantages that lead to an improvement in the side-effect profile. The results of a retrospective survey of continuous infusion of pFVIII with respect to clinical safety and efficacy are reported. Porcine FVIII stability and microbiological studies are included. It is concluded that pFVIII given by continuous infusion is safe and effective. The risk of transfusion reactions and fall in platelet count appears to be reduced, compared with bolus administration. Stability studies showed that pFVIII activity declined at room temperature, most rapidly in the dilute solution (5-10 U mL(-1)). More concentrated mixtures showed acceptable stability for up to 24 h using a variety of infusion devices. Various concentrations of pFVIII did not support the growth of Escherichia coli or Staphylococcus aureus. These observations suggest that the porcine factor is suitable for continuous infusion (CI).

  19. Improvement of fibrin clot structure after factor VIII injection in haemophilia A patients treated on demand.

    PubMed

    Antovic, Aleksandra; Mikovic, Danijela; Elezovic, Ivo; Zabczyk, Michael; Hutenby, Kjell; Antovic, Jovan P

    2014-04-01

    Patients with haemophilia A have seriously impaired thrombin generation due to an inherited deficiency of factor (F)VIII, making them form unstable fibrin clots that are unable to maintain haemostasis. Data on fibrin structure in haemophilia patients remain limited. Fibrin permeability, assessed by a flow measurement technique, was investigated in plasma from 20 patients with severe haemophilia A treated on demand, before and 30 minutes after FVIII injection. The results were correlated with concentrations of fibrinogen, FVIII and thrombin-activatable fibrinolysis inhibitor (TAFI), and global haemostatic markers: endogenous thrombin potential (ETP) and overall haemostatic potential (OHP). Fibrin structure was visualised using scanning electron microscopy (SEM). The permeability coefficient Ks decreased significantly after FVIII treatment. Ks correlated significantly with FVIII levels and dosage, and with ETP, OHP and levels of TAFI. SEM images revealed irregular, porous fibrin clots composed of thick and short fibers before FVIII treatment. The clots had recovered after FVIII replacement almost to levels in control samples, revealing compact fibrin with smaller intrinsic pores. To the best of our knowledge, this is the first description of fibrin porosity and structure before and after FVIII treatment of selected haemophilia patients. It seems that thrombin generation is the main determinant of fibrin structure in haemophilic plasma.

  20. Eradication of neutralizing antibodies to factor VIII in canine hemophilia A after liver gene therapy.

    PubMed

    Finn, Jonathan D; Ozelo, Margareth C; Sabatino, Denise E; Franck, Helen W G; Merricks, Elizabeth P; Crudele, Julie M; Zhou, Shangzhen; Kazazian, Haig H; Lillicrap, David; Nichols, Timothy C; Arruda, Valder R

    2010-12-23

    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.

  1. Expression of human factor VIII under control of the platelet-specific alphaIIb promoter in megakaryocytic cell line as well as storage together with VWF.

    PubMed

    Shi, Q; Wilcox, D A; Fahs, S A; Kroner, P A; Montgomery, R R

    2003-05-01

    Hemophilia A, which results in defective or deficient factor VIII (FVIII) protein, is one of the genetic diseases that has been addressed through gene therapy trials. FVIII synthesis does not occur in normal megakaryocytes. In hemophilia patients who have inhibitors to FVIII activity, megakaryocytes could be a protected site of FVIII synthesis and subsequent release. Since von Willebrand factor (VWF) is a carrier protein for FVIII, we hypothesize that by directing FVIII synthesis to megakaryocytes, it would traffick together with VWF to storage in megakaryocyte alpha-granules and the platelets derived from these cells. Such synthesis would establish a protected, releasable alpha-granule pool of FVIII together with VWF. When platelets are activated in a region of local vascular damage, FVIII and VWF could potentially be released together to provide improved local hemostatic effectiveness. To direct FVIII expression to the megakaryocyte lineage, we designed a FVIII expression cassette where the human B-domain deleted FVIII cDNA was placed under the control of the megakaryocytic/platelet-specific glycoprotein IIb (alphaIIb) promoter. We demonstrated by means of a functional FVIII activity assay that the biosynthesis of FVIII occurred normally in Dami cells transfected with FVIII. FVIII production was higher when driven by the alphaIIb promoter compared to the CMV promoter, and was increased about 8-fold following PMA treatment of the transfected Dami cells. Immunofluorescence staining of the transfected cells showed that FVIII stored together with VWF in the granules. The data indicate that the megakaryocytic compartment of hematopoietic cells may represent a potential target of gene therapy for hemophilia A-especially in those patients who have developed inhibitors to plasma FVIII.

  2. Structural model of porcine factor VIII and factor VIIIa molecules based on scanning transmission electron microscope (STEM) images and STEM mass analysis.

    PubMed Central

    Mosesson, M W; Fass, D N; Lollar, P; DiOrio, J P; Parker, C G; Knutson, G J; Hainfeld, J F; Wall, J S

    1990-01-01

    Porcine plasma factor VIII (fVIII) molecules are heterodimers composed of a 76,000-mol wt light chain (-A3-C1-C2) and a heavy chain ranging in molecular weight from 82,000 (A1-A2) to 166,000 (A1-A2-B). Proteolytic activation of fVIII by thrombin results in fVIIIa heterotrimers lacking B domains (A1, A2, A3-C1-C2). In this study, immunoaffinity purified fVIII was further fractionated by mono S or mono Q chromatography to prepare heterodimers containing a light chain and an A1-A2-B heavy chain (fVIII 166/76) or an A1-A2 heavy chain (fVIII 82/76). Mass analysis of scanning transmission electron microscopic (STEM) images of fVIII 166/76 indicated that heterodimers (mass 237 +/- 20 kD) had irregularly globular core structures 10-12 nm across, and frequently displayed a diffuse, occasionally globular to ovoid satellite structure extending 5-14 nm from the core, and attached to it by a thin stalk. Factor VIII 82/76 molecules (mass 176 +/- 20 kD) had the same core structures as fVIII 166/76 molecules, but lacked the satellite structure. These findings indicate that A1-A2 domains of heavy chains and the light chains of the fVIII procofactor molecule are closely associated and constitute the globular core structure, whereas the B domainal portion of heavy chains comprises the peripheral satellite appendage. Factor VIII core structures commonly displayed a finger-like projection near the origin of the B domainal stalk that was also a consistent feature of the free heavy chains (mass 128-162 kD) found in fVIII 166/76 preparations. Factor VIII light chain monomers (mass, 76 +/- 16 kD) were globular to c-shaped particles 6-8 nm across. These chains commonly possessed a v-shaped projection originating from its middle region, that could also be observed at the periphery of fVIII core molecules. Factor VIIIa preparations contained heterotrimers (mass 162 +/- 13 kD) that had the same dimensions as fVIII core structures, lacked the B domainal appendage, and sometimes possessed the

  3. Potential for cellular stress response to hepatic factor VIII expression from AAV vector

    PubMed Central

    Zolotukhin, Irene; Markusic, David M; Palaschak, Brett; Hoffman, Brad E; Srikanthan, Meera A; Herzog, Roland W

    2016-01-01

    Hemophilia A and B are coagulation disorders resulting from the loss of functional coagulation factor VIII (FVIII) or factor IX proteins, respectively. Gene therapy for hemophilia with adeno-associated virus vectors has shown efficacy in hemophilia B patients. Although hemophilia A patients are more prevalent, the development of therapeutic adeno-associated virus vectors has been impeded by the size of the F8 cDNA and impaired secretion of FVIII protein. Further, it has been reported that over-expression of the FVIII protein induces endoplasmic reticulum stress and activates the unfolded protein response pathway both in vitro and in hepatocytes in vivo, presumably due to retention of misfolded FVIII protein within the endoplasmic reticulum. Engineering of the F8 transgene, including removal of the B domain (BDD-FVIII) and codon optimization, now allows for the generation of adeno-associated virus vectors capable of expressing therapeutic levels of FVIII. Here we sought to determine if the risks of inducing the unfolded protein response in murine hepatocytes extend to adeno-associated virus gene transfer. Although our data show a mild activation of unfolded protein response markers following F8 gene delivery at a certain vector dose in C57BL/6 mice, it was not augmented upon further elevated dosing, did not induce liver pathology or apoptosis, and did not impact FVIII immunogenicity. PMID:27738644

  4. IDO1 suppresses inhibitor development in hemophilia A treated with factor VIII

    PubMed Central

    Matino, Davide; Gargaro, Marco; Santagostino, Elena; Di Minno, Matteo N.D.; Castaman, Giancarlo; Morfini, Massimo; Rocino, Angiola; Mancuso, Maria E.; Di Minno, Giovanni; Coppola, Antonio; Talesa, Vincenzo N.; Volpi, Claudia; Vacca, Carmine; Orabona, Ciriana; Iannitti, Rossana; Mazzucconi, Maria G.; Santoro, Cristina; Tosti, Antonella; Chiappalupi, Sara; Sorci, Guglielmo; Tagariello, Giuseppe; Belvini, Donata; Radossi, Paolo; Landolfi, Raffaele; Fuchs, Dietmar; Boon, Louis; Pirro, Matteo; Marchesini, Emanuela; Grohmann, Ursula; Puccetti, Paolo; Iorio, Alfonso; Fallarino, Francesca

    2015-01-01

    The development of inhibitory antibodies to factor VIII (FVIII) is a major obstacle in using this clotting factor to treat individuals with hemophilia A. Patients with a congenital absence of FVIII do not develop central tolerance to FVIII, and therefore, any control of their FVIII-reactive lymphocytes relies upon peripheral tolerance mechanisms. Indoleamine 2,3-dioxygenase 1 (IDO1) is a key regulatory enzyme that supports Treg function and peripheral tolerance in adult life. Here, we investigated the association between IDO1 competence and inhibitor status by evaluating hemophilia A patients harboring F8-null mutations that were either inhibitor negative (n = 50) or positive (n = 50). We analyzed IDO1 induction, expression, and function for any relationship with inhibitor occurrence by multivariable logistic regression and determined that defective TLR9-mediated activation of IDO1 induction is associated with an inhibitor-positive status. Evaluation of experimental hemophilic mouse models with or without functional IDO1 revealed that tryptophan metabolites, which result from IDO1 activity, prevent generation of anti-FVIII antibodies. Moreover, treatment of hemophilic animals with a TLR9 agonist suppressed FVIII-specific B cells by a mechanism that involves IDO1-dependent induction of Tregs. Together, these findings indicate that strategies aimed at improving IDO1 function should be further explored for preventing or eradicating inhibitors to therapeutically administered FVIII protein. PMID:26426076

  5. Six Amino Acid Residues in a 1200 Å2 Interface Mediate Binding of Factor VIII to an IgG4κ Inhibitory Antibody

    PubMed Central

    Lin, Jasper C.; Ettinger, Ruth A.; Schuman, Jason T.; Zhang, Ai-Hong; Wamiq-Adhami, Muhammad; Nguyen, Phuong-Cac T.; Nakaya-Fletcher, Shelley M.; Puranik, Komal; Thompson, Arthur R.; Pratt, Kathleen P.

    2015-01-01

    The development of neutralizing anti-factor VIII (FVIII) antibodies complicates the treatment of many hemophilia A patients. The C-terminal C2 domain is a particularly antigenic FVIII region. A crystal structure of recombinant FVIII-C2 bound to an Fab fragment of the patient-derived monoclonal antibody BO2C11, which recognizes an immunodominant inhibitor epitope on FVIII and blocks its ability to bind von Willebrand factor (VWF) and phospholipids, revealed that 15 amino acids in FVIII contact this antibody. Forty-three recombinant FVIII-C2 proteins, each with a surface-exposed side chain mutated to alanine or another residue, were generated, and surface plasmon resonance studies were carried out to evaluate effects of these substitutions on BO2C11/FVIII-C2 binding affinity. Thermodynamic analysis of experiments carried out at three temperatures indicated that one beta hairpin turn at the antigen-antibody interface (FVIII-F2196, N2198, M2199 and F2200) plus two non-contiguous arginines (FVIII-R2215 and R2220), contributed appreciably to the affinity. B-domain-deleted (BDD) FVIII-F2196A, FVIII-F2196K and FVIII-M2199A were generated and characterized. Their pro-coagulant activities and binding to VWF were similar to those of WT-BDD-FVIII, and FVIII-F2196K avoided neutralization by BO2C11 and murine inhibitory mAb 1B5. This study suggests specific sites for amino acid substitutions to rationally design FVIII variants capable of evading immunodominant neutralizing anti-FVIII antibodies. PMID:25615825

  6. Epitope mapping of inhibitory antibodies targeting the C2 domain of coagulation factor VIII by hydrogen-deuterium exchange mass spectrometry

    PubMed Central

    Sevy, Alexander M.; Healey, John F.; Deng, Wei; Spiegel, P. Clint; Meeks, Shannon L.; Li, Renhao

    2014-01-01

    Summary Background The development of anti-factor VIII (fVIII) antibodies (inhibitors) is a significant complication in the management of patients with hemophilia A, leading to significant increases in morbidity and treatment cost. Using a panel of anti-fVIII monoclonal antibodies (MAbs) to different epitopes on fVIII, we recently have shown that epitope specificity, inhibitor kinetics, and time to maximum inhibition are more important than inhibitor titer in predicting response to fVIII and the combination of fVIII and recombinant factor VIIa. In particular, a subset of high-titer inhibitors responded to high dose fVIII, which would not be predicted based on their inhibitor titer alone. Thus the ability to quickly map the epitope spectrum of patient plasma using a clinically feasible assay may fundamentally change how clinicians approach the treatment of high-titer inhibitor patients. Objectives To map the epitopes of anti-fVIII MAbs, of which 3 are classical inhibitors and one non-classical, using hydrogen-deuterium exchange coupled with liquid chromatography-mass spectrometry (HDX-MS). Methods Binding epitopes of 4 MAbs targeting fVIII C2 domain were mapped using HDX-MS. Results The epitopes determined by HDX-MS are consistent with those obtained earlier through structural characterization and antibody competition assays. In addition classical and non-classical inhibitor epitopes could be distinguished using a limited subset of C2-derived peptic fragments. Conclusion Our results demonstrate the effectiveness and robustness of the HDX-MS method for epitope mapping and suggest a potential role of rapid mapping of fVIII inhibitor epitopes in facilitating individualized treatment of inhibitor patients. PMID:24152306

  7. Characterization of a splicing mutation in the factor VIII gene at the RNA level.

    PubMed

    David, D; Tavares, A; Lavinha, J

    1995-01-01

    Haemophilia A is an X-linked bleeding disorder caused by mutations in the coagulation factor VIII (FVIII) gene. The identification and characterization of naturally occurring disease-producing mutations allows the recognition of new mechanisms of pathogenesis in haemophilia A. Analysis of the illegitimately transcribed FVIII mRNA in a severely affected patient has revealed that the A-->G transition at position -2 of the acceptor splice site of intron 4 results in the skipping of exon 5 in 90% of the processed pre-mRNA. Another minor mRNA species arising from the skipping of exons 4 and 5 has also been observed. The skipping of exon 5 predicts the removal of the corresponding 13 amino acids from the A1 domain of FVIII. A novel missense mutation, C329S, in exon 8 of FVIII gene has been identified in another patient.

  8. Recombinant B domain deleted porcine factor VIII for the treatment of bleeding episodes in adults with acquired hemophilia A.

    PubMed

    Gomperts, Edward

    2015-08-01

    Hemophilia A is an inherited deficiency of clotting factor VIII (FVIII) often complicated by inhibitor development (CHAWI) in which neutralizing antibodies block the therapeutic benefit of replacement therapy. Inhibitors to FVIII can also be seen in an auto-immune disease known as acquired hemophilia A (AHA). 'Bypassing' therapies have been shown to provide hemostasis but dosing must be done empirically because current assays cannot measure objective markers of treatment efficacy and safety. A recombinant porcine sequence factor VIII (r-pFVIII) has been developed for the management of AHA. Preclinical, Phase I and Phase II clinical research studies in CHAWI subjects showed therapeutic potential and safety of this agent. A Phase II/III study in AHA with serious bleeding episodes shows a positive response in all subjects after administration. Based on current preclinical and clinical trial data, r-pFVIII should become the first line of treatment in the management of hemorrhage in patients with AHA.

  9. Intraosseous delivery of lentiviral vectors targeting factor VIII expression in platelets corrects murine hemophilia A.

    PubMed

    Wang, Xuefeng; Shin, Simon C; Chiang, Andy F J; Khan, Iram; Pan, Dao; Rawlings, David J; Miao, Carol H

    2015-04-01

    Intraosseous (IO) infusion of lentiviral vectors (LVs) for in situ gene transfer into bone marrow may avoid specific challenges posed by ex vivo gene delivery, including, in particular, the requirement of preconditioning. We utilized IO delivery of LVs encoding a GFP or factor VIII (FVIII) transgene directed by ubiquitous promoters (a MND or EF-1α-short element; M-GFP-LV, E-F8-LV) or a platelet-specific, glycoprotein-1bα promoter (G-GFP-LV, G-F8-LV). A single IO infusion of M-GFP-LV or G-GFP-LV achieved long-term and efficient GFP expression in Lineage(-)Sca1(+)c-Kit(+) hematopoietic stem cells and platelets, respectively. While E-F8-LV produced initially high-level FVIII expression, robust anti-FVIII immune responses eliminated functional FVIII in circulation. In contrast, IO delivery of G-F8-LV achieved long-term platelet-specific expression of FVIII, resulting in partial correction of hemophilia A. Furthermore, similar clinical benefit with G-F8-LV was achieved in animals with pre-existing anti-FVIII inhibitors. These findings further support platelets as an ideal FVIII delivery vehicle, as FVIII, stored in α-granules, is protected from neutralizing antibodies and, during bleeding, activated platelets locally excrete FVIII to promote clot formation. Overall, a single IO infusion of G-F8-LV was sufficient to correct hemophilia phenotype for long term, indicating that this approach may provide an effective means to permanently treat FVIII deficiency.

  10. Anti-CD3 antibodies modulate anti-factor VIII immune responses in hemophilia A mice after factor VIII plasmid-mediated gene therapy.

    PubMed

    Peng, Baowei; Ye, Peiqing; Rawlings, David J; Ochs, Hans D; Miao, Carol H

    2009-11-12

    One major obstacle in gene therapy is the generation of immune responses directed against transgene product. Five consecutive anti-CD3 treatments concomitant with factor VIII (FVIII) plasmid injection prevented the formation of inhibitory antibodies against FVIII and achieved persistent, therapeutic levels of FVIII gene expression in treated hemophilia A mice. Repeated plasmid gene transfer is applicable in tolerized mice without eliciting immune responses. Anti-CD3 treatment significantly depleted both CD4+ and CD8+ T cells, whereas increased transforming growth factor-beta levels in plasma and the frequency of both CD4+CD25+FoxP3+ and CD4+CD25-Foxp3+ regulatory T cells in the initial few weeks after treatment. Although prior depletion of CD4+CD25+ cells did not abrogate tolerance induction, adoptive transfer of CD4+ cells from tolerized mice at 6 weeks after treatment protected recipient mice from anti-FVIII immune responses. Anti-CD3-treated mice mounted immune responses against both T-dependent and T-independent neo-antigens, indicating that anti-CD3 did not hamper the immune systems in the long term. Concomitant FVIII plasmid + anti-CD3 treatment induced long-term tolerance specific to FVIII via a mechanism involving the increase in transforming growth factor-beta levels and the generation of adaptive FVIII-specific CD4+Foxp3+ regulatory T cells at the periphery. Furthermore, anti-CD3 can reduce the titers of preexisting anti-FVIII inhibitory antibodies in hemophilia A mice.

  11. Intensity of factor VIII treatment and inhibitor development in children with severe hemophilia A: the RODIN study.

    PubMed

    Gouw, Samantha C; van den Berg, H Marijke; Fischer, Kathelijn; Auerswald, Günter; Carcao, Manuel; Chalmers, Elizabeth; Chambost, Hervé; Kurnik, Karin; Liesner, Ri; Petrini, Pia; Platokouki, Helen; Altisent, Carmen; Oldenburg, Johannes; Nolan, Beatrice; Garrido, Rosario Pérez; Mancuso, M Elisa; Rafowicz, Anne; Williams, Mike; Clausen, Niels; Middelburg, Rutger A; Ljung, Rolf; van der Bom, Johanna G

    2013-05-16

    The objective of this study was to examine the association of the intensity of treatment, ranging from high-dose intensive factor VIII (FVIII) treatment to prophylactic treatment, with the inhibitor incidence among previously untreated patients with severe hemophilia A. This cohort study aimed to include consecutive patients with a FVIII activity < 0.01 IU/mL, born between 2000 and 2010, and observed during their first 75 FVIII exposure days. Intensive FVIII treatment of hemorrhages or surgery at the start of treatment was associated with an increased inhibitor risk (adjusted hazard ratio [aHR], 2.0; 95% confidence interval [CI], 1.3-3.0). High-dose FVIII treatment was associated with a higher inhibitor risk than low-dose FVIII treatment (aHR, 2.3; 95% CI, 1.0-4.8). Prophylaxis was only associated with a decreased overall inhibitor incidence after 20 exposure days of FVIII. The association with prophylaxis was more pronounced in patients with low-risk F8 genotypes than in patients with high-risk F8 genotypes (aHR, 0.61, 95% CI, 0.19-2.0 and aHR, 0.85, 95% CI, 0.51-1.4, respectively). In conclusion, our findings suggest that in previously untreated patients with severe hemophilia A, high-dosed intensive FVIII treatment increases inhibitor risk and prophylactic FVIII treatment decreases inhibitor risk, especially in patients with low-risk F8 mutations.

  12. Acquired factor VIII deficiency associated with a novel primary immunodeficiency suggestive of autosomal recessive hyper IgE syndrome.

    PubMed

    Ozgur, Tuba Turul; Asal, Gulten Turkkan; Gurgey, Aytemiz; Tezcan, Ilhan; Ersoy, Fugen; Sanal, Ozden

    2007-05-01

    Primary immunodeficiency diseases (PID) are associated with various autoimmune complications and several manifestations of autoimmunity can be seen in the disorders of T cells, B cells, phagocytes, and complement components. Acquired hemophilia is a rare entity in childhood. Although autoantibodies may develop in various forms of PID, Factor VIII (FVIII) inhibitors have not been described before. Herein, we present a case of acquired hemophilia resulting from FVIII inhibitors who had underlying undefined PID features suggestive of autosomal recessive hyper IgE syndrome. Our patient responded to corticosteroid treatment rather well and quickly, with an increased FVIII level and decreased FVIII inhibitors. However, FVIII inhibitor reappeared 7 months later, and disappeared spontaneously 4 months ago. Long-term and close follow-up is needed to observe the long-term prognosis in this child.

  13. Biosynthesis of FVIII in megakaryocytic cells: improved production and biochemical characterization.

    PubMed

    Rodriguez, Marie-Hélène; Plantier, Jean-Luc; Enjolras, Nathalie; Réa, Muriel; Leboeuf, Marylène; Uzan, Georges; Négrier, Claude

    2004-12-01

    Haemophilia A is an attractive target for gene therapy. We designed a haemophilia A gene therapy strategy involving the genetic modification of haematopoietic stem cells to achieve tissue-specific expression of a factor VIII (FVIII) transgene in the megakaryocytic lineage. Platelets would then serve as vehicles to store the expressed FVIII and deliver the coagulation factor at the site of vascular injury. A local correction of the haemostasis defect could, therefore, be expected following platelet activation and secretion. In this study, we demonstrated that a model of haematopoietic cell lines (Dami cells) could produce a correctly processed FVIII. FVIII transgenes were placed under the control of the human platelet glycoprotein IIb (GPIIb) promoter and used for stable transfection of the Dami megakaryocytic cell line. The highest FVIII production was obtained when the FVIII transgene contained a factor IX intron 1 gene sequence inserted in the FVIII intron 1 and 13 sites. Reverse transcription polymerase chain reaction demonstrated that the splicing of these introns was complete. Recombinant FVIII (rFVIII) produced in Dami cells was a biologically active molecule (specific activity: 5664 IU/mg) that was correctly glycosylated and sulphated. This recombinant FVIII protein exhibited biochemical characteristics after deglycosylation or thrombin activation that were comparable to a commercially available B-domainless rFVIII. These results demonstrate the advantages of a modified FVIII transgene and represent the first biochemical characterization of megakaryocyte-produced FVIII.

  14. Generation of an optimized lentiviral vector encoding a high-expression factor VIII transgene for gene therapy of hemophilia A.

    PubMed

    Johnston, J M; Denning, G; Doering, C B; Spencer, H T

    2013-06-01

    We previously compared the expression of several human factor VIII (fVIII) transgene variants and demonstrated the superior expression properties of B domain-deleted porcine fVIII. Subsequently, a hybrid human/porcine fVIII molecule (HP-fVIII) comprising 91% human amino-acid sequence was engineered to maintain the high-expression characteristics of porcine fVIII. The bioengineered construct then was used effectively to treat knockout mice with hemophilia A. In the current study, we focused on optimizing self-inactivating (SIN) lentiviral vector systems by analyzing the efficacy of various lentiviral components in terms of virus production, transduction efficiency and transgene expression. Specifically, three parameters were evaluated: (1) the woodchuck hepatitis post-transcriptional regulatory element (WPRE), (2) HIV versus SIV viral vector systems and (3) various internal promoters. The inclusion of a WPRE sequence had negligible effects on viral production and HP-fVIII expression. HIV and SIV vectors were compared and found to be similar with respect to transduction efficiency in both K562s and HEK-293T cells. However, there was an enhanced expression of HP-fVIII by the SIV system, which was evident in both K562 and BHK-M cell lines. To further compare expression of HP-fVIII from an SIV-based lentiviral system, we constructed expression vectors containing the high expression transgene and a human elongation factor-1 alpha, cytomegalovirus (CMV) or phosphoglycerate kinase promoter. Expression was significantly greater from the CMV promoter, which also yielded therapeutic levels of HP-fVIII in hemophilia A mice. Based on these studies, an optimized vector contains the HP-fVIII transgene driven by a CMV internal promoter within a SIV-based lentiviral backbone lacking a WPRE.

  15. Continuous infusion of porcine factor VIII in patients with haemophilia A and high-responding inhibitors: stability and clinical experience.

    PubMed

    O'Gorman, P; Dimichele, D M; Kasper, C K; Mannucci, P M; Santagostini, E; Hay, C R

    2001-11-01

    A multicentre retrospective survey was conducted to assess the efficacy and side-effect profile of porcine factor VIII (pFVIII:C) given by continuous infusion (CI) to patients with congenital haemophilia A and inhibitors. Twenty-nine episodes in 18 patients were treated by CI of pFVIII:C. Efficacy was graded as good in 79% of infusions and fair in 17%. There was a failed response in only one episode. Fourteen percent of patients experienced transfusion reactions with bolus doses, but no reactions were observed in patients given CI. There were no severe reactions. All the reactions resolved following interruption of the infusion and administration of steroids. Premedication did not prevent reactions. In this series the median decrease in platelet count after bolus injection of pFVIII:C was -67 X 10(9) L(-1) compared with a median decrease of -2 x 109 L(-1) during the course of CI, thus, continuous infusion of pFVIII:C appears to have less effect on platelet count than bolus injection. An anamnestic response was associated with 77% of infusions. This high rate of anamnesis reflects patient selection, in that they were all known to have high-level high-responding FVIII inhibitors with cross-reactivity to pFVIII. After reconstitution, the pFVIII:C showed little loss in factor VIII activity in solution over a 24-h period. We conclude that pFVIII:C may be effectively administered by CI to patients with haemophilia A and high-responding FVIII inhibitors. CI is the probably the mode of administration of choice for intensive replacement therapy with pFVIII.

  16. Characterization of factor VIII pharmaceutical preparations by means of MudPIT proteomic approach.

    PubMed

    Basilico, Fabrizio; Nardini, Ilaria; Mori, Filippo; Brambilla, Elena; Benazzi, Louise; De Palma, Antonella; Rosti, Enrico; Farina, Claudio; Mauri, PierLuigi

    2010-09-21

    For a good clinical outcome of Haemophilia A substitutive therapy a detailed characterization of factor VIII (FVIII) concentrates is required, in order to disclose the eventual relations between differently composed concentrates and their biological effects. This preliminary work could be a first step towards a deep structural characterization of FVIII concentrates, using the fast and simply manageable MudPIT technology, which enables the identification and characterization of protein mixtures taking advantage of both the high separation capacity of two-dimensional chromatography and the powerful peptide characterization ability of tandem mass spectrometry. The aim of this study was to evaluate the suitability of for the characterization of FVIII molecule in complex mixtures such its commercial concentrates, both plasma-derived and recombinant, and for the determination of the protein composition of different FVIII preparations. By means of Multidimensional Protein Identification Technology (MudPIT) it was possible to assess the presence of factor VIII in its preparations and to identify most of the contaminant proteins without gel separation. In particular, 125 and 42 proteins were identified in plasma-derived and recombinant concentrates, respectively. Concerning investigation of FVIII, 24 different peptides were identified in plasma-derived corresponding to 7, 29, 27, 19 and 67 of percentage coverage for A1, A2, A3, C1 and C2 domains, respectively. About its multimeric carrier von Willebrand factor (VWF), we have sequenced 42% of domain interacting with A3 and C2 domains of FVIII. Finally, it has been observed that normalized parameters, such as total peptide hits obtained by SEQUEST may be used for evaluation of the relative abundance of FVIII in different preparations.

  17. Bioengineered coagulation factor VIII enables long-term correction of murine hemophilia A following liver-directed adeno-associated viral vector delivery

    PubMed Central

    Brown, Harrison C; Wright, J Fraser; Zhou, Shangzhen; Lytle, Allison M; Shields, Jordan E; Spencer, H Trent; Doering, Christopher B

    2014-01-01

    Clinical data support the feasibility and safety of adeno-associated viral (AAV) vectors in gene therapy applications. Despite several clinical trials of AAV-based gene transfer for hemophilia B, a unique set of obstacles impede the development of a similar approach for hemophilia A. These include (i) the size of the factor VIII (fVIII) transgene, (ii) humoral immune responses to fVIII, (iii) inefficient biosynthesis of human fVIII, and (iv) AAV vector immunity. Through bioengineering approaches, a novel fVIII molecule, designated ET3, was developed and shown to improve biosynthetic efficiency 10- to 100-fold. In this study, the utility of ET3 was assessed in the context of liver-directed, AAV-mediated gene transfer into hemophilia A mice. Due to the large size of the expression cassette, AAV-ET3 genomes packaged into viral particles as partial genome fragments. Despite this potential limitation, a single peripheral vein administration of AAV-ET3 into immune-competent hemophilia A mice resulted in correction of the fVIII deficiency at lower vector doses than previously reported for similarly oversized AAV-fVIII vectors. Therefore, ET3 appears to improve vector potency and mitigate at least one of the critical barriers to AAV-based clinical gene therapy for hemophilia A. PMID:26015976

  18. Cosegregation of a factor VIII microsatellite marker with mild hemophilia A in Golden Retriever dogs.

    PubMed

    Brooks, Marjory B; Barnas, Jennifer L; Fremont, Jacqueline; Ray, Jharna

    2005-01-01

    Mild hemophilia A (factor VIII deficiency) was diagnosed in Golden Retrievers and pedigree studies were undertaken to test the cosegregation of an intragenic factor VIII marker with the disease phenotype. The study population consisted of 30 client-owned dogs (22 males and 8 females). Hemophilic males (n = 12) typically demonstrated prolonged bleeding after trauma or surgery rather than spontaneous hemorrhagic events. The affected males had a proportionate reduction in factor VIII coagulant activity (mean FVIII:C = 4%) and factor VIII protein concentration (mean FVIII:Ag = 3%). Twenty-five dogs (10 affected males, 8 clear males, 2 obligate carrier dams, and 5 suspect carrier daughters) were genotyped for a factor VIII microsatellite marker, with allele size assigned by an automated capillary electrophoresis system. Five distinct marker alleles were present in the study pedigree and a 300-base pair allele was found to segregate with the hemophilia A phenotype. The inheritance of the hemophilia-associated allele defined carrier status for 5 suspect daughters of obligate carrier dams. The limitations inherent to linkage analyses (i.e., lack of access to key family members and homozygosity at the marker locus) did not preclude carrier detection in this pedigree. We conclude that genotype analysis for the intragenic factor VIII marker can aid in control of canine hemophilia A through enhanced carrier detection.

  19. Effects of FVIII immunity on hepatocyte and hematopoietic stem cell-directed gene therapy of murine hemophilia A.

    PubMed

    Lytle, Allison M; Brown, Harrison C; Paik, Na Yoon; Knight, Kristopher A; Wright, J Fraser; Spencer, H Trent; Doering, Christopher B

    2016-01-01

    Immune responses to coagulation factors VIII (FVIII) and IX (FIX) represent primary obstacles to hemophilia treatment. Previously, we showed that hematopoietic stem cell (HSC) retroviral gene therapy induces immune nonresponsiveness to FVIII in both naive and preimmunized murine hemophilia A settings. Liver-directed adeno-associated viral (AAV)-FIX vector gene transfer achieved similar results in preclinical hemophilia B models. However, as clinical immune responses to FVIII and FIX differ, we investigated the ability of liver-directed AAV-FVIII gene therapy to affect FVIII immunity in hemophilia A mice. Both FVIII naive and preimmunized mice were administered recombinant AAV8 encoding a liver-directed bioengineered FVIII expression cassette. Naive animals receiving high or mid-doses subsequently achieved near normal FVIII activity levels. However, challenge with adjuvant-free recombinant FVIII induced loss of FVIII activity and anti-FVIII antibodies in mid-dose, but not high-dose AAV or HSC lentiviral (LV) vector gene therapy cohorts. Furthermore, unlike what was shown previously for FIX gene transfer, AAV-FVIII administration to hemophilia A inhibitor mice conferred no effect on anti-FVIII antibody or inhibitory titers. These data suggest that functional differences exist in the immune modulation achieved to FVIII or FIX in hemophilia mice by gene therapy approaches incorporating liver-directed AAV vectors or HSC-directed LV.

  20. Structural investigation of zymogenic and activated forms of human blood coagulation factor VIII: a computational molecular dynamics study

    PubMed Central

    2010-01-01

    Background Human blood coagulation factor VIII (fVIII) is a large plasma glycoprotein with sequential domain arrangement in the order A1-a1-A2-a2-B-a3-A3-C1-C2. The A1, A2 and A3 domains are interconnected by long linker peptides (a1, a2 and a3) that possess the activation sites. Proteolysis of fVIII zymogen by thrombin or factor Xa results in the generation of the activated form (fVIIIa) which serves as a critical co-factor for factor IXa (fIXa) enzyme in the intrinsic coagulation pathway. Results In our efforts to elucidate the structural differences between fVIII and fVIIIa, we developed the solution structural models of both forms, starting from an incomplete 3.7 Å X-ray crystal structure of fVIII zymogen, using explicit solvent MD simulations. The full assembly of B-domainless single-chain fVIII was built between the A1-A2 (Ala1-Arg740) and A3-C1-C2 (Ser1669-Tyr2332) domains. The structural dynamics of fVIII and fVIIIa, simulated for over 70 ns of time scale, enabled us to evaluate the integral motions of the multi-domain assembly of the co-factor and the possible coordination pattern of the functionally important calcium and copper ion binding in the protein. Conclusions MD simulations predicted that the acidic linker peptide (a1) between the A1 and A2 domains is largely flexible and appears to mask the exposure of putative fIXa enzyme binding loop (Tyr555-Asp569) region in the A2 domain. The simulation of fVIIIa, generated from the zymogen structure, predicted that the linker peptide (a1) undergoes significant conformational reorganization upon activation by relocating completely to the A1-domain. The conformational transition led to the exposure of the Tyr555-Asp569 loop and the surrounding region in the A2 domain. While the proposed linker peptide conformation is predictive in nature and warrants further experimental validation, the observed conformational differences between the zymogen and activated forms may explain and support the large body of

  1. The mild phenotype in severe hemophilia A with Arg1781His mutation is associated with enhanced binding affinity of factor VIII for factor X.

    PubMed

    Yada, Koji; Nogami, Keiji; Wakabayashi, Hironao; Fay, Philip J; Shima, Midori

    2013-06-01

    The clinical severity in some patients with haemophilia A appears to be unrelated to the levels of factor (F)VIII activity (FVIII:C), but mechanisms are poorly understood. We have investigated a patient with a FVIII gene mutation at Arg1781 to His (R1781H) presenting with a mild phenotype despite FVIII:C of 0.9 IU/dl. Rotational thromboelastometry using the patient's whole blood demonstrated that the clot time and clot firmness were comparable to those usually observed at FVIII:C 5-10 IU/dl. Thrombin and FXa assays using plasma samples also showed that the peak levels of thrombin formation and the initial rate of FXa generation were comparable to those observed at FVIII:C 5-10 IU/dl. The results suggested a significantly greater haemostatic potential in this individual than in those with severe phenotype. The addition of incremental amounts of FX to control plasma with FVIII:C 0.9 IU/dl in clot waveform analyses suggested that the enhanced functional tenase assembly might have been related to changes in association between FVIII and FX. To further investigate this mechanism, we prepared a stably expressed, recombinant, B-domainless FVIII R1781H mutant. Thrombin generation assays using mixtures of control plasma and FVIII revealed that the coagulation function observed with the R1781H mutant (0.9 IU/dl) was comparable to that seen with wild-type FVIII:C at ~5 IU/dl. In addition, the R1781H mutant demonstrated an ~1.9-fold decrease in Km for FX compared to wild type. These results indicated that relatively enhanced binding affinity of FVIII R1781H for FX appeared to moderate the severity of the haemophilia A phenotype.

  2. Prolonged activity of a recombinant factor VIII-Fc fusion protein in hemophilia A mice and dogs

    PubMed Central

    Dumont, Jennifer A.; Liu, Tongyao; Low, Susan C.; Zhang, Xin; Kamphaus, George; Sakorafas, Paul; Fraley, Cara; Drager, Douglas; Reidy, Thomas; McCue, Justin; Franck, Helen W. G.; Merricks, Elizabeth P.; Nichols, Timothy C.; Bitonti, Alan J.; Pierce, Glenn F.

    2012-01-01

    Despite proven benefits, prophylactic treatment for hemophilia A is hampered by the short half-life of factor VIII. A recombinant factor VIII-Fc fusion protein (rFVIIIFc) was constructed to determine the potential for reduced frequency of dosing. rFVIIIFc has an ∼ 2-fold longer half-life than rFVIII in hemophilia A (HemA) mice and dogs. The extension of rFVIIIFc half-life requires interaction of Fc with the neonatal Fc receptor (FcRn). In FcRn knockout mice, the extension of rFVIIIFc half-life is abrogated, and is restored in human FcRn transgenic mice. The Fc fusion has no impact on FVIII-specific activity. rFVIIIFc has comparable acute efficacy as rFVIII in treating tail clip injury in HemA mice, and fully corrects whole blood clotting time (WBCT) in HemA dogs immediately after dosing. Furthermore, consistent with prolonged half-life, rFVIIIFc shows 2-fold longer prophylactic efficacy in protecting HemA mice from tail vein transection bleeding induced 24-48 hours after dosing. In HemA dogs, rFVIIIFc also sustains partial correction of WBCT 1.5- to 2-fold longer than rFVIII. rFVIIIFc was well tolerated in both species. Thus, the rescue of FVIII by Fc fusion to provide prolonged protection presents a novel pathway for FVIII catabolism, and warrants further investigation. PMID:22246033

  3. Parameters influencing FVIII pharmacokinetics in patients with severe and moderate haemophilia A.

    PubMed

    Kepa, S; Horvath, B; Reitter-Pfoertner, S; Schemper, M; Quehenberger, P; Grundbichler, M; Heistinger, M; Neumeister, P; Mannhalter, C; Pabinger, I

    2015-05-01

    In haemophilia A patients factor VIII (FVIII) recovery and half-life can vary substantially. There are parameters known to modulate FVIII pharmacokinetics (PK), but they explain only about 34% of the variability. The aim of this study was to identify new parameters that influence FVIII PK and thus to expand the current knowledge. FVIII PK were determined in 42 haemophilia A patients (37 severe, 5 moderate) without inhibitor. Patients' characteristics and laboratory parameters were evaluated for an association with FVIII PK. We analysed plasma levels of low-density lipoprotein receptor-related protein 1 (LRP1) and protein C (PC) activity, which had been hypothesized to influence FVIII activity. Furthermore, four variations in intron 6 of the LRP1 gene, which had been shown to influence LRP1, were investigated. FVIII half-life differed widely from 6.2 to 20.7 h, with a median of 10.0 h. Patients with blood group O had shorter FVIII half-life compared to patients with non-O blood group (median FVIII half-life 9.0 h vs. 10.4 h, P = 0.018). Age was significantly associated with FVIII half-life (r = 0.32, P = 0.035). Besides age, also VWF antigen (r = 0.52, P < 0.001) and blood group (r = -0.37, P = 0.015) was associated with FVIII half-life. No correlation was found with FVIII- or LRP1-genotype, LRP1 or PC concentrations. Our data showed large differences in FVIII PK between individual patients and revealed age, blood group and VWF levels as important determining factors for FVIII half-life. FVIII genotype or levels of LRP1 or PC had no influence on FVIII PK.

  4. Evaluation of the biological differences of canine and human factor VIII in gene delivery: Implications in human hemophilia treatment

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The canine is the most important large animal model for testing novel hemophilia A(HA) treatment. It is often necessary to use canine factor VIII (cFIII) gene or protein for the evaluation of HA treatment in the canine model. However, the different biological properties between cFVIII and human FVII...

  5. [Ssp DnaB intein-mediated ligation of heavy and light chains of coagulation factor VIII in Escherichia coli].

    PubMed

    Zhu, Fuxiang; Liu, Zelong; Qu, Huige; Xin, Xiaolin; Dong, Hongxin; Liu, Xiangqin

    2009-07-01

    We studied the ligation of coagulation factor VIII heavy and light chains in Escherichia coli by utilizing the intein-mediated protein trans-splicing. A B-domain deleted factor VIII (BDD-FVIII) gene was broken into two halves of heavy and light chains before Ser1657 which meets the splicing required conserved residue and then fused to 106 and 48 amino acid-containing N-part termed Int-N and C-part termed Int-C coding sequences of split mini Ssp DnaB intein respectively. These two fusion genes were constructed into a prokaryotic expression vector pBV220. Through induction for expression of recombinant protein it displayed an obvious protein band as predicted size of BDD-FVIII protein on SDS-PAGE gel. Western blotting using factor VIII specific antibodies confirmed that this protein band is BDD-FVIII produced by protein trans-splicing. It demonstrated that the heavy and light chains of BDD-FVIII can be efficiently ligated with the Ssp DnaB intein-mediated protein trans-splicing. These results provided evidence for encouraging our ongoing investigation with intein as a means in dual AAV vectors carrying the factor VIII gene to overcome the packaging size limitation of a single AAV vector in hemophilia A gene therapy.

  6. Epitope specificity of anti-factor VIII antibodies from inhibitor positive acquired and congenital haemophilia A patients using synthetic peptides spanning A and C domains.

    PubMed

    Gharagozlou, Soheila; Sharifian, Ramazan A; Khoshnoodi, Jalal; Karimi, Katayoon; Milani, Monica; Okita, David K; Shokri, Fazel; Conti-Fine, Bianca M

    2009-05-01

    Development of antibodies (Ab) that either block the function of coagulation factor VIII (FVIII) (inhibitors) or clear it from circulation, seriously complicate the treatment of haemophilia A patients with FVIII products. Autoantibodies which develop in subjects without congenital FVIII defects, cause acquired haemophilia, a rare but life-threatening coagulopathy. Identification of the FVIII epitopes to which inhibitor Abs bind will help understanding the mechanisms of inhibitor activity, and perhaps development of new therapies. Here, we examined the FVIII peptide sequence regions recognised by anti-FVIII Ab in the plasma of six congenital and one acquired haemophilia patients with high inhibitor titers (24.4-2000 BU/ml). We used indirect ELISA and overlapping synthetic peptides, 20 residues long, spanning the sequence of the A and C FVIII domains. None of the plasma samples reacted with A1, A3 or C1 domain peptides. Six plasmas reacted with A2 and/or C2 peptides. Peptides spanning residues A2-521-690 and C2-2251-2332 were recognised most frequently and strongly. They include residues that contribute to the binding sites for activated factor IX and phosphatidyl serine/von Willebrand factor. These results suggest that anti-FVIII Abs share a pattern of antigen specificity in our panel of patients, and that exposed regions of the FVIII molecule that form functionally important binding sites elicit an intense Ab response.

  7. In vivo expansion of regulatory T cells with IL-2/IL-2 mAb complexes prevents anti-factor VIII immune responses in hemophilia A mice treated with factor VIII plasmid-mediated gene therapy.

    PubMed

    Liu, Chao-Lien; Ye, Peiqing; Yen, Benjamin C; Miao, Carol H

    2011-08-01

    Generation of transgene-specific immune responses can constitute a major complication following gene therapy treatment. An in vivo approach to inducing selective expansion of Regulatory T (Treg) cells by injecting interleukin-2 (IL-2) mixed with a specific IL-2 monoclonal antibody (JES6-1) was adopted to modulate anti-factor VIII (anti-FVIII) immune responses. Three consecutive IL-2 complexes treatments combined with FVIII plasmid injection prevented anti-FVIII formation and achieved persistent, therapeutic-level of FVIII expression in hemophilia A (HemA) mice. The IL-2 complexes treatment expanded CD4(+)CD25(+)Foxp3(+) Treg cells five- to sevenfold on peak day, and they gradually returned to normal levels within 7-14 days without changing other lymphocyte populations. The transiently expanded Treg cells are highly activated and display suppressive function in vitro. Adoptive transfer of the expanded Treg cells protected recipient mice from generation of high-titer antibodies following FVIII plasmid challenge. Repeated plasmid transfer is applicable in tolerized mice without eliciting immune responses. Mice treated with IL-2 complexes mounted immune responses against both T-dependent and T-independent neoantigens, indicating that IL-2 complexes did not hamper the immune system for long. These results demonstrate the important role of Treg cells in suppressing anti-FVIII immune responses and the potential of developing Treg cell expansion therapies that induce long-term tolerance to FVIII.

  8. Suppression of FVIII Inhibitor Formation in Hemophilic Mice by Delivery of Transgene Modified Apoptotic Fibroblasts

    PubMed Central

    Su, Rui-Jun; Epp, Angela; Latchman, Yvette; Bolgiano, Doug; Pipe, Steven W; Josephson, Neil C

    2009-01-01

    The development of inhibitory antibodies to factor VIII (FVIII) is currently the most significant complication of FVIII replacement therapy in the management of patients with severe hemophilia A. Immune tolerance protocols for the eradication of inhibitors require daily delivery of intravenous FVIII for at least 6 months and are unsuccessful in 20–40% of treated patients. We hypothesize that tolerance can be induced more efficiently and reliably by delivery of FVIII antigen within autologous apoptotic cells (ACs). In this study, we demonstrated suppression of the T cell and inhibitor responses to FVIII by infusion of FVIII expression vector modified apoptotic syngeneic fibroblasts in both naive and preimmunized hemophilia A mice. ACs without FVIII antigen exerted modest generalized immune suppression mediated by anti-inflammatory signals. However, FVIII expressing apoptotic syngeneic fibroblasts produced much stronger antigen-specific immune suppression. Mice treated with these fibroblasts generated CD4+ T cells that suppressed the immune response to FVIII after adoptive transfer into naive recipients and antigen-specific CD4+CD25+ regulatory T cells (Tregs) that inhibited the proliferation of FVIII responsive effector T cells in vitro. These preclinical results demonstrate the potential for using FVIII vector modified autologous ACs to treat high-titer inhibitors in patients with hemophilia A. PMID:19755963

  9. Some Effects of Calcium on the Activation of Human Factor VIII/Von Willebrand Factor Protein by Thrombin

    PubMed Central

    Switzer, Mary Ellen; McKee, Patrick A.

    1977-01-01

    When Factor VIII/von Willebrand factor (FVIII/vWF) protein is rechromatographed on 4% agarose in 0.25 M CaCl2, the protein and vWF activity appear in the void volume, but most of the FVIII procoagulant activity elutes later. Recent evidence suggests that the delayed FVIII procoagulant activity is a proteolytically modified form of FVIII/vWF protein that filters anomalously from agarose in 0.25 M CaCl2. To test whether or not thrombin is the protease involved, the effect of 0.25 M CaCl2 on FVIII/vWF and its reaction with thrombin was examined. About 30% of the FVIII procoagulant activity was lost immediately when solutions of FVIII/vWF protein were made 0.25 M in CaCl2. When FVIII in 0.15 M NaCl was activated with 0.04 U thrombin/ml and then made 0.25 M in CaCl2, the procoagulant activity of a broad range of FVIII/vWF protein concentrations remained activated for at least 6 h. But, in 0.25 M CaCl2, the increase in FVIII procoagulant activity in response to thrombin was much more gradual and once activated, the procoagulant activity was stabilized by 0.25 M CaCl2. When thrombin-activated FVIII/vWF protein was filtered on 4% agarose in 0.15 M NaCl, there was considerable inactivation of FVIII procoagulant activity; however, the procoagulant activity that did remain eluted in the void volume. In contrast, when thrombin-activated FVIII/vWF protein was filtered in 0.25 M CaCl2, the FVIII procoagulant activity eluted well after the void volume and remained activated for 6 h. The procoagulant peak isolated by filtering nonthrombin-activated FVIII/vWF protein on agarose in 0.25 M CaCl2 was compared to that isolated from thrombin-activated FVIII/vWF protein. Both procoagulant activity peak proteins had about the same specific vWF activity as the corresponding void volume protein. Before reduction, the sodium dodecyl sulfate gel patterns for the two procoagulant activity peak proteins were the same. After reduction, the gel pattern for the nonthrombin-activated procoagulant

  10. Human von Willebrand factor/factor VIII concentrates in the management of pediatric patients with von Willebrand disease/hemophilia A

    PubMed Central

    Castaman, Giancarlo; Linari, Silvia

    2016-01-01

    Several plasma-derived intermediate and high-purity concentrates containing von Willebrand factor (VWF) and factor VIII (FVIII) are currently available. The main role of these products in the management of the pediatric population is represented by the replacement therapy in patients with severe or intermediate forms of von Willebrand disease, in whom other treatments are ineffective or contraindicated. Another important role of VWF/FVIII concentrates in children may be their use in immune tolerance induction (ITI) protocols. ITI is particularly recommended for hemophilia A children who have developed an inhibitor against FVIII, currently the most serious complication of substitutive treatment in hemophilia. Although recombinant concentrates may represent the preferred option in children with hemophilia A, VWF/FVIII concentrates may offer an advantage in rescuing patients who failed previous ITI. PMID:27445481

  11. Human von Willebrand factor/factor VIII concentrates in the management of pediatric patients with von Willebrand disease/hemophilia A.

    PubMed

    Castaman, Giancarlo; Linari, Silvia

    2016-01-01

    Several plasma-derived intermediate and high-purity concentrates containing von Willebrand factor (VWF) and factor VIII (FVIII) are currently available. The main role of these products in the management of the pediatric population is represented by the replacement therapy in patients with severe or intermediate forms of von Willebrand disease, in whom other treatments are ineffective or contraindicated. Another important role of VWF/FVIII concentrates in children may be their use in immune tolerance induction (ITI) protocols. ITI is particularly recommended for hemophilia A children who have developed an inhibitor against FVIII, currently the most serious complication of substitutive treatment in hemophilia. Although recombinant concentrates may represent the preferred option in children with hemophilia A, VWF/FVIII concentrates may offer an advantage in rescuing patients who failed previous ITI.

  12. Characterization of the anti-factor VIII immunoglobulin profile in patients with hemophilia A by use of a fluorescence-based immunoassay

    PubMed Central

    Boylan, Brian; Rice, Anne S.; Dunn, Amy L.; Tarantino, Michael D.; Brettler, Doreen B.; Barrett, John C.; Miller, Connie H.

    2015-01-01

    Summary Background The development of neutralizing antibodies, referred to as inhibitors, against factor VIII (FVIII) is a major complication associated with FVIII infusion therapy for the treatment of hemophilia A (HA). Previous studies have shown that a subset of HA patients and a low percentage of healthy individuals harbor non-neutralizing anti-FVIII antibodies that do not elicit the clinical manifestations associated with inhibitor development. Objective Assess HA patients' anti-FVIII antibody profiles as potential predictors of clinical outcomes. Methods A fluorescence immunoassay (FLI) was used to detect anti-FVIII antibodies in 491 samples from 371 HA patients. Results Assessments of antibody profiles showed that the presence of anti-FVIII IgG1, IgG2, or IgG4 correlated qualitatively and quantitatively with the presence of a FVIII inhibitor as reported by the Nijmegen-Bethesda assay (NBA). Forty-eight patients with a negative inhibitor history contributed serial samples to the study, including seven patients who had negative NBA titers initially and later converted to NBA-positive. The FLI detected anti-FVIII IgG1 in five of those seven patients prior to their conversion to NBA-positive. Five of 15 serial-sample patients who had a negative inhibitor history and a positive anti-FVIII IgG1 later developed an inhibitor, compared to 2 of 33 patients with a negative inhibitor history without anti-FVIII IgG1. Conclusions These data provide a rationale for future studies designed both to monitor the dynamics of anti-FVIII antibody profiles in HA patients as a potential predictor of future inhibitor development and to assess the value of the anti-FVIII FLI as a supplement to traditional inhibitor testing. PMID:25354263

  13. Exposure of FVIII in the Presence of Phosphatidyl Serine Reduces Generation of Memory B-Cells and Induces Regulatory T-Cell-Mediated Hyporesponsiveness in Hemophilia A Mice.

    PubMed

    Ramakrishnan, Radha; Davidowitz, Andrew; Balu-Iyer, Sathy V

    2015-08-01

    A major complication of replacement therapy with Factor VIII (FVIII) for hemophilia A (HA) is the development of unwanted immune responses. Previous studies showed that administration of FVIII in the presence of phosphatidyl serine (PS) reduced the development of anti-FVIII antibodies in HA mice. However, the impact of PS-mediated effects on immunological memory, such as generation of memory B-cells, is not clear. The effect of PS on memory B-cells was therefore investigated using adoptive transfer approach in FVIII(-/-) HA mice. Adoptive transfer of memory B-cells from a PS-FVIII-treated group to naïve mice followed by challenge of the recipient mice with FVIII showed a significantly reduced anti-FVIII antibody response in the recipient mice, compared with animals that received memory B-cells from free FVIII and FVIII-charge matched phosphatidyl glycerol (PG) group. The decrease in memory B-cell response is accompanied by an increase in FoxP3 expressing regulatory T-cells (Tregs). Flow cytometry studies showed that the generation of Tregs is higher in PS-treated animals as compared with FVIII and FVIII-PG treated animals. The PS-mediated hyporesponsiveness was found to be antigen-specific. The PS-FVIII immunization showed hyporesponsiveness toward FVIII rechallenge but not against ovalbumin (OVA) rechallenge, an unrelated antigen. This demonstrates that PS reduces immunologic memory of FVIII and induces antigen-specific peripheral tolerance in HA mice.

  14. Sustaining expression of B domain-deleted human factor VIII mediated by using lentiviral vectors in NOD/SCID mouse.

    PubMed

    Li, Yan-Jie; Chen, Chong; Zeng, Ling-Yu; Cao, Jiang; Xu, Kai-Lin

    2012-06-01

    Recently, gene therapy has been become a promising approach to cure hemophilia A, a most common recessive bleeding disease. The aim of this study was to determine the perspective of lentiviral vector in hemophilia A gene therapy in vitro and in NOD/SCID mice. Lentivirus transfer vector pXZ9/BDDFVIII containing human B-domain-deleted Factor VIII-IRES-eGFP coding sequence and mock control pXZ9 were constructed. Lentivirus was prepared by co-transfecting 3 plasmids into 293FT cells. 293FT, HLF, human bone marrow mesenchymal stem cells and Chang-liver cells were transfected with the prepared virus. Coagulant activity of human FVIII, human FVIII antigen, human FVIII mRNA transcription and genomic integration were assayed by ELISA, one-step method, RT-PCR and PCR after infection. Lentiviral particles were concentrated by ultracentrifugation and NOD/SCID mice were transfected via portal vein injection. Human FVIII antigen in mouse blood plasma was analyzed by ELISA. eGFP expression was observed by fluorescent microscopy and human FVIII transcription in mouse liver was analyzed by RT-PCR at one month after transduction. The results showed that the high titer of recombinant virus was prepared and used to efficiently transduce the target cells in vitro. At 72 h after transfection, high levels of FVIII activity and FVIII antigen were detected. Human FVIII gene transcription could be detected in the liver of NOD/SCID mice received lentiviral particles carrying FVIII gene. Mouse hepatocytes were transfected with recombinant lentivirus efficiently in vivo. Human FVIII level in mouse blood plasma reached to (49 ± 6) mU, (54 ± 8) mU and (23 ± 4) mU at 72 h, one week and one month after transfection respectively. It is concluded that the lentiviral particles carrying BDDhFVIII gene can high efficiently transfect the target cells both in vitro and in vivo, and the transfected target cells can secrete hFVIII efficiently. The sustained expression of human FVIII in NOD/SCID mice is

  15. Transient blockade of the inducible costimulator pathway generates long-term tolerance to factor VIII after nonviral gene transfer into hemophilia A mice.

    PubMed

    Peng, Baowei; Ye, Peiqing; Blazar, Bruce R; Freeman, Gordon J; Rawlings, David J; Ochs, Hans D; Miao, Carol H

    2008-09-01

    Formation of inhibitory antibodies is a common problem encountered in clinical treatment for hemophilia. Human factor VIII (hFVIII) plasmid gene therapy in hemophilia A mice also leads to strong humoral responses. We demonstrate that short-term therapy with an anti-ICOS monoclonal antibody to transiently block the inducible costimulator/inducible costimulator ligand (ICOS/ICOSL) signaling pathway led to sustained tolerance to hFVIII in hFVIII plasmid-treated hemophilia A mice and allowed persistent, high-level FVIII functional activity (100%-300% of normal). Anti-ICOS treatment resulted in depletion of ICOS(+)CD4(+) T cells and activation of CD25(+)Foxp3(+) Tregs in the peripheral blood, spleen, and lymph nodes. CD4(+) T cells from anti-ICOS-treated mice did not proliferate in response to hFVIII stimulation and produced high levels of regulatory cytokines, including interleukin-10 and transforming growth factor-beta. Moreover, CD4(+)CD25(+) Tregs from tolerized mice adoptively transferred dominant tolerance in syngeneic hFVIII plasmid-treated hemophilia A mice and reduced the production of antibodies against FVIII. Anti-ICOS-treated mice tolerized to hFVIII generated normal primary and secondary antibody responses after immunization with the T-dependent antigen, bacteriophage Phix 174, indicating maintenance of immune competency. Our data indicate that transient anti-ICOS monoclonal antibody treatment represents a novel single-agent immunomodulatory strategy to overcome the immune responses against transgene product after gene therapy.

  16. Phenotypic correction and stable expression of factor VIII in hemophilia A mice by embryonic stem cell therapy.

    PubMed

    Wang, J J; Kuang, Y; Zhang, L L; Shen, C L; Wang, L; Lu, S Y; Lu, X B; Fei, J; Gu, M M; Wang, Z G

    2013-05-13

    Hereditary deficiency of factor VIII (FVIII) leads to hemophilia A, a severe X-linked bleeding disorder. Current therapies include fixed-dose FVIII prophylaxis, factor replacement therapy, and most recently, gene therapy. Prophylaxis and FVIII replacement therapies are limited by incomplete efficacy, high cost, restricted availability, and development of neutralizing antibodies in chronically treated individuals. Limited success has been obtained in preclinical trials using gene therapy for the treatment of hemophilia. Therefore, new options for therapy for hemophilia A are needed. We evaluated the potential of embryonic stem cells for correcting hemophilia A in mice. FVIII-deficient mouse blastocysts were collected and injected with mouse embryonic stem cells stably expressing green-fluorescent protein (GFP) and transferred to pseudopregnant recipient mice. Expression of FVIII was measured in the liver and plasma of the 5 chimeric mice that were produced. Three of these mice were GFP-positive at the age of 6 months. The plasma FVIII activity levels were equal to those of wild-type mice. These data demonstrate that embryonic stem cell transplantation at an early embryonic stage has potential as therapy for this progressively debilitating, life-threatening bleeding disorder.

  17. Neonatal helper-dependent adenoviral vector gene therapy mediates correction of hemophilia A and tolerance to human factor VIII.

    PubMed

    Hu, Chuhong; Cela, Racel G; Suzuki, Masataka; Lee, Brendan; Lipshutz, Gerald S

    2011-02-01

    Neonatal gene therapy is a promising strategy for treating a number of congenital diseases diagnosed shortly after birth as expression of therapeutic proteins during postnatal life may limit the pathologic consequences and result in a potential "cure." Hemophilia A is often complicated by the development of antibodies to recombinant protein resulting in treatment failure. Neonatal administration of vectors may avoid inhibitory antibody formation to factor VIII (FVIII) by taking advantage of immune immaturity. A helper-dependent adenoviral vector expressing human factor VIII was administered i.v. to neonatal hemophilia A knockout mice. Three days later, mice produced high levels of FVIII. Levels declined rapidly with animal growth to 5 wk of age with stable factor VIII expression thereafter to >1 y of age. Decline in factor VIII expression was not related to cell-mediated or humoral responses with lack of development of antibodies to capsid or human factor VIII proteins. Subsequent readministration and augmentation of expression was possible as operational tolerance was established to factor VIII without development of inhibitors; however, protective immunity to adenovirus remained.

  18. A population pharmacokinetic model for perioperative dosing of factor VIII in hemophilia A patients

    PubMed Central

    Hazendonk, Hendrika; Fijnvandraat, Karin; Lock, Janske; Driessens, Mariëtte; van der Meer, Felix; Meijer, Karina; Kruip, Marieke; Gorkom, Britta Laros-van; Peters, Marjolein; de Wildt, Saskia; Leebeek, Frank; Cnossen, Marjon; Mathôt, Ron

    2016-01-01

    The role of pharmacokinetic-guided dosing of factor concentrates in hemophilia is currently a subject of debate and focuses on long-term prophylactic treatment. Few data are available on its impact in the perioperative period. In this study, a population pharmacokinetic model for currently registered factor VIII concentrates was developed for severe and moderate adult and pediatric hemophilia A patients (FVIII levels <0.05 IUmL−1) undergoing elective, minor or major surgery. Retrospective data were collected on FVIII treatment, including timing and dosing, time point of FVIII sampling and all FVIII plasma concentrations achieved (trough, peak and steady state), brand of concentrate, as well as patients’ and surgical characteristics. Population pharmacokinetic modeling was performed using non-linear mixed-effects modeling. Population pharmacokinetic parameters were estimated in 75 adults undergoing 140 surgeries (median age: 48 years; median weight: 80 kg) and 44 children undergoing 58 surgeries (median age: 4.3 years; median weight: 18.5 kg). Pharmacokinetic profiles were best described by a two-compartment model. Typical values for clearance, intercompartment clearance, central and peripheral volume were 0.15 L/h/68 kg, 0.16 L/h/68 kg, 2.81 L/68 kg and 1.90 L/68 kg. Interpatient variability in clearance and central volume was 37% and 27%. Clearance decreased with increasing age (P<0.01) and increased in cases with blood group O (26%; P<0.01). In addition, a minor decrease in clearance was observed when a major surgical procedure was performed (7%; P<0.01). The developed population model describes the perioperative pharmacokinetics of various FVIII concentrates, allowing individualization of perioperative FVIII therapy for severe and moderate hemophilia A patients by Bayesian adaptive dosing. PMID:27390359

  19. [Recurrent intestinal ischemia due to factor VIII].

    PubMed

    Castellanos Monedero, Jesús Javier; Legaz Huidobro, María Luisa; Galindo Andugar, María Angeles; Rodríguez Pérez, Alvaro; Mantrana del Valle, José María

    2008-01-01

    Intestinal ischemia is difficult to diagnose and can be caused by several etiologic processes. We report the case of a female patient with recurrent bowel ischemia due to small vessel thrombosis, which is caused by factor VIII, a procoagulant factor.

  20. Factor VIII Is Synthesized in Human Endothelial Cells, Packaged in Weibel-Palade Bodies and Secreted Bound to ULVWF Strings.

    PubMed

    Turner, Nancy A; Moake, Joel L

    2015-01-01

    The cellular synthesis site and ensuing storage location for human factor VIII (FVIII), the coagulation protein deficient in hemophilia A, has been elusive. FVIII stability and half-life is dependent on non-covalent complex formation with von Willebrand factor (VWF) to avoid proteolysis and clearance. VWF is synthesized in megakaryocytes and endothelial cells, and is stored and secreted from platelet alpha granules and Weibel-Palade bodies of endothelial cells. In this paper we provide direct evidence for FVIII synthesis in 2 types of primary human endothelial cells: glomerular microvascular endothelial cells (GMVECs) and umbilical vein endothelial cells (HUVECs). Gene expression quantified by real time PCR revealed that levels of F8 and VWF are similar in GMVECs and HUVECs. Previous clinical studies have shown that stimulation of vasopressin V2 receptors causes parallel secretion of both proteins. In this study, we found that both endothelial cell types express AVPR2 (vasopressin V2 receptor gene) and that AVPR2 mRNA levels are 5-fold higher in GMVECs than HUVECs. FVIII and VWF proteins were detected by fluorescent microscopy in Weibel-Palade bodies within GMVECs and HUVECs using antibodies proven to be target specific. Visual presence of FVIII and VWF in Weibel-Palade bodies was confirmed by correlation measurements. The high extent of correlation was compared with negative correlation values obtained from FVIII detection with cytoplasmic proteins, β-actin and Factor H. FVIII activity was positive in GMVEC and HUVEC cell lysates. Stimulated GMVECs and HUVECs were found to secrete cell-anchored ultra-large VWF strings covered with bound FVIII.

  1. Assessing patients' and caregivers' perspectives on stability of factor VIII products for haemophilia A: a web-based study in the United States and Canada.

    PubMed

    DiBenedetti, D B; Coles, T M; Sharma, T; Pericleous, L; Kulkarni, R

    2014-07-01

    Haemophilia A is a rare inherited bleeding disorder characterized by an inability of the blood to clot normally. Patients can experience spontaneous or trauma-induced joint and soft tissue bleeding and must keep coagulation factor VIII (FVIII) accessible at all times; thus, FVIII product storage and stability are critical. Our primary objective was to assess haemophilia A patients' and caregivers' experiences and preferences with FVIII product storage and stability. A secondary objective was to evaluate the use of the social media site Facebook in recruitment. In this cross-sectional study, 145 English-speaking adult patients and caregivers of children with haemophilia A were recruited through two state-based haemophilia organizations in the United States (US) and one national organization in Canada for a web-based survey assessing demographics and FVIII product ordering, usage, and storage practices. Of the 101 individuals who completed the survey, 60% resided in Canada; 57% were recruited through Facebook. Caregivers and patients responded similarly to questions about ordering practices and product usage, with some distinction between groups in storage practices. Two-thirds of participants noted challenges with storing FVIII products, especially storage away from home. More than half preferred storing FVIII products at room temperature vs. in the refrigerator for long periods of time. FVIII product accessibility, usage and storage affect disease management. Results support the need for more convenient and accessible FVIII products for patients in daily life and while travelling. In addition, the use of social media has potential value in recruiting this population.

  2. Factor VIII ectopically targeted to platelets is therapeutic in hemophilia A with high-titer inhibitory antibodies

    PubMed Central

    Shi, Qizhen; Wilcox, David A.; Fahs, Scot A.; Weiler, Hartmut; Wells, Clive W.; Cooley, Brian C.; Desai, Drashti; Morateck, Patricia A.; Gorski, Jack; Montgomery, Robert R.

    2006-01-01

    Inhibitory immune response to exogenously infused factor VIII (FVIII) is a major complication in the treatment of hemophilia A. Generation of such inhibitors has the potential to disrupt gene therapy for hemophilia A. We explore what we believe to be a novel approach to overcome this shortcoming. Human B-domain–deleted FVIII (hBDDFVIII) was expressed under the control of the platelet-specific αIIb promoter in platelets of hemophilic (FVIIInull) mice to create 2bF8trans mice. The FVIII transgene product was stored in platelets and released at the site of platelet activation. In spite of the lack of FVIII in the plasma of 2bF8trans mice, the bleeding phenotype of FVIIInull mice was corrected. More importantly, the bleeding phenotype was corrected in the presence of high inhibitory antibody titers introduced into the mice by infusion or by spleen cell transfer from recombinant hBDDFVIII–immunized mice. Our results demonstrate that this approach to the targeted expression of FVIII in platelets has the potential to correct hemophilia A, even in the presence of inhibitory immune responses to infused FVIII. PMID:16823491

  3. A novel cell-sheet technology that achieves durable factor VIII delivery in a mouse model of hemophilia A.

    PubMed

    Tatsumi, Kohei; Sugimoto, Mitsuhiko; Lillicrap, David; Shima, Midori; Ohashi, Kazuo; Okano, Teruo; Matsui, Hideto

    2013-01-01

    Gene- or cell-based therapies aimed at creating delivery systems for coagulation factor VIII (FVIII) protein have emerged as promising options for hemophilia A treatment. However, several issues remain to be addressed regarding the efficacies and adverse events of these new classes of therapies. To improve an existing cell-based therapy involving the subcutaneous transplantation of FVIII-transduced blood outgrowth endothelial cells (BOECs), we employed a novel cell-sheet technology that allows individual dispersed cells to form a thin and contiguous monolayer without traditional bioabsorbable scaffold matrices. Compared to the traditional methodology, our cell-sheet approach resulted in longer-term and 3-5-fold higher expression of FVIII (up to 11% of normal) in recipient hemophilia A mice that lacked a FVIII humoral immune response due to transient immunosuppression with cyclophosphamide. Histological studies revealed that the transplanted BOEC sheets were structured as flat clusters, supporting the long-term expression of therapeutic FVIII in plasma from an ectopic subcutaneous space. Our novel tissue-engineering approach using genetically modified BOEC sheets could aid in development of cell-based therapy that will allow safe and effective in vivo delivery of functional FVIII protein in patients with hemophilia A.

  4. Evaluation of the biological differences of canine and human factor VIII in gene delivery: implications in human hemophilia treatment

    PubMed Central

    Wang, Qizhao; Dong, Biao; Firrman, Jenni; Wu, Wenman; Roberts, Sean; Moore, Andrea Rossi; Liu, LinShu; Chin, Mario P.S.; Diao, Yong; Kost, Joseph; Xiao, Weidong

    2016-01-01

    The canine is the most important large animal model for testing novel hemophilia A(HA) treatment. It is often necessary to use canine factor VIII (cFIII) gene or protein for the evaluation of HA treatment in the canine model. However, the different biological properties between cFVIII and human FVIII(hFVIII) indicated that the development of novel HA treatment may require careful characterization of non-human FVIII. To investigate whether the data obtained using cFVIII can translate to HA treatment in human, we analyzed the differential biological properties of canine heavy chain (cHC) and light chain (cLC) by comparing with human HC (hHC) and LC (hLC). The secretion of cHC was 5~30 fold higher than hHC, with or without LCs. cHC+hLC group exhibited ~18-fold increase in coagulation activity compared with hHC+hLC delivery by recombinant adeno-associated viral vectors. Unlike hHC, the secretion of cHC was independent of LCs. cLC improves the specific activity of FVIII by 2~3-fold compared with hLC. Moreover, the cLC but not cHC, contributes the high stability of cFVIII. Our results suggested that the cFVIII expression results in the canine model should be interpreted with caution as the cHC secreted more efficiently than hHC and cLC exhibited a more active and stable phenotype than hLC. PMID:27064790

  5. Evaluation of the biological differences of canine and human factor VIII in gene delivery: implications in human hemophilia treatment.

    PubMed

    Wang, Q; Dong, B; Firrman, J; Wu, W; Roberts, S; Moore, A R; Liu, L S; Chin, M P S; Diao, Y; Kost, J; Xiao, W

    2016-07-01

    The canine is the most important large animal model for testing novel hemophilia A (HA) treatment. It is often necessary to use canine factor VIII (cFIII) gene or protein for the evaluation of HA treatment in the canine model. However, different biological properties between cFVIII and human FVIII (hFVIII) indicated that the development of novel HA treatment may require careful characterization of non-human FVIII. To investigate whether the data obtained using cFVIII can translate to HA treatment in human, we analyzed the differential biological properties of canine heavy chain (cHC) and light chain (cLC) by comparing with human heavy chain (hHC) and light chain (hLC). The secretion of cHC was 5-30-fold higher than hHC, with or without light chains (LCs). cHC+hLC group exhibited ~18-fold increase in coagulation activity compared with hHC+hLC delivery by recombinant adeno-associated viral vectors. Unlike hHC, the secretion of cHC was independent of LCs. cLC improves the specific activity of FVIII by two- to threefold compared with hLC. Moreover, the cLC, but not cHC, contributes to the higher stability of cFVIII. Our results suggested that the cFVIII expression results in the canine model should be interpreted with caution as the cHC secreted more efficiently than hHC and cLC exhibited a more active and stable phenotype than hLC.

  6. Computer-predicted peptides that mimic discontinuous epitopes on the A2 domain of factor VIII.

    PubMed

    Lebreton, A; Simon, N; Moreau, V; Demolombe, V; Cayzac, C; Nguyen, C; Schved, J F; Granier, C; Lavigne-Lissalde, G

    2015-05-01

    Development of antibodies (Abs) against factor VIII (FVIII) is a severe complication of haemophilia A treatment. Recent publications suggest that domain specificity of anti-FVIII antibodies, particularly during immune tolerance induction (ITI), might be related to the outcome of the treatment. Obtaining suitable tools for a fine mapping of discontinuous epitopes could thus be helpful. The aim of this study was to map discontinuous epitopes on FVIII A2 domain using a new epitope prediction functionality of the PEPOP bioinformatics tool and a peptide inhibition assay based on the Luminex technology. We predicted, selected and synthesized 40 peptides mimicking discontinuous epitopes on the A2 domain of FVIII. A new inhibition assays using Luminex technology was performed to identify peptides able to inhibit the binding of anti-A2 Abs to A2 domain. We identified two peptides (IFKKLYHVWTKEVG and LYSRRLPKGVKHFD) able to block the binding of anti-A2 allo-antibodies to this domain. The three-dimensional representation of these two peptides on the A2 domain revealed that they are localized on a limited region of A2. We also confirmed that residues 484-508 of the A2 domain define an antigenic site. We suggest that dissection of the antibody response during ITI using synthetic peptide epitopes could provide important information for the management of patients with inhibitors.

  7. ADAMTS13 content in plasma-derived factor VIII/von Willebrand factor concentrates.

    PubMed

    Peyvandi, Flora; Mannucci, Pier M; Valsecchi, Carla; Pontiggia, Silvia; Farina, Claudio; Retzios, Anastassios D

    2013-10-01

    Thrombotic thrombocytopenic purpura (TTP) is a microangiopathy syndrome caused by a congenital or acquired deficiency of ADAMTS13, a plasma metalloprotease that cleaves von Willebrand factor (VWF) and thus prevents the formation of platelet-rich thrombi in the microcirculation. TTP can be fatal if not appropriately and timely treated with the infusion of fresh frozen plasma (FFP) or exchange plasmapheresis, that reverse the process of microangiopathy by removing anti-ADAMTS13 autoantibodies and replacing functional ADAMTS13. The treatment of TTP with FFP is not free from risks and must be administered in hospitals or clinics, owing to the substantial amount of plasma volume infused or exchanged and the frequent need of catheter application. Moreover, most FFPs are not subjected to treatments to remove or inactivate blood-borne infectious agents. A number of recent reports indicate that certain plasma-derived VWF-factor VIII (FVIII) concentrates are clinically effective in the treatment of congenital TTP. In this study, we measured ADAMTS13 levels in various plasma-derived VWF-FVIII concentrates, showing that Koate(®) -DVI (Grifols), contained relatively high amounts of ADAMTS13 and that Alphanate(®) (Grifols) was the closest other product in terms of protease content. Koate(®) -DVI contains, on average (five lots tested), 0.091 ± 0.007 Units of ADAMTS13 activity per IU of FVIII. On the basis of this analysis and other reports of VWF-FVIII concentrate utilization in congenital TTP, potential dosing, and future clinical developments are discussed.

  8. Thrombomodulin as a marker for vascular tumors. Comparative study with factor VIII and Ulex europaeus I lectin.

    PubMed

    Yonezawa, S; Maruyama, I; Sakae, K; Igata, A; Majerus, P W; Sato, E

    1987-10-01

    Thrombomodulin (TM) is a newly described endothelial cell-associated protein that functions as a potent natural anticoagulant by converting thrombin from a procoagulant protease to an anticoagulant. Various vascular tumors were characterized with immunoperoxidase staining with the use of a polyclonal anti-TM serum. The staining patterns of TM were compared with those of Factor VIII-related antigen (FVIII-RAG) and Ulex europaeus agglutinin-I (UEA-I), which have been used as markers for endothelial cells. The results showed that TM is a specific and a highly sensitive marker for angiosarcomas in comparison with FVIII-RAG or UEA-I. In contrast, UEA-I is more sensitive for benign vascular tumors than TM or FVIII-RAG. The other mesenchymal tumors of nonvascular origin showed negative staining for three endothelial markers. These results indicate that TM is a new specific and sensitive tool for the diagnosis of angiosarcomas.

  9. Renal Vein Thrombosis in a Newborn With Abnormal Factor VIII Level

    PubMed Central

    Szafranska, Agnieszka; Pajak, Agata; Kilis-Pstrusinska, Katarzyna; Królak-Olejnik, Barbara

    2015-01-01

    Abstract Renal vein thrombosis (RVT) in neonates is a rare condition of low mortality but significant morbidity due to renal impairment. We report the case of a male term newborn with left RVT and elevated serum factor VIII (FVIII). The main symptoms of the patient and the important clinical findings: prompt diagnosis of RVT was possible because the classic clinical presentation of macroscopic hematuria, thrombocytopenia, and palpable flank mass were present in this newborn infant. The main diagnoses: finally, the reason of RVT was established when the infant was 3 months of age: the increased level of FVIII was confirmed. We discuss the diagnosis, therapy, and outcome of the patient and compare with the literature. Therapeutics interventions: however, despite anticoagulant therapy the left kidney developed areas of scarring and then atrophy. Conclusions and outcomes: Prothrombotic defects should be considered in all patients with perinatal RVT. Elevated factor VIII as a reason of RVT in neonatal period is particularly rare. Given a poor renal outcome in children associated with elevated levels of factor VIII, consideration could be given to more aggressive antithrombotic therapy in such cases. PMID:26252276

  10. Interferon-gamma secretion defects in haemophilia A patients receiving highly purified plasma-derived or recombinant factor VIII.

    PubMed

    Newton-Nash, D K; Tollerud, D; Guevarra, L; Gill, J C

    1996-12-01

    The outcome of developing immune responses is influenced by interactions among a large and complex network of secreted cytokines. T-cell secretion of interferon-gamma (IFN-gamma), tumour necrosis factor alpha (TNF-alpha) and TNF-beta, or lymphotoxin contributes to the development of cell-mediated immunity, whereas secretion of interleukin (IL)-4, IL-5 and IL-6 contributes to development of humoral immunity. Humoral immunity to factor VIII (FVIII) develops in approximately 25% of severe haemophilia A patients. The aim of our research was to understand the underlying immune response to FVIII in patients with FVIII inhibitors. We report a defect in IFN-gamma secretion by peripheral blood mononuclear cells (PBMC) derived from haemophilia A patients, which was accompanied by a low level of mitogen-induced proliferation and a significant decrease in the percentage of natural killer (NK) cells. All of the observed defects were found in haemophilia A patients, both with and without FVIII inhibitors, who were free of viral infection and had been treated predominantly or exclusively with monoclonal antibody-purified or recombinant FVIII.

  11. Assessment of in vitro cytokine response in hemophilia A patients with or without factor VIII inhibitory antibody.

    PubMed

    Towfighi, Farzaneh; Gharagozlou, Soheila; Kardar, Gholam-Ali; Sharifian, Ramazan-Ali; Karimi, Katayoon; Lak, Manijheh; Pourfathollah, Ali-Akbar; Soleimani, Sedigheh; Shokri, Fazel

    2007-08-01

    Factor VIII (FVIII) inhibitor antibodies are produced in a proportion of hemophilia A patients. Development of anti-FVIII inhibitor antibodies is a T cell-dependent response, mediated by FVIII specific CD4(+) T cells. This study was performed to investigate the contribution of T helper (Th) cell-mediated cytokine response in inhibitor production. Peripheral blood mononuclear cells (PBMCs) were obtained from hemophilia A patients with (n = 14) or without inhibitor (n = 14) and from normal individuals (n = 14). Following stimulation of PBMCs with rFVIII and phytohemagglutinin (PHA) mitogen, the secreted cytokines, interferon-gamma (IFN-gamma), interleukin-10 (IL-10), and transforming growth factor-beta1 (TGF-beta1), in culture supernatant and the proliferative response were assessed using sandwich ELISA and (3)H-thymidine incorporation, respectively. No significant proliferative response to FVIII was observed, whereas PHA induced a strong response in all groups. No cytokine secretion was observed in response to FVIII stimulation. Although PHA induced IL-10, TGF-beta1 and IFN-gamma secretion in all groups, the level of IFN-gamma was significantly lower in hemophilia A patients than in normal individuals (p < 0.0001). The levels of TGF-beta1 and IL-10 were similarly higher in patients compared with normal subjects, but the difference was not statistically significant. Lack of FVIII-induced proliferative response and cytokine production together with reduced secretion of PHA-induced IFN-gamma in both groups of patients suggest involvement of nonspecific immunosuppression possibly due to hepatitis C virus (HCV) infection observed in the majority of patients.

  12. Genotype and phenotype report on patients with combined deficiency of factor V and factor VIII in Iran.

    PubMed

    Karimi, Mehran; Cairo, Andrea; Safarpour, Mohammad M; Haghpanah, Sezaneh; Ekramzadeh, Maryam; Afrasiabi, Abdolreza; Shahriari, Mahdi; Menegatti, Marzia

    2014-06-01

    Combined factor V (FV) and factor VIII (FVIII) deficiency is a rare autosomal recessive bleeding disorder characterized by mild-to-moderate bleeding. Epistaxis, postsurgical bleeding and menorrhagia are the most common symptoms. The aim of this study is to report the phenotype-genotype characterization carried out in patients affected with combined FV and FVIII deficiency from Iran. A cross-sectional study was conducted in Shiraz Hemophilia Center, southern Iran. Twelve cases, seven men and five women coming from eight families were included in our study after taking consent form. Coagulation activity for all patients was measured. All exons and intron-exon junctions of lectin mannose binding protein 1 (LMAN1) gene and multiple coagulation factor deficiency 2 genes were amplified by PCR, and subsequently sequenced by the Sanger method. Patients[Combining Acute Accent] age ranged from 6 to 59 years mean ± SD: 23.8 ± 15.4 years and median: 22 years. No patient presented with severe bleeding symptom. Only one patient had severe FV and FVIII deficiency (both factor levels <1%). Four different type of mutations (duplication, insertion, splice site and nonsense), occurring in different locuses, were identified on LMAN1 gene in 12 Iranian patients. There was a significant correlation between FV and FVIII levels, which is indicative of association with loss of function of LMAN1 gene, and reduced plasma levels of both factors. Our study showed that all of our characterized patients with combined FV and FVIII deficiency present different homozygous mutations on LMAN1 gene introducing a premature stop codon. Larger studies are needed to calculate the correlation between factor levels, genetic and bleeding symptoms.

  13. Nanocapsule-delivered Sleeping Beauty mediates therapeutic Factor VIII expression in liver sinusoidal endothelial cells of hemophilia A mice.

    PubMed

    Kren, Betsy T; Unger, Gretchen M; Sjeklocha, Lucas; Trossen, Alycia A; Korman, Vicci; Diethelm-Okita, Brenda M; Reding, Mark T; Steer, Clifford J

    2009-07-01

    Liver sinusoidal endothelial cells are a major endogenous source of Factor VIII (FVIII), lack of which causes the human congenital bleeding disorder hemophilia A. Despite extensive efforts, gene therapy using viral vectors has shown little success in clinical hemophilia trials. Here we achieved cell type-specific gene targeting using hyaluronan- and asialoorosomucoid-coated nanocapsules, generated using dispersion atomization, to direct genes to liver sinusoidal endothelial cells and hepatocytes, respectively. To highlight the therapeutic potential of this approach, we encapsulated Sleeping Beauty transposon expressing the B domain-deleted canine FVIII in cis with Sleeping Beauty transposase in hyaluronan nanocapsules and injected them intravenously into hemophilia A mice. The treated mice exhibited activated partial thromboplastin times that were comparable to those of wild-type mice at 5 and 50 weeks and substantially shorter than those of untreated controls at the same time points. Further, plasma FVIII activity in the treated hemophilia A mice was nearly identical to that in wild-type mice through 50 weeks, while untreated hemophilia A mice exhibited no detectable FVIII activity. Thus, Sleeping Beauty transposon targeted to liver sinusoidal endothelial cells provided long-term expression of FVIII, without apparent antibody formation, and improved the phenotype of hemophilia A mice.

  14. The mesenchymal stem cells derived from transgenic mice carrying human coagulation factor VIII can correct phenotype in hemophilia A mice.

    PubMed

    Wang, Qing; Gong, Xiuli; Gong, Zhijuan; Ren, Xiaoyie; Ren, Zhaorui; Huang, Shuzhen; Zeng, Yitao

    2013-12-20

    Hemophilia A (HA) is an inherited X-linked recessive bleeding disorder caused by coagulant factor VIII (FVIII) deficiency. Previous studies showed that introduction of mesenchymal stem cells (MSCs) modified by FVIII-expressing retrovirus may result in phenotypic correction of HA animals. This study aimed at the investigation of an alternative gene therapy strategy that may lead to sustained FVIII transgene expression in HA mice. B-domain-deleted human FVIII (hFVIIIBD) vector was microinjected into single-cell embryos of wild-type mice to generate a transgenic mouse line, from which hFVIIIBD-MSCs were isolated, followed by transplantation into HA mice. RT-PCR and real-time PCR analysis demonstrated the expression of hFVIIIBD in multi-organs of recipient HA mice. Immunohistochemistry showed the presence of hFVIIIBD positive staining in multi-organs of recipient HA mice. ELISA indicated that plasma hFVIIIBD level in recipient mice reached its peak (77 ng/mL) at the 3rd week after implantation, and achieved sustained expression during the 5-week observation period. Plasma FVIII activities of recipient HA mice increased from 0% to 32% after hFVIIIBD-MSCs transplantation. APTT (activated partial thromboplastin time) value decreased in hFVIIIBD-MSCs transplanted HA mice compared with untreated HA mice (45.5 s vs. 91.3 s). Our study demonstrated an effective phenotypic correction in HA mice using genetically modified MSCs from hFVIIIBD transgenic mice.

  15. Treatment of patients with hemophilia A and inhibitors to factor FVIII with cimetidine.

    PubMed

    Ambriz Fernandez, R; Quintana Gonzalez, S; Martinez Murillo, C; Dominguez Garcia, V; Rodriguez Moyado, H; Collazo Jaloma, J

    1996-01-01

    In this study, cimetidine was used to treat patients with hemophilia A and inhibitors to factor VIII who presented with acute hemorrhages (Group A) and those without hemorrhages (Group B). The dose of cimetidine was 15 mg/kg/day. Group A consisted of five patients with inhibitors between 156 and > 10,000 Bethesda Units (BU), all with serious hemorrhagic problems. The control of hemorrhaging was effective in 100% of these patients, although inhibitor levels remained high (25-380 BU). Group B consisted of seven patients who did not have hemorrhages, whose inhibitor levels were 41-358 BU. Five of these patients no longer had anamnestic responses to Factor VIII after several months of treatment with cimetidine. No difference in the response to cimetidine was seen between HIV positive and HIV negative patients. The results suggest that cimetidine is useful to suppress inhibitors to Factor VIII in patients with hemophilia A.

  16. Haemorrhagic symptoms in patients with combined factors V and VIII deficiency in north-eastern Iran.

    PubMed

    Mansouritorgabeh, H; Rezaieyazdi, Z; Pourfathollah, A A; Rezai, J; Esamaili, H

    2004-05-01

    Of the six types of dual coagulation factors deficiency, combined factors V and VIII are the most common type, a few cases of this disease have been reported in different populations. This accounts for the relatively low number of cases reported so far. Our report, which included 19 patients, is the second largest group that has been reported from one centre in north-eastern Iran. The most frequent spontaneous bleeding symptoms were epistaxis and haemarthrosis, and the most frequent traumatic bleeding symptoms were bleeding after dental extraction and bleeding after cutting any part of the body. It seemed that dual coagulation FV and FVIII deficiency is as severe as single coagulation factor (V or VIII) deficiency.

  17. Adsorption and function of recombinant factor VIII at solid-water interfaces in the presence of Tween-80.

    PubMed

    Joshi, Omkar; McGuire, Joseph; Wang, D Q

    2008-11-01

    The adsorption, structural alteration and biological activity of a recombinant Factor VIII was investigated in the presence of the surfactant Tween-80, at hydrophilic and hydrophobic solid-water interfaces. Hydrophilic and silanized, hydrophobic silica surfaces were used as substrates for protein and surfactant adsorption, which was monitored in situ, with ellipsometry. At the hydrophobic surface, the presence of Tween in the protein solution resulted in a reduction in amount of protein adsorbed, while rFVIII adsorption at the hydrophilic surface was entirely unaffected by the presence of Tween. These observations were attributed to high binding strength between Tween and the hydrophobic surface, and low binding strength between Tween and the hydrophilic surface. Colloidal particles bearing hydrophilic and hydrophobic surfaces, and net positive or negative surface charge, were used as substrates for rFVIII adsorption in evaluation of tertiary structure change and biological activity retention at interfaces. Fluorescence emission spectroscopy showed that rFVIII tertiary structure was changed upon exposure to hydrophobic nanoparticle surfaces. Similarly, the biological activity of rFVIII (based on the activated partial thromboplastin time) was reduced at hydrophobic surfaces. At high surfactant concentration, these properties were better preserved. This was attributed to Tween adsorption sterically inhibiting rFVIII adsorption. While hydrophilic surfaces were associated with relatively high rFVIII adsorption, they did not induce large changes in structure or activity. This was attributed to the formation of a tightly packed, ordered adsorbed layer on these surfaces, governed by electrostatic attraction and not mediated by the rFVIII active site.

  18. Is some better than none: are TEG and TGA profiles different in severe FVIII-deficient patients with inhibitors?

    PubMed

    Salinas, V; Carmona, R; Mohammed, B M; Martin, E J; Brophy, D F; Young, G

    2015-05-01

    Severe factor VIII (FVIII)-deficient patients with and without FVIII inhibitors cannot be distinguished using FVIII levels. The FVIII assay is sensitive to detect factor levels below 1%. While severe FVIII-deficient, non-inhibitor patients have FVIII < 1%, they may retain unmeasurable residual factor activity. In contrast, inhibitor patients have a FVIII antibody that presumably fully eliminates FVIII activity. It is unknown whether thromboelastography (TEG) and thrombin generation assay (TGA) can differentiate between patients with FVIII < 1% with and without the presence of FVIII inhibitors. The primary objective was to discern whether TEG and TGA could differentiate between severe FVIII-deficient patients with and without the presence of FVIII inhibitors. A secondary objective was to correlate TEG and TGA to annualized bleeding rates. This observational study performed TEG and TGA in healthy volunteers (N = 15), severe FVIII-deficient (N = 15) and severe FVIII-deficient patients with inhibitors (N = 15). Kaolin-activated TEG was better at differentiating reaction time (31.3 vs. 120 min respectively, P = 0.004) and kinetics time (6.1 vs. 23.1 min respectively, P = 0.028) between the non-inhibitor and inhibitor patients. TEG activated by tissue factor in plasma-containing corn trypsin inhibitor failed to differentiate groups. The TGA failed to differentiate peak thrombin, endogenous thrombin potential and lag time between groups. There was no correlation between TEG and TGA with annualized bleeding rates. Kaolin-activated TEG, but not TGA, differentiated between severe FVIII-deficient patients with and without inhibitors. These assays did not find a correlation to annualized bleeding rate.

  19. Construction of a mouse model of factor VIII deficiency by gene targeting

    SciTech Connect

    Bi, L.; Lawler, A.; Gearhart, J.

    1994-09-01

    To develop a small animal model of hemophilia A for gene therapy experiments, we set out to construct a mouse model for factor VIII deficiency by gene targeting. First, we screened a mouse liver cDNA library using a human FVIII cDNA probe. We cloned a 2.6 Kb partial mouse factor VIII cDNA which extends from 800 base pairs of the 3{prime} end of exon 14 to the 5{prime} end of exon 26. A mouse genomic library made from strain 129 was then screened to obtain genomic fragments covering the exons desired for homologous recombination. Two genomic clones were obtained, and one covering exon 15 through 22 was used for gene targeting. To make gene targeting constructs, a 5.8 Kb genomic DNA fragment covering exons 15 to 19 of the mouse FVIII gene was subcloned, and the neo expression cassette was inserted into exons 16 and 17 separately by different strategies. These two constructs were named MFVIIIC-16 and MFVIIIC-17. The constructs were linearized and transfected into strain 129 mouse ES cells by electroporation. Factor VIII gene-knockout ES cell lines were selected by G-418 and screened by genomic Southern blots. Eight exon 16 targeted cell lines and five exon 17 targeted cell lines were obtained. Three cell lines from each construct were injected into blastocysts and surgically transferred into foster mothers. Multiple chimeric mice with 70-90% hair color derived from the ES-cell genotype were seen with both constructs. Germ line transmission of the ES-cell genotype has been obtained for the MFVIIIC-16 construct, and multiple hemophilia A carrier females have been identified. Factor VIII-deficient males will be conceived soon.

  20. Prolonged Activated Partial Thromboplastin Time: Difficulties in Discriminating Coexistent Factor VIII Inhibitor and Lupus Anticoagulant.

    PubMed

    Ames, Paul R J; Graf, Maria; Archer, Jeremy; Scarpato, Nicola; Iannaccone, Luigi

    2015-03-01

    To review the diagnostic difficulties of a prolonged activated partial thromboplastin time (aPTT) when 2 inhibitors with opposite clinical presentations coexist, we searched MEDLINE from January 1970 to November 2013 using acquired, factor VIII (FVIII), factor IX, hemophilia A and B, inhibitor, lupus anticoagulant (LA), antiphospholipid, anticardiolipin, anti-β2-glycoprotein I, antibodies, syndrome, bleeding, and thrombosis. We identified 13 articles for a total of 15 cases of possible coexistence of FVIII inhibitor and LA. The presenting clinical manifestation was thrombosis in 6 cases and bleeding in 9 cases. Activated partial thromboplastin time was the presenting laboratory abnormality in all cases, and first-line investigations suggested the coexistence of LA and acquired FVIII inhibitor. None of the articles addressed the diagnostic accuracy of the screening tests by performing "second line" assays. We reviewed the diagnostic pitfalls of the cases under study and provide some guidance for alternative tests when facing a prolonged aPTT that may have a double meaning.

  1. Laboratory identification of factor VIII inhibitors in the real world: the experience from Australasia.

    PubMed

    Favaloro, E J; Bonar, R; Kershaw, G; Mohammed, S; Duncan, E; Marsden, K

    2010-07-01

    The laboratory has a key role in the initial detection of factor inhibitors and an ongoing role in the measurement of inhibitor titres during the course of inhibitor eradication therapy. The most commonly seen factor inhibitors are those directed against factor VIII (FVIII), usually detected either using the original or Nijmegen-modified Bethesda assay. In view of previously demonstrated high variability in laboratory results for inhibitor assays, we have more extensively examined laboratory performance in the identification of FVIII inhibitors. Over the past 3 years, we conducted two questionnaire-based surveys and two wet-challenge surveys utilizing eight samples comprising no FVIII inhibitor (n = 1), or low-titre (n = 2), medium-titre (n = 3) or high-titre (n = 2) FVIII inhibitor. Four samples were tested by 42 laboratories in 2007, and four by 52 laboratories in 2009. High inter-laboratory variation was evident, with CVs around 50% not uncommon, and some 10% of all laboratories (or around 15% of laboratories using Bethesda method) failed to detect low-level inhibitors of around 1 BU mL(-1). Laboratories using the Nijmegen method appeared to perform better than those using a standard Bethesda assay, with lower evident assay variation and no false negatives. There was a wide variety of laboratory practice, with no two laboratories using exactly the same process for testing and interpretation of factor inhibitor findings. In conclusion, our study indicates that there is still much need for standardization and improvement in factor inhibitor detection, and we hope that our findings provide a basis for future improvements in this area.

  2. [vWF improves secretion and activity of intein spliced BDD-FVIII].

    PubMed

    Zhu, Fu-Xiang; Yang, Shu-De; Liu, Ze-Long; Miao, Jing; Qu, Hui-Ge; Chi, Xiao-Yan

    2010-05-01

    As synthesized by vascular endothelial cells and megakaryocytes, the von Willebrand factor (vWF) plays an important hemostatic role in the binding to and stabilizing blood coagulation factor VIII (FVIII) and preventing its enzymatic degradation. Our recent work demonstrated intein can efficiently ligate BDD-FVIII (B-domaim deleted FVIII) posttranslationally by protein trans-splicing after transfer of split BDD-FVIII gene by a dual-vector system. In this study we investigated the effect of vWF on secretion and activity of intein-ligated BDD-FVIII. We observed the levels of full-length BDD-FVIII antigen secreted into culture supernatant by ELISA and their activity by Coatest assay after transfection of cultured 293 cells with intein-fused BDD-FVIII heavy- and light-chain genes simultaneously with the vWF gene co-transfected. The data showed that the amount of full-length BDD-FVIII protein and their bioactivity in vWF gene co-transfected cell supernatant were 235 +/- 21 ng x mL(-1) and 1.98 +/- 0.2 u x mL(-1), respectively, greater than that of non-vWF co-transfected cell (110 +/- 18) ng x mL(-1) and 1.10 +/- 0.15 u x nL(-1)) or just BDD-FVIII gene transfected control cell (131 +/- 25 ng x mL(-1) and 1.22 +/- 0.18 u x mL(-1)) indicating the benefit of vWF gene co-transfection in the secretion and activity of intein-spliced BDD-FVIII protein. It provided evidence that vWF gene co-transfer may be useful to improve efficacy of gene therapy for hemophilia A in protein splicing-based split FVIII gene transfer.

  3. Evolution of recombinant factor VIII safety: KOGENATE and Kogenate FS/Bayer.

    PubMed

    Lusher, Jeanne M; Scharrer, Inge

    2009-11-01

    The use of factor VIII (FVIII) concentrates in the treatment of hemophilia A has raised important safety issues, historically of pathogen transmission and increasingly of inhibitor development to FVIII treatment. While manufacturing processes of current recombinant FVIII products have been shaped entirely around preventing pathogen transmission, the same modifications that afford a greater margin of safety could affect immunogenicity of the product, consequences of which could only be seen through long-term clinical experience. This review summarizes pathogen safety and inhibitor reports from clinical trials, post-marketing surveillance studies, and study reports on KOGENATE and its successor, Kogenate FS/Bayer. Although KOGENATE and Kogenate FS/Bayer are nearly identical products, subtle manufacturing improvements to address the need for greater margins of safety from a pathogen transmission perspective have also led to a potentially improved immunogenicity profile (15% in previously untreated/minimally treated patients with severe hemophilia A for Kogenate FS/Bayer). Notably, there has been no occurrence of pathogen contamination, and minimal de novo inhibitor formation in previously treated patients throughout the use of both products. Overall, KOGENATE and Kogenate FS/Bayer have a long history of safety in a variety of clinical settings, including treatment of bleeding, surgical management, and prophylaxis therapy.

  4. Common and rare von Willebrand factor (VWF) coding variants, VWF levels, and factor VIII levels in African Americans: the NHLBI Exome Sequencing Project.

    PubMed

    Johnsen, Jill M; Auer, Paul L; Morrison, Alanna C; Jiao, Shuo; Wei, Peng; Haessler, Jeffrey; Fox, Keolu; McGee, Sean R; Smith, Joshua D; Carlson, Christopher S; Smith, Nicholas; Boerwinkle, Eric; Kooperberg, Charles; Nickerson, Deborah A; Rich, Stephen S; Green, David; Peters, Ulrike; Cushman, Mary; Reiner, Alex P

    2013-07-25

    Several rare European von Willebrand disease missense variants of VWF (including p.Arg2185Gln and p.His817Gln) were recently reported to be common in apparently healthy African Americans (AAs). Using data from the NHLBI Exome Sequencing Project, we assessed the association of these and other VWF coding variants with von Willebrand factor (VWF) and factor VIII (FVIII) levels in 4468 AAs. Of 30 nonsynonymous VWF variants, 6 were significantly and independently associated (P < .001) with levels of VWF and/or FVIII. Each additional copy of the common VWF variants encoding p.Thr789Ala or p.Asp1472His was associated with 6 to 8 IU/dL higher VWF levels. The VWF variant encoding p.Arg2185Gln was associated with 7 to 13 IU/dL lower VWF and FVIII levels. The type 2N-related VWF variant encoding p.His817Gln was associated with 17 IU/dL lower FVIII level but normal VWF level. A novel, rare missense VWF variant that predicts disruption of an O-glycosylation site (p.Ser1486Leu) and a rare variant encoding p.Arg2287Trp were each associated with 30 to 40 IU/dL lower VWF level (P < .001). In summary, several common and rare VWF missense variants contribute to phenotypic differences in VWF and FVIII among AAs.

  5. Omental implantation of BOECs in hemophilia dogs results in circulating FVIII antigen and a complex immune response.

    PubMed

    Ozelo, Margareth C; Vidal, Barbara; Brown, Christine; Notley, Colleen; Hegadorn, Carol; Webster, Sandra; Harpell, Lori; Ahlin, James; Winterborn, Andrew; Handforth, Janine; Arruda, Valder R; Hough, Christine; Lillicrap, David

    2014-06-26

    Ex vivo gene therapy strategies avoid systemic delivery of viruses thereby mitigating the risk of vector-associated immunogenicity. Previously, we delivered autologous factor VIII (FVIII)-expressing blood outgrowth endothelial cells (BOECs) to hemophilia A mice and showed that these cells remained sequestered within the implanted matrix and provided therapeutic levels of FVIII. Prior to translating this strategy into the canine (c) model of hemophilia A, we increased cFVIII transgene expression by at least 100-fold with the use of the elongation factor 1 alpha (EF1α) promoter and a strong endothelial enhancer element. BOECs isolated from hemophilia A dogs transduced with this lentiviral vector express levels of cFVIII ranging between 1.0 and 1.5 U/mL per 10(6) cells over 24 hours. Autologous BOECs have been implanted into the omentum of 2 normal and 3 hemophilia A dogs. These implanted cells formed new vessels in the omentum. All 3 hemophilia A dogs treated with FVIII-expressing autologous BOECs developed anti-FVIII immunoglobulin G2 antibodies, but in only 2 of the dogs were these antibodies inhibitory. FVIII antigen levels >40% in the absence of FVIII coagulant function were detected in the circulation for up to a year after a single gene therapy treatment, indicating prolonged cellular viability and synthesis of FVIII.

  6. The pharmacokinetics and pharmacodynamics of desmopressin: effect on plasma factor VIII:C and von Willebrand factor.

    PubMed

    Argenti, D; Jensen, B K; Heald, D

    1997-01-01

    The relationship between plasma concentrations of desmopressin and clotting factors Factor VIII:C (FVIII:C) and von Willebrand Factor (vWF) were explored after a single 15-minute intravenous infusion of desmopressin (0.3 microg/kg) to 28 healthy male subjects. Individual plasma desmopressin-vWF and desmopressin-FVIII:C concentration/response-time data were fitted to a pharmacodynamic sigmoid E ( max ) model linked to a two-compartment open pharmacokinetic model (Ke0 link). The model demonstrated that the onset rate of pharmacodynamic activity for FVIII:C and vWF was relatively rapid following intravenous administration. However, the offset rate of pharmacodynamic activity was rate-limited by the elimination rate of desmopressin. Mean maximum pharmacodynamic activity for both factors was estimated to be three- to four-times higher than baseline activity, and the mean desmopressin concentrations that produce half-maximal effects were approximately 250 to 300 pg/mL. Interindividual variation in pharmacodynamic-parameter estimates were of the magnitude that suggests a wide range of pharmacodynamic responses are possible for a fixed desmopressin dose.

  7. Delivery of Full-Length Factor VIII Using a piggyBac Transposon Vector to Correct a Mouse Model of Hemophilia A

    PubMed Central

    Matsui, Hideto; Fujimoto, Naoko; Sasakawa, Noriko; Ohinata, Yasuhide; Shima, Midori; Yamanaka, Shinya; Sugimoto, Mitsuhiko; Hotta, Akitsu

    2014-01-01

    Viral vectors have been used for hemophilia A gene therapy. However, due to its large size, full-length Factor VIII (FVIII) cDNA has not been successfully delivered using conventional viral vectors. Moreover, viral vectors may pose safety risks, e.g., adverse immunological reactions or virus-mediated cytotoxicity. Here, we took advantages of the non-viral vector gene delivery system based on piggyBac DNA transposon to transfer the full-length FVIII cDNA, for the purpose of treating hemophilia A. We tested the efficiency of this new vector system in human 293T cells and iPS cells, and confirmed the expression of the full-length FVIII in culture media using activity-sensitive coagulation assays. Hydrodynamic injection of the piggyBac vectors into hemophilia A mice temporally treated with an immunosuppressant resulted in stable production of circulating FVIII for over 300 days without development of anti-FVIII antibodies. Furthermore, tail-clip assay revealed significant improvement of blood coagulation time in the treated mice.piggyBac transposon vectors can facilitate the long-term expression of therapeutic transgenes in vitro and in vivo. This novel gene transfer strategy should provide safe and efficient delivery of FVIII. PMID:25126862

  8. Delivery of full-length factor VIII using a piggyBac transposon vector to correct a mouse model of hemophilia A.

    PubMed

    Matsui, Hideto; Fujimoto, Naoko; Sasakawa, Noriko; Ohinata, Yasuhide; Shima, Midori; Yamanaka, Shinya; Sugimoto, Mitsuhiko; Hotta, Akitsu

    2014-01-01

    Viral vectors have been used for hemophilia A gene therapy. However, due to its large size, full-length Factor VIII (FVIII) cDNA has not been successfully delivered using conventional viral vectors. Moreover, viral vectors may pose safety risks, e.g., adverse immunological reactions or virus-mediated cytotoxicity. Here, we took advantages of the non-viral vector gene delivery system based on piggyBac DNA transposon to transfer the full-length FVIII cDNA, for the purpose of treating hemophilia A. We tested the efficiency of this new vector system in human 293T cells and iPS cells, and confirmed the expression of the full-length FVIII in culture media using activity-sensitive coagulation assays. Hydrodynamic injection of the piggyBac vectors into hemophilia A mice temporally treated with an immunosuppressant resulted in stable production of circulating FVIII for over 300 days without development of anti-FVIII antibodies. Furthermore, tail-clip assay revealed significant improvement of blood coagulation time in the treated mice. piggyBac transposon vectors can facilitate the long-term expression of therapeutic transgenes in vitro and in vivo. This novel gene transfer strategy should provide safe and efficient delivery of FVIII.

  9. Low-dose continuous infusion of factor VIII in patients with haemophilia A

    PubMed Central

    Prelog, Tomaž; Dolničar, Majda Benedik; Kitanovski, Lidija

    2016-01-01

    Background Patients with haemophilia A (HA) or B (HB) can be given prophylactic or on-demand treatment administered by continuous infusion or bolus injections of factor VIII (FVIII) or IX (FIX). In this study we evaluated the efficacy and safety of low-dose continuous infusion of FVIII or FIX. Material and methods We studied all eligible patients with HA or HB treated with continuous infusion of factor concentrates over an 18-year period in a single Slovenian Haemophilia Comprehensive Care Centre. Treatment started with a bolus injection of FVIII or FIX, followed by continuous infusion at the initial rate of 2 IU/kg/h of FVIII in HA patients and 4.5 IU/kg/h of FIX in HB patients. The infusion rate was subsequently adjusted according to the indication for therapy. Results A total of 66 continuous infusions (40 in major surgery, 10 in minor surgery and 16 with bleeding episode) in 46 HA patients and 16 (15 in severe and 1 in mild HA) in eight HB patients were included in the study. During the first week of treatment, the median continuous infusion rates in HA patients undergoing major surgery, minor surgery and a bleeding event were 2.18 (0.75–3.68), 1.48 (1.0–2.54) and 2.24 (1.33–3.93) IU/kg/h, respectively. The median FVIII activities were 0.69 (0.37–1.19), 0.47 (0.39–0.84) and 0.52 (0.36–1.06) IU/mL. After the first week of treatment, even lower doses of FVIII were needed. Red blood cell transfusions had to be administered to three patients (2 with severe and 1 with moderate HA) during the continuous infusion and inhibitors developed in five patients. In HB patients, the median continuous infusion rate was 1.85 (1.07–2.94) IU/kg/h and the median FIX activity was 0.62 (0.30–1.04) IU/mL. Red blood cell transfusions were not required, and thrombophlebitis and inhibitors did not appear. Discussion Overall, low-dose continuous infusion was shown to be an effective and safe way of treating patients with HA. The protocol used also proved efficient and

  10. In vitro and in vivo characterization of a high-purity, solvent/detergent-treated factor VIII concentrate: evidence for its therapeutic efficacy in von Willebrand's disease.

    PubMed

    Mazurier, C; De Romeuf, C; Parquet-Gernez, A; Goudemand, M

    1989-07-01

    A factor VIII (FVIII) concentrate, virus-inactivated by the solvent/detergent procedure, was studied in vitro. In contrast with most high-purity, virus-inactivated FVIII concentrates, it contains not only high levels of von Willebrand factor (vWF) antigen and ristocetin cofactor activity but also high molecular weight forms of von Willebrand factor. Furthermore, it is able to promote platelet adhesion on collagen in a perfusion system. In vivo studies performed in patients with different types of von Willebrand's disease provided evidence that this concentrate corrects Duke's bleeding time and prevents or stops haemorrhages. Thus, the particular advantages of this FVIII/vWF preparation are safety, low content of contamination proteins, and efficacy in von Willebrand's disease.

  11. Von Willebrand factor and coagulation factor VIII in Moyamoya disease associated with Graves' disease: A case report

    PubMed Central

    Ren, Shou-Chen; Gao, Bao-Qin; Yang, Wei-Li; Feng, Wei-Xin; Xu, Jian; Li, Shao-Wu; Wang, Yong-Jun

    2016-01-01

    The present study reported the case of a Chinese boy who was diagnosed with Moyamoya disease (MMD) associated with Graves' disease (GD). An overactivation of von Willebrand factor (vWF) and coagulation factor VIII (FVIII) was identified in the plasma of the patient. Thiamazole and metoprolol treatment was thus administrated. After 2 months of treatment, the patient's thyroid function returned to normal and the neurological symptoms improved gradually. At the same time, the activities of vWF and FVIII were depressed. During the 20-month follow-up, information regarding the neurological symptoms, cerebrovascular imaging, thyroid function, thyroid autoantibodies and coagulation parameters was collected. High levels of thyroid autoantibodies persisted throughout the follow-up period, while other coagulation parameters remained in the normal range. In conclusion, considering the vital role of vWF and FVIII in vascular diseases, it is hypothesized that these two factors may serve an important role in the occurrence of GD associated with MMD. PMID:27882137

  12. Prospective surveillance study of haemophilia A patients switching from moroctocog alfa or other factor VIII products to moroctocog alfa albumin-free cell culture (AF-CC) in usual care settings.

    PubMed

    Parra Lopez, Rafael; Nemes, Laszlo; Jimenez-Yuste, Victor; Rusen, Luminita; Cid, Ana R; Charnigo, Robert J; Baumann, James A; Smith, Lynne; Korth-Bradley, Joan M; Rendo, Pablo

    2015-10-01

    This prospective, open-label, postauthorisation safety surveillance study assessed clinically significant inhibitor development in patients with severe haemophilia A transitioning from moroctocog alfa or other factor VIII (FVIII) replacement products to reformulated moroctocog alfa (AF-CC). Males aged ≥ 12 years with severe haemophilia A (FVIII:C) < 1 IU/dl), > 150 exposure days (EDs) to recombinant or plasma-derived FVIII products, and no detectable inhibitor at screening were enrolled. Primary end point was the incidence of clinically significant FVIII inhibitor development. Secondary end points included annualised bleeding rate (ABR), less-than-expected therapeutic effect (LETE), and FVIII recovery. Patients were assigned to one of two cohorts based on whether they were transitioning to moroctocog alfa (AF-CC) from moroctocog alfa (cohort 1; n=146) or from another recombinant or plasma-derived FVIII product (cohort 2; n=62). Mean number of EDs on study was 94 (range, 1-139). Six positive FVIII inhibitor results, as determined by local laboratories, were reported in four patients; none were confirmed by a central laboratory, no inhibitor-related clinical manifestations were reported, and all anti-FVIII antibody assays were negative. Median ABRs were 23.4 and 3.4 in patients categorised at baseline as following on-demand and prophylactic regimens, respectively; 86.5% of bleeding episodes resolved after one infusion. LETE incidence was 0.06% and 0.19% in the on-demand and prophylaxis settings, respectively. FVIII recovery remained constant throughout the study. No new safety concerns were identified. This study found no increased risk of clinically significant FVIII inhibitor development in patients transitioning from moroctocog alfa or other FVIII replacement products to moroctocog alfa (AF-CC).

  13. Assessment of the frequency of regulatory T cells (CD4+CD25+CD127-) in children with hemophilia A: relation to factor VIII inhibitors and disease severity.

    PubMed

    El-Asrar, Mohamed Abo; Hamed, Ahmed El-Saeed; Darwish, Yasser Wagih; Ismail, Eman Abdel Rahman; Ismail, Noha Ali

    2016-01-01

    A rapidly growing evidence showed that regulatory T cells (Tregs) play a crucial role in tolerance to coagulation factors and may be involved in the pathogenesis of inhibitor formation in patients with hemophilia. We determined the percentage of Tregs (CD4CD25CD127) in 45 children with hemophilia A compared with 45 healthy controls, and assessed their relation to the clinical characteristics of patients and factor VIII (FVIII) inhibitors. Patients were studied stressing on frequency of bleeding attacks, joint pain, history of viral hepatitis, and the received therapy (FVIII precipitate/cryotherapy). FVIII activity and FVIII inhibitors were assessed with flow cytometric analysis of CD4CD25CD127 Tregs. According to residual FVIII activity levels, 30 patients (66.7%) had mild/moderate hemophilia A, whereas 15 (33.3%) patients had severe hemophilia A. The frequency of Tregs was significantly lower among all patients with hemophilia A compared with controls (2.59 ± 1.1 versus 3.73 ± 1.12%; P = 0.002). Tregs were significantly decreased among patients with FVIII inhibitors compared with the inhibitor-negative group (P < 0.001). Patients with hematuria or severe hemophilia A had lower Tregs levels than those without (P = 0.34 and P = 0.011, respectively). A significant positive correlation was found between the percentage of Tregs and FVIII among hemophilia A patients. ROC curve analysis revealed that the cut-off value of Tregs at 1.91% could differentiate patients with and without FVIII inhibitors, with a sensitivity of 100% and a specificity of 91.3%. We suggest that alteration in the frequency of Tregs in young patients with hemophilia A may contribute to inhibitor formation and disease severity.

  14. Trp[superscript 2313]-His[superscript 2315] of Factor VIII C2 Domain Is Involved in Membrane Binding Structure of a Complex Between the C[subscript 2] Domain and an Inhibitor of Membrane Binding

    SciTech Connect

    Liu, Zhuo; Lin, Lin; Yuan, Cai; Nicolaes, Gerry A.F.; Chen, Liqing; Meehan, Edward J.; Furie, Bruce; Furie, Barbara; Huang, Mingdong

    2010-11-03

    Factor VIII (FVIII) plays a critical role in blood coagulation by forming the tenase complex with factor IXa and calcium ions on a membrane surface containing negatively charged phospholipids. The tenase complex activates factor X during blood coagulation. The carboxyl-terminal C2 domain of FVIII is the main membrane-binding and von Willebrand factor-binding region of the protein. Mutations of FVIII cause hemophilia A, whereas elevation of FVIII activity is a risk factor for thromboembolic diseases. The C2 domain-membrane interaction has been proposed as a target of intervention for regulation of blood coagulation. A number of molecules that interrupt FVIII or factor V (FV) binding to cell membranes have been identified through high throughput screening or structure-based design. We report crystal structures of the FVIII C2 domain under three new crystallization conditions, and a high resolution (1.15 {angstrom}) crystal structure of the FVIII C2 domain bound to a small molecular inhibitor. The latter structure shows that the inhibitor binds to the surface of an exposed {beta}-strand of the C2 domain, Trp{sup 2313}-His{sup 2315}. This result indicates that the Trp{sup 2313}-His{sup 2315} segment is an important constituent of the membrane-binding motif and provides a model to understand the molecular mechanism of the C2 domain membrane interaction.

  15. Enhanced factor VIII heavy chain for gene therapy of hemophilia A.

    PubMed

    Chen, Lingxia; Lu, Hui; Wang, Jinhui; Sarkar, Rita; Yang, Xiao; Wang, Hongli; High, Katherine A; Xiao, Weidong

    2009-03-01

    Hemophilia A gene therapy using recombinant adenovirus-associated virus (AAV) vectors has been hampered by the size of the factor VIII (FVIII) cDNA. Previously, splitting the FVIII coding sequence into a heavy-chain (HC) fragment and a light-chain (LC) fragment for dual recombinant AAV vector delivery has been successfully explored. However, the main disadvantage of this approach is a "chain imbalance" problem in which LC secretion is approximately 1-2 logs higher than that of HC, and therefore, the majority of protein synthesized is nonfunctional. To improve HC secretion, we constructed alternate FVIII HCs based on our observation that LC facilitates HC secretion. To our surprise, most of the new HC molecules exhibited enhanced expression over the traditional HC molecule (HC(745)). The optimized HC mutein, HC(HL), including additional acidic-region-3 (ar3) sequences, exhibited three- to fivefold higher activity in both enzyme-linked immunosorbent assay (ELISA) and activated partial thromboplastin time (aPTT) assay in in vitro testing. Further characterization suggested ar3 sequences increased HC secretion, rather than promoting HC synthesis. Intravenous delivery of AAV8-HC(HL)+AAV8-LC or AAV8-HC(745)+AAV8-LC achieved phenotypic correction in hemophilia A mice. Mice receiving AAV8-HC(HL)+AAV8-LC achieved three- to fourfold higher HC expression than AAV8-HC(745)+AAV8-LC, consistent with the FVIII functional assays. HC(HL) should be substituted for HC(745) in a dual AAV vector strategy due to its enhanced expression.

  16. Factoring in Factor VIII With Acute Ischemic Stroke.

    PubMed

    Siegler, James E; Samai, Alyana; Albright, Karen C; Boehme, Amelia K; Martin-Schild, Sheryl

    2015-10-01

    There is growing research interest into the etiologies of cryptogenic stroke, in particular as it relates to hypercoagulable states. An elevation in serum levels of the procoagulant factor VIII is recognized as one such culprit of occult cerebral infarctions. It is the objective of the present review to summarize the molecular role of factor VIII in thrombogenesis and its clinical use in the diagnosis and prognosis of acute ischemic stroke. We also discuss the utility of screening for serum factor VIII levels among patients at risk for, or those who have experienced, ischemic stroke.

  17. Phase I study of BAY 94-9027, a PEGylated B-domain-deleted recombinant factor VIII with an extended half-life, in subjects with hemophilia A

    PubMed Central

    Coyle, T E; Reding, M T; Lin, J C; Michaels, L A; Shah, A; Powell, J

    2014-01-01

    Background BAY 94-9027 is a B-domain-deleted recombinant factor VIII (rFVIII) with site-specific attachment of poly(ethylene glycol) that has shown an extended half-life in animal models of hemophilia. Objectives To assess the pharmacokinetics and safety of BAY 94-9027 after single and repeated administration in subjects with severe hemophilia A. Patients/Methods This 8-week, prospective, multicenter, open-label, phase I trial was conducted in 14 subjects aged 21–58 years with FVIII of < 1%, ≥ 150 days of exposure to FVIII, and no history of FVIII inhibitors. After a ≥ 3-day washout, subjects received a single dose of sucrose-formulated rFVIII (rFVIII-FS) (cohort 1 [n = 7], 25 IU kg−1; cohort 2 [n = 7], 50 IU kg−1) for a 48-h pharmacokinetic (PK) study. After another ≥ 3-day washout, cohort 1 received twice-weekly BAY 94-9027 at 25 IU kg−1 (16 doses), and cohort 2 received once-weekly BAY 94-9027 at 60 IU kg−1 (nine doses). A 168-h PK study was performed after the first and last BAY 94-9027 doses. Results BAY 94-9027 showed equivalent recovery and an improved PK profile vs. rFVIII-FS, with a half-life of ∼ 19 h (vs. ∼ 13.0 h for rFVIII-FS). BAY 94-9027 was well tolerated, and no immunogenicity was observed. Conclusions This phase I study demonstrates that BAY 94-9027 has an extended half-life in subjects with hemophilia A and, after multiple dosing, was well tolerated with no immunogenicity during the 8-week trial. A phase III study in a larger number of subjects is underway to fully characterize how this prolonged half-life will permit less frequent prophylaxis dosing for patients with hemophilia. PMID:24843882

  18. Evaluation of the activated partial thromboplastin time assay for clinical monitoring of PEGylated recombinant factor VIII (BAY 94-9027) for haemophilia A.

    PubMed

    Gu, J-M; Ramsey, P; Evans, V; Tang, L; Apeler, H; Leong, L; Murphy, J E; Laux, V; Myles, T

    2014-07-01

    Patients with haemophilia (PWH) are usually monitored by the one-stage activated partial thromboplastin time (aPTT) factor VIII (FVIII) assay. Different aPTT activators may affect clotting time (CT) and FVIII:C levels in patients treated with PEGylated FVIII. To evaluate the characteristics of PEGylated FVIII (BAY 94-9027) in various aPTT clotting assays, and to identify suitable aPTT reagents for monitoring BAY 94-9027 during the treatment of PWH, BAY 94-9027 and World Health Organization (WHO) 8th FVIII standards (WHO-8) were spiked into pooled and individual severe haemophilia A plasma at 1.0, 0.25 and 0.05 IU mL(-1) . Five commercial aPTT reagents widely used in clinical laboratories were compared and evaluated for BAY 94-9027 activity in plasma from PWH. BAY 94-9027 and WHO-8 bestowed similar CT and excellent precision when ellagic acid (SynthAFax, Dade Actin, and Cephascreen) aPTT reagents were used. In contrast, BAY 94-9027 showed significantly prolonged CT and poor precision compared with WHO-8 using silica aPTT reagents (APTT-SP and STA PTT 5). Furthermore, free 60-kDa polyethylene glycol (PEG), used for the conjugation of FVIII, showed a dose-dependent prolongation of CT in the APTT-SP assay. There was no effect on the SynthAFax-APTT, prothrombin time, or FXIa-initiated thrombin generation assay, demonstrating that the PEG moiety on FVIII has no general effect on the coagulation cascade. In summary, ellagic aPTT reagents (SynthAFax, Dade Actin, and Cephascreen) are most suitable for evaluating potency of BAY 94-9027 and should be the preferred aPTT reagents used in clinical laboratories for monitoring FVIII activity after infusion of BAY 94-9027 to PWH.

  19. [Separation of coagulation factor VIII with high activity using gigaporous anion exchange chromatography].

    PubMed

    Kang, Limei; Zhang, Yan; Luo, Jian; Li, You; Zhou, Yuefang; Yu, Rong; Su, Zhiguo

    2012-06-01

    A purification process to obtain coagulation factor VIII (F VIII) with high activity from human plasma was established. Based on the analysis of the size ratio between F VIII and matrix porous medium and its effect on the protein activity, a novel purification process designed was superporous ion exchange chromatography (IEC). The operating conditions of gigaporous and traditional anion exchange chromatography were optimized separately. The chromogenic substrate, gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to monitor the bioactivity and purity of the chromatographic products. The results showed that the superporous medium could not only protect structure of macro-protein but also enhance its mass transfer, finally giving FVIII product with high activity. The yield of F VIII in superporous chromatography was about five times of commercially agarose chromatography and the specific activity was up to 154 IU/mg protein. Furthermore, we studied the regeneration process of the superporous medium, washing the column with 5 column volumes of 1 mol/L NaOH at a low flow rate, to ensure the chromatographic stability. This purification process is simple, reproducible and suitable for large-scale production.

  20. Factor VIII brand and the incidence of factor VIII inhibitors in previously untreated UK children with severe hemophilia A, 2000-2011.

    PubMed

    Collins, Peter W; Palmer, Benedict P; Chalmers, Elizabeth A; Hart, Daniel P; Liesner, Ri; Rangarajan, Savita; Talks, Katherine; Williams, Michael; Hay, Charles R M

    2014-11-27

    The effect of recombinant factor VIII (rFVIII) brand on inhibitor development was investigated in all 407 severe hemophilia A previously untreated patients born in the United Kingdom (UK) between 1 January 2000 and 31 December 2011. Eighty-eight (22%) had been in the RODIN study. Information was extracted from the National Haemophilia Database. Because exposure days (EDs) were not known for some patients, time from first treatment was used as a surrogate for rFVIII exposure. An inhibitor developed in 118 (29%) patients, 60 high and 58 low titer, after a median (interquartile range) of 7.8 (3.3-13.5) months from first exposure and 16 (9-30) EDs. Of 128 patients treated with Kogenate Bayer/Helixate NexGen, 45 (35.2%, 95% confidence interval [CI] 27.4-43.8) developed an inhibitor compared with 42/172 (24.4%, 95% CI 18.6% to 31.4%) with Advate (P = .04). The adjusted hazard ratio (HR) (95% CI) for Kogenate Bayer/Helixate NexGen compared with Advate was 2.14 (1.12-4.10) (P = .02) for high titer and 1.75 (1.11-2.76) (P = .02) for all inhibitors. When excluding UK-RODIN patients, the adjusted HR (95% CI) for high-titer inhibitors was 2.00 (0.93-4.34) (P = .08). ReFacto AF was associated with a higher incidence of all, but not high-titer, inhibitors than Advate. These results will help inform debate around the relative immunogenicity and use of rFVIII brands.

  1. Limited blood sampling for pharmacokinetic dose tailoring of FVIII in the prophylactic treatment of haemophilia A.

    PubMed

    Björkman, S

    2010-07-01

    The aim of this study was to evaluate the use of limited blood sampling and Bayesian analysis to estimate the pharmacokinetics (PK) and tailor the dose of factor VIII (FVIII) in an individual patient. In a Bayesian analysis, PK parameters are estimated from only a few plasma concentration measurements, using a previously established PK model. First the necessary model was created using intense blood sampling FVIII data from 10 patients. Then FVIII data from another 21 patients were used for 'clinical' evaluation. Three scenarios were created retrospectively by reduction of the original 7-sample data set; blood sampling at 4, 24 and 48 h, at 8 and 30 h and at 24 h after the infusion. PK parameters were estimated for each individual using Bayesian analysis and compared with those obtained using conventional methods from the full data. The accuracy of predictions of FVIII levels during prophylactic treatment 5-17 months later and implications for dose tailoring were also investigated. Blood sampling at 4, 24 and 48 h was found to give practically the same PK information as a full, conventional (7-10-sample) study. Even a single 24-h FVIII level provided adequate data for initial dose tailoring and gave predictions of FVIII levels 5-17 months later that were not appreciably worse than predictions based on the full PK analysis. By contrast, dose tailoring based on body weight failed completely. In conclusion, PK-based dose tailoring of FVIII can be performed using limited blood sampling during prophylactic treatment.

  2. Potential role of a new PEGylated recombinant factor VIII for hemophilia A.

    PubMed

    Wynn, Tung Thanh; Gumuscu, Burak

    2016-01-01

    Hemophilia A, a deficiency in the activity of coagulation factor (F) VIII, is an X-linked bleeding disorder with an approximate incidence of one in 5,000 male infants. Bleeding-related complications often result in greater severity of disease, poor quality of life, surgical interventions for severe joint destruction, and shortened life span. With the availability of plasma-derived and recombinant FVIII products, the benefits of primary prophylaxis were demonstrated and is now the standard of care for patients with severe factor deficiencies. Current hemophilia research is focusing on the creation of new factor replacement therapies with longer half-lives; accessing alternative mechanisms to achieve desired hemostasis and enhance bypassing activity; and limiting the immunogenicity of the protein. PEGylation involves the covalent attachment of polyethylene glycol (PEG) to a protein, peptide, or a small molecule drug. PEG effectively increases the molecular weight and size of the protein by creating a hydrophilic cloud around the molecule. This molecular change may reduce susceptibility of the molecule to proteolytic activity and degradation. It is also believed that PEGylation changes the surface charge of the protein that ultimately interferes with some receptor-mediated clearance processes. The half-life of PEGylated factor is more prolonged when compared to non-PEGylated full-length recombinant FVIII. The dawn of a new era in the care of hemophilia patients is upon us with the release of recombinant FVIII products with extended half-lives, and products with even more extended half-life will become available in a very short time. With all the promise of these new agents, many questions still remain.

  3. Efficacy of engineered FVIII-producing skeletal muscle enhanced by growth factor-releasing co-axial electrospun fibers.

    PubMed

    Liao, I-Chien; Leong, Kam W

    2011-02-01

    Co-axial electrospun fibers can offer both topographical and biochemical cues for tissue engineering applications. In this study, we demonstrate the sustained treatment of hemophilia through a non-viral, tissue engineering approach facilitated by growth factor-releasing co-axial electrospun fibers. FVIII-producing skeletal myotubes were first engineered on aligned electrospun fibers in vitro, followed by implantation in hemophilic mice with or without a layer of core-shell electrospun fibers designed to provide sustained delivery of angiogenic or lymphangiogenic growth factors, which serves to stimulate the lymphatic or vascular systems to enhance the FVIII transport from the implant site into systemic circulation. Upon subcutaneous implantation into hemophilic mice, the construct seamlessly integrated with the host tissue within one month, and specifically induced either vascular or lymphatic network infiltration in accordance with the growth factors released from the electrospun fibers. Engineered constructs that induced angiogenesis resulted in sustained elevation of plasma FVIII and significantly reduced blood coagulation time for at least 2-months. Biomaterials-assisted functional tissue engineering was shown in this study to offer protein replacement therapy for a genetic disorder such as hemophilia.

  4. Synthesis of factor VIII antigen by cultured guinea pig megakaryocytes.

    PubMed

    Nachman, R; Levine, R; Jaffe, E A

    1977-10-01

    Immunoprecipitates containing guinea pig Factor VIII antigen were prepared from guinea pig plasma with a cross-reacting rabbit anti-human Factor VIII. Monospecific antisera to guinea pig Factor VIII antigen were produced in rabbits by using these washed immunoprecipitates as immunogens. The resulting antisera to guinea pig Factor VIII antigen detected Factor VIII antigen in guinea pig plasma and inhibited the von Willebrand factor activity in guinea pig plasma. This antibody also detected Factor VIII antigen in a solubilized protein mixture prepared from isolated cultured guinea pig megakaryocytes. Cultured guinea pig megakaryocytes were labeled with radio-active leucine. By radioautography, 96.2% of the radio-activity was present in megakaryocytes. The radio-active Factor VIII antigen present in the solubilized cell protein mixture was isolated by immunoprecipitation and characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The results demonstrate that cultured guinea pig megakaryocytes synthesize Factor VIII antigen which contains the same polypeptide subunit (mol wt 200,000) present in guinea pig plasma Factor VIII antigen.

  5. Early eradication of factor VIII inhibitor in patients with congenital hemophilia A by immune tolerance induction with a high dose of immunoglobulin.

    PubMed

    Mizoguchi, Yoko; Furue, Aya; Kagawa, Reiko; Chijimatsu, Ikue; Tomioka, Keita; Shimomura, Maiko; Imanaka, Yusuke; Nishimura, Shiho; Saito, Satoshi; Miki, Mizuka; Ono, Atsushi; Konishi, Nakao; Kawaguchi, Hiroshi; Kobayashi, Masao

    2016-04-01

    The production of factor VIII (FVIII) inhibitory antibodies is a serious problem in patients with hemophilia A. Immune tolerance induction (ITI) is the only strategy proven to eradicate persistent inhibitors and has been shown to be successful in 70 % of patients with hemophilia A. However, a minority of hemophilia patients present life-long inhibitors. To eliminate such inhibitors, we designed an intravenous immunoglobulin (IVIG) strategy in combination with high dose recombinant FVIII for ITI in hemophilia A children with inhibitors. Four previously untreated patients produced inhibitors within 16 exposures to FVIII. The peak inhibitor titers in these patients ranged from 3 to 14 BU/mL. The patients received ITI combined with IVIG within 1.5 months after the inhibitors were detected. All patients showed a negative titer for inhibitors by 28 days, with no anamnestic responses. The recovery of FVIII in the plasma concentration was normalized within three months after initiation of ITI. An additional course of IVIG administration led to induction of complete tolerance by 20 months after initiation of ITI therapy in all patients. ITI treatment with high-dose FVIII combined with IVIG may be effective for the early elimination of inhibitors.

  6. A novel B-domain O-glycoPEGylated FVIII (N8-GP) demonstrates full efficacy and prolonged effect in hemophilic mice models

    PubMed Central

    Kjalke, Marianne; Karpf, Ditte M.; Balling, Kristoffer W.; Johansen, Peter B.; Elm, Torben; Øvlisen, Kristine; Möller, Flemming; Holmberg, Heidi L.; Gudme, Charlotte N.; Persson, Egon; Hilden, Ida; Pelzer, Hermann; Rahbek-Nielsen, Henrik; Jespersgaard, Christina; Bogsnes, Are; Pedersen, Anette A.; Kristensen, Anne K.; Peschke, Bernd; Kappers, Wendy; Rode, Frederik; Thim, Lars; Tranholm, Mikael; Ezban, Mirella; Olsen, Eva H. N.; Bjørn, Søren E.

    2013-01-01

    Frequent infusions of intravenous factor VIII (FVIII) are required to prevent bleeding associated with hemophilia A. To reduce the treatment burden, recombinant FVIII with a longer half-life was developed without changing the protein structure. FVIII–polyethylene glycol (PEG) conjugates were prepared using an enzymatic process coupling PEG (ranging from 10 to 80 kDa) selectively to a unique O-linked glycan in the FVIII B-domain. Binding to von Willebrand factor (VWF) was maintained for all conjugates. Upon cleavage by thrombin, the B-domain and the associated PEG were released, generating activated FVIII (FVIIIa) with the same primary structure and specific activity as native FVIIIa. In both FVIII- and VWF-deficient mice, the half-life was found to increase with the size of PEG. In vivo potency and efficacy of FVIII conjugated with a 40-kDa PEG (N8-GP) and unmodified FVIII were not different. N8-GP had a longer duration of effect in FVIII-deficient mouse models, approximately a twofold prolonged half-life in mice, rabbits, and cynomolgus monkeys; however, the prolongation was less pronounced in rats. Binding capacity of N8-GP on human monocyte-derived dendritic cells was reduced compared with unmodified FVIII, resulting in several-fold reduced cellular uptake. In conclusion, N8-GP has the potential to offer efficacious prevention and treatment of bleeds in hemophilia A at reduced dosing frequency. PMID:23335368

  7. [Intein-mediated F309SfVIII ligation with enhanced secretion of its heavy chain.].

    PubMed

    Zhu, Fu-Xiang; Liu, Ze-Long; Qu, Hui-Ge; Chi, Xiao-Yan

    2009-12-25

    Coagulation factor VIII (fVIII) is a secretion protein and plays a crucial role in the coagulation cascade. Hemophilia A resulted from deficiency of fVIII is the most common X-linked recessive bleeding disorder. Gene therapy is recognized as an attractive strategy for the eventual cure of this disease. However, the gene therapy is hampered by the big size of fVIII gene when using the most promising gene vectors, adeno-associated virus (AAV) vectors. In this study we explored the intein-mediated protein trans-splicing to deliver a Phe(309)-->Ser mutant full-length fVIII (F309SfVIII) gene by using a dual-vector system. An intein is a protein sequence embedded within a precursor protein and can excise itself through protein splicing. The F309SfVIII is proven to be beneficial to its secretion. The F309SfVIII gene was broken into heavy and light chains before Ser(1239) in B domain and fused with the coding sequences of Ssp DnaB intein respectively to construct a pair of plasmid vectors by inserting them into the pcDNA3.1 vectors. Forty-eight hours after co- or separate transfection of 293 cells, the co-transfected cell lysate showed an obvious ligated F309SfVIII protein band by Western blot with a polyclonal antibody against fVIII. The amounts of secreted F309SfVIII protein in culture supernatants and their bioactivities were (71+/-9) ng/mL and (0.38+/-0.09) IU/mL determined by ELISA and Coatest assay respectively. The supernatant from combined cells with separate transfections also displayed lower levels of F309SfVIII antigen and fVIII activity [(25+/-6) ng/mL and (0.12+/-0.05) IU/mL], indicating the F309SfVIII could be formed by splicing both before and after secretion. The content of F309SfVIII heavy chain protein from co-transfected cell supernatant was higher than that of intein-fused heavy chain transfection alone [(135+/-10) ng/mL vs (37+/-7) ng/mL, P<0.01)]. These data demonstrated that intein could be used as a technical strategy in a dual-vector system

  8. Influences of ABO blood group, age and gender on plasma coagulation factor VIII, fibrinogen, von Willebrand factor and ADAMTS13 levels in a Chinese population

    PubMed Central

    Wang, Zongkui; Dou, Miaomiao; Du, Xi; Ma, Li; Sun, Pan; Cao, Haijun; Ye, Shengliang; Jiang, Peng; Liu, Fengjuan; Lin, Fangzhao

    2017-01-01

    Background ABO blood group is a hereditary factor of plasma levels of coagulation factor VIII (FVIII) and von Willebrand factor (VWF). Age and gender have been shown to influence FVIII, VWF, fibrinogen (Fbg), and ADAMTS13 (A disintegrin and metalloprotease with thrombospondin type 1 motif, 13). We investigated the effects of ABO type, age, and gender on plasma levels of FVIII, Fbg, VWF, and ADAMTS13 in a Chinese population. Methods A total of 290 healthy volunteers were eligible for this study. ABO blood group was determined by indirect technique. FVIII:C and Fbg were measured by clotting assays. VWF antigen (VWF:Ag), collagen-binding activity (VWF:CBA), and ADAMTS13 antigen were assessed by ELISA, whereas VWF ristocetin cofactor activity (VWF:Rcof) was performed by agglutination of platelets with ristocetin. Results Mean FVIII:C and VWF levels (VWF:Ag, VWF:CBA, and VWF:Rcof) were significantly higher in non-O than in O type subjects (p < 0.05 for all comparison). ADAMTS13 antigen decreased with increasing age, whereas the other parameters increased. Other than ADAMTS13 (p < 0.01), no gender-related variations were observed in the other parameters. Moreover, FVIII:C, Fbg, VWF:Ag, VWF:CBA, and VWF:Rcof showed significant and positive relationships with age (r = 0.421, 0.445, 0.410, 0.401, and 0.589, resp.; all p < 0.001), whereas a negative relationship was observed for ADAMTS13 antigen (r = 0.306; p = 0.006). Furthermore, FVIII:C were strongly correlated with VWF:Ag, VWF:CBA, and VWF:Rcof (r = 0.746, r = 0.746, and r = 0.576, resp.; p < 0.0001). VWF parameters were also strongly correlated with each other (r = 0.0.847 for VWF:Ag and VWF:CBA; r = 0.722 for VWF:Ag and VWF:Rcof; p < 0.0001). Conclusions ABO blood group, age, and gender showed different effects on plasma levels of FVIII:C, Fbg, VWF:Ag, VWF:CBA, VWF:Rcof, and ADAMTS13 antigen. These new data on a Chinese population are quite helpful to compare with other ethnic groups. PMID

  9. Safety and efficacy of BAY 94-9027, a prolonged-half-life factor VIII.

    PubMed

    Reding, M T; Ng, H J; Poulsen, L H; Eyster, M E; Pabinger, I; Shin, H-J; Walsch, R; Lederman, M; Wang, M; Hardtke, M; Michaels, L A

    2017-03-01

    Essentials Recombinant factor VIII BAY 94-9027 conjugates in a site-specific manner with polyethylene glycol. BAY 94-9027 was given to patients with severe hemophilia A as prophylaxis and to treat bleeds. BAY 94-9027 prevented bleeds at dose intervals up to every 7 days and effectively treated bleeds. BAY 94-9027 treatment was mainly well tolerated and no patient developed factor VIII inhibitors. Click to hear Dr Tiede's perspective on half-life extended factor VIII for the treatment of hemophilia A SUMMARY: Background BAY 94-9027 is a B-domain-deleted prolonged-half-life recombinant factor VIII (FVIII) that conjugates in a site-specific manner with polyethylene glycol. Objective Assess efficacy and safety of BAY 94-9027 for prophylaxis and treatment of bleeds in patients with severe hemophilia A. Patients/methods In this multinational, phase 2/3, partially randomized, open-label trial, men aged 12-65 years with FVIII < 1% and ≥ 150 exposure days to FVIII received BAY 94-9027 for 36 weeks on demand or prophylactically at intervals determined following a 10-week run-in period on 25 IU kg(-1) body weight two times per week. Patients with > 1 bleed during the run-in subsequently received 30-40 IU kg(-1) two times per week; patients with ≤ 1 bleed were eligible for randomization to every-5-days (45-60 IU kg(-1) ) or every-7-days (60 IU kg(-1) ) prophylaxis (1 : 1) for 26 additional weeks until randomization arms were filled. Patients who were eligible but not randomized continued twice-weekly prophylaxis. The primary efficacy outcome was annualized bleeding rate (ABR). Results The intent-to-treat population included 132 patients (prophylaxis, n = 112; on demand, n = 20). Median ABR (quartile [Q1; Q3]) for patients treated two times per week who were not eligible for randomization (n = 13) improved after dose increase (17.4 [14.3; 26.0] to 4.1 [2.0; 10.6]). Median ABR for patients randomized to every-5-days treatment (n = 43) was 1.9 (0; 4.2), similar to patients

  10. Allele frequencies of three factor VIII gene polymorphisms in Iranian populations and their application in hemophilia A carrier detection.

    PubMed

    Azimifar, S Babak; Seyedna, S Yoosef; Zeinali, Sirous

    2006-05-01

    Hemophilia A is an X-linked recessive bleeding disorder caused by a quantitative or qualitative deficiency of blood coagulation factor VIII (FVIII). ARMS (amplification refractory mutation system) primers were designed to determine allele frequencies of three FVIII gene linked markers, IVS7 nt 27 G/A SNP, BclI/intron 18, and HindIII/intron 19 among 85 normal Iranian women from unrelated families. Then same method was applied to perform carrier detection for hemophilia A families. The allele frequencies of IVS7 nt 27 "G"/"A" allele, BclI "T"/"A" allele, and HindIII "C"/"T" allele among normal women were 0.88/0.12, 0.52/0.48, and 0.48/0.52, respectively. The three polymorphisms were found to be in strong linkage disequilibrium, which decreased the overall heterozygosity to 51%. Twenty-one women from 15 unrelated hemophilia A families were referred to us for hemophilia A carrier detection. Taking advantage of these three biallelic polymorphisms in conjunction with multiallelic St14 VNTR (locus DXS52), IVS13 (CA)n STR, and IVS22 (CA)n STR, carrier status was determined in 16 women (16/21 or 76% of the at-risk women) from 11 families (11/15 or 73% of the families). The used ARMS methods are rapid and can easily be applied in conjunction with other FVIII gene linked polymorphisms for indirect mutation detection of hemophilia A where they are informative.

  11. Combined deficiency of factor V and factor VIII is due to mutations in either LMAN1 or MCFD2

    PubMed Central

    Zhang, Bin; McGee, Beth; Yamaoka, Jennifer S.; Guglielmone, Hugo; Downes, Katharine A.; Minoldo, Salvador; Jarchum, Gustavo; Peyvandi, Flora; de Bosch, Norma B.; Ruiz-Saez, Arlette; Chatelain, Bernard; Olpinski, Marian; Bockenstedt, Paula; Sperl, Wolfgang; Kaufman, Randal J.; Nichols, William C.; Tuddenham, Edward G. D.; Ginsburg, David

    2006-01-01

    Mutations in LMAN1 (ERGIC-53) or MCFD2 cause combined deficiency of factor V and factor VIII (F5F8D). LMAN1 and MCFD2 form a protein complex that functions as a cargo receptor ferrying FV and FVIII from the endoplasmic reticulum to the Golgi. In this study, we analyzed 10 previously reported and 10 new F5F8D families. Mutations in the LMAN1 or MCFD2 genes accounted for 15 of these families, including 3 alleles resulting in no LMAN1 mRNA accumulation. Combined with our previous reports, we have identified LMAN1 or MCFD2 mutations as the causes of F5F8D in 71 of 76 families. Among the 5 families in which no mutations were identified, 3 were due to misdiagnosis, with the remaining 2 likely carrying LMAN1 or MCFD2 mutations that were missed by direct sequencing. Our results suggest that mutations in LMAN1 and MCFD2 may account for all cases of F5F8D. Immunoprecipitation and Western blot analysis detected a low level of LMAN1-MCFD2 complex in lymphoblasts derived from patients with missense mutations in LMAN1 (C475R) or MCFD2 (I136T), suggesting that complete loss of the complex may not be required for clinically significant reduction in FV and FVIII. PMID:16304051

  12. Cofactor Activity in Factor VIIIa of the Blood Clotting Pathway Is Stabilized by an Interdomain Bond between His281 and Ser524 Formed in Factor VIII*

    PubMed Central

    Wakabayashi, Hironao; Monaghan, Morgan; Fay, Philip J.

    2014-01-01

    The factor VIII (FVIII) crystal structure suggests a possible bonding interaction of His281 (A1 domain) with Ser524 (A2 domain), although the resolution of the structure (∼4 Å) does not firmly establish this bonding. To establish that side chains of these residues participate in an interdomain bond, we prepared and examined the functional properties of a residue swap variant (H281S/S524H) where His281 and Ser524 residues were exchanged with one another and a disulfide-bridged variant (H281C/S524C) where the two residues were replaced with Cys. The latter variant showed efficient disulfide bonding of the A1 and A2 domains. The swap variant showed WT-like FVIII and FVIIIa stability, which were markedly reduced for H281A and S524A variants in an earlier study. The disulfide-bridged variant showed ∼20% increased FVIII stability, and FVIIIa did not decay during the time course measured. This variant also yielded 35% increased thrombin peak values compared with WT in a plasma-based thrombin generation assay. Binding analyses of H281S-A1/A3C1C2 dimer with S524H-A2 subunit yielded a near WT-like affinity value, whereas combining the variant dimer or A2 subunit with the WT complement yielded ∼5- and ∼10-fold reductions, respectively, in affinity. Other functional properties including thrombin generation potential, FIXa binding affinity, Km for FX of FXase complexes, thrombin activation efficiency, and down-regulation by activated protein C showed similar results for the two variants compared with WT FVIII. These results indicate that the side chains of His281 and Ser524 are in close proximity and contribute to a bonding interaction in FVIII that is retained in FVIIIa. PMID:24692542

  13. Continuous infusion of factor VIII for surgery and major bleeding.

    PubMed

    Hay, C R; Doughty, H I; Savidge, G F

    1996-03-01

    In a clinical trial, 24 patients with haemophilia A who needed surgery or had suffered severe bleeding were treated by continuous infusion of Monoclate P, a factor VIII concentrate that is immunopurified by monoclonal antibodies. Continuous infusion of Monoclate P began with a dose of 2 U/kg per h that was adjusted according to the results of factor VIII assays to achieve a factor VIII target level of 100 IU/dl for 2 days and then 80 IU/dl for 5 days. The safety, efficacy, and economics of this approach were assessed. No haemorrhagic episodes were observed. The continuous infusion was convenient and had the advantage of producing steady-state levels of factor VIII. With a single-compartment model, we found median factor VIII clearance values of 3.11 (range 1.79-7.78) x 10(3) litres/kg per h, elimination rates of 5.0-19.4 x 10(-2)/h and a median half-life of 9.9 h (range 4.8-20.0 h). Clearance and the elimination rate appeared to decline over the infusion period, as judged by the decreasing infusion rate required to maintain the target concentration of factor VIII. An economic comparison with bolus therapy, using theoretically derived bolus dosages, indicated that the potential saving was related inversely to the factor VIII half-life. Potential savings of 75% were predicted on the first postoperative day, averaging 35% over the full course of therapy.

  14. Stable recombinant expression and characterization of the two haemophilic factor VIII variants C329S (CRM(-)) and G1948D (CRM(r)).

    PubMed

    David, D; Saenko, E L; Santos, I M; Johnson, D J; Tuddenham, E G; McVey, J H; Kemball-Cook, G

    2001-06-01

    In haemophilia A, the functional defect at the molecular level of most factor VIII (FVIII) missense mutations remains unknown. Site-directed mutagenesis of B domain-deleted FVIII cDNA (FVIIISQ) was used to introduce two mutations associated with severe cross-reacting material (CRM)-negative (FVIII-C329S) or mild/moderate CRM-reduced (FVIII-G1948D) haemophilia A. Wild-type (FVIIISQ-WT) and variant FVIIISQ proteins were successfully expressed after stable transfection in Chinese hamster ovary (CHO) cells, and partially characterized at the intracellular, molecular and functional levels. Reverse transcription polymerase chain reaction analysis confirmed that both transcription and mRNA processing appeared normal in CHO cells transfected with both the wild-type and two variant constructs. In contrast to FVIIISQ-WT, immunofluorescence analysis of both CRM(-) and CRM(r) variants showed intracellular FVIII accumulation within the rough endoplasmic reticulum, suggesting secretion defects in transfected CHO cells. Immunoblot analysis of the FVIIISQ variant proteins that were secreted showed that they were expressed as mixed populations of uncleaved 170 kDa polypeptides, processed 90 kDa heavy chains and 80 kDa light chains, similar to FVIIISQ-WT. Phenotypic analysis of the B domain-deleted FVIIISQ variants expressed in CHO cells correlated well with the patients' reduced FVIII activity and, in addition, surface plasmon resonance studies demonstrated that both missense mutations were associated with increased rates of A2 domain dissociation following thrombin activation. We conclude that the mutations found are responsible for the haemophilia A phenotype, through intracellular retention and decreased stability of the active cofactor FVIIIa.

  15. BAY 81-8973, a full-length recombinant factor VIII: Human heat shock protein 70 improves the manufacturing process without affecting clinical safety.

    PubMed

    Maas Enriquez, Monika; Thrift, John; Garger, Stephen; Katterle, Yvonne

    2016-11-01

    BAY 81-8973 is a full-length, unmodified recombinant human factor VIII (FVIII) approved for the treatment of hemophilia A. BAY 81-8973 has the same amino acid sequence as the currently marketed sucrose-formulated recombinant FVIII (rFVIII-FS) product and is produced using additional advanced manufacturing technologies. One of the key manufacturing advances for BAY 81-8973 is introduction of the gene for human heat shock protein 70 (HSP70) into the rFVIII-FS cell line. HSP70 facilitates proper folding of proteins, enhances cell survival by inhibiting apoptosis, and potentially impacts rFVIII glycosylation. HSP70 expression in the BAY 81-8973 cell line along with other manufacturing advances resulted in a higher-producing cell line and improvements in the pharmacokinetics of the final product as determined in clinical studies. HSP70 protein is not detected in the harvest or in the final BAY 81-8973 product. However, because this is a new process, clinical trial safety assessments included monitoring for anti-HSP70 antibodies. Most patients, across all age groups, had low levels of anti-HSP70 antibodies before exposure to the investigational product. During BAY 81-8973 treatment, 5% of patients had sporadic increases in anti-HSP70 antibody levels above a predefined threshold (cutoff value, 239 ng/mL). No clinical symptoms related to anti-HSP70 antibody development occurred. In conclusion, addition of HSP70 to the BAY 81-8973 cell line is an innovative technology for manufacturing rFVIII aimed at improving protein folding and expression. Improved pharmacokinetics and no effect on safety of BAY 81-8973 were observed in clinical trials in patients with hemophilia A.

  16. Evaluation of factor VIII polymorphic short tandem repeat markers in linkage analysis for carrier diagnosis of hemophilia A

    PubMed Central

    Shrestha, Sabina; Dong, Sufang; Li, Zuhua; Huang, Zhuliang; Zheng, Fang

    2016-01-01

    Hemophilia A (HA) is the most common inherited X-linked recessive bleeding disorder caused by heterogeneous mutations in the factor VIII gene (FVIII). Diagnosis of the carrier is critical for preventing the birth of children affected by this coagulation disorder, which ultimately facilitates its management. Due to the heterogeneous nature of mutations, the large inversions and the complexity of the FVIII gene, direct recognition of the disease-associated mutation in HA is complex. Indirect linkage analysis using highly informative heterozygous polymorphic markers is an alternative method for determining the co-segregation of the mutant gene within a family for carrier detection of HA. The aim of the present study was to perform carrier diagnosis in a family with HA. Rapid multifluorescent polymerase chain reaction (PCR) was performed with six extragenic short tandem repeats (STRs), DXS1073, DXS15, DXS8091, DXS1227, DXS991, DXS993 and one intragenic marker, STR22 for linkage analysis in the HA family. All the STR markers employed in the present study were informative for linkages of pathogenic and healthy haplotypes among family members, particularly STR22, DXS1073 and DXS15. The STR marker, STR22, is within the FVIII gene while the DXS1073 and DXS15 markers are very close to the FVIII gene, where the chances of recombination are comparatively low, and provided the most accurate interpretation analysis, indicating that the proband's sister may have been the HA carrier. Rapid multifluorescent PCR using STR markers and linkage analysis was identified to be a simple method for performing HA carrier diagnosis. PMID:27446547

  17. Repeated infusions of VWF/FVIII concentrate: impact of VWF:FVIII ratio on FVIII trough and peak levels in a rabbit model.

    PubMed

    Raquet, E; Stockschläder, M; Dickneite, G

    2011-09-01

    The ratio of von Willebrand factor (VWF) to FVIII differs among available VWF/FVIII concentrates. Repeated infusions of concentrates with a low VWF:FVIII ratio may expose patients with von Willebrand disease to supranormal FVIII levels. The aim of this study was to determine the effects of repeated infusions with two VWF/FVIII concentrates differing in VWF:FVIII ratio on attained FVIII trough and peak levels as well as other pharmacokinetic parameters. Rabbits were randomized to receive multiple 150 IU kg⁻¹ VWF:RCo infusions at 4 h intervals with VWF/FVIII concentrates of a high (Haemate® P/Humate-P®) or low (Wilate®) VWF:FVIII ratio. Trough plasma FVIII and VWF levels were measured after each infusion. Pharmacokinetic analysis was performed using samples collected frequently after infusion. Mean FVIII trough level after the first Wilate infusion was 50.6 IU dL⁻¹ with a 95% confidence interval (CI) of 43.1-58.2 IU dL⁻¹, compared with 31.8 IU dL⁻¹ (CI, 24.4-39.1 IU dL⁻¹) for Haemate P (P<0.001). Trough levels progressively increased over the 24 h treatment period in both groups. After the final infusion, mean trough FVIII remained significantly higher (P = 0.002) in recipients of Wilate. Mean peak FVIII concentration after infusion was 67% higher in the Wilate group (167 vs. 100 IU dL⁻¹ , respectively; P = 0.002). Mean cumulative exposure to FVIII, as measured by area under the curve, was 84% greater in Wilate-treated animals. Half-life did not differ between the two concentrates. Animal model data suggest that exposure to elevated FVIII levels can be reduced through use of VWF/FVIII concentrates with higher VWF:FVIII ratios.

  18. [Comparative study of concentrated blood derivatives of factor VIII].

    PubMed

    Baklaja, R; Miletić, V; Stajić, M; Cvetković, V; Grozdanić, V

    1984-01-01

    The work presents results of the investigations of blood derivatives--F VIII concentrates: commercial cryoprecipitate, concentrate of intermediary purity and derivatives of high purity: Kriobulin--Immuno, Octobulin--Landerlan, Profilate--Alfa, Factor VIII--Behring, Hemofil--Hyland, Factorate--Armour Pharma, AHF--Kaote Cutter. The following parameters were investigated: VIII: C, VIIIR: Ag, total protein, protein electrophoresis, IgG, IgA and IgM immunoglobulins and anti-A and anti-B isoagglutinins. All derivatives except cyroprecipitate have considerably higher VIIIR: RAg value compared with VIII: C, which indicated inactivation of labile VIII: C component during concentrate preparation. Specific activity varied depending on purity of preparations, but ranged from 1,72 to 22. High isoagglutinin titer of anti-A was noted in preparations of high purity, as well as the presence of immunoglobulins. Despite considerable differences in vitro, all concentrated derivatives F VIII have similar immediate clinical effect and recovery from 0,87 to 1,36. All results indicate that new ways of derivative F VIII purification should be found with lower degree of contamination of other plasma proteins and less risk of hepatitis virus transmission. When certain indications are recognized, cryoprecipitate produced in our country in all blood transfusion services should be used.

  19. A novel mutation (4040-4045 nt. delA) in exon 14 of the factor VIII gene causing severe hemophilia A.

    PubMed

    Onsori, Habib; Feizi, Mohammad Ali Hosseinpour; Feizi, Abbas Ali Hosseinpour

    2011-09-01

    Hemophilia A is an X-linked congenital bleeding disorder caused by Factor VIII deficiency. Different mutations including point mutations, deletions, insertions and inversions have been reported in the FVIII gene, which cause hemophilia A. In the current study, with the use of conformational sensitive gel electrophoresis (CSGE) analysis, we report a novel 1-nt deletion in the A6 sequence at codons 1328-1330 (4040-4045 nt delA) occurring in exon 14 of the FVIII gene in a seven-year-old Iranian boy with severe hemophilia A. This mutation that causes frameshift and premature stop-codon at 1331 has not previously been reported in the F8 Hemophilia A Mutation, Structure, Test and Resource Site (HAMSTeRS) database.

  20. Phenotypes of allo- and autoimmune antibody responses to FVIII characterized by surface plasmon resonance.

    PubMed

    Lewis, Kenneth B; Hughes, Richard J; Epstein, Melinda S; Josephson, Neil C; Kempton, Christine L; Kessler, Craig M; Key, Nigel S; Howard, Tom E; Kruse-Jarres, Rebecca; Lusher, Jeanne M; Walsh, Christopher E; Watts, Raymond G; Ettinger, Ruth A; Pratt, Kathleen P

    2013-01-01

    Evidence of antibody isotype/subtype switching may provide prognostic value regarding the state of immune responses to therapeutic proteins, e.g. anti-factor VIII (FVIII) antibodies that develop in many hemophilia A patients, clinically termed "inhibitors". A sensitive, high- information-content surface plasmon resonance (SPR) assay has been developed to quantify IgG subtype distributions and the domain specificity of anti-drug antibodies. Plasma samples from 22 subjects with an allo- or auto-immune reaction to FVIII were analyzed. Pre-analytical treatment protocols were developed to minimize non-specific binding and specific matrix interference due to von Willebrand factor-FVIII interactions. The dynamic range for IgG quantification was 0.2-5 µg/ml (∼1-33 nM), allowing characterization of inhibitor-positive samples. Subtype-specific monoclonal antibodies were used to quantify the IgG subtype distribution of FVIII-specific antibodies. Most samples obtained from multiply-infused inhibitor subjects contained IgG₄ antibodies. Several distinct phenotypes were assigned based on the IgG subtype distribution: IgG₁, IgG₄, IgG₁ & IgG₄, and IgG₁, IgG₂ & IgG₄. An IgG₁-only response was found in mild/moderate HA subjects during early FVIII infusions, and analysis of serial samples followed antibody class switching as several subjects' immune responses developed. Competition studies utilizing a recombinant FVIII-C2 domain indicated 40-80% of FVIII-specific antibodies in most samples were directed against this domain.

  1. Multidimensional Genome-wide Analyses Show Accurate FVIII Integration by ZFN in Primary Human Cells

    PubMed Central

    Sivalingam, Jaichandran; Kenanov, Dimitar; Han, Hao; Nirmal, Ajit Johnson; Ng, Wai Har; Lee, Sze Sing; Masilamani, Jeyakumar; Phan, Toan Thang; Maurer-Stroh, Sebastian; Kon, Oi Lian

    2016-01-01

    Costly coagulation factor VIII (FVIII) replacement therapy is a barrier to optimal clinical management of hemophilia A. Therapy using FVIII-secreting autologous primary cells is potentially efficacious and more affordable. Zinc finger nucleases (ZFN) mediate transgene integration into the AAVS1 locus but comprehensive evaluation of off-target genome effects is currently lacking. In light of serious adverse effects in clinical trials which employed genome-integrating viral vectors, this study evaluated potential genotoxicity of ZFN-mediated transgenesis using different techniques. We employed deep sequencing of predicted off-target sites, copy number analysis, whole-genome sequencing, and RNA-seq in primary human umbilical cord-lining epithelial cells (CLECs) with AAVS1 ZFN-mediated FVIII transgene integration. We combined molecular features to enhance the accuracy and activity of ZFN-mediated transgenesis. Our data showed a low frequency of ZFN-associated indels, no detectable off-target transgene integrations or chromosomal rearrangements. ZFN-modified CLECs had very few dysregulated transcripts and no evidence of activated oncogenic pathways. We also showed AAVS1 ZFN activity and durable FVIII transgene secretion in primary human dermal fibroblasts, bone marrow- and adipose tissue-derived stromal cells. Our study suggests that, with close attention to the molecular design of genome-modifying constructs, AAVS1 ZFN-mediated FVIII integration in several primary human cell types may be safe and efficacious. PMID:26689265

  2. Factor VIII mutation and desmopressin-responsiveness in 62 patients with mild haemophilia A.

    PubMed

    Nance, D; Fletcher, S N; Bolgiano, D C; Thompson, A R; Josephson, N C; Konkle, B A

    2013-09-01

    Utilization of the synthetic vasopressin analogue (1-deamino-8-D-arginine-vasopressin, DDAVP) in treatment of mild haemophilia A (MHA, specific clotting factor VIII activity level 0.05-0.4 IU mL(-1) ) is convenient and effective for many but not all patients. Genetic testing for patients with MHA is increasingly recognized as providing valuable information for patient care beyond informing reproductive decisions, and as more patients are genotyped, mutation data can be utilized to individualize treatment decisions. To determine if genetic information informs response to DDAVP, a retrospective chart review was performed under Institutional Review Board approval to extract patient data with MHA, genetic mutation results, and response to DDAVP challenge. 62 patients met inclusion criteria. Complete responses (C) presented in mean value IU mL(-1) (range), were recorded for 32 of 62(52%) subjects: pre 0.19(0.04-0.45) and post 0.78(0.5-1.95); partial responses (P) were recorded for 15 of 62(24%) subjects: pre 0.1(0.06-0.15) and post 0.4(0.3-0.47); responses that were not clinically significant (N) were recorded for 15 of 62(24%) subjects: pre 0.17(0.02-0.34) and post 0.25(0.03-0.44). Subjects (related and unrelated) with the same mutation showed a trend towards a similar response to DDAVP. Eight genotypes were common to two or more subjects (n = 26). Two genotypes were concordant in all subjects [p.Ser2192Ile n = 3(C), p.Ala2220Pro n = 2(P)]. Of mutations in the C1 or C2 domains, 13 of 15(87%) subjects responded to DDAVP [C = 9(60%); P = 4(27%); n = 2(13%)]. Baseline FVIII:C did not predict magnitude of response to DDAVP. Genetic mutation results can assist with predicting DDAVP responsiveness, but baseline FVIII:C may not.

  3. The human clotting factor VIII cDNA contains an autonomously replicating sequence consensus- and matrix attachment region-like sequence that binds a nuclear factor, represses heterologous gene expression, and mediates the transcriptional effects of sodium butyrate.

    PubMed Central

    Fallaux, F J; Hoeben, R C; Cramer, S J; van den Wollenberg, D J; Briët, E; van Ormondt, H; van Der Eb, A J

    1996-01-01

    Expression of the human blood-clotting factor VIII (FVIII) cDNA is hampered by the presence of sequences located in the coding region that repress transcription. We have previously identified a 305-bp fragment within the FVIII cDNA that is involved in the repression (R.C. Hoeben, F.J. Fallaux, S.J. Cramer, D.J.M. van den Wollenberg, H. van Ormondt, E. Briet, and A.J. van der Eb, Blood 85:2447-2454, 1995). Here, we show that this 305-bp region of FVIII cDNA contains sequences that resemble the yeast (Saccharomyces cerevisiae) autonomously replicating sequence consensus. Two of these DNA elements coincide with AT-rich sequences that are often found in matrix attachment regions or scaffold-attached regions. One of these elements, consisting of nucleotides 1569 to 1600 of the FVIII cDNA (nucleotide numbering is according to the system of Wood et al. (W.I. Wood, D.J. Capon, C.C. Simonsen, D.L. Eaton, J. Gitschier, D. Keyt, P.H. Seeburg, D.H. Smith, P. Hollingshead, K.L. Wion, et al., Nature [London] 312:330-337,1984), binds a nuclear factor in vitro but loses this capacity after four of its base pairs have been changed. A synthetic heptamer of this segment can repress the expression of a chloramphenicol acetyltransferase (CAT) reporter gene and also loses this capacity upon mutation. Furthermore, we demonstrate that repression by FVIII sequences can be relieved by sodium butyrate. We demonstrate that the synthetic heptamer (FVIII nucleotides 1569 to 1600), when placed upstream of the Moloney murine leukemia virus long terminal repeat promoter that drives the CAT reporter, can render the CAT reporter inducible by butyrate. This effect was absent when the same element was mutated. The stimulatory effect of butyrate could not be attributed to butyrate-responsive elements in the studied long terminal repeat promoters. Our data provide a functional characterization of the sequences that repress expression of the FVIII cDNA. These data also suggest a link between

  4. Endogenous factor VIII synthesis from the intron 22-inverted F8 locus may modulate the immunogenicity of replacement therapy for hemophilia A.

    PubMed

    Pandey, Gouri Shankar; Yanover, Chen; Miller-Jenkins, Lisa M; Garfield, Susan; Cole, Shelley A; Curran, Joanne E; Moses, Eric K; Rydz, Natalia; Simhadri, Vijaya; Kimchi-Sarfaty, Chava; Lillicrap, David; Viel, Kevin R; Przytycka, Teresa M; Pierce, Glenn F; Howard, Tom E; Sauna, Zuben E

    2013-10-01

    Neutralizing antibodies (inhibitors) to replacement factor VIII (FVIII, either plasma derived or recombinant) impair the effective management of hemophilia A. Individuals with hemophilia A due to major deletions of the FVIII gene (F8) lack antigenically cross-reactive material in their plasma ("CRM-negative"), and the prevalence of inhibitors in these individuals may be as high as 90%. Conversely, individuals with hemophilia A caused by F8 missense mutations are CRM-positive, and their overall prevalence of inhibitors is <10% (ref. 2). Individuals with the F8 intron 22 inversion (found in ∼50% of individuals with severe hemophilia A) have been grouped with the former on the basis of their genetic defect and CRM-negative status. However, only ∼20% of these individuals develop inhibitors. Here we demonstrate that the levels of F8 mRNA and intracellular FVIII protein in B lymphoblastoid cells and liver biopsies from individuals with the intron 22 inversion are comparable to those in healthy controls. These results support the hypothesis that most individuals with the intron 22 inversion are tolerized to FVIII and thus do not develop inhibitors. Furthermore, we developed a new pharmacogenetic algorithm that permits the stratification of inhibitor risk for individuals and subpopulations by predicting the immunogenicity of replacement FVIII using, as input, the number of putative T cell epitopes in the infused protein and the competence of major histocompatibility complex class II molecules to present such epitopes. This algorithm showed statistically significant accuracy in predicting the presence of inhibitors in 25 unrelated individuals with the intron 22 inversion.

  5. Collagen-bound von Willebrand factor has reduced affinity for factor VIII.

    PubMed

    Bendetowicz, A V; Wise, R J; Gilbert, G E

    1999-04-30

    von Willebrand factor (vWf) is a multimeric adhesive glycoprotein that serves as a carrier for factor VIII in plasma. Although each vWf subunit displays a high affinity binding site for factor VIII in vitro, in plasma, only 2% of the vWf sites for factor VIII are occupied. We investigated whether interaction of plasma proteins with vWf or adhesion of vWf to collagen may alter the affinity or availability of factor VIII-binding sites on vWf. When vWf was immobilized on agarose-linked monoclonal antibody, factor VIII bound to vWf with high affinity, and neither the affinity nor binding site availability was influenced by the presence of 50% plasma. Therefore, plasma proteins do not alter the affinity or availability of factor VIII-binding sites. In contrast, when vWf was immobilized on agarose-linked collagen, its affinity for factor VIII was reduced 4-fold, with KD increasing from 0.9 to 3.8 nM. However, one factor VIII-binding site remained available on each vWf subunit. A comparable reduction in affinity for factor VIII was observed when vWf was a constituent of the subendothelial cell matrix and when it was bound to purified type VI collagen. In parallel with the decreased affinity for factor VIII, collagen-bound vWf displayed a 6-fold lower affinity for monoclonal antibody W5-6A, with an epitope composed of residues 78-96 within the factor VIII-binding motif of vWf. We conclude that collagen induces a conformational change within the factor VIII-binding motif of vWf that lowers the affinity for factor VIII.

  6. Loss of factor VIII activity during storage in PVC containers due to adsorption.

    PubMed

    McLeod, A G; Walker, I R; Zheng, S; Hayward, C P

    2000-03-01

    Recombinant factor VIII concentrates are stable when administered in a reconstituted form according to the manufacturer's specifications, and undiluted via infusion with syringe mini-pumps. However many Haemophilia centres administer recombinant factor VIII further diluted in intravenous fluids for greater ease of administration. To investigate the stability of recombinant factor VIII during administration as a diluted infusion, reconstituted factor VIII was stored in polyvinylchloride (PVC) mini-bags undiluted (146 IU mL-1) and at factor VIII concentrations of 10 IU mL-1 and 2 IU mL-1. After 48 h of storage at room temperature in PVC mini-bags, the recoveries of factor VIII activity were 41.9% of the initial activity for the undiluted (146 IU mL-1) product and 43.7% of the initial activity for factor VIII diluted to 10 IU mL-1. For factor VIII diluted to 2 IU mL-1, the amount of factor VIII activity remaining at 48 h was only 1.8% of the initial activity. In contrast, 100% of factor VIII activity was recovered after 48 h when undiluted reconstituted product (146 IU mL-1) was stored in a syringe. To investigate the mechanism of factor VIII activity loss during storage, factor VIII samples collected after 0, 3 and 48 h of storage were analysed by immunoblotting with factor VIII antibodies. No evidence of factor VIII proteolytic degradation during storage was found, however, large amounts of factor VIII antigen were recovered from the empty PVC mini-bags following elution with denaturing detergent. We conclude that clinically significant losses of factor VIII activity occur during storage in PVC mini-bags and that the loss of activity is most likely due to protein adsorption onto the plastic surface. This loss of factor VIII activity during storage in PVC containers may substantially affect the safety and potential cost savings of administering recombinant factor VIII by continuous infusion.

  7. Isolation, subunit structure, and proteolytic modification of bovine factor VIII.

    PubMed

    Legaz, M E; Weinstein, M J; Heldebrant, C M; Davie, E W

    1975-01-20

    A new method has been described for the isolation of factor VIII. The method results in a high yield of factor VIII that is homogeneous by several different criteria. The purified protein is very stable and is not dissociated in the presence of 1 M NaCl or 0.25 M CaCl2. The highly purified protein is readily activated and inactivated by various proteolytic enzymes, such as thrombin, plasmin, and trypsin. The molecular events that lead to the activation reaction, however, have not been established.

  8. In silico calculated affinity of FVIII-derived peptides for HLA class II alleles predicts inhibitor development in haemophilia A patients with missense mutations in the F8 gene.

    PubMed

    Pashov, A D; Calvez, T; Gilardin, L; Maillère, B; Repessé, Y; Oldenburg, J; Pavlova, A; Kaveri, S V; Lacroix-Desmazes, S

    2014-03-01

    Forty per cent of haemophilia A (HA) patients have missense mutations in the F8 gene. Yet, all patients with identical mutations are not at the same risk of developing factor VIII (FVIII) inhibitors. In severe HA patients, human leucocyte antigen (HLA) haplotype was identified as a risk factor for onset of FVIII inhibitors. We hypothesized that missense mutations in endogenous FVIII alter the affinity of the mutated peptides for HLA class II, thus skewing FVIII-specific T-cell tolerance and increasing the risk that the corresponding wild-type FVIII-derived peptides induce an anti-FVIII immune response during replacement therapy. Here, we investigated whether affinity for HLA class II of wild-type FVIII-derived peptides that correspond to missense mutations described in the Haemophilia A Mutation, Structure, Test and Resource database is associated with inhibitor development. We predicted the mean affinity for 10 major HLA class II alleles of wild-type FVIII-derived peptides that corresponded to 1456 reported cases of missense mutations. Linear regression analysis confirmed a significant association between the predicted mean peptide affinity and the mutation inhibitory status (P = 0.006). Significance was lost after adjustment on mutation position on FVIII domains. Although analysis of the A1-A2-A3-C1 domains yielded a positive correlation between predicted HLA-binding affinity and inhibitory status (OR = 0.29 [95% CI: 0.14-0.60] for the high affinity tertile, P = 0.002), the C2 domain-restricted analysis indicated an inverse correlation (OR = 3.56 [1.10-11.52], P = 0.03). Our data validate the importance of the affinity of FVIII peptides for HLA alleles to the immunogenicity of therapeutic FVIII in patients with missense mutations.

  9. Successful immune tolerance induction with low-dose coagulation factor VIII in a patient with hemophilia A from a developing country.

    PubMed

    Ay, Yilmaz; Ersin, Toret; Yesim, Oymak; Hilkay, Karapinar Tuba; Dilek, Ince; Gulcihan, Ozek; Ahmet, Koc

    2016-09-01

    Inhibitor development is the most frequent and serious complication of the treatment in patients with hemophilia. Immune tolerance induction (ITI) is the only option of treatment for the eradication of factor VIII (FVIII) inhibitor. We would like to present our case with hemophilia whose FVIII inhibitor eradication was done by a low-dose ITI regimen. Our patient has been applied on-demand therapy until 8 years of age. Secondary prophylaxis was began because of having hemophilic arthropathy. A low titer of FVIII inhibitor (4.2 BU/ml) was detected in the fifth month of the prophylaxis. The peak inhibitor titer of patient was 4.6 BU/ml, and there was no decrease in inhibitor titer in the follow-up duration. The low-dose ITI (50 IU/kg, 3 days a week) was started. His inhibitor level was detected negative and the recovery test was ameliorated in the 15th of the ITI therapy. High-dose regimen ITI could not be given particularly in developing countries such as Turkey in view of the high cost of treatment. Patients who had good risk factors might be successfully treated by using low-dose ITI regimen as effective as high-dose ITI regimen.

  10. Gene transfer to hemophilia A mice via oral delivery of FVIII-chitosan nanoparticles.

    PubMed

    Bowman, Katherine; Sarkar, Rita; Raut, Sanj; Leong, Kam W

    2008-12-18

    Effective oral delivery of a non-viral gene carrier would represent a novel and attractive strategy for therapeutic gene transfer. To evaluate the potential of this approach, we studied the oral gene delivery efficacy of DNA polyplexes composed of chitosan and Factor VIII DNA. Transgene DNA was detected in both local and systemic tissues following oral administration of the chitosan nanoparticles to hemophilia A mice. Functional factor VIII protein was detected in plasma by chromogenic and thrombin generation assays, reaching a peak level of 2-4% FVIII at day 22 after delivery. In addition, a bleeding challenge one month after DNA administration resulted in phenotypic correction in 13/20 mice given 250-600 microg of FVIII DNA in chitosan nanoparticles, compared to 1/13 mice given naked FVIII DNA and 0/6 untreated mice. While further optimization would be required to render this type of delivery system practical for hemophilia A gene therapy, the findings suggest the feasibility of oral, non-viral delivery for gene medicine applications.

  11. Efficacy and safety of a new generation von Willebrand factor/factor VIII concentrate (Wilate®) in the management of perioperative haemostasis in von Willebrand disease patients undergoing surgery.

    PubMed

    Windyga, J; von Depka-Prondzinski, M

    2011-06-01

    The aim of this study was to assess the efficacy of Wilate®, a new generation, plasma-derived, high-purity, double virus-inactivated von Willebrand factor (VWF) and factor VIII (FVIII) concentrate (ratio close to physiological 1:1) in the perioperative management of haemostasis in von Willebrand disease (VWD). Data for VWD patients who received Wilate® for perioperative management were obtained from four European, prospective, open-label, non-controlled, non-randomised, multicentre phase II or III clinical trials. A total of 57 surgical procedures were performed (major: n = 27; minor n = 30) in 32 patients. The majority of patients (n = 19, 59.4%) had type 3 VWD, 9 (28.1%) had type 2 VWD and four (12.5%) had type 1 VWD. During major surgery, median daily FVIII dose and mean number of infusions were 25 IU•kg-1 FVIII (VWF:RCo ~23 IU•kg-1) and 11.0, respectively. Corresponding values for minor surgery were 35 IU•kg-1 (VWF:RCo ~32 IU•kg-1) and 1.5. The efficacy of Wilate® was rated by the investigator as excellent or good in 51 of 53 (96%) procedures. Tolerability was rated as very good or good in 100% of major surgeries (27 of 27) and minor surgeries (29 of 29). Wilate® is an effective and well-tolerated VWF/FVIII replacement therapy in the perioperative management of haemostasis in patients with VWD. It can be administered at a similar FVIII dose, but at a lower VWF dose, as compared to older generation products. Clinical benefits were shown in a population with a high proportion of type 3 VWD patients.

  12. Infection risk and stability of a continuous 8-h 250 mL rFVIII infusion.

    PubMed

    Lambing, A; Kuriakose, P; Mueller, L M

    2014-03-01

    This study seeks to identify the delivery method of continuous infusion using a 250 cc IV bag via pump, change every 8 h. Additionally, the study will examine the infection risk with the use of 8 h infusions. Ten hemophilia A patients were identified for the study. Each patient received a bolus factorVIII (FVIII) infusion with a pre FVIII level and 1 h post FVIII level to determine recovery levels for optimal dosing. On the day of 8-h continuous infusion, the pt received a bolus VIII (Kogenate FS (™)) for correction to 100% followed by individually calculated continuous infusion (Kogenate FS (™)) FVIII. FVIII levels were drawn from the IV bag and peripherally from the patient in the opposite arm at time points: pre infusion, 1, 2, 3, 4, 5, 6 and 8 h. Additionally, blood cultures were drawn from the IV bag and from the IV tubing at time points pre infusion, 4 and 8 h. Fourteen subjects agreed to participate in the study; 4 failed to follow up, hence 10 subjects were included in the analysis of data; 7 severe, 2 moderate, and 1 mild hemophilia A. Age range was 26-62 years. Ethnic breakdown included 5 African American, 4 Caucasian, 1 Hispanic. With all infusions, the range of FVIII was 65-135% (blood) and 62-200% (bag). After the start of infusion, there were no significant differences noted between the hourly FVIII levels in the subjects and the IV values (P-value range 0.36-0.9). Additionally, given three time points with six cultures per patient, totaling 60 points of cultures drawn for the study, all cultures from the IV bag and patient were negative. The effective delivery method and safety of an 8-h continuous infusion of FVIII (Kogenate FS (™)) has been confirmed. This method can be helpful given that many hospitals may not carry the required mini-pumps, allowing a standard safe delivery of FVIII (Kogenate FS (™)) continuous infusion by available means.

  13. Suppression of inhibitor formation against FVIII in a murine model of hemophilia A by oral delivery of antigens bioencapsulated in plant cells.

    PubMed

    Sherman, Alexandra; Su, Jin; Lin, Shina; Wang, Xiaomei; Herzog, Roland W; Daniell, Henry

    2014-09-04

    Hemophilia A is the X-linked bleeding disorder caused by deficiency of coagulation factor VIII (FVIII). To address serious complications of inhibitory antibody formation in current replacement therapy, we created tobacco transplastomic lines expressing FVIII antigens, heavy chain (HC) and C2, fused with the transmucosal carrier, cholera toxin B subunit. Cholera toxin B-HC and cholera toxin B-C2 fusion proteins expressed up to 80 or 370 µg/g in fresh leaves, assembled into pentameric forms, and bound to GM1 receptors. Protection of FVIII antigen through bioencapsulation in plant cells and oral delivery to the gut immune system was confirmed by immunostaining. Feeding of HC/C2 mixture substantially suppressed T helper cell responses and inhibitor formation against FVIII in mice of 2 different strain backgrounds with hemophilia A. Prolonged oral delivery was required to control inhibitor formation long-term. Substantial reduction of inhibitor titers in preimmune mice demonstrated that the protocol could also reverse inhibitor formation. Gene expression and flow cytometry analyses showed upregulation of immune suppressive cytokines (transforming growth factor β and interleukin 10). Adoptive transfer experiments confirmed an active suppression mechanism and revealed induction of CD4(+)CD25(+) and CD4(+)CD25(-) T cells that potently suppressed anti-FVIII formation. In sum, these data support plant cell-based oral tolerance for suppression of inhibitor formation against FVIII.

  14. Characterization of five partial deletions of the factor VIII gene

    SciTech Connect

    Youssoufian, H.; Antonarakis, S.E.; Aronis, S.; Tsiftis, G.; Phillips, D.G.; Kazazian, H.H. Jr.

    1987-06-01

    Hemophilia A is an X-linked disorder of coagulation caused by a deficiency of factor VIII. By using cloned DNA probes, the authors have characterized the following five different partial deletions of the factor VIII gene from a panel of 83 patients with hemophilia A: (i) a 7-kilobase (kb) deletion that eliminates exon 6; (ii) a 2.5-kb deletion that eliminates 5' sequences of exon 14; (iii) a deletion of at least 7 kb that eliminates exons 24 and 25; (iv) a deletion of at least 16 kb that eliminates exons 23-25; and (v) a 5.5-kb deletion that eliminates exon 22. The first four deletions are associated with severe hemophilia A. By contrast, the last deletion is associated with moderate disease, possibly because of in-frame splicing from adjacent exons. None of those patients with partial gene deletions had circulating inhibitors to factor VIII. One deletion occurred de novo in a germ cell of the maternal grandmother, while a second deletion occurred in a germ cell of the maternal grandfather. These observations demonstrate that de novo deletions of X-linked genes can occur in either male or female gametes.

  15. Rituximab and intermediate-purity plasma-derived factor VIII concentrate (Koate®) as adjuncts to therapeutic plasma exchange for thrombotic thrombocytopenic purpura in patients with an ADAMTS13 inhibitor.

    PubMed

    Pandey, Soumya; Nakagawa, Mayumi; Rosenbaum, Eric R; Arnaoutakis, Konstantinos; Hutchins, Laura F; Makhoul, Issam; Milojkovic, Natasha; Cottler-Fox, Michele

    2015-02-01

    Thrombotic thrombocytopenic purpura (TTP) results from a congenital or acquired deficiency of the von Willebrand factor (vWF)-cleaving protease ADAMTS13. The disease can be fatal and hence treatment should be initiated promptly. Therapeutic plasma exchange (TPE) remains the standard treatment along with adjunct therapies including steroids and immunosuppressive drugs. Addition of rituximab to TPE has been shown to be beneficial in refractory/relapsing TTP; however, TPE results in removal of rituximab from the circulation requiring more frequent dosing of rituximab to achieve a favorable outcome. The intermediate-purity plasma-derived Factor VIII concentrate (FVIII) Koate® contains the highest amount of ADAMTS13 activity yet reported and has been used successfully in treating congenital TTP. Here we report our experience with addition of this FVIII concentrate to rituximab, corticosteroids and TPE in three TTP patients with an ADAMTS13 inhibitor to permit withholding TPE for 48 h after rituximab infusion.

  16. Mutating factor VIII: lessons from structure to function.

    PubMed

    Fay, Philip J; Jenkins, P Vincent

    2005-01-01

    Factor VIII, a metal ion-dependent heterodimer, circulates in complex with von Willebrand factor. At sites of vessel wall damage, this procofactor is activated to factor VIIIa by limited proteolysis and assembles onto an anionic phospholipid surface in complex with factor IXa to form the intrinsic factor Xase; an enzyme complex that efficiently converts factor X to factor Xa during the propagation phase of coagulation. Factor Xase activity is down-regulated by mechanisms that include self-dampening by dissociation of a critical factor VIIIa subunit and proteolytic inactivation by the activated protein C pathway. Recent studies identify putative metal ion coordination sites as well as ligands involved in the catabolism of the activated and procofactor forms of the protein. Our knowledge of these multiple intra- and inter-molecular interactions has been facilitated by the application of naturally occurring and site-directed mutations to study factor VIII structure and function. In this review, we document important and novel contributions following this line of investigation.

  17. Analysing two dinucleotide repeats of FVIII gene in Iranian population.

    PubMed

    Rabbani, B; Rezaeian, A; Khanahmad, H; Bagheri, R; Kamali, E; Zeinali, S

    2007-11-01

    Using dinucleotide repeats for carrier detection and prenatal diagnosis of haemophilia A patients, led us to find different alleles and their frequencies in Iranian population. Polymerase chain reaction (PCR) amplification of two short tandem repeat (STR) loci of factor VIII (FVIII) gene was performed, and the PCR products were resolved on 10% native polyacrylamide gel, and samples were analysed with sequenced DNA markers made of PCR cloning of the dinucleotide FVIII gene fragments. Seven different alleles were observed for intron 13 STR, having 18-24 (CA) repeating units and five alleles for intron 22 STR having 24-28 repeating units of (CACT). Bands produced during dinucleotide study were defined in detail so this could improve the genotyping of heterozygotes and homozygotes. Conformational band produced were characterized to specify the dinucleotide pattern. Our results confirm the Hardy-Weinberg proportions of the heterozygosity rate of the 85 analysed individuals. The observed heterozygosity rate for intron 13 and 22 was 52% and 59% respectively. Our data also indicate that our population is closer to caucasians than to any other populations. Finding different dinucleotide repeat alleles and their frequencies has made it possible to identify carriers and provide prenatal diagnosis with more confidence. This allows antenatal diagnosis to be performed in the vast majority of carriers.

  18. Efficacy and safety of rVIII-SingleChain: results of a phase 1/3 multicenter clinical trial in severe hemophilia A

    PubMed Central

    Mahlangu, Johnny; Kuliczkowski, Kazimierz; Karim, Faraizah Abdul; Stasyshyn, Oleksandra; Kosinova, Marina V.; Lepatan, Lynda Mae; Skotnicki, Aleksander; Boggio, Lisa N.; Klamroth, Robert; Oldenburg, Johannes; Hellmann, Andrzej; Santagostino, Elena; Baker, Ross I.; Fischer, Kathelijn; Gill, Joan C.; P’Ng, Stephanie; Chowdary, Pratima; Escobar, Miguel A.; Khayat, Claudia Djambas; Rusen, Luminita; Bensen-Kennedy, Debra; Blackman, Nicole; Limsakun, Tharin; Veldman, Alex; St. Ledger, Katie

    2016-01-01

    Recombinant VIII (rVIII)-SingleChain is a novel B-domain–truncated recombinant factor VIII (rFVIII), comprised of covalently bonded factor VIII (FVIII) heavy and light chains. It was designed to have a higher binding affinity for von Willebrand factor (VWF). This phase 1/3 study investigated the efficacy and safety of rVIII-SingleChain in the treatment of bleeding episodes, routine prophylaxis, and surgical prophylaxis. Participants were ≥12 years of age, with severe hemophilia A (endogenous FVIII <1%). The participants were allocated by the investigator to receive rVIII-SingleChain in either an on-demand or prophylaxis regimen. Of the 175 patients meeting study eligibility criteria, 173 were treated with rVIII-SingleChain, prophylactically (N = 146) or on-demand (N = 27). The total cumulative exposure was 14 306 exposure days (EDs), with 120 participants reaching ≥50 EDs and 52 participants having ≥100 EDs. Hemostatic efficacy was rated by the investigator as excellent or good in 93.8% of the 835 bleeds treated and assessed. Across all prophylaxis regimens, the median annualized spontaneous bleeding rate was 0.00 (Q1, Q3: 0.0, 2.4) and the median overall annualized bleeding rate (ABR) was 1.14 (Q1, Q3: 0.0, 4.2). Surgical hemostasis was rated as excellent/good in 100% of major surgeries by the investigator. No participant developed FVIII inhibitors. In conclusion, rVIII-SingleChain is a novel rFVIII molecule showing excellent hemostatic efficacy in surgery and in the control of bleeding events, low ABR in patients on prophylaxis, and a favorable safety profile in this large clinical study. This trial was registered at www.clinicaltrials.gov as #NCT01486927. PMID:27330001

  19. Efficacy and safety of rVIII-SingleChain: results of a phase 1/3 multicenter clinical trial in severe hemophilia A.

    PubMed

    Mahlangu, Johnny; Kuliczkowski, Kazimierz; Karim, Faraizah Abdul; Stasyshyn, Oleksandra; Kosinova, Marina V; Lepatan, Lynda Mae; Skotnicki, Aleksander; Boggio, Lisa N; Klamroth, Robert; Oldenburg, Johannes; Hellmann, Andrzej; Santagostino, Elena; Baker, Ross I; Fischer, Kathelijn; Gill, Joan C; P'Ng, Stephanie; Chowdary, Pratima; Escobar, Miguel A; Khayat, Claudia Djambas; Rusen, Luminita; Bensen-Kennedy, Debra; Blackman, Nicole; Limsakun, Tharin; Veldman, Alex; St Ledger, Katie; Pabinger, Ingrid

    2016-08-04

    Recombinant VIII (rVIII)-SingleChain is a novel B-domain-truncated recombinant factor VIII (rFVIII), comprised of covalently bonded factor VIII (FVIII) heavy and light chains. It was designed to have a higher binding affinity for von Willebrand factor (VWF). This phase 1/3 study investigated the efficacy and safety of rVIII-SingleChain in the treatment of bleeding episodes, routine prophylaxis, and surgical prophylaxis. Participants were ≥12 years of age, with severe hemophilia A (endogenous FVIII <1%). The participants were allocated by the investigator to receive rVIII-SingleChain in either an on-demand or prophylaxis regimen. Of the 175 patients meeting study eligibility criteria, 173 were treated with rVIII-SingleChain, prophylactically (N = 146) or on-demand (N = 27). The total cumulative exposure was 14 306 exposure days (EDs), with 120 participants reaching ≥50 EDs and 52 participants having ≥100 EDs. Hemostatic efficacy was rated by the investigator as excellent or good in 93.8% of the 835 bleeds treated and assessed. Across all prophylaxis regimens, the median annualized spontaneous bleeding rate was 0.00 (Q1, Q3: 0.0, 2.4) and the median overall annualized bleeding rate (ABR) was 1.14 (Q1, Q3: 0.0, 4.2). Surgical hemostasis was rated as excellent/good in 100% of major surgeries by the investigator. No participant developed FVIII inhibitors. In conclusion, rVIII-SingleChain is a novel rFVIII molecule showing excellent hemostatic efficacy in surgery and in the control of bleeding events, low ABR in patients on prophylaxis, and a favorable safety profile in this large clinical study. This trial was registered at www.clinicaltrials.gov as #NCT01486927.

  20. Acquired factor VIII deficiency after consuming the dried gallbladder of a cobra, Naja naja

    PubMed Central

    Kim, Hyun Ju; Lee, Won Sik; Lee, Young Jin; Jun, Hyun Soo; Seo, Su-Kil

    2010-01-01

    Acquired factor VIII deficiency is very rare, often fatal. It is associated with pregnancy, autoimmune diseases, malignancy, and drugs, although no underlying cause is found in 50%. A 49-year-old male was referred with right shoulder bruising. The coagulation test showed a prolonged activated partial thromboplastin time. The factor VIII level was less than 1%, and the factor VIII inhibitor antibody titer was 246 Bethesda units/mL. The findings were compatible with acquired factor VIII deficiency. He had consumed the dried gallbladder of a cobra, Naja naja, for two weeks, it contained venom. After the initial treatment with factor VIII, he did not take supplemental coagulation factor VIII. The patient was readmitted with left forearm swelling. He lost consciousness suddenly and brain computed tomography (CT) revealed a subdural hematoma. Despite administering recombinant factor VII, his bleeding was not controlled and he died. PMID:21120211

  1. Comparison of human coagulation factor VIII expression directed by cytomegalovirus and mammary gland-specific promoters in HC11 cells and transgenic mice.

    PubMed

    Wang, Qing; Hao, Siguo; Ma, Liyuan; Zhang, Wenhao; Wan, Jiangbo; Deng, Xiaohui

    2015-10-01

    Hemophilia A is an inherited X-linked recessive bleeding disorder caused by coagulant factor VIII (FVIII) deficiency. The conventional treatment involves the administration of recombinant human FVIII (rhFVIII) preparations. In this study, the mammary gland 'bioreactor' is designed to specifically and efficiently express a foreign protein hFVIII in the mammary glands of transgenic mice. We constructed a P1A3-hFVIIIBD vector directed by the mammary gland-specific P1A3 promoter, and transiently transfected HC11 cells and mouse mammary glands with P1A3-hFVIIIBD or CMV-hFVIIIBD vectors directed by a ubiquitous cytomegalovirus (CMV) promoter, respectively. We also generated P1A3-hFVIIIBD and CMV-hFVIIIBD transgenic mice by microinjection, respectively. Our data indicated that both vectors effectively expressed hFVIIIBD in HC11 cells at the transcription level, and hFVIIIBD protein was efficiently expressed in mouse milk after the injection of the hFVIIIBD vectors into mouse mammary glands during lactation. In both CMV-hFVIIIBD and P1A3-hFVIIIBD transgenic mice, hFVIIIBD proteins were efficiently expressed in the mammary glands at the mRNA and protein levels. No significant difference was observed in hFVIIIBD levels between the CMV-hFVIIIBD and P1A3-hFVIIIBD transgenic mice (P > 0.05). However, the activity of hFVIII in CMV-directed transgenic mice was slightly higher than that in P1A3-directed transgenic mice (P < 0.05). While hFVIIIBD was present in multiple organs in CMV-hFVIIIBD mice, P1A3-hFVIIIBD mice showed negligible hFVIIIBD expression in organs other than the mammary glands. This study demonstrated that the mammary gland-specific P1A3-hFVIIIBD vector was more suitable for the generation of hFVIIIBD mammary gland bioreactor.

  2. Deep sequencing reveals different compositions of mRNA transcribed from the F8 gene in a panel of FVIII-producing CHO cell lines.

    PubMed

    Kaas, Christian S; Bolt, Gert; Hansen, Jens J; Andersen, Mikael R; Kristensen, Claus

    2015-07-01

    Coagulation factor VIII (FVIII) is one of the most complex biopharmaceuticals due to the large size, poor protein stability and extensive post-translational modifications. As a consequence, efficient production of FVIII in mammalian cells poses a major challenge, with typical yields two to three orders of magnitude lower than for antibodies. In the present study we investigated CHO DXB11 cells transfected with a plasmid encoding human coagulation factor VIII. Single cell clones were isolated from the pool of transfectants and a panel of 14 clones representing a dynamic range of FVIII productivities was selected for RNA sequencing analysis. The analysis showed distinct differences in F8 RNA composition between the clones. The exogenous F8-dhfr transcript was found to make up the most abundant transcript in the present clones. No correlation was seen between F8 mRNA levels and the measured FVIII productivity. It was found that three MTX resistant, nonproducing clones had different truncations of the F8 transcripts. We find that by using deep sequencing, in contrast to microarray technology, for determining the transcriptome from CHO transfectants, we are able to accurately deduce the mature mRNA composition of the transgene and identify significant truncations that would probably otherwise have remained undetected.

  3. von Willebrand factor binds to the surface of dendritic cells and modulates peptide presentation of factor VIII

    PubMed Central

    Sorvillo, Nicoletta; Hartholt, Robin B.; Bloem, Esther; Sedek, Magdalena; Brinke, Anja ten; van der Zwaan, Carmen; van Alphen, Floris P.; Meijer, Alexander B.; Voorberg, Jan

    2016-01-01

    It has been proposed that von Willebrand factor might affect factor VIII immunogenicity by reducing factor VIII uptake by antigen presenting cells. Here we investigate the interaction of recombinant von Willebrand factor with immature monocyte-derived dendritic cells using flow cytometry and confocal microscopy. Surprisingly, von Willebrand factor was not internalized by immature dendritic cells, but remained bound to the cell surface. As von Willebrand factor reduces the uptake of factor VIII, we investigated the repertoire of factor VIII presented peptides when in complex with von Willebrand factor. Interestingly, factor VIII-derived peptides were still abundantly presented on major histocompatibility complex class II molecules, even though a reduction of factor VIII uptake by immature dendritic cells was observed. Inspection of peptide profiles from 5 different donors showed that different core factor VIII peptide sequences were presented upon incubation with factor VIII/von Willebrand factor complex when compared to factor VIII alone. No von Willebrand factor peptides were detected when immature dendritic cells were pulsed with different concentrations of von Willebrand factor, confirming lack of von Willebrand factor endocytosis. Several von Willebrand factor derived peptides were recovered when cells were pulsed with von Willebrand factor/factor VIII complex, suggesting that factor VIII promotes endocytosis of small amounts of von Willebrand factor by immature dendritic cells. Taken together, our results establish that von Willebrand factor is poorly internalized by immature dendritic cells. We also show that von Willebrand factor modulates the internalization and presentation of factor VIII-derived peptides on major histocompatibility complex class II. PMID:26635035

  4. The C1 and C2 domains of blood coagulation factor VIII mediate its endocytosis by dendritic cells

    PubMed Central

    Gangadharan, Bagirath; Ing, Mathieu; Delignat, Sandrine; Peyron, Ivan; Teyssandier, Maud; Kaveri, Srinivas V.; Lacroix-Desmazes, Sébastien

    2017-01-01

    The development of inhibitory antibodies to therapeutic factor VIII is the major complication of replacement therapy in patients with hemophilia A. The first step in the initiation of the anti-factor VIII immune response is factor VIII interaction with receptor(s) on antigen-presenting cells, followed by endocytosis and presentation to naïve CD4+ T cells. Recent studies indicate a role for the C1 domain in factor VIII uptake. We investigated whether charged residues in the C2 domain participate in immunogenic factor VIII uptake. Co-incubation of factor VIII with BO2C11, a monoclonal C2-specific immunoglobulin G, reduced factor VIII endocytosis by dendritic cells and presentation to CD4+ T cells, and diminished factor VIII immunogenicity in factor VIII-deficient mice. The mutation of basic residues within the BO2C11 epitope of C2 replicated reduced in vitro immunogenic uptake, but failed to prevent factor VIII immunogenicity in mice. BO2C11 prevents factor VIII binding to von Willebrand factor, thus potentially biasing factor VIII immunogenicity by perturbing its half-life. Interestingly, a factor VIIIY1680C mutant, that does not bind von Willebrand factor, demonstrated unaltered endocytosis by dendritic cells as well as immunogenicity in factor VIII-deficient mice. Co-incubation of factor VIIIY1680C with BO2C11, however, resulted in decreased factor VIII immunogenicity in vivo. In addition, a previously described triple C1 mutant showed decreased uptake in vitro, and reduced immunogenicity in vivo, but only in the absence of endogenous von Willebrand factor. Taken together, the results indicate that residues in the C1 and/or C2 domains of factor VIII are implicated in immunogenic factor VIII uptake, at least in vitro. Conversely, in vivo, the binding to endogenous von Willebrand factor masks the reducing effect of mutations in the C domains on factor VIII immunogenicity. PMID:27758819

  5. Prevalence of IVS10nt-18G/A in Calabrian patients with moderate/mild hemophilia A and relation with Factor VIII inhibitor antibodies.

    PubMed

    Prejanò, Simona; Santoro, Rita C; Iannaccaro, Piergiorgio

    2015-10-01

    Hemophilia A is an X-linked bleeding disorder caused by widespread mutations in the factor VIII gene. In the course of a screening to research some hemophilia A mutations, our team has identified and posted a previously unreported nucleotide change in intron 10 in 20 patients with hemophilia A. We tried to identify a possible blood relationship between the people with this mutation, performing a backwards study of every family tree. First, we interviewed the patients and, if possible, parents and grandparents. When direct memory was no longer available, we consulted Registries of Births, Marriages and Deaths, and if these data were not sufficient, going backwards in time, we consulted registries of parish churches where newborns were baptized. The studied mutation was present in 33 hemophilic patients living in Calabria, 28 of them related. Three patients, carriers of this mutation, developed an FVIII inhibitor. In all the cases, the inhibitor development followed intensive treatments, after many days of exposure. Our study displayed the presence of a responsible moderate hemophilia A mutation, limited apparently to our country, probably because of a single ancestral event, and connected with FVIII inhibitor development.

  6. Definition of the affinity of binding between human von Willebrand factor and coagulation factor VIII.

    PubMed

    Ganz, P R; Atkins, J S; Palmer, D S; Dudani, A K; Hashemi, S; Luison, F

    1991-10-15

    Factor VIII and von Willebrand factor are two plasma proteins essential for effective hemostasis. In vivo, they form a non-covalent complex whose association appears to be metal ion dependent. However, a precise definition of the nature of the molecular forces governing their association remains to be defined, as does their binding affinity. In this paper we have determined the dissociation constant and stoichiometry for Factor VIII binding to immobilized von Willebrand factor. The data demonstrate that these proteins interact saturably and with relatively high affinity. Computer assisted analyses of the Scatchard data favour a two site binding model. The higher affinity site was found to have a Kd of 62 (+/- 13) x 10(-12) M while that of the lower affinity site was 380 (+/- 92) x 10(-12) M. The density of Factor VIII binding sites (Bmax) present on von Willebrand factor was 31 (+/- 3) pM for the high affinity binding site and 46 (+/- 6) pM for the lower site, corresponding to a calculated Factor VIII: von Willebrand factor binding ratio of 1:33 and 1:23, respectively.

  7. Abnormal factor VIII coagulant antigen in patients with renal dysfunction and in those with disseminated intravascular coagulation.

    PubMed Central

    Weinstein, M J; Chute, L E; Schmitt, G W; Hamburger, R H; Bauer, K A; Troll, J H; Janson, P; Deykin, D

    1985-01-01

    Factor VIII antigen (VIII:CAg) exhibits molecular weight heterogeneity in normal plasma. We have compared the relative quantities of VIII:CAg forms present in normal individuals (n = 22) with VIII:CAg forms in renal dysfunction patients (n = 19) and in patients with disseminated intravascular coagulation (DIC; n = 7). In normal plasma, the predominant VIII: CAg form, detectable by sodium dodecyl sulfate polyacrylamide gel electrophoresis, was of molecular weight 2.4 X 10(5), with minor forms ranging from 8 X 10(4) to 2.6 X 10(5) D. A high proportion of VIII:CAg in renal dysfunction patients, in contrast, was of 1 X 10(5) mol wt. The patients' high 1 X 10(5) mol wt VIII: CAg level correlated with increased concentrations of serum creatinine, F1+2 (a polypeptide released upon prothrombin activation), and with von Willebrand factor. Despite the high proportion of the 1 X 10(5) mol wt VIII:CAg form, which suggests VIII:CAg proteolysis, the ratio of Factor VIII coagulant activity to total VIII:CAg concentration was normal in renal dysfunction patients. These results could be simulated in vitro by thrombin treatment of normal plasma, which yielded similar VIII:CAg gel patterns and Factor VIII coagulant activity to antigen ratios. DIC patients with high F1+2 levels but no evidence of renal dysfunction had an VIII:CAg gel pattern distinct from renal dysfunction patients. DIC patients had elevated concentrations of both the 1 X 10(5) and 8 X 10(4) mol wt VIII:CAg forms. We conclude that an increase in a particular VIII:CAg form correlates with the severity of renal dysfunction. The antigen abnormality may be the result of VIII:CAg proteolysis by a thrombinlike enzyme and/or prolonged retention of proteolyzed VIII:CAg fragments. Images PMID:3932466

  8. Successful Pregnancy in a Patient with Combined Deficiency of Factor V and Factor VIII.

    PubMed

    El Adib, Ahmed Ghassan; Majdi, Farah; Dilai, Mohamed Othmane; Asmouki, Hamid; Bassir, Ahlam; Harou, Karam; Soumani, Abderraouf; Younous, Said; Mahmal, Lahoucine

    2014-01-01

    Inherited combined factor V and factor VIII deficiency (F5F8D) is autosomal recessive transmission disorder. Epistaxis, postsurgical bleeding, and menorrhagia are the most common symptoms. The risk of miscarriage and placental abruption is consequent. We report a case of successful pregnancy in a patient with F5F8D. 20-year-old woman, born of consanguineous parents, third gestate, first parity, two miscarriages, admitted for child birth of a spontaneous pregnancy estimated at 38 weeks and was diagnosed with F5F8D. At admission, patient was hemodynamically stable, with good obstetric conditions. The biologic results showed low levels of PT (52%), factor V (7%), and factor VIII (5%), and the activated partial thromboplastin time was prolonged (68,6%). Parturient was admitted in intensive care unit, maternal and fetal monitoring was performed. Fresh frozen plasma (FFP) and factor VIII concentrates were perfused at the induction of labor. Analgesia used fentanyl titration. The delivery gave birth to a newborn male, with Apgar 10/10 and 3000 g. The puerperium was simple without any important bleeding. Laboratory tests for the newborn were acceptable. Little literature is available on this subject and there are no guidelines available concerning pregnancy; we chose to prescribe a combination of factor VIII concentrate and FFP in pre-, per- and postpartum. The same protocol was successfully used in a patient before dental extraction and prostatectomy. Vaginal delivery is possible, as our case. Management by multidisciplinary team is recommended.

  9. Crystal Structure of Human Factor VIII: Implications for the Formation of the Factor IXa-Factor VIIIa Complex

    SciTech Connect

    Ngo, J.C.; Huang, M.; Roth, D.A.; Furie, B.C.; Furie, B.

    2008-06-03

    Factor VIII is a procofactor that plays a critical role in blood coagulation, and is missing or defective in hemophilia A. We determined the X-ray crystal structure of B domain-deleted human factor VIII. This protein is composed of five globular domains and contains one Ca{sup 2+} and two Cu{sup 2+} ions. The three homologous A domains form a triangular heterotrimer where the A1 and A3 domains serve as the base and interact with the C2 and C1 domains, respectively. The structurally homologous C1 and C2 domains reveal membrane binding features. Based on biochemical studies, a model of the factor IXa-factor VIIIa complex was constructed by in silico docking. Factor IXa wraps across the side of factor VIII, and an extended interface spans the factor VIII heavy and light chains. This model provides insight into the activation of factor VIII and the interaction of factor VIIIa with factor IXa on the membrane surface.

  10. Crystal Structure of Human Factor VIII: Implications for the Formation of the Factor IXa-Factor VIIIa Complex

    SciTech Connect

    Chi Ki Ngo,J.; Huang, M.; Roth, D.; Furie, B.; Furie, B.

    2008-01-01

    Factor VIII is a procofactor that plays a critical role in blood coagulation, and is missing or defective in hemophilia A. We determined the X-ray crystal structure of B domain-deleted human factor VIII. This protein is composed of five globular domains and contains one Ca(2+) and two Cu(2+) ions. The three homologous A domains form a triangular heterotrimer where the A1 and A3 domains serve as the base and interact with the C2 and C1 domains, respectively. The structurally homologous C1 and C2 domains reveal membrane binding features. Based on biochemical studies, a model of the factor IXa-factor VIIIa complex was constructed by in silico docking. Factor IXa wraps across the side of factor VIII, and an extended interface spans the factor VIII heavy and light chains. This model provides insight into the activation of factor VIII and the interaction of factor VIIIa with factor IXa on the membrane surface.

  11. Synthesis of FVIII in Hemophilia-A patients with the intron-22-inversion may modulate immunogenicity

    PubMed Central

    Pandey, Gouri Shankar; Yanover, Chen; Miller-Jenkins, Lisa M.; Garfield, Susan; Cole, Shelley A.; Curran, Joanne E.; Moses, Eric K.; Rydz, Natalia; Simhadri, Vijaya; Kimchi-Sarfaty, Chava; Lillicrap, David; Viel, Kevin; Przytycka, Teresa M.; Pierce, Glenn F.; Howard, Tom E.; Sauna, Zuben E.

    2013-01-01

    Neutralizing antibodies (inhibitors) to replacement Factor-VIII impair the effective management of hemophilia-A1. Individuals with hemophilia-A due to major F8 gene disruptions lack antigenically cross-reactive material in their plasma (CRM-negative) and prevalence of inhibitors is >60%. Conversely, subjects with missense mutations are CRM-positive and the prevalence of inhibitors is <10%2. Individuals with the intron-22-inversion (~50% of individuals with severe hemophilia-A) should be in the former group based on the genetic defect. Although these individuals are CRM-negative, only 20% of them develop inhibitors3. Here we demonstrate the presence of comparable levels of F8 mRNA and intracellular Factor-VIII protein in B-lymphoblastoid cells and liver biopsies from healthy controls and subjects with the intron-22-inversion. These results support the hypothesis that most individuals with the intron-22-inversion are tolerized to Factor-VIII and thus do not develop inhibitors. Furthermore we developed a pharmacogenetic algorithm that permits the stratification of inhibitor risk for sub-populations by predicting immunogenicity using, as input, the number of putative T-cell epitopes in the infused FVIII and the competence of MHC-Class-II molecules to present such epitopes. The algorithm exhibited significant accuracy in predicting inhibitors in 25 unrelated individuals with the intron-22-inversion (AUC = 0.890; P = 0.001). PMID:24037092

  12. Protected by nature? Effects of strenuous physical exercise on FVIII activity in moderate and mild haemophilia A patients: a pilot study.

    PubMed

    Groen, W G; den Uijl, I E M; van der Net, J; Grobbee, D E; de Groot, Ph G; Fischer, K

    2013-07-01

    Increase of factor VIII activity (FVIII) after physical exercise has been reported in healthy subjects and small-scale studies in patients with coagulopathies. The aim was to study whether moderate and mild haemophilia A patients are able to increase their endogenous FVIII activity levels by physical activity. We studied changes in FVIII activity levels after high-intensity exercise in 15 haemophilia A patients, 20-39 years, eight with moderate, seven with mild haemophilia. Patients cycled until volitional exhaustion, blood samples were drawn before and 10 min after the exercise test. FVIII activity increased 2.5 times (range 1.8-7.0 times), for both severities. Absolute increases were markedly different: median 7 IU dL(-1) (range 3-9 IU dL(-1) ) in patients with moderate, compared to 15 IU dL(-1) (range 6-62 IU dL(-1) ) in mild haemophilia patients. VWF and VWFpp increased independently of severity; median 50% (range 8-123%) and median 165% (range 48-350%), respectively, reflecting acute release of VWF. These observations may be used to promote high-intensity activities before participating in sports for moderate and mild haemophilia A patients, to reduce bleeding risk. Further studies are warranted to fully appreciate the clinical significance of exercise on different levels of intensity in patients with mild and moderate haemophilia A.

  13. Importance of a quality assurance scheme for factor VIII assays in quality monitoring of human plasma destined for fractionation into factor VIII concentrate.

    PubMed

    Gabra, G S; Prowse, C V; Boulton, F E

    1989-01-01

    A national quality assurance scheme has been established to monitor the validity of factor VIII assays performed by the various laboratories of the Scottish National Blood Transfusion Service engaged in collection and processing of donor plasma destined for fractionation. The results over the first 3-year period show that comparable assay values can be obtained by participating centres using a common standard, despite differences in equipment, methods or substrate chosen for the one-stage assay. The results also showed that chromogenic factor VIII assays correlated well with the one-stage method. Random factor VIII assays performed on plasma, harvested and frozen within 18 h from collection, were analysed to validate recently proposed Scottish specifications which stipulate that 70% of plasma donations destined for fractionation should contain at least 0.7 IU/ml. Plasma harvested and frozen between 8 and 18 h from collection did not meet the specified level in any of the regional centres. This nationally specified level was also not met by plasma harvested and frozen within 8 h from collection in spite of being achieved individually by three regional centres. Assays performed on large plasma pools at the Fractionation Centre suggested loss of some factor VIII during storage, transportation and thawing of plasma prior to bulk processing.

  14. Enhanced glycosylation of factor VIII:C in capillary endothelial cells following. beta. -adrenoreceptor stimulation is not due to increased sugar transport

    SciTech Connect

    Tavarez-Pagan, J.J.; Oliveira, C.M.; Banerjee, D.K. )

    1990-02-26

    Factor VIII:C (Antihemophilia Factor A) in capillary endothelial cells from bovine adrenal medulla found to contain M{sub r} 200,000 and M{sub r} 46,000 dalton protein species when analyzed by SDS-PAGE under reduced condition. But the secretory FVIII:C under non-reduced condition gave rise to a protein of M{sub r} 270,000 dalton indicating that the heavy and light chains are held together by S-S bridge. Upon stimulation of these cells with {beta}-agonist, isoproterenol (10{sup {minus}7} M), a 2-fold increase in the ratio of ({sup 3}H)-mannose to ({sup 35}S)-methionine incorporation in immunoprecipitated FVIII:C was observed. In order to understand the molecular mechanism of this increase, a detail study of sugar transport was carried out. The transport of 2-deoxy-D-({sup 3}H)-glucose in these cells was time and temperature dependent. Furthermore, inhibition of 2-deoxy-D-({sup 3}H)-glucose uptake by cytochalasin B strongly supported for a carrier mediated process. However, when the transport studies were conducted in the presence of isoproterenol at 10{sup {minus}5} M or 10{sup {minus}7} M or 8 Br-cAMP (2 mM) no increase was observed. The kinetic studies indicated that the K{sub m} and V{sub max} for 2 deoxy-D-({sup 3}H)-glucose were 0.24 mM and 0.88 nmol/mg protein/minute, respectively under normal conditions but these values were changed to 0.37 mM and 0.69 nmol/mg protein/minute when stimulated with isoproterenol (10{sup {minus}7}M).

  15. Lower inhibitor development in hemophilia A mice following administration of recombinant factor VIII-O-phospho-L-serine complex.

    PubMed

    Purohit, Vivek S; Ramani, Karthik; Sarkar, Rita; Kazazian, Haig H; Balasubramanian, Sathyamangalam V

    2005-05-06

    Factor VIII is a multidomain protein composed of A1, A2, B, A3, C1, and C2 domains. Deficiency or dysfunction of factor VIII causes hemophilia A, a bleeding disorder. Administration of exogenous recombinant factor VIII as a replacement leads to development of inhibitory antibodies against factor VIII in 15-30% of hemophilia A patients. Hence, less immunogenic preparations of factor VIII are highly desirable. Inhibitory antibodies against factor VIII are mainly directed against immunodominant epitopes in C2, A3, and A2 domains. Further, several universal epitopes for CD4+ T-cells have been identified within the C2 domain. The C2 domain is also known to interact specifically with phosphatidylserine-rich lipid vesicles. Here, we have investigated the hypothesis that complexation of O-phospho-l-serine, the head group of phosphatidylserine, with the C2 domain can reduce the overall immunogenicity of factor VIII. The biophysical (circular dichroism and fluorescence) and biochemical studies (ELISA and size exclusion chromatography) showed that O-phospho-l-serine binds to the phospholipid-binding region in the C2 domain, and this interaction causes subtle changes in the tertiary structure of the protein. O-Phospho-l-serine also prevented aggregation of the protein under thermal stress. The immunogenicity of the factor VIII-O-phospho-l-serine complex was evaluated in hemophilia A mice. The total and inhibitory antibody titers were lower for factor VIII-O-phospho-l-serine complex compared with factor VIII alone. Moreover, factor VIII administered as a complex with O-phospho-l-serine retained in vivo activity in hemophilia A mice. Our results suggest that factor VIII-O-phospho-l-serine complex may be beneficial to increase the physical stability and reduce immunogenicity of recombinant factor VIII preparations.

  16. Extrahepatic sources of factor VIII potentially contribute to the coagulation cascade correcting the bleeding phenotype of mice with hemophilia A.

    PubMed

    Zanolini, Diego; Merlin, Simone; Feola, Maria; Ranaldo, Gabriella; Amoruso, Angela; Gaidano, Gianluca; Zaffaroni, Mauro; Ferrero, Alessandro; Brunelleschi, Sandra; Valente, Guido; Gupta, Sanjeev; Prat, Maria; Follenzi, Antonia

    2015-07-01

    A large fraction of factor VIII in blood originates from liver sinusoidal endothelial cells although extrahepatic sources also contribute to plasma factor VIII levels. Identification of cell-types other than endothelial cells with the capacity to synthesize and release factor VIII will be helpful for therapeutic approaches in hemophilia A. Recent cell therapy and bone marrow transplantation studies indicated that Küpffer cells, monocytes and mesenchymal stromal cells could synthesize factor VIII in sufficient amount to ameliorate the bleeding phenotype in hemophilic mice. To further establish the role of blood cells in expressing factor VIII, we studied various types of mouse and human hematopoietic cells. We identified factor VIII in cells isolated from peripheral and cord blood, as well as bone marrow. Co-staining for cell type-specific markers verified that factor VIII was expressed in monocytes, macrophages and megakaryocytes. We additionally verified that factor VIII was expressed in liver sinusoidal endothelial cells and endothelial cells elsewhere, e.g., in the spleen, lungs and kidneys. Factor VIII was well expressed in sinusoidal endothelial cells and Küpffer cells isolated from human liver, whereas by comparison isolated human hepatocytes expressed factor VIII at very low levels. After transplantation of CD34(+) human cord blood cells into NOD/SCIDγNull-hemophilia A mice, fluorescence activated cell sorting of peripheral blood showed >40% donor cells engrafted in the majority of mice. In these animals, plasma factor VIII activity 12 weeks after cell transplantation was up to 5% and nine of 12 mice survived after a tail clip-assay. In conclusion, hematopoietic cells, in addition to endothelial cells, express and secrete factor VIII: this information should offer further opportunities for understanding mechanisms of factor VIII synthesis and replenishment.

  17. Influence of factor VIII level and its inhibitor titer on the therapeutic response to corticosteroids alone in the management of acquired hemophilia

    PubMed Central

    Vautier, Mathieu; de Boysson, Hubert; Creveuil, Christian; Repesse, Yohan; Borel-Derlon, Annie; Troussard, Xavier; Damaj, Gandhi L.; Bienvenu, Boris; Gautier, Philippe; Aouba, Achille

    2016-01-01

    Abstract The treatment of acquired hemophilia (AH) involves discussing whether corticosteroids should be administered alone or combined with immunosuppressant drugs, which increase the risk of infection especially in elderly patients and/or those with autoimmunity or neoplastic diseases, who represent the target population of the disease. Prognostic factors highlighting adequate responses to corticosteroids alone must be identified for satisfactory clinical response and lower infectious risk. We aimed to evaluating the efficacy of corticosteroids alone in the management of AH depending on factor VIII (FVIII, ≥ or <1 IU/dL) levels and/or inhibitor (INH, ≤ or >20 Bethesda units per milliliter [BU/mL]) titer. We conducted a retrospective single-center study including 24 patients treated for AH with corticosteroids alone. Time to achieve partial remission (PR: absence of hemorrhage and FVIII levels >50 IU/dL) was significantly shorter in the FVIII ≥ 1 IU/dL group than in the FVIII < 1 IU/dL group (20 [10–55] vs 39 [20–207] days, P = 0.044) and in the INH ≤ 20 BU/mL and FVIII ≥ 1 IU/dL group than in the FVIII < 1 IU/dL and/or INH > 20 BU/mL group (15 [11–35] vs 41 [20–207] days, P = 0.003). In both subgroups, time to achieve complete remission (CR: negative INH and corticosteroids below 10 mg/d) was also significantly shorter than that observed in the opposite subgroups. INH titer, considered alone, did not affect the length of time to onset of PR or CR. CR and PR rates did not differ significantly depending on these variables. Our study suggests that in AH, patients with FVIII levels ≥1 IU/dL considered alone or combined with INH titer ≤20 BU/mL could be treated by corticosteroids alone, given that this subgroup of patients displayed faster therapeutic responses to this strategy. PMID:27902587

  18. Switching clotting factor concentrates: considerations in estimating the risk of immunogenicity.

    PubMed

    Matino, D; Lillicrap, D; Astermark, J; Dolan, G; Kessler, C; Lambert, T; Makris, M; O'Donnell, J; Pipe, S; Santagostino, E; Saint-Remy, J-M; Schramm, W; Iorio, A

    2014-03-01

    The development of neutralizing antibodies to factor VIII (FVIII) is the most serious complication of therapy for haemophilia A. There is now excellent documentation that a large number of both genetic and environmental factors contribute to the risk of FVIII inhibitor incidence. One of the environmental factors that has been proposed as an influence on this complication is the occurrence of FVIII product switching. There are only a small number of clinical studies that have addressed this question, and thus, the amount of objective information available to assess this association is limited. In this review, in addition to summarizing past evidence pertinent to this subject, we present the results of a complementary strategy, a Delphi analysis, to add to the considerations of product switching and FVIII immunogenicity. With the imminent arrival in the clinic of several new FVIII products, the haemophilia community must be prepared to collect prospectively controlled data to better address this important management issue.

  19. Matching-adjusted indirect comparisons of efficacy of BAY 81-8973 vs two recombinant factor VIII for the prophylactic treatment of severe hemophilia A

    PubMed Central

    Pocoski, Jennifer; Li, Nanxin; Ayyagari, Rajeev; Church, Nikki; Maas Enriquez, Monika; Xiang, Quer; Kelkar, Sneha; Du, Ella X; Wu, Eric Q; Xie, Jipan

    2016-01-01

    Background No head-to-head trials comparing recombinant factor VIII (rFVIII) products currently exist. This was a matching-adjusted indirect comparison (MAIC) study of efficacy of BAY 81-8973 with antihemophilic factor (recombinant) plasma/albumin-free method (rAHF-PFM) and turoctocog alfa for the prophylaxis of severe hemophilia A. Methods A systematic literature review was conducted to identify trials of rAHF-PFM and turoctocog alfa. Comparisons were conducted using BAY 81-8973 individual patient data (IPD) from LEOPOLD trials and published data from rAHF-PFM and turoctocog alfa trials. Differences in outcome reporting were reconciled using transformation of BAY 81-8973 IPD. Patients in pooled LEOPOLD trials were weighted to match baseline characteristics for rAHF-PFM or turoctocog alfa trials using MAICs. After matching, annualized bleed rates (ABRs) were compared using weighted t-tests. Results Two rAHF-PFM trials and one turoctocog alfa trial were identified. In these trials, rFVIIIs were dosed thrice weekly or every other day; in LEOPOLD trials, BAY 81-8973 was dosed twice- or thrice weekly. Three MAICs were conducted because the two rAHF-PFM trials calculated ABRs differently, matching for age, race, and weight (turoctocog alfa only). BAY 81-8973 had similar ABR of all bleeds vs rAHF-PFM (two trials: 4.8 vs 6.3, 1.9 vs 1.8 [square root transform]) and lower ABR of spontaneous bleeds and trauma bleeds (2.6 vs 4.1, 2.1 vs 4.7; both P<0.05). BAY 81-8973 showed lower ABR of all bleeds and spontaneous bleeds vs turoctocog alfa (4.3 vs 6.5, 2.8 vs 4.3; both P<0.05) and similar ABR of trauma bleeds (1.5 vs 1.6). In subgroup analysis, twice-weekly BAY 81-8973 had similar ABRs of all bleeds, spontaneous bleeds, and trauma bleeds compared to rAHF-PFM and turoctocog alfa. Conclusion This indirect comparison found that prophylaxis with BAY 81-8973, even including the lower frequency of two times a week and lower factor VIII consumption, has efficacy comparable to r

  20. ADAMTS13 content and VWF multimer and triplet structure in commercially available VWF/FVIII concentrates.

    PubMed

    Kannicht, Christoph; Fisseau, Claudine; Hofmann, Werner; Kröning, Mario; Fuchs, Birte

    2015-03-01

    ADAMTS13 is a metalloproteinase that cleaves von Willebrand factor (VWF) into smaller multimers in vivo. This cleavage creates both the typical multimeric size distribution and the characteristic triplet band distribution of VWF. Here we analysed ADAMTS13 content, VWF multimeric size distribution and VWF triplet structure in five commercial VWF/factor VIII (FVIII) concentrates. The relative distribution of ADAMTS13 activity values corresponded well to the ADAMTS13 antigen values for all examined concentrates except Haemate HS®, which had markedly higher ADAMTS13 antigen/activity ratio, with Fanhdi® and Haemate HS® displaying the most intense ADAMTS13 signal. Interestingly, ADAMTS13 levels did not correlate with the high molecular weight multimer content of the concentrates, but did correlate with VWF triplet distribution. Densitometric quantification showed that Wilate®, Immunate® and Willfact® displayed human plasma-like VWF triplet distribution, whereas Fanhdi® and Haemate HS® showed enhanced content of the faster migrating triplet band, which corresponded well to their higher ADAMTS13 content. In summary, Immunate®, Willfact® and Wilate® had lower levels of ADAMTS13 antigen and activity and exhibited a plasma-like VWF triplet structure. Fanhdi® and Haemate HS® had higher ADAMTS13 content and an altered triplet structure. The possible impact of these observations on function and clinical efficacy of VWF/FVIII concentrates is discussed.

  1. Relation of factor VIII and IX inhibitors with ABO blood groups in 150 patients with haemophilia A and B.

    PubMed

    Torghabeh, Hassan Mansouri; Pourfathollah, Aliakbar; Shooshtari, Mahmood Mahmoodian; Yazdi, Zahra Rezaie

    2006-03-01

    Many investigations have proved relations between ABO blood groups with some diseases and factor VIII and von willebrand level in plasma. In this study we investigated a relation between ABO blood groups and factor VIII and IX inhibitors in 102 patients with haemophilia A and 48 patients with haemophilia B. The assay of inhibitor was done by Bethesda method. There were no relation between ABO blood groups and factor VIII and IX inhibitors.

  2. Nattokinase decreases plasma levels of fibrinogen, factor VII, and factor VIII in human subjects.

    PubMed

    Hsia, Chien-Hsun; Shen, Ming-Ching; Lin, Jen-Shiou; Wen, Yao-Ke; Hwang, Kai-Lin; Cham, Thau-Ming; Yang, Nae-Cherng

    2009-03-01

    Nattokinase, a serine proteinase from Bacillus subtilis, is considered to be one of the most active functional ingredients found in natto. In this study, we hypothesized that nattokinase could reduce certain factors of blood clotting and lipids that are associated with an increase risk for cardiovascular disease (CVD). Thus, an open-label, self-controlled clinical trial was conducted on subjects of the following groups: healthy volunteers (Healthy Group), patients with cardiovascular risk factors (Cardiovascular Group), and patients undergoing dialysis (Dialysis Group). All subjects ingested 2 capsules of nattokinase (2000 fibrinolysis units per capsule) daily orally for 2 months. The laboratory measurements were performed on the screening visit and, subsequently, regularly after the initiation of the study. The intent-to-treat analysis was performed on all 45 enrolled subjects. By use of mixed model analysis, a significant time effect, but not group effect, was observed in the change from baseline of fibrinogen (P = .003), factor VII (P < .001), and factor VIII (P < .001), suggesting that the plasma levels of the 3 coagulation factors continuously declined during intake; also, the extents of decrease were similar between groups. After 2 months of administration, fibrinogen, factor VII, and factor VIII decreased 9%, 14%, and 17%, respectively, for the Healthy Group; 7%, 13%, and 19%, respectively, for the Cardiovascular Group; and 10%, 7%, and 19%, respectively, for the Dialysis Group, whereas blood lipids were unaffected by nattokinase. No significant changes of uric acid or notable adverse events were observed in any of the subjects. In summary, this study showed that oral administration of nattokinase could be considered as a CVD nutraceutical by decreasing plasma levels of fibrinogen, factor VII, and factor VIII.

  3. Comparison of Clot-based, Chromogenic, and Fluorescence Assays for Measurement of Factor VIII Inhibitors in the U.S. Hemophilia Inhibitor Research Study

    PubMed Central

    Miller, Connie H.; Rice, Anne S.; Boylan, Brian; Shapiro, Amy D.; Lentz, Steven R.; Wicklund, Brian M.; Kelly, Fiona M.; Soucie, J. Michael

    2015-01-01

    Summary Background Detection and validation of inhibitors (antibodies) to hemophilia treatment products are important for clinical care, evaluation of product safety, and assessment of population trends. Methods Centralized monitoring for factor VIII (FVIII) inhibitors was conducted for patients in the Hemophilia Inhibitor Research Study using a previously reported modified Nijmegen-Bethesda clotting assay (NBA), a chromogenic Bethesda assay (CBA), and a novel fluorescence immunoassay (FLI). Results NBA and CBA were performed on 1005 specimens and FLI on 272 specimens. CBA was negative on 880/883 specimens (99.7%) with Nijmegen-Bethesda units (NBU)<0.5 and positive on 42/42 specimens (100%) with NBU≥2.0 and 43/80 specimens (53.8%) with NBU 0.5–1.9. Among specimens with positive NBA and negative CBA, 58.1% were FLI-negative, 12.9% had evidence of lupus anticoagulant, and 35.5% had non-time-dependent inhibition. CBA and FLI were positive on 72.4% and 100% of 1.0–1.9 NBU specimens and 43.1% and 50.0% of 0.5–0.9 NBU specimens. FLI detected antibodies in 98.0% of CBA-positive and 81.6% of NBA-positive specimens (P=0.004). Among 21 new inhibitors detected by NBA, 5 (23.8%) with 0.7–1.3 NBU did not react in CBA or FLI. Among previously positive patients with 0.5–1.9 NBU, 7/25 (28%) were not CBA or FLI positive. FLI was positive on 36/169 NBU-negative specimens (21.3%). Conclusions FVIII specificity could not be demonstrated by CBA or FLI for 26% of inhibitors of 0.5–1.9 NBU; such results must be interpreted with caution. Low titer inhibitors detected in clot-based assays should always be repeated, with consideration given to evaluating their reactivity with FVIII using more specific assays. PMID:23601690

  4. Two distinct forms of Factor VIII coagulant protein in human plasma. Cleavage by thrombin, and differences in coagulant activity and association with von Willebrand factor.

    PubMed Central

    Weinstein, M J; Chute, L E

    1984-01-01

    We have characterized Factor VIII coagulant protein, present in normal human plasma, that reacts with a specific human 125I-labeled anti-human VIII:C antigen Fab antibody fragment. Two major Factor VIII coagulant antigen populations were present. The first, approximately 85% of the total antigen, was bound to von Willebrand factor and when tested in a standard one-stage assay had Factor VIII coagulant activity. The second antigenic population, eluting near fibrinogen when plasma was gel filtered, was not bound to von Willebrand protein, did not have Factor VIII coagulant activity unless activated, but did block anti-VIII:C Fab neutralization of clotting activity. The two antigenic populations were separable by cryoprecipitation and agarose gel electrophoresis. Although the two antigenic populations differed in their Factor VIII coagulant activity and in their binding to von Willebrand factor, the principal member of both populations is of mol wt 2.4 X 10(5). Both antigens, when proteolyzed by thrombin, were quickly converted to a 1 X 10(5)-mol wt form in association with the appearance of VIII:C activity. The 1 X 10(5)-mol wt antigen was further slowly degraded to an 8 X 10(4)-mol wt form while Factor VIII coagulant activity declined. These results demonstrate the presence of an inactive Factor VIII coagulant protein in plasma, not associated with von Willebrand factor, that can react with thrombin to yield Factor VIII coagulant activity. Images PMID:6421875

  5. The maintenance of tolerance after successful immune tolerance induction in hemophilia A and B: the North American Registry. Factor VIII/IX Subcommittee of the International Society for Thrombosis and Hemostasis.

    PubMed

    DiMichele, D; Kroner, B

    2000-10-01

    The North American Immune Tolerance Registry (NAITR) was initiated with the goal of determining, by questionnaire, immune tolerance (ITT) practices in hemophilia treatment centers in Canada and the United States. Sixty-eight centers (40%) responded. Of the 130 registry subjects with hemophilia A who completed ITT, 93 (72%) achieved tolerance. Of the 11 completed ITT courses in patients with hemophilia B, 4 (36%) were successful. Maintenance therapy was defined as any dotting factor regimen administered subsequent to the patient achieving the treating physician's criteria for successful immune tolerance. Seventy-five (81%) of 93 individuals in the hemophilia A cohort who successfully achieved tolerance were maintained on a regular (prophylactic factor VIII (FVIII) regimen for a variable time period post-ITT. The median dose used was 150 units/kg/week (range: 17-700). Forty-eight (64%) subjects remained tolerant while receiving regular doses of FVIII for a median observation period of 13 months (range 0-129 months). Of 27 patients whose maintenance therapy had been stopped, 17 (68%) remained tolerant over a median period of 19 months (range 1-54 months) and 9 relapsed. Among the relapses, 3 occurred after maintenance therapy was stopped; 6 were noted on prophylactic FVIII at a median time of 11 months (range 2-61 months). The definition of tolerance was reviewed for the 9 subjects who relapsed and was defined by a normal recovery and survival in only 1/9 patients. Among the 11 hemophilia B subjects in the cohort who completed tolerance, 4 had a successful outcome. Four individuals were placed on maintenance regimens of 25-100 units FIX/kg/day and all remained tolerant.

  6. Idiotypes of murine monoclonal antibodies to clotting factor VIII:C

    SciTech Connect

    Pechet, L.; Tiarks, C.Y.; Ghalili, K.; Humphreys, R.E.

    1986-03-05

    The authors goal is to study idiotypic immunoregulation of inhibitors to clotting factor VIII:C. To this end, they used monoclonal antibodies (MoAbs) against VIII:C: Synbiotics, C7F7, and C5, directed against epitopes on the C terminal fragment of VIII:C; C2, C6, C8 directed against epitopes on the N terminal fragment of VIII:C; C10, directed against a non-functional epitope; IB3, Chemicon and Hybritech, to undetermined epitopes. Anti-idiotypic antibodies against C7F7, C8, Synbiotics and Hybritech were produced in rabbits. Competitive radioimmunoassays (RIA) tested cross-reactivity between each immunogen and the other MoAbs. Synbiotics cross-reacted with Chemicon and IB3, indicating they were directed against the same epitope on the C terminal fragment of VIII:C. They did not cross-react with Hybritech, C7F7, C2, C5, C6, C8, or C10. C7F7 showed no cross-reactivities. C8 cross-reacted with C6 but not with C2, C5, C10, C7F7, Synbiotics, or Hybritech. Hybritech did not did not cross-react with any of the other MoAbs. In conclusion, with four anti-idiotypic antibodies and ten MoAbs to VIII:C, they defined at least five functional epitopes and one non-functional epitope on the factor VIII:C molecule to which inhibitors may develop: C2, C6-C8 (N terminal), C7F7, C5, Synbiotics (C terminal), Hybritech (undetermined epitope) and C10 (non-functional).

  7. Remarks on small sample size taken from reference population for examination of factor VIII.

    PubMed

    Sikorová, J; Zvárová, J

    1988-01-01

    Investigation of plasma factor VIII activities in 20 reference (normal) individuals (N = 20) is frequently used to evaluate the reference distribution parameters by means of simple statistical method. Two examples demonstrate the inaccuracy of conclusions of such a small sample of data. The first one shows the comparison of estimate of F.VIII:C reference limits obtained from examination of 6 different groups randomly chosen (N = 20 each) with a group of N = 120 reference individuals. The second example presents the balance of haemophilia A carrier detection rate performed in 30 obligatory carriers and 42 normal women by examination of F.VIII:C and VWF-Ag using universal discriminant. The correction figures typical for specific conditions of our laboratory are obtained from examination of N = 20 and N = 80 individuals and using heuristic optimalization.

  8. Chromogenic assay of human coagulation factor VIII: statistical comparison of 2 working dilution procedures.

    PubMed

    Alonso, C; Gonzalez, A; Frutos, G

    2005-08-01

    The effect of 2 different practices for preparation of working dilutions in the chromogenic substrate method for potency assay of factor VIII was evaluated. In this study the potency of several concentrate materials was shown to be statistically equivalent, whether performing the assay with independent or serial working dilutions.

  9. Analysis of mutations in the entire coding sequence of the factor VIII gene

    SciTech Connect

    Bidichadani, S.I.; Lanyon, W.G.; Connor, J.M.

    1994-09-01

    Hemophilia A is a common X-linked recessive disorder of bleeding caused by deleterious mutations in the gene for clotting factor VIII. The large size of the factor VIII gene, the high frequency of de novo mutations and its tissue-specific expression complicate the detection of mutations. We have used a combination of RT-PCR of ectopic factor VIII transcripts and genomic DNA-PCRs to amplify the entire essential sequence of the factor VIII gene. This is followed by chemical mismatch cleavage analysis and direct sequencing in order to facilitate a comprehensive search for mutations. We describe the characterization of nine potentially pathogenic mutations, six of which are novel. In each case, a correlation of the genotype with the observed phenotype is presented. In order to evaluate the pathogenicity of the five missense mutations detected, we have analyzed them for evolutionary sequence conservation and for their involvement of sequence motifs catalogued in the PROSITE database of protein sites and patterns.

  10. Analysis of the essential sequences of the factor VIII gene in twelve haemophilia A patients by single-stranded conformation polymorphism.

    PubMed

    David, D; Moreira, I; Lalloz, M R; Rosa, H A; Schwaab, R; Morais, S; Diniz, M J; de Deus, G; Campos, M; Lavinha, J

    1994-04-01

    We report the analysis by single-stranded conformation polymorphism of the essential sequences of the factor VIII(FVIII) gene (total length about 14 kb) including the entire coding sequence, flanking intronic sequences and the putative regulatory sequences 5' to the gene, in twelve unselected haemophilia A patients of Portuguese origin. Direct sequencing of the fragments with an altered migration pattern led to the identification of the disease-producing mutations in five patients. Three of these mutations, namely a 1 bp insertion in a motif of eight consecutive A residues at codon 1439 (FVIIIPorto3); a C to T transition at codon 1966 (Arg-->Stop), found in an inhibitor-positive patient (FVIIIMontijo); and a G to A transition at codon 479 (Gly-->Arg; FVIIIPorto1), have been reported in other ethnic groups. The two novel mutations are the substitution of AG by GG at the 3' end of intron 4 (FVIIILisboa1) destroying the invariant splice acceptor sequence, and a G to A transition at codon 1948 resulting in an aspartic acid substitution for glycine (FVIIIPorto2).

  11. Binding of factor VIII to von willebrand factor is enabled by cleavage of the von Willebrand factor propeptide and enhanced by formation of disulfide-linked multimers.

    PubMed

    Bendetowicz, A V; Morris, J A; Wise, R J; Gilbert, G E; Kaufman, R J

    1998-07-15

    von Willebrand factor (vWF) is a multimeric adhesive glycoprotein with one factor VIII binding site/subunit. Prior reports suggest that posttranslational modifications of vWF, including formation of N-terminal intersubunit disulfide bonds and subsequent cleavage of the propeptide, influence availability and/or affinity of factor VIII binding sites. We found that deletion of the vWF propeptide produced a dimeric vWF molecule lacking N-terminal intersubunit disulfide bonds. This molecule bound fluorescein-labeled factor VIII with sixfold lower affinity than multimeric vWF in an equilibrium flow cytometry assay (approximate KDs, 5 nmol/L v 0.9 nmol/L). Coexpression of propeptide-deleted vWF with the vWF propeptide in trans yielded multimeric vWF that displayed increased affinity for factor VIII. Insertion of an alanine residue at the N-terminus of the mature vWF subunit destroyed binding to factor VIII, indicating that the native mature N-terminus is required for factor VIII binding. The requirement for vWF propeptide cleavage was shown by (1) a point mutation of the vWF propeptide cleavage site yielding pro-vWF that was defective in factor VIII binding and (2) correlation between efficiency of intracellular propeptide cleavage and factor VIII binding. Furthermore, in a cell-free system, addition of the propeptide-cleaving enzyme PACE/furin enabled factor VIII binding in parallel with propeptide cleavage. Our results indicate that high-affinity factor VIII binding sites are located on N-terminal disulfide-linked vWF subunits from which the propeptide has been cleaved.

  12. Comparative field study evaluating the activity of recombinant factor VIII Fc fusion protein in plasma samples at clinical haemostasis laboratories

    PubMed Central

    Sommer, J M; Moore, N; McGuffie-Valentine, B; Bardan, S; Buyue, Y; Kamphaus, G D; Konkle, B A; Pierce, G F

    2014-01-01

    Discrepancies exist for some of the modified coagulation factors when assayed with different one-stage clotting and chromogenic substrate assay reagents. The aim of this study was to evaluate the performance of a recombinant factor VIII Fc fusion protein (rFVIIIFc), currently in clinical development for the treatment of severe haemophilia A, in a variety of one-stage clotting and chromogenic substrate assays in clinical haemostasis laboratories. Haemophilic plasma samples spiked with rFVIIIFc or Advate® at 0.05, 0.20 or 0.80 IU mL−1 were tested by 30 laboratories using their routine procedures and plasma standards. Data were evaluated for intra- and inter-laboratory variation, accuracy and possible rFVIIIFc-specific assay discrepancies. For the one-stage assay, mean recovery was 95% to 100% of expected for both Advate® and rFVIIIFc at 0.8 IU mL−1. Intra-laboratory percent coefficient of variance (CV) ranged from 6.3% to 7.8% for Advate®, and 6.0% to 10.3% for rFVIIIFc. Inter-laboratory CV ranged from 10% for Advate® and 16% for rFVIIIFc at 0.8 IU mL−1, to over 30% at 0.05 IU mL−1 for both products. For the chromogenic substrate assay, the average FVIII recovery was 107% ± 5% and 124% ± 8% of label potency across the three concentrations of Advate® and rFVIIIFc, respectively. Plasma rFVIIIFc levels can be monitored by either the one-stage or the chromogenic substrate assay routinely performed in clinical laboratories without the need for a product-specific rFVIIIFc laboratory standard. Accuracy by the one-stage assay was comparable to that of Advate®, while marginally higher results may be observed for rFVIIIFc when using the chromogenic assay. PMID:24261554

  13. Rare and low-frequency variants and their association with plasma levels of fibrinogen, FVII, FVIII, and vWF.

    PubMed

    Huffman, Jennifer E; de Vries, Paul S; Morrison, Alanna C; Sabater-Lleal, Maria; Kacprowski, Tim; Auer, Paul L; Brody, Jennifer A; Chasman, Daniel I; Chen, Ming-Huei; Guo, Xiuqing; Lin, Li-An; Marioni, Riccardo E; Müller-Nurasyid, Martina; Yanek, Lisa R; Pankratz, Nathan; Grove, Megan L; de Maat, Moniek P M; Cushman, Mary; Wiggins, Kerri L; Qi, Lihong; Sennblad, Bengt; Harris, Sarah E; Polasek, Ozren; Riess, Helene; Rivadeneira, Fernando; Rose, Lynda M; Goel, Anuj; Taylor, Kent D; Teumer, Alexander; Uitterlinden, André G; Vaidya, Dhananjay; Yao, Jie; Tang, Weihong; Levy, Daniel; Waldenberger, Melanie; Becker, Diane M; Folsom, Aaron R; Giulianini, Franco; Greinacher, Andreas; Hofman, Albert; Huang, Chiang-Ching; Kooperberg, Charles; Silveira, Angela; Starr, John M; Strauch, Konstantin; Strawbridge, Rona J; Wright, Alan F; McKnight, Barbara; Franco, Oscar H; Zakai, Neil; Mathias, Rasika A; Psaty, Bruce M; Ridker, Paul M; Tofler, Geoffrey H; Völker, Uwe; Watkins, Hugh; Fornage, Myriam; Hamsten, Anders; Deary, Ian J; Boerwinkle, Eric; Koenig, Wolfgang; Rotter, Jerome I; Hayward, Caroline; Dehghan, Abbas; Reiner, Alex P; O'Donnell, Christopher J; Smith, Nicholas L

    2015-09-10

    Fibrinogen, coagulation factor VII (FVII), and factor VIII (FVIII) and its carrier von Willebrand factor (vWF) play key roles in hemostasis. Previously identified common variants explain only a small fraction of the trait heritabilities, and additional variations may be explained by associations with rarer variants with larger effects. The aim of this study was to identify low-frequency (minor allele frequency [MAF] ≥0.01 and <0.05) and rare (MAF <0.01) variants that influence plasma concentrations of these 4 hemostatic factors by meta-analyzing exome chip data from up to 76,000 participants of 4 ancestries. We identified 12 novel associations of low-frequency (n = 2) and rare (n = 10) variants across the fibrinogen, FVII, FVIII, and vWF traits that were independent of previously identified associations. Novel loci were found within previously reported genes and had effect sizes much larger than and independent of previously identified common variants. In addition, associations at KCNT1, HID1, and KATNB1 identified new candidate genes related to hemostasis for follow-up replication and functional genomic analysis. Newly identified low-frequency and rare-variant associations accounted for modest amounts of trait variance and therefore are unlikely to increase predicted trait heritability but provide new information for understanding individual variation in hemostasis pathways.

  14. Rare and low-frequency variants and their association with plasma levels of fibrinogen, FVII, FVIII, and vWF

    PubMed Central

    Huffman, Jennifer E.; de Vries, Paul S.; Morrison, Alanna C.; Sabater-Lleal, Maria; Kacprowski, Tim; Auer, Paul L.; Brody, Jennifer A.; Chasman, Daniel I.; Chen, Ming-Huei; Guo, Xiuqing; Lin, Li-An; Marioni, Riccardo E.; Müller-Nurasyid, Martina; Yanek, Lisa R.; Pankratz, Nathan; Grove, Megan L.; de Maat, Moniek P. M.; Cushman, Mary; Wiggins, Kerri L.; Qi, Lihong; Sennblad, Bengt; Harris, Sarah E.; Polasek, Ozren; Riess, Helene; Rivadeneira, Fernando; Rose, Lynda M.; Goel, Anuj; Taylor, Kent D.; Teumer, Alexander; Uitterlinden, André G.; Vaidya, Dhananjay; Yao, Jie; Tang, Weihong; Levy, Daniel; Waldenberger, Melanie; Becker, Diane M.; Folsom, Aaron R.; Giulianini, Franco; Greinacher, Andreas; Hofman, Albert; Huang, Chiang-Ching; Kooperberg, Charles; Silveira, Angela; Starr, John M.; Strauch, Konstantin; Strawbridge, Rona J.; Wright, Alan F.; McKnight, Barbara; Franco, Oscar H.; Zakai, Neil; Mathias, Rasika A.; Psaty, Bruce M.; Ridker, Paul M.; Tofler, Geoffrey H.; Völker, Uwe; Watkins, Hugh; Fornage, Myriam; Hamsten, Anders; Deary, Ian J.; Boerwinkle, Eric; Koenig, Wolfgang; Rotter, Jerome I.; Hayward, Caroline; Dehghan, Abbas; Reiner, Alex P.; O’Donnell, Christopher J.

    2015-01-01

    Fibrinogen, coagulation factor VII (FVII), and factor VIII (FVIII) and its carrier von Willebrand factor (vWF) play key roles in hemostasis. Previously identified common variants explain only a small fraction of the trait heritabilities, and additional variations may be explained by associations with rarer variants with larger effects. The aim of this study was to identify low-frequency (minor allele frequency [MAF] ≥0.01 and <0.05) and rare (MAF <0.01) variants that influence plasma concentrations of these 4 hemostatic factors by meta-analyzing exome chip data from up to 76 000 participants of 4 ancestries. We identified 12 novel associations of low-frequency (n = 2) and rare (n = 10) variants across the fibrinogen, FVII, FVIII, and vWF traits that were independent of previously identified associations. Novel loci were found within previously reported genes and had effect sizes much larger than and independent of previously identified common variants. In addition, associations at KCNT1, HID1, and KATNB1 identified new candidate genes related to hemostasis for follow-up replication and functional genomic analysis. Newly identified low-frequency and rare-variant associations accounted for modest amounts of trait variance and therefore are unlikely to increase predicted trait heritability but provide new information for understanding individual variation in hemostasis pathways. PMID:26105150

  15. The prevalence of factor VIII and IX inhibitors among Saudi patients with hemophilia

    PubMed Central

    Owaidah, Tarek; Momen, Abdulkareem Al; Alzahrani, Hazzaa; Almusa, Abdulrahman; Alkasim, Fawaz; Tarawah, Ahmed; Nouno, Randa Al; Batniji, Fatima Al; Alothman, Fahad; Alomari, Ali; Abu-Herbish, Saud; Abu-Riash, Mahmoud; Siddiqui, Khawar; Ahmed, Mansor; Mohamed, SY; Saleh, Mahasen

    2017-01-01

    Abstract Hemophilia A and B are X-linked diseases that predominantly affect male patients. Patients can develop coagulation factor inhibitors, which exponentially increases the treatment cost. However, the prevalence of factor VIII and IX inhibitors in Saudi Arabia is unclear. This study aimed to determine the Saudi prevalence of factor VIII and IX inhibitors. This 4-year, 7-center, cross-sectional study evaluated the Saudi prevalences of hemophilia A and B. We collected the patients’ clinical data, evaluated their disease, and tested for factor inhibitors. We included 202 patients with hemophilia (median age at diagnosis: 0.13 years, range: birth–34.8 years). The patients included 198 male patients (98%), 148 patients with hemophilia A (73.3%), and 54 patients with hemophilia B (26.7%). The patients exhibited severe factor VIII activity (<1%; 121 patients; 5.2%), moderate activity (1–5%; 7 patients; 4.9%), and mild activity (14 patients; 9.9%). Among the patients with care-related data, most patients were treated for episodic bleeding (76.8%) or received prophylaxis (22.6%); 1 patient received both treatments. Among the patients with source-related data, the factor replacements were derived from plasma (48.4%), recombinant concentrates (22.9%), both sources (14.6%), or fresh frozen plasma (14.1%). Factor VIII inhibitors were observed in 43 (29.3%) of the 147 patients, and only 1 of the 54 patients developed factor IX inhibitors. Most patients who developed inhibitors had severe hemophilia (40/44; 90.9%), and inhibitors were also common among patients who received recombinant products (14/43; 32.6%). The Saudi prevalence of factor inhibitors was similar to those among other ethnic populations. PMID:28079788

  16. Specific membrane binding of factor VIII is mediated by O-phospho-L-serine, a moiety of phosphatidylserine.

    PubMed

    Gilbert, G E; Drinkwater, D

    1993-09-21

    Phosphatidylserine, a negatively charged lipid, is exposed on the platelet membrane following cell stimulation, correlating with the expression of factor VIII receptors. We have explored the importance of the negative electrostatic potential of phosphatidylserine vs chemical moieties of phosphatidylserine for specific membrane binding of factor VIII. Fluorescein-labeled factor VIII bound to membranes containing 15% phosphatidic acid, a negatively charged phospholipid, with low affinity compared to phosphatidylserine-containing membranes. Binding was not specific as it was inhibited by other proteins in plasma. Factor VIII bound to membranes containing 10% phosphatidylserine in spite of a varying net charge provided by 0-15% stearylamine, a positively charged lipid. The soluble phosphatidylserine moiety, O-phospho-L-serine, inhibited factor VIII binding to phosphatidylserine-containing membranes with a Ki of 20 mM, but the stereoisomer, O-phospho-D-serine, was 5-fold less effective. Furthermore, binding of factor VIII to membranes containing synthetic phosphatidyl-D-serine was 5-fold less than binding to membranes containing phosphatidyl-L-serine. Membranes containing synthetic phosphatidyl-L-homoserine, differing from phosphatidylserine by a single methylene, supported high-affinity binding, but it was not specific as factor VIII was displaced by other plasma proteins. O-Phospho-L-serine also inhibited the binding of factor VIII to platelet-derived microparticles with a Ki of 20 mM, and the stereoisomer was 4-fold less effective. These results indicate that membrane binding of factor VIII is mediated by a stereoselective recognition O-phospho-L-serine of phosphatidylserine and that negative electrostatic potential is of lesser importance.

  17. Molecular Analysis of Factor VIII and Factor IX Genes in Hemophilia Patients: Identification of Novel Mutations and Molecular Dynamics Studies

    PubMed Central

    Al-Allaf, Faisal A.; Taher, Mohiuddin M.; Abduljaleel, Zainularifeen; Bouazzaoui, Abdellatif; Athar, Mohammed; Bogari, Neda M.; Abalkhail, Halah A.; Owaidah, Tarek MA.

    2017-01-01

    Background Hemophilias A and B are X-linked bleeding disorders caused by mutations in the factor VIII and factor IX genes, respectively. Our objective was to identify the spectrum of mutations of the factor VIII and factor IX genes in Saudi Arabian population and determine the genotype and phenotype correlations by molecular dynamics (MD) simulation. Methods For genotyping, blood samples from Saudi Arabian patients were collected, and the genomic DNA was amplified, and then sequenced by Sanger method. For molecular simulations, we have used softwares such as CHARMM (Chemistry at Harvard Macromolecular Mechanics; http://www.charmm-gui.org) and GROMACS. In addition, the secondary structure was determined based on the solvent accessibility for the confirmation of the protein stability at the site of mutation. Results Six mutations (three novel and three known) were identified in factor VIII gene, and six mutations (one novel and five known) were identified in factor IX gene. The factor VIII novel mutations identified were c.99G>T, p. (W33C) in exon 1, c.2138 DelA, p. (N713Tfs*9) in eon14, also a novel mutation at splicing acceptor site of exon 23 c.6430 - 1G>A. In factor IX, we found a novel mutation c.855G>C, p. (E285D) in exon 8. These novel mutations were not reported in any factor VIII or factor IX databases previously. The deleterious effects of these novel mutations were confirmed by PolyPhen2 and SIFT programs. Conclusion The protein functional and structural studies and the models built in this work would be appropriate for predicting the effects of deleterious amino acid substitutions causing these genetic disorders. These findings are useful for genetic counseling in the case of consanguineous marriages which is more common in the Saudi Arabia. PMID:28270892

  18. Prediction of Giant Thermoelectric Power Factor in Type-VIII Clathrate Si46

    PubMed Central

    Norouzzadeh, Payam; Myles, Charles W.; Vashaee, Daryoosh

    2014-01-01

    Clathrate materials have been the subject of intense interest and research for thermoelectric application. Nevertheless, from the very large number of conceivable clathrate structures, only a small fraction of them have been examined. Since the thermal conductivity of clathrates is inherently small due to their large unit cell size and open-framework structure, the current research on clathrates is focused on finding the ones with large thermoelectric power factor. Here we predict an extraordinarily large power factor for type-VIII clathrate Si46. We show the existence of a large density of closely packed elongated ellipsoidal carrier pockets near the band edges of this so far hypothetical material structure, which is higher than that of the best thermoelectric materials known today. The high crystallographic symmetry near the energy band edges for Si46-VIII clathrates is responsible for the formation of such a large number of carrier pockets. PMID:25391971

  19. Pattern of factor VIII inhibitors in patients with hemophilia A in the north east of Iran.

    PubMed

    Modaresi, A R; Torghabeh, H Mansouri; Pourfathollah, A A; Shooshtari, M Mahmoodian; Yazdi, Z Rezaie

    2006-06-01

    This survey was conducted to evaluate coagulation factor VIII:C inhibitors among 102 hemophilia A patients from different cities of Khorasan province in north east of Iran in order to identify and characterize the pattern of inhibitor formation in these patients population. For this purpose, we randomly obtained plasma samples of 102 hemophilia A patients (44 patients with severe, 28 patients with intermediate and 30 patients with mild hemophilia A) and studied them using two tests: the APTT mix and Bethesda test were performed. In the whole group 20 patients (19.6%) factor VIII inhibitors were detected. These were in 11 patients with severe, five patients with intermediate and four patients with mild hemophilia A. None of patients with hemophilia A had previously been studied for the presence of an inhibitor, so there was no existing history of inhibitor evaluation.

  20. Efficient production of dual recombinant adeno-associated viral vectors for factor VIII delivery.

    PubMed

    Wang, Qizhao; Dong, Biao; Firrman, Jenni; Roberts, Sean; Moore, Andrea Rossi; Cao, Wenjing; Diao, Yong; Kapranov, Philipp; Xu, Ruian; Xiao, Weidong

    2014-08-01

    Recombinant adeno-associated viral (rAAV) vectors have gained attention for human gene therapy because of their high safety and clinical efficacy profile. For factor VIII gene delivery, splitting the coding region between two AAV vectors remains a viable strategy to avoid the packaging capacity limitation (∼5.0 kb). However, it is time-consuming and labor-intensive to produce two rAAV vectors in separate batches. Here we demonstrated successful production of dual rAAV vectors for hemophilia A gene therapy in a single preparation. When the AAV vector plasmids carrying the human factor VIII heavy chain (hHC) and the light chain (hLC) expression cassettes were cotransfected into 293 cells along with the AAV rep&cap and mini-adenovirus helper plasmids, both rAAV-hHC and rAAV-hLC were produced at the desired ratio and in high titer. Interestingly, the rAAV-hHC vectors always yielded higher titers than rAAV-hLC vectors as a result of more efficient replication of rAAV-hHC genomes. The resulting vectors were effective in transducing the tissue culture cells in vitro. When these vectors were administered to hemophilia A mice, factor VIII was detected in the mouse plasma by both the activated partial thromboplastin time assay and enzyme-linked immunosorbent assay. The functional activity as well as the antigen levels of secreted factor VIII were similar to those of vectors produced by the traditional method. The dual-vector production method has been successfully extended to both AAV2 and AAV8 serotypes. In conclusion, cotransfection of vector plasmids presents an efficient method for producing dual or multiple AAV vectors at significantly reduced cost and labor.

  1. Mutations and a polymorphism in the factor VIII gene discovered by denaturing gradient gel electrophoresis

    SciTech Connect

    Kogan, S.; Gitschier, J. )

    1990-03-01

    Hemophilia A results from mutations in the gene coding for coagulation factor VIII. The authors gradient gel electrophoresis to screen for mutations in the region of the factor VIII gene coding for the first acidic domain. Amplification primers were designed employing the MELTMAP computer program to optimize the ability to detect mutations. Screening of amplified DNA from 228 unselected hemophilia A patients revealed two mutations and one polymorphism. Rescreening the same population by making heteroduplexes between amplified patient and control samples prior to electrophoresis revealed one additional mutation. The mutations include two missense and one 4-base-pair deletion, and each mutation was found in patients with severe hemophilia. The polymorphism, located adjacent to the adenine branch site in intron 7, is useful for genetic prediction in some cases where the Bcl I and Xba I polymorphisms are uninformative. These results suggest that DNA amplification and denaturing gradient gel electrophoresis should be an excellent strategy for identifying mutations and polymorphisms in defined regions of the factor VIII gene and other large genes.

  2. Factor VIII gene (F8) mutation and risk of inhibitor development in nonsevere hemophilia A.

    PubMed

    Eckhardt, Corien L; van Velzen, Alice S; Peters, Marjolein; Astermark, Jan; Brons, Paul P; Castaman, Giancarlo; Cnossen, Marjon H; Dors, Natasja; Escuriola-Ettingshausen, Carmen; Hamulyak, Karly; Hart, Daniel P; Hay, Charles R M; Haya, Saturnino; van Heerde, Waander L; Hermans, Cedric; Holmström, Margareta; Jimenez-Yuste, Victor; Keenan, Russell D; Klamroth, Robert; Laros-van Gorkom, Britta A P; Leebeek, Frank W G; Liesner, Ri; Mäkipernaa, Anne; Male, Christoph; Mauser-Bunschoten, Evelien; Mazzucconi, Maria G; McRae, Simon; Meijer, Karina; Mitchell, Michael; Morfini, Massimo; Nijziel, Marten; Oldenburg, Johannes; Peerlinck, Kathelijne; Petrini, Pia; Platokouki, Helena; Reitter-Pfoertner, Sylvia E; Santagostino, Elena; Schinco, Piercarla; Smiers, Frans J; Siegmund, Berthold; Tagliaferri, Annarita; Yee, Thynn T; Kamphuisen, Pieter Willem; van der Bom, Johanna G; Fijnvandraat, Karin

    2013-09-12

    Neutralizing antibodies (inhibitors) toward factor VIII form a severe complication in nonsevere hemophilia A, profoundly aggravating the bleeding pattern. Identification of high-risk patients is hampered by lack of data that take exposure days to therapeutic factor VIII concentrates into account. In the INSIGHT study, we analyzed the association between F8 mutation and inhibitor development in patients with nonsevere hemophilia A (factor VIII 2-40 IU/dL). This analysis included 1112 nonsevere hemophilia A patients from 14 centers in Europe and Australia that had genotyped at least 70% of their patients. Inhibitor risk was calculated as Kaplan-Meier incidence with cumulative number of exposure days as the time variable. During 44 800 exposure days (median, 24 exposure days per patient; interquartile range [IQR], 7-90), 59 of the 1112 patients developed an inhibitor; cumulative incidence of 5.3% (95% confidence interval [CI], 4.0-6.6) after a median of 28 exposure days (IQR, 12-71). The inhibitor risk at 50 exposure days was 6.7% (95% CI, 4.5-8.9) and at 100 exposure days the risk further increased to 13.3% (95% CI, 9.6-17.0). Among a total of 214 different F8 missense mutations 19 were associated with inhibitor development. These results emphasize the importance of F8 genotyping in nonsevere hemophilia A.

  3. Cleavage at Arg-1689 influences heavy chain cleavages during thrombin-catalyzed activation of factor VIII.

    PubMed

    Newell, Jennifer L; Fay, Philip J

    2009-04-24

    The procofactor, factor VIII, is activated by thrombin or factor Xa-catalyzed cleavage at three P1 residues: Arg-372, Arg-740, and Arg-1689. The catalytic efficiency for thrombin cleavage at Arg-740 is greater than at either Arg-1689 or Arg-372 and influences reaction rates at these sites. Because cleavage at Arg-372 appears rate-limiting and dependent upon initial cleavage at Arg-740, we investigated whether cleavage at Arg-1689 influences catalysis at this step. Recombinant B-domainless factor VIII mutants, R1689H and R1689Q were prepared and stably expressed to slow and eliminate cleavage, respectively. Specific activity values for the His and Gln mutations were approximately 50 and approximately 10%, respectively, that of wild type. Thrombin activation of the R1689H variant showed an approximately 340-fold reduction in the rate of Arg-1689 cleavage, whereas the R1689Q variant was resistant to thrombin cleavage at this site. Examination of heavy chain cleavages showed approximately 4- and 11-fold reductions in A2 subunit generation and approximately 3- and 7-fold reductions in A1 subunit generation for the R1689H and R1689Q mutants, respectively. These results suggest a linkage between light chain cleavage and cleavages in heavy chain. Results obtained evaluating proteolysis of the factor VIII mutants by factor Xa revealed modest rate reductions (<5-fold) in generating A2 and A1 subunits and in cleaving light chain at Arg-1721 from either variant, suggesting little dependence upon prior cleavage at residue 1689 as compared with thrombin. Overall, these results are consistent with a competition between heavy and light chains for thrombin exosite binding and subsequent proteolysis with binding of the former chain preferred.

  4. Dry-heat treatment process for enhancing viral safety of an antihemophilic factor VIII concentrate prepared from human plasma.

    PubMed

    Kim, In Seop; Choi, Yong Woon; Kang, Yong; Sung, Hark Mo; Shin, Jeong Sup

    2008-05-01

    Viral safety is a prerequisite for manufacturing clinical antihemophilic factor VIII concentrates from human plasma. With particular regard to the hepatitis A virus (HAV), a terminal dry-heat treatment (100 degrees for 30 min) process, following lyophilization, was developed to improve the virus safety of a solvent/detergent-treated antihemophilic factor VIII concentrate. The loss of factor VIII activity during dry-heat treatment was of about 5%. No substantial changes were observed in the physical and biochemical characteristics of the dry-heat-treated factor VIII compared with those of the factor VIII before dry-heat treatment. The dry-heat-treated factor VIII was stable for up to 24 months at 4oC. The dry-heat treatment after lyophilization was an effective process for inactivating viruses. The HAV, murine encephalomyocarditis virus (EMCV), and human immunodeficiency virus (HIV) were completely inactivated to below detectable levels within 10 min of the dry-heat treatment. Bovine herpes virus (BHV) and bovine viral diarrhea virus (BVDV) were potentially sensitive to the treatment. However porcine parvovirus (PPV) was slightly resistant to the treatment. The log reduction factors achieved during lyophilization and dry-heat treatment were > or =5.55 for HAV, > or =5.87 for EMCV, > or =5.15 for HIV, 6.13 for BHV, 4.46 for BVDV, and 1.90 for PPV. These results indicate that dry-heat treatment improves the virus safety of factor VIII concentrates, without destroying the activity. Moreover, the treatment represents an effective measure for the inactivation of non-lipid-enveloped viruses, in particular HAV, which is resistant to solvent/detergent treatment.

  5. Analysis of inversions in the factor VIII gene in Spanish hemophilia A patients and families

    SciTech Connect

    Domenech, M.; Tizzano, E.; Baiget, M.; Altisent, C.

    1994-09-01

    Intron 22 is the largest intron of the factor VIII gene and contains a CpG island from which two additional transcripts originate. One of these transcripts corresponds to the F8A gene which have telomeric extragenic copies in the X chromosome. An inversion involving homologous recombination between the intragenic and the distal or proximal copies of the F8A gene has been recently described as a common cause of severe hemophilia A (HA). We analyzed intron 22 rearrangements in 195 HA patients (123 familial and 72 sporadic cases). According to factor VIII levels, our sample was classified as severe in 114 cases, moderate in 29 cases and mild in 52 cases. An intron 22 (F8A) probe was hybridized to Southern blots of BcII digested DNA obtained from peripheral blood. A clear pattern of altered bands identifies distal or proximal inversions. We detected an abnormal pattern identifying an inversion in 49 (25%) of the analyzed cases. 43% of severe HA patients (49 cases) showed an inversion. As expected, no inversion was found in the moderate and mild group of patients. We found a high proportion (78%) of the distal rearrangement. From 49 identified inversions, 33 were found in familial cases (27%), while the remaining 15 were detected in sporadic patients (22%) in support that this mutational event occurs with a similar frequency in familial or sporadic cases. In addition, we detected a significant tendency of distal inversion to occur more frequently in familial cases than in sporadic cases. Inhibitor development to factor VIII was documented in approximately 1/3 of the patients with inversion. The identification of such a frequent molecular event in severe hemophilia A patients has been applied in our families to carrier and prenatal diagnosis, to determine the origin of the mutation in the sporadic cases and to detect the presence of germinal mosaicism.

  6. DNA polymorphisms associated with the factor VIII:C gene in the Portuguese population.

    PubMed

    David, D; Mergulhão, C; Capucho, I; Lavinha, J

    1992-01-01

    We have determined the allele frequencies of seven restriction fragment length polymorphisms within or close to factor VIII:C gene in the Portuguese population. The allele frequency of all studied intra- and extragenic biallelic polymorphisms does not differ significantly from those found in other European or Asian populations. On the contrary, the distribution of the different alleles of the TaqI RFLP at the DXS52 locus revealed similarity only with Algerians. We observed a correspondence between the TaqI and BclI alleles at this locus.

  7. Hemophilia as a defect of the tissue factor pathway of blood coagulation: Effect of factors VIII and IX on factor X activation in a continuous-flow reactor

    SciTech Connect

    Repke, D.; Gemmell, C.H.; Guha, A.; Turitto, V.T.; Nemerson, Y. ); Broze, G.J. Jr. )

    1990-10-01

    The effect of factors VIII and IX on the ability of the tissue factor-factor VIIa complex to activate factor X was studied in a continuous-flow tubular enzyme reactor. Tissue factor immobilized in a phospholipid bilayer on the inner surface of the tube was exposed to a perfusate containing factors VIIa, VIII, IX, and X flowing at a wall shear rate of 57, 300, or 1130 sec{sup {minus}1}. The addition of factors VIII and IX at their respective plasma concentrations resulted in a further 2{endash}-to 3{endash}fold increase. The direct activation of factor X by tissue factor-factor VIIa could be virtually eliminated by the lipoprotein-associated coagulation inhibitor. These results suggest that the tissue factor pathway, mediated through factors VIII and IX, produces significant levels of factor Xa even in the presence of an inhibitor of the tissue factor-factor VIIa complex; moreover, the activation is dependent on local shear conditions. These findings are consistent both with a model of blood coagulation in which initiation of the system results from tissue factor and with the bleeding observed in hemophilia.

  8. Outcome of Clinical Trials with New Extended Half-Life FVIII/IX Concentrates.

    PubMed

    Mancuso, Maria Elisa; Santagostino, Elena

    2017-03-28

    The development of a new generation of coagulation factors with improved pharmacokinetic profile will change the paradigm of treatment of persons with hemophilia (PWH). The standard treatment in PWH is represented by regular long-term prophylaxis that, given intravenously twice or thrice weekly, is associated with a not-negligible burden on patients' quality of life. The availability of drugs with improved pharmacokinetic profile may improve prophylaxis feasibility and protection against bleeding episodes. This article summarizes the main results obtained from clinical trials with modified factor VIII (FVIII) and factor IX (FIX) molecules. Published literature on new molecules for replacement treatment in hemophilia A and B was retrieved using PubMed search, and all ongoing clinical trials have been researched via www.clinicaltrials.gov. Such new molecules are usually engineered to have a longer plasma half-life than that which has been obtained by chemical modification (i.e., conjugation with polyethylene glycol, PEG) or by creating recombinant fusion proteins. Results from phase I/III studies in previously treated adults and children are now available for the vast majority of new products, including the results of their use in a surgical setting. On the contrary, trials involving previously untreated patients are still ongoing for all and results not yet available.

  9. Risk factors for high-titer inhibitor development in children with hemophilia A: results of a cohort study.

    PubMed

    Halimeh, Susan; Bidlingmaier, Christoph; Heller, Christine; Gutsche, Sven; Holzhauer, Susanne; Kenet, Gili; Kurnik, Karin; Manner, Daniela; Iorio, Alfonso; Nowak-Göttl, Ulrike

    2013-01-01

    Among the discussed risk factors for high-titre inhibitor (HRI) development in patients with hemophilia A (HA) are high dose FVIII replacement therapy and use of recombinant FVIII concentrates (rFVIII). The aim of this study was to evaluate the aforementioned risk factors for HRI development in children with hemophilia A ≤2%. About 288 ascertained PUPs (Israel and Germany) were followed after initial HA diagnosis over 200 exposure days. Inhibitor-free survival, hazard ratios (HR), and 95% confidence intervals (CIs) were calculated. Adjustment was performed for factor VIII concentrates, median single dose over the first three months of treatment, first FVIII administration before the age of three months, presence of risk HA gene mutations, "intensive treatment moments" and "year of birth" (proxy for different treatment periods). HRI occurred in 71/288 children (24.7%). In multivariate analysis adjusted for "year of birth", underlying risk gene mutations (HR/CI: 2.37/1.40-3.99), FVIII dose, measured per one IU increase per kgbw (HR/CI: 1.05/1.04-1.07), and first FVIII administration before the age of three months showed a significant impact on HR development. The risk of HRI development was similar for recombinant or plasmatic FVIII products. Children at risk should be treated with carefully calculated lower dose regimens, adapted to individual bleeding situations.

  10. Portal vein thrombosis associated with protein C deficiency and elevated Factor VIII in hepatosplenic schistosomiasis.

    PubMed

    Leite, Luiz Arthur Calheiros; Ferreira, Rita de Cássia dos Santos; Hatzlhofer, Betânia Lucena Domingues; Correia, Maria Conceição Barros; Bandeira, Ângela Pontes; Owen, James Stuart; Lima, Vera Lúcia de Menezes; Domingues, Ana Lúcia Coutinho; Lopes, Edmundo Pessoa

    2016-03-01

    Portal vein thrombosis is considered a vaso-occlusive process that can appear during the course of hepatosplenic Schistosoma mansoni, but may result from impaired portal blood flow or be associated with acquired or inherited thrombophilic factors. Here, we report the case of a 67-year-old woman who developed thrombocytopenia as a result of hypersplenism. Following the diagnosis of hepatosplenic schistosomiasis, portal vein thrombosis was detected by ultrasound examination, while haematological tests revealed low levels of protein C (43.3%) and high levels of factor VIII (183.1%). The pathogenesis of portal vein thrombosis remains unclear in some patients with S. mansoni. We recommend, therefore, that early clinical and haemostatic investigations are done to evaluate risk of portal vein thrombosis and hence avoid further complications.

  11. Prognostic factors for remission of and survival in acquired hemophilia A (AHA): results from the GTH-AH 01/2010 study.

    PubMed

    Tiede, Andreas; Klamroth, Robert; Scharf, Rüdiger E; Trappe, Ralf U; Holstein, Katharina; Huth-Kühne, Angela; Gottstein, Saskia; Geisen, Ulrich; Schenk, Joachim; Scholz, Ute; Schilling, Kristina; Neumeister, Peter; Miesbach, Wolfgang; Manner, Daniela; Greil, Richard; von Auer, Charis; Krause, Manuela; Leimkühler, Klaus; Kalus, Ulrich; Blumtritt, Jan-Malte; Werwitzke, Sonja; Budde, Eva; Koch, Armin; Knöbl, Paul

    2015-02-12

    Acquired hemophilia A (AHA) is caused by autoantibodies against factor VIII (FVIII). Immunosuppressive treatment (IST) results in remission of disease in 60% to 80% of patients over a period of days to months. IST is associated with frequent adverse events, including infections as a leading cause of death. Predictors of time to remission could help guide IST intensity but have not been established. We analyzed prognostic factors in 102 prospectively enrolled patients treated with a uniform IST protocol. Partial remission (PR; defined as no active bleeding, FVIII restored >50 IU/dL, hemostatic treatment stopped >24 hours) was achieved by 83% of patients after a median of 31 days (range 7-362). Patients with baseline FVIII <1 IU/dL achieved PR less often and later (77%, 43 days) than patients with ≥1 IU/dL (89%, 24 days). After adjustment for other baseline characteristics, low FVIII remained associated with a lower rate of PR (hazard ratio 0.52, 95% confidence interval 0.33-0.81, P < .01). In contrast, PR achieved on steroids alone within ≤21 days was more common in patients with FVIII ≥1 IU/dL and inhibitor concentration <20 BU/mL (odds ratio 11.2, P < .0001). Low FVIII was also associated with a lower rate of complete remission and decreased survival. In conclusion, presenting FVIII and inhibitor concentration are potentially useful to tailor IST in AHA.

  12. Structure of the human factor VIII C2 domain in complex with the 3E6 inhibitory antibody

    SciTech Connect

    Wuerth, Michelle E.; Cragerud, Rebecca K.; Spiegel, P. Clint

    2015-11-24

    Blood coagulation factor VIII is a glycoprotein cofactor that is essential for the intrinsic pathway of the blood coagulation cascade. Inhibitory antibodies arise either spontaneously or in response to therapeutic infusion of functional factor VIII into hemophilia A patients, many of which are specific to the factor VIII C2 domain. The immune response is largely parsed into “classical” and “non-classical” inhibitory antibodies, which bind to opposing faces cooperatively. In this study, the 2.61 Å resolution structure of the C2 domain in complex with the antigen-binding fragment of the 3E6 classical inhibitory antibody is reported. The binding interface is largely conserved when aligned with the previously determined structure of the C2 domain in complex with two antibodies simultaneously. Further inspection of the B factors for the C2 domain in various X-ray crystal structures indicates that 3E6 antibody binding decreases the thermal motion behavior of surface loops in the C2 domain on the opposing face, thereby suggesting that cooperative antibody binding is a dynamic effect. Furthermore, understanding the structural nature of the immune response to factor VIII following hemophilia A treatment will help lead to the development of better therapeutic reagents.

  13. Structure of the human factor VIII C2 domain in complex with the 3E6 inhibitory antibody

    DOE PAGES

    Wuerth, Michelle E.; Cragerud, Rebecca K.; Spiegel, P. Clint

    2015-11-24

    Blood coagulation factor VIII is a glycoprotein cofactor that is essential for the intrinsic pathway of the blood coagulation cascade. Inhibitory antibodies arise either spontaneously or in response to therapeutic infusion of functional factor VIII into hemophilia A patients, many of which are specific to the factor VIII C2 domain. The immune response is largely parsed into “classical” and “non-classical” inhibitory antibodies, which bind to opposing faces cooperatively. In this study, the 2.61 Å resolution structure of the C2 domain in complex with the antigen-binding fragment of the 3E6 classical inhibitory antibody is reported. The binding interface is largely conservedmore » when aligned with the previously determined structure of the C2 domain in complex with two antibodies simultaneously. Further inspection of the B factors for the C2 domain in various X-ray crystal structures indicates that 3E6 antibody binding decreases the thermal motion behavior of surface loops in the C2 domain on the opposing face, thereby suggesting that cooperative antibody binding is a dynamic effect. Furthermore, understanding the structural nature of the immune response to factor VIII following hemophilia A treatment will help lead to the development of better therapeutic reagents.« less

  14. Structure of the Human Factor VIII C2 Domain in Complex with the 3E6 Inhibitory Antibody.

    PubMed

    Wuerth, Michelle E; Cragerud, Rebecca K; Spiegel, P Clint

    2015-11-24

    Blood coagulation factor VIII is a glycoprotein cofactor that is essential for the intrinsic pathway of the blood coagulation cascade. Inhibitory antibodies arise either spontaneously or in response to therapeutic infusion of functional factor VIII into hemophilia A patients, many of which are specific to the factor VIII C2 domain. The immune response is largely parsed into "classical" and "non-classical" inhibitory antibodies, which bind to opposing faces cooperatively. In this study, the 2.61 Å resolution structure of the C2 domain in complex with the antigen-binding fragment of the 3E6 classical inhibitory antibody is reported. The binding interface is largely conserved when aligned with the previously determined structure of the C2 domain in complex with two antibodies simultaneously. Further inspection of the B factors for the C2 domain in various X-ray crystal structures indicates that 3E6 antibody binding decreases the thermal motion behavior of surface loops in the C2 domain on the opposing face, thereby suggesting that cooperative antibody binding is a dynamic effect. Understanding the structural nature of the immune response to factor VIII following hemophilia A treatment will help lead to the development of better therapeutic reagents.

  15. Prospective evaluation of Protein C and Factor VIII in prediction of cancer-associated thrombosis

    PubMed Central

    Tafur, AJ; Dale, G; Cherry, M; Wren, JD; Mansfield, AS; Comp, P; Rathbun, S; Stoner, JA

    2015-01-01

    Venous thromboembolism (VTE) is a preventable disease, yet it is one of the leading causes of death among patients with cancer. Improving risk stratification mechanisms will allow us to personalize thromboprophylaxis strategies. We sought to evaluate Collagen and Thrombin Activated Platelets (COAT-platelets) as well as protein C and factor VIII as biomarkers predictive of cancer-associated thrombosis in a prospective cohort of patients with cancer. Protein C was selected as a candidate based on bioinformatics prediction. Blood samples were collected before chemotherapy. All specimen processing was blinded to clinical data. Surveillance and adjudication of the main outcome of VTE was performed for up to 1 year. We used Cox proportional hazard regression to measure the association of biomarkers and incident events using SAS 9.2 for all statistical analysis. Death was modeled as a competing event. Among 241 patients followed for an average of 10.4 months, 15% died and 13% developed a VTE. COAT-platelets were not predictive of VTE. Low levels of pre-chemotherapy protein C (< 118 %) (HR 2.5; 95%CI 1.1–5.5) and high baseline factor VIII (> 261 % I) (HR 3.0; 95%CI 1.1–8.0) were predictive of VTE after adjusting for age, Khorana prediction risk, metastatic disease and D dimer. In addition, low protein C was predictive of overall mortality independent of age, metastatic disease and functional status (HR 2.8; 95%CI 1.3–6.0). Addition of these biomarkers to Cancer-VTE risk prediction models may add to risk stratification and patient selection to optimize thrombo-prophylaxis. PMID:26475410

  16. Haemophilia A: database of nucleotide substitutions, deletions, insertions and rearrangements of the factor VIII gene, second edition.

    PubMed Central

    Tuddenham, E G; Schwaab, R; Seehafer, J; Millar, D S; Gitschier, J; Higuchi, M; Bidichandani, S; Connor, J M; Hoyer, L W; Yoshioka, A

    1994-01-01

    A large number of different mutations in the factor VIII (F8) gene have been identified as a cause of haemophilia A. This compilation lists known single base-pair substitutions, deletions and insertions in the F8 gene and reviews the status of the inversional events which account for a substantial proportion of mutations causing severe haemophilia A. PMID:7984443

  17. Haemophilia A: database of nucleotide substitutions, deletions, insertions and rearrangements of the factor VIII gene, second edition.

    PubMed Central

    Tuddenham, E G; Schwaab, R; Seehafer, J; Millar, D S; Gitschier, J; Higuchi, M; Bidichandani, S; Connor, J M; Hoyer, L W; Yoshioka, A

    1994-01-01

    A large number of different mutations in the factor VIII (F8) gene have been identified as a cause of haemophilia A. This compilation lists known single base-pair substitutions, deletions and insertions in the F8 gene and reviews the status of the inversional events which account for a substantial proportion of mutations causing severe haemophilia A. PMID:7937051

  18. Analysis of factor VIII gene inversions in 164 unrelated hemophilia A families

    SciTech Connect

    Vnencak-Jones, L.; Phillips, J.A. III; Janco, R.L.

    1994-09-01

    Hemophilia A is an X-linked recessive disease with variable phenotype and both heterogeneous and wide spread mutations in the factor VIII (F8) gene. As a result, diagnostic carrier or prenatal testing often relies upon laborious DNA linkage analysis. Recently, inversion mutations resulting from an intrachromosomal recombination between DNA sequences in one of two A genes {approximately}500 kb upstream from the F8 gene and a homologous A gene in intron 22 of the F8 gene were identified and found in 45% of severe hemophiliacs. We have analyzed banked DNA collected since 1986 from affected males or obligate carrier females representing 164 unrelated hemophilia A families. The disease was sporadic in 37%, familial in 54% and in 10% of families incomplete information was given. A unique deletion was identified in 1/164, a normal pattern was observed in 110/164 (67%), and 53/164 (32%) families had inversion mutations with 43/53 (81%) involving the distal A gene (R3 pattern) and 10/53 (19%) involving the proximal A gene (R2 pattern). While 19% of all rearrangements were R2, in 35 families with severe disease (< 1% VIII:C activity) all 16 rearrangements seen were R3. In 18 families with the R3 pattern and known activities, 16 (89%) had levels < 1%, with the remaining 2 families having {le} 2.4% activity. Further, 18 referrals specifically noted the production of inhibitors and 8/18 (45%) had the R3 pattern. Our findings demonstrate that the R3 inversion mutation patterns is (1) only seen with VIII:C activity levels of {le} 2.4%, (2) seen in 46% of families with severe hemophilia, (3) seen in 45% of hemophiliacs known to have inhibitors, (4) not correlated with sporadic or familial disease and (5) not in disequilibrium with the Bcl I or Taq I intron 18 or ST14 polymorphisms. Finally, in families positive for an inversion mutation, direct testing offers a highly accurate and less expensive alternative to DNA linkage analysis.

  19. Mice deficient in LMAN1 exhibit FV and FVIII deficiencies and liver accumulation of α1-antitrypsin

    PubMed Central

    Zheng, Chunlei; Zhu, Min; Tao, Jiayi; Vasievich, Matthew P.; Baines, Andrea; Kim, Jinoh; Schekman, Randy; Kaufman, Randal J.; Ginsburg, David

    2011-01-01

    The type 1-transmembrane protein LMAN1 (ERGIC-53) forms a complex with the soluble protein MCFD2 and cycles between the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment (ERGIC). Mutations in either LMAN1 or MCFD2 cause the combined deficiency of factor V (FV) and factor VIII (FVIII; F5F8D), suggesting an ER-to-Golgi cargo receptor function for the LMAN1-MCFD2 complex. Here we report the analysis of LMAN1-deficient mice. Levels of plasma FV and FVIII, and platelet FV, are all reduced to ∼ 50% of wild-type in Lman1−/− mice, compared with the 5%-30% levels typically observed in human F5F8D patients. Despite previous reports identifying cathepsin C, cathepsin Z, and α1-antitrypsin as additional potential cargoes for LMAN1, no differences were observed between wild-type and Lman1−/− mice in the levels of cathepsin C and cathepsin Z in liver lysates or α1-antitrypsin levels in plasma. LMAN1 deficiency had no apparent effect on COPII-coated vesicle formation in an in vitro assay. However, the ER in Lman1−/− hepatocytes is slightly distended, with significant accumulation of α1-antitrypsin and GRP78. An unexpected, partially penetrant, perinatal lethality was observed for Lman1−/− mice, dependent on the specific inbred strain genetic background, suggesting a potential role for other, as yet unidentified LMAN1-dependent cargo proteins. PMID:21795745

  20. Plasmin-induced procoagulant effects in the blood coagulation: a crucial role of coagulation factors V and VIII.

    PubMed

    Ogiwara, Kenichi; Nogami, Keiji; Nishiya, Katsumi; Shima, Midori

    2010-09-01

    Plasminogen activators provide effective treatment for patients with acute myocardial infarction. However, paradoxical elevation of thrombin activity associated with failure of clot lysis and recurrent thrombosis has been reported. Generation of thrombin in these circumstances appears to be owing to plasmin (Plm)-induced activation of factor (F) XII. Plm catalyzes proteolysis of several coagulant factors, but the roles of these factors on Plm-mediated procoagulant activity remain to be determined. Recently developed global coagulation assays were used in this investigation. Rotational thromboelastometry using whole blood, clot waveform analysis and thrombin generation tests using plasma, showed that Plm (> or =125 nmol/l) shortened the clotting times in similar dose-dependent manners. In particular, the thrombin generation test, which was unaffected by products of fibrinolysis, revealed the enhanced coagulation with an approximately two-fold increase of peak level of thrombin generation. Studies using alpha2-antiplasmin-deficient plasma revealed that much lower dose of Plm (> or =16 nmol/l) actually contributed to enhancing thrombin generation. The shortening of clotting time could be observed even in the presence of corn trypsin inhibitor, supporting that Plm exerted the procoagulant activity independently of FXII. In addition, using specific coagulation-deficient plasmas, the clot waveform analysis showed that Plm did not shorten the clotting time in only FV-deficient or FVIII-deficient plasma in prothrombin time-based or activated partial thromboplastin time-based assay, respectively. Our results indicated that Plm did possess procoagulant activity in the blood coagulation, and this effect was likely attributed by multicoagulation factors, dependent on FV and/or FVIII.

  1. Polymorphisms in the F8 Gene and MHC-II Variants as Risk Factors for the Development of Inhibitory Anti-Factor VIII Antibodies during the Treatment of Hemophilia A: A Computational Assessment

    PubMed Central

    Pandey, Gouri Shankar; Yanover, Chen; Howard, Tom E.; Sauna, Zuben E.

    2013-01-01

    The development of neutralizing anti-drug-antibodies to the Factor VIII protein-therapeutic is currently the most significant impediment to the effective management of hemophilia A. Common non-synonymous single nucleotide polymorphisms (ns-SNPs) in the F8 gene occur as six haplotypes in the human population (denoted H1 to H6) of which H3 and H4 have been associated with an increased risk of developing anti-drug antibodies. There is evidence that CD4+ T-cell response is essential for the development of anti-drug antibodies and such a response requires the presentation of the peptides by the MHC-class-II (MHC-II) molecules of the patient. We measured the binding and half-life of peptide-MHC-II complexes using synthetic peptides from regions of the Factor VIII protein where ns-SNPs occur and showed that these wild type peptides form stable complexes with six common MHC-II alleles, representing 46.5% of the North American population. Next, we compared the affinities computed by NetMHCIIpan, a neural network-based algorithm for MHC-II peptide binding prediction, to the experimentally measured values and concluded that these are in good agreement (area under the ROC-curve of 0.778 to 0.972 for the six MHC-II variants). Using a computational binding predictor, we were able to expand our analysis to (a) include all wild type peptides spanning each polymorphic position; and (b) consider more MHC-II variants, thus allowing for a better estimation of the risk for clinical manifestation of anti-drug antibodies in the entire population (or a specific sub-population). Analysis of these computational data confirmed that peptides which have the wild type sequence at positions where the polymorphisms associated with haplotypes H3, H4 and H5 occur bind MHC-II proteins significantly more than a negative control. Taken together, the experimental and computational results suggest that wild type peptides from polymorphic regions of FVIII constitute potential T-cell epitopes and thus

  2. A factor VIII-derived peptide enables von Willebrand factor (VWF)-binding of artificial platelet nanoconstructs without interfering with VWF-adhesion of natural platelets

    NASA Astrophysics Data System (ADS)

    Haji-Valizadeh, Hassan; Modery-Pawlowski, Christa L.; Sen Gupta, Anirban

    2014-04-01

    There is substantial clinical interest in synthetic platelet analogs for potential application in transfusion medicine. To this end, our research is focused on self-assembled peptide-lipid nanoconstructs that can undergo injury site-selective adhesion and subsequently promote site-directed active platelet aggregation, thus mimicking platelet's primary hemostatic actions. For injury site-selective adhesion, we have utilized a coagulation factor FVIII-derived VWF-binding peptide (VBP). FVIII binds to VWF's D'-D3 domain while natural platelet GPIbα binds to VWF's A1 domain. Therefore, we hypothesized that the VBP-decorated nanoconstructs will adhere to VWF without mutual competition with natural platelets. We further hypothesized that the adherent VBP-decorated constructs can enhance platelet aggregation when co-decorated with a fibrinogen-mimetic peptide (FMP). To test these hypotheses, we used glycocalicin to selectively block VWF's A1 domain and, using fluorescence microscopy, studied the binding of fluorescently labeled VBP-decorated nanoconstructs versus platelets to ristocetin-treated VWF. Subsequently, we co-decorated the nanoconstructs with VBP and FMP and incubated them with human platelets to study construct-mediated enhancement of platelet aggregation. Decoration with VBP resulted in substantial construct adhesion to ristocetin-treated VWF even if the A1-domain was blocked by glycocalicin. In comparison, such A1-blocking resulted in significant reduction of platelet adhesion. Without A1-blocking, the VBP-decorated constructs and natural platelets could adhere to VWF concomitantly. Furthermore, the constructs co-decorated with VBP and FMP enhanced active platelet aggregation. The results indicate significant promise in utilizing the FVIII-derived VBP in developing synthetic platelet analogs that do not interfere with VWF-binding of natural platelets but allow site-directed enhancement of platelet aggregation when combined with FMP.There is substantial

  3. More than a decade of international experience with a pdFVIII/VWF concentrate in immune tolerance.

    PubMed

    Santagostino, E

    2013-01-01

    Approximately 20-30% of patients with severe haemophilia A develop alloantibodies ('inhibitors') to infused FVIII rendering use of such replacement therapy ineffective. Once an inhibitor emerges, immune tolerance induction (ITI) is the standard treatment. ITI involves giving regular doses of FVIII concentrate to eradicate the inhibitor and achieve immunogenic acceptance of administered FVIII. In the early 2000s, a retrospective analysis of inhibitor patients treated at a single centre in Germany indicated that success rates were higher when patients were treated with von Willebrand factor (VWF)-containing plasma-derived FVIII (pdFVIII/VWF) concentrate compared with recombinant or non-VWF-containing pdFVIII products. Importantly, pdFVIII/VWF as rescue therapy was able to convert 8 of 10 patients who had failed primary ITI with recombinant or non-VWF-containing pdFVIII product. A subsequent study from Italy in patients with poor prognostic factors for ITI success also reported good success rates with pdFVIII/VWF as rescue therapy (53% success; 41% partial success). The Grifols-Immune Tolerance Induction (G-ITI) Study represents the largest group of haemophilia A inhibitor patients treated with a single pdFVIII/VWF concentrate (Alphanate(®)/Fanhdi(®)) to be reported to date. Data have been collected for 95 patients who underwent primary or rescue ITI at 46 centres in Europe and the US. Currently, published data are available for 33 patients in the US cohort (11 centres), and data from the European cohort are being analysed. Both groups contained patients with poor prognostic factors and most patients received a high-dose regimen (≥ 100 IU pdFVIII/VWF kg(-1) daily). As expected, the success rate was better for primary vs. rescue ITI and for patients with good vs. poor prognostic factors. However, more than half the patients in the US cohort receiving rescue ITI achieved success (33% complete success; 20% partial success). These results should encourage clinicians

  4. Characteristics of hemophilia patients with factor VIII inhibitors detected by prospective screening

    PubMed Central

    Miller, Connie H.; Rice, Anne S.; Boylan, Brian; Payne, Amanda B.; Kelly, Fiona M.; Escobar, Miguel A.; Gill, Joan; Leissinger, Cindy; Soucie, J. Michael

    2015-01-01

    Characteristics of inhibitors identified by prospective screening may differ from those detected clinically. In a prospective study at 17 hemophilia centers with central inhibitor measurement by Nijmegen-Bethesda assay, 23 (2.8%) of 824 hemophilia A patients had new inhibitors detected: nine high-titer inhibitors (HTI: 7 ≥5.0 NBU plus 2 of 2.6 and 3.4 NBU at immune tolerance induction initiation) and 14 low-titer inhibitors (LTI: 0.5–1.9 NBU). HTI occurred at an earlier age (median 2 years, range 1–18, vs. median 11 years, range 2–61, P = 0.016). Both HTI (22%) and LTI (43%) occurred in non-severe patients. All HTI, but only 64% of LTI, were found to be FVIII-specific by chromogenic Bethesda assay or fluorescence immunoassay (FLI), indicating a high rate of false-positive LTI. Repeat specimens confirmed all HTI, 7/9 LTI, and 7/7 FVIII-specific LTI. FLI results were similar between HTI and FVIII-specific LTI; all included IgG1 and IgG4 subclasses. A comparable prospective study conducted from 1975 to 1979 at 13 U.S. centers found 31 (2.4%) new inhibitors among 1,306 patients. In both studies, one-third of inhibitors occurred in non-severe patients and one-quarter after 150 exposure days (ED). Significant differences were seen in the age at which inhibitors occurred (median 16 years in the older study vs. 5 years currently, P = 0.024) and in ED before inhibitor development, 10% in the older study and 43% currently study occurring within 20 ED, suggesting a temporal change in inhibitor development. Prospective screening detects inhibitors in patients of all severities, ages, and ED. Some LTI, however, are false positives. PMID:26147783

  5. Partial correction of a severe molecular defect in hemophilia A, because of errors during expression of the factor VIII gene

    SciTech Connect

    Young, M.; Antonarakis, S.E.; Inaba, Hiroshi

    1997-03-01

    Although the molecular defect in patients in a Japanese family with mild to moderately severe hemophilia A was a deletion of a single nucleotide T within an A{sub 8}TA{sub 2} sequence of exon 14 of the factor VIII gene, the severity of the clinical phenotype did not correspond to that expected of a frameshift mutation. A small amount of functional factor VIII protein was detected in the patient`s plasma. Analysis of DNA and RNA molecules from normal and affected individuals and in vitro transcription/translation suggested a partial correction of the molecular defect, because of the following: (i) DNA replication/RNA transcription errors resulting in restoration of the reading frame and/or (ii) {open_quotes}ribosomal frameshifting{close_quotes} resulting in the production of normal factor VIII polypeptide and, thus, in a milder than expected hemophilia A. All of these mechanisms probably were promoted by the longer run of adenines, A{sub 10} instead of A{sub 8}TA{sub 2}, after the delT. Errors in the complex steps of gene expression therefore may partially correct a severe frameshift defect and ameliorate an expected severe phenotype. 36 refs., 6 figs.

  6. Pasteurized, monoclonal antibody factor VIII concentrate: establishing a new standard for purity and viral safety of plasma-derived concentrates.

    PubMed

    Goldsmith, J C

    2000-03-01

    A factor VIII concentrate (Monoclate-P) manufactured using a combination of pasteurization and immunoaffinity chromatography has been chosen to compare and contrast manufacturing aspects of plasma-derived factor VIII concentrates. Pasteurization is a virucidal method with a long safety record in clinical practice, while immuno-affinity chromatography selectively isolates and purifies the procoagulant protein of factor VIII, and partitions potential viral contaminants and nonessential proteins to the unbound fraction. The complete Monoclate-P production process reduces human immunodeficiency virus by > or = 10.5 log10, Sindbis (a model for hepatitis C virus) by > or = 6.5 log10, and murine encephalomyocarditis virus (a non-enveloped model virus) by 7.1 log10. The viral safety of Monoclate-P has been further demonstrated in clinical studies in patients not previously treated with blood or plasma-derived products. Additionally, the manufacture of Monoclate-P includes careful donor screening and plasma testing for antibodies to syphilis and human immunodeficiency, hepatitis B, and hepatitis C viruses to enhance source plasma safety. Combined with donor selection and plasma testing, multiple viral reduction steps effectively eliminate both lipid-enveloped viruses (e.g. human immunodeficiency, hepatitis B and C) and non-lipid-enveloped viruses (e.g. hepatitis A). In addition, polymerase chain reaction-based nucleic acid detection tests for hepatitis B and C viruses and for human immunodeficiency virus-1 have been introduced as part of an investigational new drug mechanism.

  7. Identification of a novel missense mutation in exon 4 of the human factor VIII gene associated with sever hemophilia A patient.

    PubMed

    Onsori, Habib; Hosseinpour, Mohammad Ali; Montaser-Kouhsari, Sheideh; Asgharzadeh, Mohammad; Hosseinpour, Abbas Ali

    2007-12-01

    Hemophilia A is an X-linked congenital bleeding disorder caused by factor VIII deficiency. The factor VIII gene is on the long arm of the X chromosome at Xq28 spans 186 kb and consists of 26 exons. In this study to identify defects in the factor VIII gene, Single-Stranded Conformation Polymorphism (SSCP) analysis was used. A novel missense mutation due to T --> C transition at codon 153 (TGC) of the factor VIII gene which replace a cysteine with an arginine residue, was found in a patient of North-Western of Iran with sever hemophilia A. Direct sequencing of the amplified fragment was performed to confirm the mutation. This study shows that we can use of Polymerase Chain Reaction (PCR) and silver staining of SSCP methods for detecting most of the point mutations causative hemophilia A.

  8. Pharmacokinetics and safety of a novel recombinant human von Willebrand factor manufactured with a plasma-free method: a prospective clinical trial

    PubMed Central

    Mannucci, Pier Mannuccio; Kempton, Christine; Millar, Carolyn; Romond, Edward; Shapiro, Amy; Birschmann, Ingvild; Ragni, Margaret V.; Gill, Joan Cox; Yee, Thynn Thynn; Klamroth, Robert; Wong, Wing-Yen; Chapman, Miranda; Engl, Werner; Turecek, Peter L.; Suiter, Tobias M.

    2013-01-01

    Safety and pharmacokinetics (PK) of recombinant von Willebrand factor (rVWF) combined at a fixed ratio with recombinant factor VIII (rFVIII) were investigated in 32 subjects with type 3 or severe type 1 von Willebrand disease (VWD) in a prospective phase 1, multicenter, randomized clinical trial. rVWF was well tolerated and no thrombotic events, inhibitors, or serious adverse events were observed. The PK of rVWF ristocetin cofactor activity, VWF antigen, and collagen-binding activity were similar to those of the comparator plasma-derived (pd) VWF-pdFVIII. In vivo cleavage of ultra-large molecular-weight rVWF multimers by ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13; the endogenous VWF protease) and generation of characteristic satellite bands were demonstrated. In 2 subjects with specific nonneutralizing anti-VWF–binding antibodies already detectable before rVWF infusion, a reduction in VWF multimers and VWF activity was observed. Stabilization of endogenous FVIII was enhanced following post–rVWF-rFVIII infusion as shown by the difference in area under the plasma concentration curve compared with pdVWF-pdFVIII (AUC0-∞) (P < .01). These data support the concept of administering rVWF alone once a therapeutic level of endogenous FVIII is achieved. This trial was registered at www.clinicaltrials.gov as #NCT00816660. PMID:23777763

  9. Structure of the factor VIII C2 domain in a ternary complex with 2 inhibitor antibodies reveals classical and nonclassical epitopes.

    PubMed

    Walter, Justin D; Werther, Rachel A; Brison, Caileen M; Cragerud, Rebecca K; Healey, John F; Meeks, Shannon L; Lollar, Pete; Spiegel, P Clint

    2013-12-19

    The factor VIII C2 domain is a highly immunogenic domain, whereby inhibitory antibodies develop following factor VIII replacement therapy for congenital hemophilia A patients. Inhibitory antibodies also arise spontaneously in cases of acquired hemophilia A. The structural basis for molecular recognition by 2 classes of anti-C2 inhibitory antibodies that bind to factor VIII simultaneously was investigated by x-ray crystallography. The C2 domain/3E6 FAB/G99 FAB ternary complex illustrates that each antibody recognizes epitopes on opposing faces of the factor VIII C2 domain. The 3E6 epitope forms direct contacts to the C2 domain at 2 loops consisting of Glu2181-Ala2188 and Thr2202-Arg2215, whereas the G99 epitope centers on Lys2227 and also makes direct contacts with loops Gln2222-Trp2229, Leu2261-Ser2263, His2269-Val2282, and Arg2307-Gln2311. Each binding interface is highly electrostatic, with positive charge present on both C2 epitopes and complementary negative charge on each antibody. A new model of membrane association is also presented, where the 3E6 epitope faces the negatively charged membrane surface and Arg2320 is poised at the center of the binding interface. These results illustrate the potential complexities of the polyclonal anti-factor VIII immune response and further define the "classical" and "nonclassical" types of antibody inhibitors against the factor VIII C2 domain.

  10. Frequencies of VNTR and RFLP polymorphisms associated with factor VIII gene in Singapore

    SciTech Connect

    Fong, I.; Lai, P.S.; Ouah, T.C.

    1994-09-01

    The allelic frequency of any polymorphism within a population determines its usefulness for genetic counselling. This is important in populations of non-Caucasian origin as RFLPs may significantly differ among ethnic groups. We report a study of five intragenic polymorphisms in factor VIII gene carried out in Singapore. The three PCR-based RFLP markers studied were Intron 18/Bcl I, Intron 19/Hind III and Intron 22/Xba I. In an analysis of 148 unrelated normal X chromosomes, the allele frequencies were found to be A1 = 0.18, A2 = 0.82 (Bcl I RFLP), A1 = 0.80, A2 = 0.20 (Hind III RFLP) and A1 = 0.58, and A2 = 0.42 (Xba I RFLP). The heterozygosity rates of 74 females analyzed separately were 31%, 32% and 84.2%, respectively. Linkage disequilibrium was also observed to some degree between Bcl I and Hind III polymorphism in our population. We have also analyzed a sequence polymorphism in Intron 7 using hybridization with radioactive-labelled {sup 32}P allele-specific oligonucleotide probes. This polymorphism was not very polymorphic in our population with only 2% of 117 individuals analyzed being informative. However, the use of a hypervariable dinucleotide repeat sequence (VNTR) in Intron 13 showed that 25 of our of 27 (93%) females were heterozygous. Allele frequencies ranged from 1 to 55 %. We conclude that a viable strategy for molecular analysis of Hemophilia A families in our population should include the use of Intron 18/Bcl I and Intron 22/Xba I RFLP markers and the Intron 13 VNTR marker.

  11. Joint bleeding in factor VIII deficient mice causes an acute loss of trabecular bone and calcification of joint soft tissues which is prevented with aggressive factor replacement

    PubMed Central

    Lau, Anthony G.; Sun, Junjiang; Hannah, William B.; Livingston, Eric W.; Heymann, Dominique; Bateman, Ted A.; Monahan, Paul E.

    2015-01-01

    Introduction While chronic degenerative arthropathy is the main morbidity of hemophilia, a very high prevalance of low bone density is also seen in men and boys with hemophilia. The current study investigates bone degradation in the knee joint of hemophilic mice resulting from hemarthrosis and the efficacy of aggressive treatment with factor VIII in the period surrounding injury to prevent bone pathology. Methods Skeletally mature factor VIII knock-out mice were subjected to knee joint hemorrhage induced by puncture of the left knee joint capsule. Mice received either intravenous Factor VIII treatment or placebo immediately prior to injury and at hours 4, 24, 48, 72 and 96 after hemorrhage. Mice were euthanized two-weeks after injury and the joint morphology and loss of bone in the proximal tibia was assessed using microCT imaging. Results Quantitative microCT imaging of the knee joint found acute bone loss at the proximal tibia following injury including loss of trabecular bone volumetric density and bone mineral density, as well as trabecular connectivity density, number, and thickness. Unexpectedly, joint injury also resulted in calcification of the joint soft tissues including the tendons, ligaments, menisci, and cartilage. Treatment with factor VIII prevented this bone and soft tissue degeneration. Conclusion Knee joint hemorrhage resulted in acute changes of adjacent bone including loss of bone density and mineralization of joint soft tissues. The rapid calcification and loss of bone has implications for the initiation and progression of osteoarthritic degradation following joint bleeding. PMID:24712867

  12. Severe Hemophilia A in a Male Old English Sheep Dog with a C→T Transition that Created a Premature Stop Codon in Factor VIII

    PubMed Central

    Lozier, Jay N; Kloos, Mark T; Merricks, Elizabeth P; Lemoine, Nathaly; Whitford, Margaret H; Raymer, Robin A; Bellinger, Dwight A; Nichols, Timothy C

    2016-01-01

    Animals with hemophilia are models for gene therapy, factor replacement, and inhibitor development in humans. We have actively sought dogs with severe hemophilia A that have novel factor VIII mutations unlike the previously described factor VIII intron 22 inversion. A male Old English Sheepdog with recurrent soft-tissue hemorrhage and hemarthrosis was diagnosed with severe hemophilia A (factor VIII activity less than 1% of normal). We purified genomic DNA from this dog and ruled out the common intron 22 inversion; we then sequenced all 26 exons. Comparing the results with the normal canine factor VIII sequence revealed a C→T transition in exon 12 of the factor VIII gene that created a premature stop codon at amino acid 577 in the A2 domain of the protein. In addition, 2 previously described polymorphisms that do not cause hemophilia were present at amino acids 909 and 1184. The hemophilia mutation creates a new TaqI site that facilitates rapid genotyping of affected offspring by PCR and restriction endonuclease analyses. This mutation is analogous to the previously described human factor VIII mutation at Arg583, which likewise is a CpG dinucleotide transition causing a premature stop codon in exon 12. Thus far, despite extensive treatment with factor VIII, this dog has not developed neutralizing antibodies (‘inhibitors’) to the protein. This novel mutation in a dog gives rise to severe hemophilia A analogous to a mutation seen in humans. This model will be useful for studies of the treatment of hemophilia. PMID:27780008

  13. Pharmacokinetics, efficacy, and safety of a plasma-derived VWF/FVIII concentrate (VONCENTO) for on-demand and prophylactic treatment in patients with von Willebrand disease (SWIFT-VWD study).

    PubMed

    Lissitchkov, Toshko J; Buevich, Evgeny; Kuliczkowski, Kazimierz; Stasyshyn, Oleksandra; Cerqueira, Monica Hermida; Klukowska, Anna; Joch, Christine; Seifert, Wilfried

    2017-03-01

    VONCENTO (CSL Behring Gmbh, Marburg, Germany) is a plasma-derived, high concentration, lower volume [relative to HAEMATE P (CSL Behring)], high-purity von Willebrand factor (VWF)/factor VIII (FVIII) concentrate with a VWF/FVIII ratio similar to HAEMATE P. This open-label, multicentre study investigated the pharmacokinetic, haemostatic efficacy, and safety profiles of VONCENTO in study participants at least 12 years of age with von Willebrand disease (VWD) who required treatment of nonsurgical bleeding (NSB) events or underwent surgery or prophylaxis. The first 12-month on-demand treatment period comprised a pharmacokinetic investigation and an efficacy analysis. After 12 months, qualifying study participants were switched to prophylactic therapy and included in a further 12-month efficacy analysis. In total, 21 study participants (including three adolescents, and 13 study participants with VWD type 3) received VONCENTO as on-demand treatment for 12 months. 'Excellent'/'good' haemostatic efficacy was achieved in 98.3% of the 407 NSB events assessed by investigators. Following the switch to prophylactic treatment, the total number of NSBs in eight patients markedly decreased from 304 to 10 (with haemostatic efficacy judged to be 'excellent' for all). The annualised bleeding rate also significantly decreased from a median of 26.5 events to one event. Safety assessments showed no inhibitory antibodies to either FVIII or VWF, no transmission of infectious agents, no thromboembolic events and no treatment-related serious adverse events. VONCENTO was shown to be well tolerated and provided excellent haemostatic efficacy in the treatment of bleeds or during prophylaxis in study participants with VWD, including also those with type 3, the severest form of VWD.

  14. Pharmacokinetics, efficacy, and safety of a plasma-derived VWF/FVIII concentrate (VONCENTO) for on-demand and prophylactic treatment in patients with von Willebrand disease (SWIFT-VWD study)

    PubMed Central

    Lissitchkov, Toshko J.; Buevich, Evgeny; Kuliczkowski, Kazimierz; Stasyshyn, Oleksandra; Cerqueira, Monica Hermida; Klukowska, Anna; Joch, Christine; Seifert, Wilfried

    2017-01-01

    VONCENTO (CSL Behring Gmbh, Marburg, Germany) is a plasma-derived, high concentration, lower volume [relative to HAEMATE P (CSL Behring)], high-purity von Willebrand factor (VWF)/factor VIII (FVIII) concentrate with a VWF/FVIII ratio similar to HAEMATE P. This open-label, multicentre study investigated the pharmacokinetic, haemostatic efficacy, and safety profiles of VONCENTO in study participants at least 12 years of age with von Willebrand disease (VWD) who required treatment of nonsurgical bleeding (NSB) events or underwent surgery or prophylaxis. The first 12-month on-demand treatment period comprised a pharmacokinetic investigation and an efficacy analysis. After 12 months, qualifying study participants were switched to prophylactic therapy and included in a further 12-month efficacy analysis. In total, 21 study participants (including three adolescents, and 13 study participants with VWD type 3) received VONCENTO as on-demand treatment for 12 months. ‘Excellent’/‘good’ haemostatic efficacy was achieved in 98.3% of the 407 NSB events assessed by investigators. Following the switch to prophylactic treatment, the total number of NSBs in eight patients markedly decreased from 304 to 10 (with haemostatic efficacy judged to be ‘excellent’ for all). The annualised bleeding rate also significantly decreased from a median of 26.5 events to one event. Safety assessments showed no inhibitory antibodies to either FVIII or VWF, no transmission of infectious agents, no thromboembolic events and no treatment-related serious adverse events. VONCENTO was shown to be well tolerated and provided excellent haemostatic efficacy in the treatment of bleeds or during prophylaxis in study participants with VWD, including also those with type 3, the severest form of VWD. PMID:27203734

  15. Therapeutic and routine prophylactic properties of rFactor VIII Fc (efraloctocog alfa, Eloctate®) in hemophilia A

    PubMed Central

    Chowdary, Pratima; Fosbury, Emma; Riddell, Anne; Mathias, Mary

    2016-01-01

    rFVIIIFc (efraloctocog alfa, Eloctate®) is an extended half-life (EHL) factor VIII licensed for use in patients with hemophilia A for prophylaxis and treatment of bleeding and surgical episodes. Pharmacokinetic studies in adults have shown a mean 1.5-fold increase in half-life compared to full-length factor VIII. When compared to adults, the half-life is decreased by 8% in adolescents between 12 and 17 years, by 18% in children 6 to <12 years, and by 33% in children between the ages of 2 and <6 years. There is a considerable interindividual variation in the prolongation of the half-life particularly in children and across the age groups, the range extending from no increase to a 2.5-fold increase. In addition to age, von willebrand factor (VWF) antigen level has demonstrated a significant impact on rFVIIIFc half-life, with higher VWF levels associated with greater prolongation of half-life. The pivotal and pediatric clinical trials have demonstrated the efficacy and safety of rFVIIIFc for use in regular prophylaxis and in management of bleeds and surgery. In these studies, just under half the participants showed a zero annualized bleed rate (ABR), and the median ABR (1.6 in the pivotal study for the individualized prophylaxis arm) showed a further decrease in the extension study. On average, the patients required fewer infusions (reduced by at least a third), and the mean weekly consumption seems to be in keeping with standard recombinant factor VIII. EHL rFVIIIFc has made decreased infusion frequency a possibility. However, the interindividual variability in dose and infusion frequency highlights the need for a personalized approach based on individual patient’s half-life and/or response to treatment. PMID:27695377

  16. TGF-β1 along with other platelet contents augments Treg cells to suppress anti-FVIII immune responses in hemophilia A mice

    PubMed Central

    Haribhai, Dipica; Luo, Xiaofeng; Chen, Juan; Jia, Shuang; Shi, Linzheng; Schroeder, Jocelyn A.; Weiler, Hartmut; Aster, Richard H.; Hessner, Martin J.; Hu, Jianda; Williams, Calvin B.; Shi, Qizhen

    2017-01-01

    Platelets are a rich source of many cytokines and chemokines including transforming growth factor β 1 (TGF-β1). TGF-β1 is required to convert conventional CD4+ T (Tconv) cells into induced regulatory T (iTreg) cells that express the transcription factor Foxp3. Whether platelet contents will affect Treg cell properties has not been explored. In this study, we show that unfractionated platelet lysates (pltLys) containing TGF-β1 efficiently induced Foxp3 expression in Tconv cells. The common Treg cell surface phenotype and in vitro suppressive activity of unfractionated pltLys-iTreg cells were similar to those of iTreg cells generated using purified TGF-β1 (purTGFβ-iTreg) cells. However, there were substantial differences in gene expression between pltLys-iTreg and purTGFβ-iTreg cells, especially in granzyme B, interferon γ, and interleukin-2 (a 30.99-, 29.18-, and 17.94-fold difference, respectively) as determined by gene microarray analysis. In line with these gene signatures, we found that pltLys-iTreg cells improved cell recovery after transfer and immune suppressive function compared with purTGFβ-iTreg cells in factor VIII (FVIII)–deficient (F8null, hemophilia A model) mice after recombinant human FVIII (rhF8) infusion. Acute antibody-mediated platelet destruction in F8null mice followed by rhF8 infusion increased the number of Treg cells and suppressed the antibody response to rhF8. Consistent with these data, ex vivo proliferation of F8-specific Treg cells from platelet-depleted animals increased when restimulated with rhF8. Together, our data suggest that pltLys-iTreg cells may have advantages in emerging clinical applications and that platelet contents impact the properties of iTreg cells induced by TGF-β1. PMID:28164173

  17. Analysis of the cell death-inducing ability of the ethylene response factors in group VIII of the AP2/ERF family.

    PubMed

    Ogata, Takuya; Kida, Yuma; Tochigi, Mayuko; Matsushita, Yasuhiko

    2013-08-01

    The ethylene response factor (ERF) family is one of the largest families of plant-specific transcription factors. We have shown previously that the overexpression of the gene for NtERF3, a tobacco transcriptional repressor containing the ERF-associated amphiphilic repression (EAR) motif in the C-terminal region, induces hypersensitive reaction (HR)-like cell death. Many EAR motif-containing ERFs, including NtERF3, are clustered in group VIII of the ERF family. In this study, we aimed at revealing the cell death-inducing ability of group VIII ERFs and the correlation between ERFs and HR. The results showed that many of the EAR motif-containing ERFs classified into subgroup VIII-a of Arabidopsis, rice, and tobacco had cell death-inducing ability in tobacco leaves. Seven AtERFs in subgroup VIII-b did not induce cell death; however, some ERFs in subgroup VIII-b of rice and tobacco showed cell death-inducing ability. An expression analysis of group VIII ERFs in HR-inducing tobacco suggested that the cell death-inducing ability of NtERFs was not necessarily associated with induction of HR. In addition, it was revealed that the EAR motif-containing AtERFs in subgroup II-a also showed cell death-inducing ability. The influence of sequence variation in the EAR motif on the ability to induce cell death is also discussed.

  18. Hemostatic efficacy, safety, and pharmacokinetics of a recombinant von Willebrand factor in severe von Willebrand disease

    PubMed Central

    Gill, Joan C.; Castaman, Giancarlo; Windyga, Jerzy; Kouides, Peter; Ragni, Margaret; Leebeek, Frank W. G.; Obermann-Slupetzky, Ortrun; Chapman, Miranda; Fritsch, Sandor; Pavlova, Borislava G.; Presch, Isabella

    2015-01-01

    This phase 3 trial evaluated the safety and hemostatic efficacy of a recombinant von Willebrand factor (rVWF) for treatment of bleeds in severe von Willebrand disease (VWD). rVWF was initially administered together with recombinant factor VIII (rFVIII) and subsequently alone, as long as hemostatic factor VIII activity (FVIII:C) levels were maintained. Pharmacokinetics (PK) were evaluated in a randomized cross-over design (rVWF vs rVWF:rFVIII at 50 IU VWF:ristocetin cofactor activity [RCo]/kg). Bleed control for all treated bleeds (N = 192 bleeds in 22 subjects) was rated good or excellent (96.9% excellent; 119 of 122 minor, 59 of 61 moderate, and 6 of 7 major bleeds) on a 4-point scale (4 = none to 1 = excellent). A single infusion was effective in 81.8% of bleeds. Treatment success, defined as the number of subjects with a mean efficacy rating of <2.5, was 100%. The PK profile of rVWF was not influenced by rFVIII (mean VWF:RCo terminal half-life: 21.9 hours for rVWF and 19.6 hours for rVWF:rFVIII). FVIII:C levels increased rapidly after rVWF alone, with hemostatic levels achieved within 6 hours and sustained through 72 hours after infusion. Eight adverse events (AEs; 6 nonserious AEs in 4 subjects and 2 serious AEs [chest discomfort and increased heart rate, without cardiac symptomatology] concurrently in 1 subject) were associated with rVWF. There were no thrombotic events or severe allergic reactions. No VWF or FVIII inhibitors, anti-VWF binding antibodies, or antibodies against host cell proteins were detected. These results show that rVWF was safe and effective in treating bleeds in VWD patients and stabilizes endogenous FVIII:C, which may eliminate the need for rFVIII after the first infusion. This trial was registered at www.clinicaltrials.gov as #NCT01410227. PMID:26239086

  19. Hemostatic efficacy, safety, and pharmacokinetics of a recombinant von Willebrand factor in severe von Willebrand disease.

    PubMed

    Gill, Joan C; Castaman, Giancarlo; Windyga, Jerzy; Kouides, Peter; Ragni, Margaret; Leebeek, Frank W G; Obermann-Slupetzky, Ortrun; Chapman, Miranda; Fritsch, Sandor; Pavlova, Borislava G; Presch, Isabella; Ewenstein, Bruce

    2015-10-22

    This phase 3 trial evaluated the safety and hemostatic efficacy of a recombinant von Willebrand factor (rVWF) for treatment of bleeds in severe von Willebrand disease (VWD). rVWF was initially administered together with recombinant factor VIII (rFVIII) and subsequently alone, as long as hemostatic factor VIII activity (FVIII : C) levels were maintained. Pharmacokinetics (PK) were evaluated in a randomized cross-over design (rVWF vs rVWF:rFVIII at 50 IU VWF:ristocetin cofactor activity [RCo]/kg). Bleed control for all treated bleeds (N = 192 bleeds in 22 subjects) was rated good or excellent (96.9% excellent; 119 of 122 minor, 59 of 61 moderate, and 6 of 7 major bleeds) on a 4-point scale (4 = none to 1 = excellent). A single infusion was effective in 81.8% of bleeds. Treatment success, defined as the number of subjects with a mean efficacy rating of <2.5, was 100%. The PK profile of rVWF was not influenced by rFVIII (mean VWF:RCo terminal half-life: 21.9 hours for rVWF and 19.6 hours for rVWF:rFVIII). FVIII : C levels increased rapidly after rVWF alone, with hemostatic levels achieved within 6 hours and sustained through 72 hours after infusion. Eight adverse events (AEs; 6 nonserious AEs in 4 subjects and 2 serious AEs [chest discomfort and increased heart rate, without cardiac symptomatology] concurrently in 1 subject) were associated with rVWF. There were no thrombotic events or severe allergic reactions. No VWF or FVIII inhibitors, anti-VWF binding antibodies, or antibodies against host cell proteins were detected. These results show that rVWF was safe and effective in treating bleeds in VWD patients and stabilizes endogenous FVIII : C, which may eliminate the need for rFVIII after the first infusion. This trial was registered at www.clinicaltrials.gov as #NCT01410227.

  20. Crystal Structure of the Bovine lactadherin C2 Domain, a Membrane Binding Motif, Shows Similarity to the C2 Domains of Factor V and Factor VIII

    SciTech Connect

    Lin,L.

    2007-01-01

    Lactadherin, a glycoprotein secreted by a variety of cell types, contains two EGF domains and two C domains with sequence homology to the C domains of blood coagulation proteins factor V and factor VIII. Like these proteins, lactadherin binds to phosphatidylserine (PS)-containing membranes with high affinity. We determined the crystal structure of the bovine lactadherin C2 domain (residues 1 to 158) at 2.4 {angstrom}. The lactadherin C2 structure is similar to the C2 domains of factors V and VIII (rmsd of C{sub {alpha}} atoms of 0.9 {angstrom} and 1.2 {angstrom}, and sequence identities of 43% and 38%, respectively). The lactadherin C2 domain has a discoidin-like fold containing two {beta}-sheets of five and three antiparallel {beta}-strands packed against one another. The N and C termini are linked by a disulfide bridge between Cys1 and Cys158. One {beta}-turn and two loops containing solvent-exposed hydrophobic residues extend from the C2 domain {beta}-sandwich core. In analogy with the C2 domains of factors V and VIII, some or all of these solvent-exposed hydrophobic residues, Trp26, Leu28, Phe31, and Phe81, likely participate in membrane binding. The C2 domain of lactadherin may serve as a marker of cell surface phosphatidylserine exposure and may have potential as a unique anti-thrombotic agent.

  1. Crystal Structure of the Bovine lactadherin C2 Domain, a Membrane Binding Motif, Shows Similarity of the C2 Domains of Factor V and Factor VIII

    SciTech Connect

    Lin,L.; Huai, Q.; Huang, M.; Furie, B.; Furie, B.

    2007-01-01

    Lactadherin, a glycoprotein secreted by a variety of cell types, contains two EGF domains and two C domains with sequence homology to the C domains of blood coagulation proteins factor V and factor VIII. Like these proteins, lactadherin binds to phosphatidylserine (PS)-containing membranes with high affinity. We determined the crystal structure of the bovine lactadherin C2 domain (residues 1 to 158) at 2.4 Angstroms. The lactadherin C2 structure is similar to the C2 domains of factors V and VIII (rmsd of C? atoms of 0.9 Angstroms and 1.2 Angstroms, and sequence identities of 43% and 38%, respectively). The lactadherin C2 domain has a discoidin-like fold containing two ?-sheets of five and three antiparallel ?-strands packed against one another. The N and C termini are linked by a disulfide bridge between Cys1 and Cys158. One ?-turn and two loops containing solvent-exposed hydrophobic residues extend from the C2 domain ?-sandwich core. In analogy with the C2 domains of factors V and VIII, some or all of these solvent-exposed hydrophobic residues, Trp26, Leu28, Phe31, and Phe81, likely participate in membrane binding. The C2 domain of lactadherin may serve as a marker of cell surface phosphatidylserine exposure and may have potential as a unique anti-thrombotic agent.

  2. Influence of heat treatment on FVIII:C recovery from freeze dried cryoprecipitate.

    PubMed Central

    Benny, A G; Ockelford, P A; Johns, A S; Scott, R H; Woodfield, D G; Berry, E W

    1988-01-01

    A standard lyophilised triple cryoprecipitate preparation, stabilised by the addition of Synthamin 17, was heat treated at 60 degrees C for 48 hours. The total protein content, factor VIII concentration, and factor VIII recovery were not affected by the heat treatment procedure. Heat treatment did not influence the reconstitution characteristics of the freeze dried preparation and there were no side effects during or after administration. The mean in vivo rise of factor VIII from infused heat treated triple cryoprecipitate was 2.5 (SD 0.9)%/unit/kg with a half life of 13.1 (3.1) hours. These results compare favourably with those obtained using non-heated triple cryoprecipitate. Cryoprecipitate can be heat treated without adversely influencing factor VIII recovery, and the ability to prepare a heat treated cryoprecipitate means that a small pool high yield factor VIII preparation can again be used in routine clinical practice. PMID:3142936

  3. Recurrent myocardial infarctions in a young football player secondary to thrombophilia, associated with elevated factor VIII activity

    PubMed Central

    Vacek, Thomas P; Yu, Shipeng; Rehman, Shahnaz; Grubb, Blair P; Kosinski, Daniel; Verghese, Cherian; Eltahawy, Ehab A; Shafiq, Qaiser

    2014-01-01

    Myocardial infarction (MI) due to coronary atherosclerosis in young adults is uncommon; rare causes such as cocaine abuse, arterial dissection, and thromboembolism should be considered. A 21-year-old football player, and otherwise healthy African American man, developed chest pain during exercise while bench-pressing 400 lbs. Acute MI was diagnosed based on physical examination, electrocardiography findings, and elevated cardiac enzymes. Coronary arteriography showed a thrombus occluding the proximal left anterior descending artery (LAD). Aggressive antiplatelet therapy with aspirin, clopidogrel, and eptifibatide was pursued, in addition to standard post-MI care. This led to the successful resolution of symptoms and dissolution of the thrombus, demonstrated by repeat coronary arteriography. Five months later, he presented with similar symptoms during exercise after lifting heavy weights, and was found to have another acute MI. Coronary arteriography again showed a thrombus occluding the LAD. No evidence of coronary artery dissection or vasospasm was found. Only mild atherosclerotic plaque burden was observed on both occasions by intravascular ultrasound. A bare metal stent was placed at the site as it was thought this site had acted as a nidus for small plaque rupture and thrombus formation. Elevated serum factor VIII activity at 205% (reference range 60%–140%) was found, a rare cause of hypercoagulability. Further workup revealed a patent foramen ovale during a Valsalva maneuver by transesophageal echocardiography. Both events occurred during weight lifting, which can transiently increase right heart pressure in a similar way to the Valsalva maneuver. In light of all the findings, we concluded that an exercise-related increase in factor VIII activity led to coronary arterial thrombosis in the presence of a small ruptured plaque. Alternatively, venous clots may have traversed the patent foramen ovale and occluded the LAD. In addition to continuing aggressive risk

  4. Staining for factor VIII related antigen and Ulex europaeus agglutinin I (UEA-I) in 230 tumours. An assessment of their specificity for angiosarcoma and Kaposi's sarcoma.

    PubMed

    Leader, M; Collins, M; Patel, J; Henry, K

    1986-11-01

    In this study we examined the staining reactivity of commercially available antisera to factor VIII related antigen (F VIII RAg) and Ulex europaeus agglutinin I (UEA-I) on sections from 230 formalin fixed paraffin embedded tumours. These included 196 sarcomas, 20 carcinomas and 14 angiomas. All angiomas showed positive staining for F VIII RAg; all carcinomas showed negative staining; the vasoformative areas of all angiosarcomas stained positively but only four of six angiosarcomas showed positive staining of their solid areas; of seven Kaposi's sarcomas, all showed positive staining of vessels and six showed positive staining of the spindle cell component. In the remaining 181 non-vascular sarcomas there was a false positive result in four tumours (2.2%), three of which had a history of irradiation. Pre-radiotherapy biopsies of these three tumours stained negatively with anti-F VIII RAg. UEA-I was demonstrated in all the angiomas studied, in all angiosarcomas (including the solid components) and in well-formed vessels of all Kaposi's sarcomas, but only in the spindle cell component of 3/6. However, there was an unacceptably high rate of false positive staining amongst the carcinomas and non-vascular sarcomas. In conclusion, F VIII RAg is a specific but not a sensitive marker of angiosarcomas; UEA-I is a sensitive but not a specific marker of angiosarcomas.

  5. Tolerogenic nanoparticles to induce immunologic tolerance: Prevention and reversal of FVIII inhibitor formation.

    PubMed

    Zhang, Ai-Hong; Rossi, Robert J; Yoon, Jeongheon; Wang, Hong; Scott, David W

    2016-03-01

    The immune response of hemophilia A patients to administered FVIII is a major complication that obviates this very therapy. We have recently described the use of synthetic, biodegradable nanoparticles carrying rapamycin and FVIII peptide antigens, to induce antigen-specific tolerance. Herein we test the tolerogenicity of nanoparticles that contains full length FVIII protein in hemophilia A mice, focusing on anti-FVIII humoral immune response. As expected, recipients of tolerogenic nanoparticles remained unresponsive to FVIII despite multiple challenges for up to 6 months. Furthermore, therapeutic treatments in FVIII-immunized mice with pre-existing anti-FVIII antibodies resulted in diminished antibody titers, albeit efficacy required longer therapy with the tolerogenic nanoparticles. Interestingly, durable FVIII-specific tolerance was also achieved in animals co-administered with FVIII admixed with nanoparticles encapsulating rapamycin alone. These results suggest that nanoparticles carrying rapamycin and FVIII can be employed to induce specific tolerance to prevent and even reverse inhibitor formation.

  6. Improvement of virus safety of a S/D-treated factor VIII concentrate by additional dry heat treatment at 100 degrees C.

    PubMed

    Dichtelmüller, H; Rudnick, D; Breuer, B; Kotitschke, R; Kloft, M; Darling, A; Watson, E; Flehmig, B; Lawson, S; Frösner, G

    1996-06-01

    In order to increase the virus safety of a solvent/detergent-treated Factor VIII concentrate in regard to non-lipid coated viruses and to respond to the continuous discussion about reports on hepatitis A transmission by Factor VIII preparations, we have investigated the effect of a terminal dry heat treatment (30 min 100 degrees C) on HAV and various other viruses. By this treatment Hepatitis A virus was inactivated below detectable level after a few minutes (> 5.3 log10). Other RNA viruses such as the Human Immunodeficiency Virus (> 6.6 log10), bovine viral diarrhoea virus (> 6.6 log10) and vesicular stomatitis virus (> 5.8 log10) were also inactivated below detectable level. Pseudo rabies virus and reovirus Type 3 are inactivated by 5.7 and > 6.0 log10, respectively. SV40 and bovine parvo virus showed significant resistance to dry heat treatment. We conclude that the involvement of two strong virus inactivation steps, acting by different mechanisms, improves the virus safety of Factor VIII concentrates without destroying the Factor VIII activity. Moreover, the terminal 100 degrees C heat treatment for 30 min represents an effective measure to inactivate non-lipid enveloped viruses, in particular hepatitis A, which is resistant to solvent/detergent treatment.

  7. Association of Single Nucleotide Polymorphisms in the ST3GAL4 Gene with VWF Antigen and Factor VIII Activity

    PubMed Central

    Song, Jaewoo; Xue, Cheng; Preisser, John S.; Cramer, Drake W.; Houck, Katie L.; Liu, Guo; Folsom, Aaron R.; Couper, David; Yu, Fuli; Dong, Jing-fei

    2016-01-01

    VWF is extensively glycosylated with biantennary core fucosylated glycans. Most N-linked and O-linked glycans on VWF are sialylated. FVIII is also glycosylated, with a glycan structure similar to that of VWF. ST3GAL sialyltransferases catalyze the transfer of sialic acids in the α2,3 linkage to termini of N- and O-glycans. This sialic acid modification is critical for VWF synthesis and activity. We analyzed genetic and phenotypic data from the Atherosclerosis Risk in Communities (ARIC) study for the association of single nucleotide polymorphisms (SNPs) in the ST3GAL4 gene with plasma VWF levels and FVIII activity in 12,117 subjects. We also analyzed ST3GAL4 SNPs found in 2,535 subjects of 26 ethnicities from the 1000 Genomes (1000G) project for ethnic diversity, SNP imputation, and ST3GAL4 haplotypes. We identified 14 and 1,714 ST3GAL4 variants in the ARIC GWAS and 1000G databases respectively, with 46% being ethnically diverse in their allele frequencies. Among the 14 ST3GAL4 SNPs found in ARIC GWAS, the intronic rs2186717, rs7928391, and rs11220465 were associated with VWF levels and with FVIII activity after adjustment for age, BMI, hypertension, diabetes, ever-smoking status, and ABO. This study illustrates the power of next-generation sequencing in the discovery of new genetic variants and a significant ethnic diversity in the ST3GAL4 gene. We discuss potential mechanisms through which these intronic SNPs regulate ST3GAL4 biosynthesis and the activity that affects VWF and FVIII. PMID:27584569

  8. The prevalence of factor VIII and IX inhibitors among Saudi patients with hemophilia: Results from the Saudi national hemophilia screening program.

    PubMed

    Owaidah, Tarek; Momen, Abdulkareem Al; Alzahrani, Hazzaa; Almusa, Abdulrahman; Alkasim, Fawaz; Tarawah, Ahmed; Nouno, Randa Al; Batniji, Fatima Al; Alothman, Fahad; Alomari, Ali; Abu-Herbish, Saud; Abu-Riash, Mahmoud; Siddiqui, Khawar; Ahmed, Mansor; Mohamed, S Y; Saleh, Mahasen

    2017-01-01

    Hemophilia A and B are X-linked diseases that predominantly affect male patients. Patients can develop coagulation factor inhibitors, which exponentially increases the treatment cost. However, the prevalence of factor VIII and IX inhibitors in Saudi Arabia is unclear.This study aimed to determine the Saudi prevalence of factor VIII and IX inhibitors.This 4-year, 7-center, cross-sectional study evaluated the Saudi prevalences of hemophilia A and B. We collected the patients' clinical data, evaluated their disease, and tested for factor inhibitors.We included 202 patients with hemophilia (median age at diagnosis: 0.13 years, range: birth-34.8 years). The patients included 198 male patients (98%), 148 patients with hemophilia A (73.3%), and 54 patients with hemophilia B (26.7%). The patients exhibited severe factor VIII activity (<1%; 121 patients; 5.2%), moderate activity (1-5%; 7 patients; 4.9%), and mild activity (14 patients; 9.9%). Among the patients with care-related data, most patients were treated for episodic bleeding (76.8%) or received prophylaxis (22.6%); 1 patient received both treatments. Among the patients with source-related data, the factor replacements were derived from plasma (48.4%), recombinant concentrates (22.9%), both sources (14.6%), or fresh frozen plasma (14.1%). Factor VIII inhibitors were observed in 43 (29.3%) of the 147 patients, and only 1 of the 54 patients developed factor IX inhibitors. Most patients who developed inhibitors had severe hemophilia (40/44; 90.9%), and inhibitors were also common among patients who received recombinant products (14/43; 32.6%).The Saudi prevalence of factor inhibitors was similar to those among other ethnic populations.

  9. Factors Associated with the Time of Admission among Notified Dengue Fever Cases in Region VIII Philippines from 2008 to 2014

    PubMed Central

    Gil Cuesta, Julita; Cerro, Boyd Roderick; Guha-Sapir, Debarati

    2016-01-01

    In cases of Dengue fever, late hospital admission can lead to treatment delay and even death. In order to improve early disease notification and management, it is essential to investigate the factors affecting the time of admission of Dengue cases. This study determined the factors associated with the time of admission among notified Dengue cases. The study covered the period between 2008 and 2014 in Region VIII, Philippines. The factors assessed were age, sex, hospital sector, hospital level, disease severity based on the 1997 WHO Dengue classification, and period of admission (distinguishing between the 2010 Dengue epidemic and non-epidemic time). We analysed secondary data from the surveillance of notified Dengue cases. We calculated the association through chi-square test, ordinal logistic regression and linear regression at p value < 0.05. The study included 16,357 admitted Dengue cases. The reported cases included a majority of children (70.09%), mild cases of the disease (64.00%), patients from the public sector (69.82%), and non-tertiary hospitals (62.76%). Only 1.40% of cases had a laboratory confirmation. The epidemic period in 2010 comprised 48.68% of all the admitted cases during this period. Late admission was more likely among adults than children (p<0.05). The severe type of the disease was more likely to be admitted late than the mild type (p<0.05). Late admission was also more likely in public hospitals than in private hospitals (p<0.05); and within tertiary level hospitals than non-tertiary hospitals (p<0.05). Late admission was more likely during the non-epidemic period than the 2010 epidemic period (p<0.05). A case fatality rate of 1 or greater was significantly associated with children, severe diseases, tertiary hospitals and public hospitals when admitted late (p<0.05). Data suggests that early admission among child cases was common in Region VIII. This behavior is encouraging, and should be continued. However, further study is needed on the

  10. Diagnosis and management challenges in patients with mild haemophilia A and discrepant FVIII measurements.

    PubMed

    Trossaert, M; Lienhart, A; Nougier, C; Fretigny, M; Sigaud, M; Meunier, S; Fouassier, M; Ternisien, C; Negrier, C; Dargaud, Y

    2014-07-01

    Thirty per cent of patients with mild haemophilia A (MHA) present markedly different FVIII: C level when assayed by one-stage clotting and two-stage chromogenic assays. It is, therefore, a real clinical challenge to predict the individual bleeding risk of these patients. The aim of the present work was to study the relationship between the bleeding tendency of these patients with the results of a panel of phenotypic and genotypic tools. Thirty-six patients with MHA were included in this multicentre prospective clinical study. The severity of bleeding symptoms was evaluated using the ISTH/SSC score. FVIII:C levels were measured using an activated partial thromboplastin time-based one-stage FVIII assay (FVIII: C1) and three commercial chromogenic kits (FVIII:CR). FVIII antigen levels, thrombin generation measurement and FVIII gene mutation analysis were also performed. Our results showed that a one-stage FVIII: C assay cannot rule out the diagnosis of MHA, a combined use of FVIII:C1 with a FVIII:CR is suitable for detecting MHA. We observed that FVIII:CR results better reflected the clinical bleeding tendency of patients compared to FVIII:C1. We also observed a relationship between thrombin generation (TG) capacity and FVIII:CR of these patients. FVIII gene mutation analysis showed mutations previously reported in MHA patients with discrepant FVIII:C measurements, but with no predictive value of the individual bleeding phenotype of patients. Overall, we observed a relationship between chromogenic FVIII:C results, TG assay and bleeding tendency of patients with discrepant FVIII:C measurements, while FVIII:C1 was not well correlated with clinical bleeding phenotype in this particular population.

  11. Coagulation factor V and VIII/V ratio as predictors of outcome in paracetamol induced fulminant hepatic failure: relation to other prognostic indicators.

    PubMed Central

    Pereira, L M; Langley, P G; Hayllar, K M; Tredger, J M; Williams, R

    1992-01-01

    The value of coagulation factor V and VIII/V levels as prognostic indicators was assessed in 27 patients with fulminant hepatic failure and compared with other predictive indices. Admission factor V levels were significantly reduced in 22 patients with paracetamol induced hepatic failure compared with a healthy control group (median 9.5% v 103%, respectively; p less than 0.001) and with lower values in non-A non-B hepatitis (median 2.7%). Values in the seven patients who died after paracetamol overdose, considered together with the four who underwent liver transplantation (group median 5.1%), were significantly lower than in the 11 who survived (median 11.8%; p less than 0.01). Median admission factor VIII was higher in those who died or received a transplant than in those who survived (298% v 162%; p less than 0.05), with both results higher than in healthy volunteers (median 104%; p less than 0.01) but lower than in non-A non-B hepatitis (median 340%). The ratio of factor VIII/V on admission was less than 30 in all patients who survived paracetamol overdose (median 17) with corresponding values greater than 30 in 10 of 11 of those who died (median 39). A factor V result less than or equal to 10% on admission predicted an adverse outcome in 10 of 11 fatal cases, a 91% sensitivity which was greater than for the previously defined indicator of an arterial blood pH less than 7.30 on admission (sensitivity 82%). Prothrombin time at admission or on day 4 did not usefully predict outcome in our series. Predictive accuracy was 73% and 82% for factor V and admission acidosis respectively and 95% for factor V in conjunction with admission coma grade III or IV and factor VIII (ratio > 30). These criteria may be useful in selecting patients with paracetamol induced fulminant hepatic failure for transplantation. PMID:1740285

  12. Phenotypic Correction of Hemophilia A in Sheep by Postnatal Intraperitoneal Transplantation of FVIII-Expressing MSC

    PubMed Central

    Porada, Christopher D.; Sanada, Chad; Kuo, Chung-Jung; Colletti, Evan; Mandeville, Walter; Hasenau, John; Zanjani, Esmail D.; Moot, Robert; Doering, Christopher; Spencer, H. Trent; Almeida-Porada, Graça

    2011-01-01

    We recently re-established a line of sheep that accurately mimics the clinical symptoms and genetics of severe hemophilia A (HA). Herein, we tested a novel, non-ablative transplant therapy in 2 pediatric HA animals. Paternal mesenchymal stem cells (MSC) were transduced with a porcine FVIII-encoding lentivector, and transplanted via the intraperitoneal route, without preconditioning. At the time of transplantation, these animals had received multiple hFVIII treatments for various spontaneous bleeds, and had developed debilitating hemarthroses which produced severe defects in posture and gait. Transplantation of transduced MSC resolved all existent hemarthroses, and spontaneous bleeds ceased. Damaged joints recovered fully; the animals regained normal posture and gait and resumed normal activity. Despite achieving factor-independence, a sharp rise in pre-existent Bethesda titers occurred following transplantation, decreasing the effectiveness and duration of therapy. Post-mortem examination revealed widespread engraftment, with MSC present within the lung, liver, intestine, and thymus, but particularly within joints affected at the time of transplantation, suggesting MSC homed to sites of ongoing injury/inflammation to release FVIII, explaining the dramatic improvement in hemarthrotic joints. In summary, this novel, non-ablative MSC transplantation was straightforward, safe, and converted life-threatening, debilitating HA to a moderate phenotype in a large animal model. PMID:21906573

  13. Evaluation of the expression of VIII factor and VEGF in the regeneration of non-vital teeth in dogs using propolis

    PubMed Central

    Zarei, Mina; Jafarian, Amir Hossein; Harandi, Azadeh; Javidi, Maryam; Gharechahi, Maryam

    2017-01-01

    Objective(s): The purpose of the present study was the immunohistochemical evaluation of VEGF and VII factors in dog’s teeth pulp revascularized with MTA and propolis. Materials and Methods: 144 mature and immature two rooted dog’s premolar canals were selected. Pulp necrosis and infection were established after 2 weeks and the disinfection of the canals was done with copious NaOCl irrigation and triantibiotic mixture (ciprofloxacin, metronidazole, and minocycline) for 3 weeks. Subsequently, the blood clot was evoked in the canal by periapical tissue irritation with a k-file. The samples were randomly divided into 6 experimental groups: propolis (groups 1, 2), MTA (groups 3, 4), and parafilm (groups 5, 6) in immature and mature teeth. The animals were sacrificed and samples were prepared for immunohistochemical evaluation of VEGF and the VIII factor. Results: Tissue regeneration was seen in 64.5% of MTA, 38% of propolis, and 0% of parafilm group samples. Expression of VEGF and VIII factor in the propolis group was more than the MTA group and it showed a reduction after 3 months in comparison to 1 month. VEGF and VIII factor were seen in stromal cells in addition to endothelial vessel cells. Overall, expression of angiogenic factors was more in the open apex teeth compared to close apex ones. Conclusion: According to the results of this study, propolis can induce the expression of VEGF and VIII factor in infected mature and immature dog’s teeth and is a suitable biomaterial for the revascularization technique. PMID:28293394

  14. Factor VIII alloantibody inhibitors: cost analysis of immune tolerance induction vs. prophylaxis and on-demand with bypass treatment.

    PubMed

    Earnshaw, S R; Graham, C N; McDade, C L; Spears, J B; Kessler, C M

    2015-05-01

    Development of inhibitors (alloantibodies to exogenous factor VIII) is the most significant treatment complication in patients with haemophilia A. The only proven way to eradicate inhibitors is through immune tolerance induction (ITI), while bypassing agents are typically employed to treat or prevent bleeds in patients with high titre inhibitors. Costs of these approaches have not been well studied. The aim of this study was to compare lifetime costs of treating patients with severe haemophilia A with inhibitors using on-demand or prophylaxis treatment with bypassing agents and ITI. A decision-analytic model was developed to compare the treatment costs and outcomes. Quantitation of the reduction in bleeding events for patients on prophylaxis and after eradication of inhibitors when on ITI and relapse of inhibitors was derived from published studies. Costs were obtained from standard US costing sources and are reported in 2014 US dollars. Costs and outcomes were discounted 3% per annum. Lifetime costs of treating patients with inhibitors are lower for ITI vs. on-demand or prophylaxis. Patients are also projected to live longer, have greater quality-adjusted life-years, and have fewer bleeding events than patients treated on-demand. Treating patients via ITI to eradicate inhibitors may result in lower lifetime costs and greater life-years and quality-adjusted life-years than treating with bypassing agents.

  15. Cloning, purification, crystallization and preliminary X-ray studies of RFC boxes II-VIII of replication factor C from Methanococcus jannaschii.

    PubMed

    Lee, Ick; Lokanath, Neratur K; Min, Kyeongsik; Ha, Sung Chul; Kim, Dong Young; Kim, Kyeong Kyu

    2002-03-01

    Replication factor C (RFC) is the accessory protein required to load the proliferating cell nuclear antigen (PCNA) onto DNA in replication process. RFC is composed of several subunits and each subunit contains the highly conserved sequences RFC boxes II-VIII. RFC boxes II-VIII of the large subunit of replication factor C from Methanococcus jannaschii has been overexpressed in Escherichia coli, purified and crystallized at 295 K using ammonium sulfate as precipitant. Crystals belong to the space group R32, with unit-cell parameters a = b = 238.23 (5), c = 73.17 (12) A. Native data were collected at 100 K to a resolution of 3.2 A using a synchrotron-radiation source.

  16. Evaluation of an AutoAnalyzer method for quantitating anti-A and anti-B haemagglutinins in factor VIII preparations.

    PubMed Central

    Bowell, P J; Abdalla, S; Snape, T J; Gunson, H H

    1980-01-01

    The continuous flow principle employed in the Technicon AutoAnalyzer has been adapted for the assay of anti-A and anti-B. The method has an acceptable degree of reproducibility and has been used, principally, for the quantitation of anti-A and anti-B in factor VIII concentrates of intermediate activity. It is, however, a method that can be applied to the assay of these antibodies in serum samples. PMID:6776152

  17. Evaluation of von Willebrand factor phenotypes and genotypes in Hemophilia A patients with and without identified F8 mutations

    PubMed Central

    Boylan, Brian; Rice, Anne S.; De Staercke, Christine; Eyster, M. Elaine; Yaish, Hassan M.; Knoll, Christine M.; Bean, Christopher J.; Miller, Connie H.

    2015-01-01

    Summary Background Hemophilia A (HA) is an X-linked bleeding disorder caused by a deficiency in factor VIII (FVIII). von Willebrand disease (VWD) is characterized by a quantitative or qualitative defect in von Willebrand Factor (VWF). Patients with VWD with severely low VWF or VWD Type 2N (VWD2N), a VWD subtype distinguished by defective VWF binding to FVIII, may have reduced FVIII levels secondary to their VWD. These patients superficially resemble patients with HA, and pose a potential for misdiagnosis. Objectives Investigate the unexplained cause of bleeding in HA patients without known FVIII mutations by assessing plasma VWF antigen (VWF:Ag), FVIII binding capacities, and VWF genotypes. Patients/Methods Thirty-seven of 1027 patients with HA studied as part of the Hemophilia Inhibitor Research Study lacked identifiable F8 mutations. These patients (cases) and 73 patients with identified F8 mutations (controls) were evaluated for VWF:Ag, patient's VWF capacity to bind FVIII (VWF:FVIIIB), and VWF sequence. Results Four cases had VWF:Ag <3 IU/dL and VWF mutations consistent with Type3 VWD. Six cases and one control were heterozygous for mutations previously reported to cause Type1 VWD (VWD1) (n=5 cases and 1 control) or predicted to be deleterious by Polyphen2 and SIFT prediction tools (n=1 case). One control had VWF:Ag <30 IU/dl, and seven patients (4 cases and 3 controls), including two cases who were heterozygous for a known VWD2N mutation, had reduced VWF:FVIIIB. Conclusions These data emphasize that some patients diagnosed with HA require VWF assessments in order to achieve a comprehensive diagnosis and an optimal treatment strategy. PMID:25780857

  18. Missense mutations near the N-glycosylation site of the A2 domain lead to various intracellular trafficking defects in coagulation factor VIII

    PubMed Central

    Wei, Wei; Zheng, Chunlei; Zhu, Min; Zhu, Xiaofan; Yang, Renchi; Misra, Saurav; Zhang, Bin

    2017-01-01

    Missense mutation is the most common mutation type in hemophilia. However, the majority of missense mutations remain uncharacterized. Here we characterize how hemophilia mutations near the unused N-glycosylation site of the A2 domain (N582) of FVIII affect protein conformation and intracellular trafficking. N582 is located in the middle of a short 310-helical turn (D580-S584), in which most amino acids have multiple hemophilia mutations. All 14 missense mutations found in this 310-helix reduced secretion levels of the A2 domain and full-length FVIII. Secreted mutants have decreased activities relative to WT FVIII. Selected mutations also lead to partial glycosylation of N582, suggesting that rapid folding of local conformation prevents glycosylation of this site in wild-type FVIII. Protease sensitivity, stability and degradation of the A2 domain vary among mutants, and between non-glycosylated and glycosylated species of the same mutant. Most of the mutants interact with the ER chaperone BiP, while only mutants with aberrant glycosylation interact with calreticulin. Our results show that the short 310-helix from D580 to S584 is critical for proper biogenesis of the A2 domain and FVIII, and reveal a range of molecular mechanisms by which FVIII missense mutations lead to moderate to severe hemophilia A. PMID:28327546

  19. Severe hemophilia A in a female by cryptic translocation: Order and orientation of factor VIII within Xq28

    SciTech Connect

    Migeon, B.R.; McGinniss, M.J.; Antonarakis, S.E.; Axelman, J.; Stasiowski, B.A.; Youssoufian, H.; Kearns, W.G.; Chung, A.; Pearson, P.L.; Kazazian, H.H. Jr. ); Muneer, R.S. )

    1993-04-01

    The authors report studies of a female with severe hemophilia A resulting from a complex de novo translocation of chromosomes X and 17 (46,X,t(X; 17)). Somatic cell hybrids containing the normal X, the der(X), or the der(17) were analyzed for coagulation factor VIII (F8C) sequences using Southern blots and polymerase chain reaction. The normal X, always late replicating, contains a normal F8C gene, whereas the der(X) has no F8C sequences. The der(17) chromosome containing Xq24-Xq28 carries a functional G6PD locus and a deleted F8C allele that lacks exons 1--15. Also, it lacks the DXYS64-X locus, situated between the F8C locus and the Xq telomere. These results indicate that a cryptic breakpoint within Xq28 deleted the 5[prime] end of F8C, but left the more proximal G6PD locus intact on the der(17)chromosome. As the deleted segment includes the 5[prime] half of F8C as well as the subtelomeric DXYS64 locus, F8C must be oriented on the chromosome with its 5[prime] region closest to the telomere. Therefore, the order of these loci is Xcen-G6PD-3[prime]F8C-5[prime]F8C-DXYS64-Xqtel. The analysis of somatic cell hybrids has elucidated the true nature of the F8C mutation in the pro-band, revealing a more complex rearrangement (three chromosomes involved) than that expected from cytogenetic analysis, chromosome painting, and Southern blots. A 900-kb segment within Xq28 has been translocated to another autosome. Hemophilia A in this heterozygous female is due to the decapitation of the F8C gene on the der(17) and inactivation of the intact allele on the normal X. 27 refs., 5 figs., 1 tab.

  20. Factor VIII (F8) inversions in severe hemophilia A: Male germ cell origin and diagnosis with RT-PCR

    SciTech Connect

    Antonarakis, S.E. |; Rossiter, J.P.; Young, M.

    1994-09-01

    The Factor VIII (F8) gene, which is defective in hemophilia A, is located in the most telomeric megabase of Xq. Inversions due to intrachromosomal homologous recombination between mispaired copies of gene A located within intron 22 of the gene and about 500 kb telomeric to it account for nearly half of the cases of severe hemophilia A. We hypothesized that pairing of Xq with its homolog inhibits the inversion process, and that therefore the event originates predominantly in male germ cells. In all 21 informative cases in which the inversion originated in a maternal grandparent, DNA polymorphism analysis using markers within or very closely linked to F8, determined that it occurred in the male germline. In addition, all but one of 56 mothers of sporadic cases due to inversions were carriers. The data indicate that the F8 gene inversions leading to severe hemophilia A occur almost exclusively in male germ cells. The mean age of maternal grandfathers at the birth of their carrier daughters was 29.9 years (13 cases), i.e. not different from the mean paternal age in the general population, supporting the hypothesis that the inversions occur in meiosis. The inversions can be diagnosed by Southern blot analysis. For more rapid diagnosis we have used RT-PCR of RNA ectopically expressed in blood. Oligonucleotides were used to PCR amplify, after the initial RT reaction of RNA samples using random hexamers, either the normal transcript (F8 exons 21 to 24;312 bp product) or the novel abnormal transcript that is generated after the inversion. Both type 1 and 2 inversions can be recognized in affecteds and carriers by the presence of the diagnostic PcR product of 248 bp. Correct diagnoses were made in samples from 6 patients and 2 carriers with type 1 inversions, 2 patients and 2 carriers with type 2 inversions and 5 normal controls.

  1. Long-term course of anti-factor VIII antibody in patients with hemophilia A at a single center

    PubMed Central

    Joo, Sang Chun; Choi, Yong Mook

    2016-01-01

    Background Immune tolerance induction (ITI) can reduce inhibitors against factor VIII concentrates by 70-80%. In this study, we elucidated the characteristics of inhibitors and attempted to determine the proper indications and timing for ITI. Methods Subjects included hemophilia A patients registered at the Korea Hemophilia Foundation from 1991 through 2014. Inhibitors were classified as persistent and transient. Patients were classified into groups according to peak inhibitor titer: low (<2 BU/mL), moderate (2 to <5 BU/mL), high (5 to <10 BU/mL), and very high titer (≥10 BU/mL). Results Overall, 350 (21.4%) of 1,634 hemophilia A patients developed inhibitors at least once. Of these, 100 (6.1%) and 250 (15.3%) patients developed persistent and transient inhibitors, respectively. For transient inhibitors, the median peak titer was 1.0 BU/mL, persistent for median of 11.0 months (10.0, 8.0, 13.0, and 19.0 months in the low, moderate, high, and very high titer transient inhibitor groups, respectively). Overall, 95.8% (215), 72.2% (17), 52.4% (21), and 21.7% (97) of patients in the low, moderate, high, and very high titer groups became inhibitor-negative spontaneously, without ITI. Conclusion Given the spontaneous disappearance of inhibitors and high cost of ITI, it is worthwhile to postpone ITI for 11 months unless the peak inhibitor titer is greater than 10 BU/mL. PMID:27104190

  2. Factor VIII delivery devices in haemophilia A. Barriers and drivers for treatment adherence.

    PubMed

    Fernández-Arias, Isabel; Kim, Hae Kyung

    2016-11-01

    Introducción y objetivo: Recabar la experiencia de pacientes con hemofilia A con sus dispositivos de reconstitucion de factor de coagulacion, barreras para la adherencia y determinar sus preferencias, presentando una nueva jeringa de doble camara (JDC). Método: Investigacion transversal mediante encuesta dirigida y sesion de prueba de la JDC. Resultados: Participaron 74 pacientes, el 50% en tratamiento con profilaxis, y 7 anos (mediana) con su tratamiento habitual (RIC 17,25). En la encuesta, la JDC recibio la mayor puntuacion (75/100, p < 0,001) y la mayor probabilidad de uso en profilaxis (p < 0,001). En la sesion practica (n = 29), el 62,1% prefirio la JDC y necesitaron de mediana 43 segundos (24,5-82) para la preparacion, vs. 4 minutos (1-15) con el tratamiento habitual (p < 0,001). La opinion favorable del medico respecto a la profilaxis resulto muy influyente en la actitud de los participantes hacia la adherencia (OR = 1,324, IC 95% = 1,040-1,685, p = 0,023). Conclusiones: La JDC fue el dispositivo preferido y se mostro con probabilidad de favorecer la profilaxis.

  3. Establishment of reference intervals for von Willebrand factor antigen and eight coagulation factors in a Korean population following the Clinical and Laboratory Standards Institute guidelines.

    PubMed

    Jang, Ja-Hyun; Seo, Ja-Young; Bang, Sung-Hwan; Park, In-Ae; Kim, Hee-Jin; Kim, Sun-Hee

    2010-04-01

    Establishment of reference intervals for coagulation molecules is important but is costly and sometimes not feasible. Since reference intervals from manufacturers or the literature are mostly out of date or involved Western populations, the authors determined reference intervals for VWF: Ag and eight factors in a Korean population. VWF: Ag, factor VIII (FVIII), FII, FV, FVII, FIX, FX, FXI, and FXII were determined in Korean individuals visiting for routine checkup following the CLSI (Clinical and Laboratory Standards Institute) guidelines. Reagents by Diagnostica Stago were used on the STA Compact Analyzer (Diagnostica Stago). Exclusion criteria were medical history or laboratory findings that could affect the factor levels. Influence of demographic factors was analyzed. Mean +/- 2 x SD or central 95 percentile was used, as appropriate. We obtained data from 266 adults for VWF: Ag, 371 adults for FVIII, and minimum 136 adults for the rest. Reference interval for VWF was 51-176% (52-155% in blood group O and 71-186% for non-O). Reference interval for FVIII was 64-197% (55-150% in O and 77-205% in non-O). Reference interval for FII was 77-121%, FV 81-160%, FVII 68-149%, FIX 67-154%, FX 69-126%, FXI 59-138%, and FXII 48-177%. The medians of VWF: Ag, FVIII, and FIX were significantly higher in the elderly group (> or =60 years). We established local reference intervals for VWF: Ag and eight coagulation factors in a Korean population according to the CLSI guidelines. Significantly, different reference intervals were obtained in blood group O vs. non-O for VWF: Ag and FVIII. The reference intervals obtained in this study could be adopted in other clinical laboratories after appropriate validation.

  4. The 1.7 Å X-ray crystal structure of the porcine factor VIII C2 domain and binding analysis to anti-human C2 domain antibodies and phospholipid surfaces.

    PubMed

    Brison, Caileen M; Mullen, Steven M; Wuerth, Michelle E; Podolsky, Kira; Cook, Matthew; Herman, Jacob A; Walter, Justin D; Meeks, Shannon L; Spiegel, P Clint

    2015-01-01

    The factor VIII C2 domain is essential for binding to activated platelet surfaces as well as the cofactor activity of factor VIII in blood coagulation. Inhibitory antibodies against the C2 domain commonly develop following factor VIII replacement therapy for hemophilia A patients, or they may spontaneously arise in cases of acquired hemophilia. Porcine factor VIII is an effective therapeutic for hemophilia patients with inhibitor due to its low cross-reactivity; however, the molecular basis for this behavior is poorly understood. In this study, the X-ray crystal structure of the porcine factor VIII C2 domain was determined, and superposition of the human and porcine C2 domains demonstrates that most surface-exposed differences cluster on the face harboring the "non-classical" antibody epitopes. Furthermore, antibody-binding results illustrate that the "classical" 3E6 antibody can bind both the human and porcine C2 domains, although the inhibitory titer to human factor VIII is 41 Bethesda Units (BU)/mg IgG versus 0.8 BU/mg IgG to porcine factor VIII, while the non-classical G99 antibody does not bind to the porcine C2 domain nor inhibit porcine factor VIII activity. Further structural analysis of differences between the electrostatic surface potentials suggest that the C2 domain binds to the negatively charged phospholipid surfaces of activated platelets primarily through the 3E6 epitope region. In contrast, the G99 face, which contains residue 2227, should be distal to the membrane surface. Phospholipid binding assays indicate that both porcine and human factor VIII C2 domains bind with comparable affinities, and the human K2227A and K2227E mutants bind to phospholipid surfaces with similar affinities as well. Lastly, the G99 IgG bound to PS-immobilized factor VIII C2 domain with an apparent dissociation constant of 15.5 nM, whereas 3E6 antibody binding to PS-bound C2 domain was not observed.

  5. Factor VIII assay

    MedlinePlus

    ... P, Lillicrap D. Hemophilia A and B. In: Hoffman R, Benz EJ Jr, Silberstein LE, Heslop HE, ... Laboratory evaluation of hemostatic and thrombotic disorders. In: Hoffman R, Benz EJ Jr, Silberstein LE, Heslop HE, ...

  6. Development of inhibitory antibodies to therapeutic factor VIII in severe hemophilia A is associated with microsatellite polymorphisms in the HMOX1 promoter.

    PubMed

    Repessé, Yohann; Peyron, Ivan; Dimitrov, Jordan D; Dasgupta, Suryasarathi; Moshai, Elika Farrokhi; Costa, Catherine; Borel-Derlon, Annie; Guillet, Benoit; D'Oiron, Roseline; Aouba, Achille; Rothschild, Chantal; Oldenburg, Johannes; Pavlova, Anna; Kaveri, Srinivas V; Lacroix-Desmazes, Sébastien

    2013-10-01

    Induction of heme oxygenase-1, a stress-inducible enzyme with anti-inflammatory activity, reduces the immunogenicity of therapeutic factor VIII in experimental hemophilia A. In humans, heme oxygenase-1 expression is modulated by polymorphisms in the promoter of the heme oxygenase-1-encoding gene (HMOX1). We investigated the relationship between polymorphisms in the HMOX1 promoter and factor VIII inhibitor development in severe hemophilia A. We performed a case-control study on 99 inhibitor-positive patients and 263 patients who did not develop inhibitors within the first 150 cumulative days of exposure to therapeutic factor VIII. Direct sequencing and DNA fragment analysis were used to study (GT)n polymorphism and single nucleotide polymorphisms located at -1135 and -413 in the promoter of HMOX1. We assessed associations between the individual allele frequencies or genotypes, and inhibitor development. Our results demonstrate that inhibitor-positive patients had a higher frequency of alleles with large (GT)n repeats (L: n≥30), which are associated with lesser heme oxygenase-1 expression (odds ratio 2.31; 95% confidence interval 1.46-3.66; P<0.001]. Six genotypes (L/L, L/M, L/S, M/M, M/S and S/S) of (GT)n repeats were identified (S: n<21; M: 21≤n<30). The genotype group including L alleles (L/L, L/M and L/S) was statistically more frequent among inhibitor-positive than inhibitor-negative patients, as compared to the other genotypes (33.3% versus 17.1%) (odds ratio 2.21, 95% confidence interval 1.30-3.76; P<0.01). To our knowledge, this is the first association identified between HMOX1 promoter polymorphism and development of anti-drug antibodies. Our study paves the way towards modulation of the endogenous anti-inflammatory machinery of hemophilia patients to reduce the risk of inhibitor development.

  7. Protein modification during anti-viral heat-treatment bioprocessing of factor VIII concentrates, factor IX concentrates, and model proteins in the presence of sucrose.

    PubMed

    Smales, C Mark; Pepper, Duncan S; James, David C

    2002-01-05

    To ensure the optimal safety of plasma derived and new generation recombinant proteins, heat treatment is customarily applied in the manufacturing of such biopharmaceuticals as a means of viral inactivation. In subjecting proteins to anti-viral heat-treatment it is necessary to use high concentrations of thermostabilizing excipients to prevent protein damage, and it is therefore imperative that the correct balance between bioprocessing conditions, maintenance of protein integrity and virus kill is found. In this study we have utilized model proteins (lysozyme, fetuin, and human serum albumin) and plasma-derived therapeutic proteins (factor VIII and factor IX) to investigate the protein modifications that occur during anti-viral heat treatment. Specifically, we investigated the relationship between bioprocessing conditions and the type and extent of protein modification under a variety of industrially relevant wet and lyophilized heat treatments using sucrose as a thermostabilizing agent. Heat treatment led to the formation of disulfide crosslinks and aggregates in proteins containing free cysteine residues. Terminal oligosaccharide sialic acid residues were hydrolyzed from the glycan moieties of glycoproteins during anti-viral heat treatment. Heat treatment promoted sucrose hydrolysis to yield glucose and fructose, leading, in turn, to the glycation of lysine amino groups in those proteins containing di-lysine motifs. During extended hear treatments, 1,2-dicarbonyl type advanced glycation end-products were also formed. Glycation-type modifications were more prevalent in wet heat-treated protein formulations.

  8. Molecular characterization of severe hemophilia A suggests that about half the mutations are not within the coding regions and splice junctions of the factor VIII gene

    SciTech Connect

    Higuchi, Miyoko; Kazazian, H.H. Jr.; Kasch, L.; Warren, T.C.; McGinniss, M.J.; Antonarakis, S.E. ); Phillips, J.A. III; Janco, R. ); Kasper, C. )

    1991-08-15

    Hemophilia A is an X chromosome-linked disorder resulting from deficiency of factor VIII, an important protein in blood coagulation. A large number of disease-producing mutations have been reported in the factor VIII gene. However, a comprehensive analysis of the mutations has been difficult because of the larger gene size, its many scattered exons, and the high frequency of de novo mutations. Recently, the authors have shown that nearly all mutations resulting in mild-to-moderate hemophilia A can be detected by PCR and denaturing gradient gel electrophoresis (DGGE). In this study, they attempted to discover the mutations causing severe hemophilia A by analyzing 47 unselected patients, 30 of whom had severe hemophilia and 17 of whom had mild-to-moderate disease. Using DGGE as a screening method, they analyzed 99% of the coding region, 94% of the splice junctions, the promoter region, and the polyadenylylation site of the gene. They found the mutation in 16 of 17 (94%) patients with mild-to-moderate disease but in only 16 of 30 (53%) patients with severe hemophilia A.

  9. Immune regulatory gene polymorphisms as predisposing risk factors for the development of factor VIII inhibitors in Indian severe haemophilia A patients.

    PubMed

    Pinto, P; Ghosh, K; Shetty, S

    2012-09-01

    Development of inhibitors to factor VIII, a serious complication of replacement therapy in haemophilia A patients, leads to increased bleeding, morbidity and mortality. There is no data on the risk factors for inhibitor development in Indian patients with severe haemophilia A. Our aim was to study the role of immune regulatory gene polymorphisms in the development of inhibitors. Fourteen immune regulatory gene polymorphisms (IL1β, IL4, IL10, TNFA and CTLA4) were analysed in 120 patients with severe haemophilia A, i.e. 50 inhibitor positive patients, and 70 inhibitor negative control patients, by PCR-RFLP, DNA sequencing and allele-specific PCRs. The IL10 promoter 'GCC' haplotypes overall (P: 0.002, OR: 3.452, 95% CI: 1.607-7.416), and 'GCC/ATA' (P: 0.011, OR: 3.492, 95% CI: 1.402-8.696) haplotype, associated with high and intermediate IL10 production, respectively, were significantly higher in inhibitor positive patients, whereas the 'non-GCC' haplotypes overall (P: 0.002,OR: 0.290, 95% CI 0.135-0.622) and 'ATA/ATA' haplotype (P: 0.025, OR: 0.278, 95% CI: 0.096-0.802), associated with low IL10 synthesis, were significantly higher among inhibitor negative patients. The TNFA rs1799724 C/T heterozygote prevalence was significantly higher in the inhibitor positive group (P: 0.021, OR: 3.190, 95% CI: 1.273-7.990), whereas the other polymorphisms showed no statistically significant association with the presence of inhibitors. Different immune regulatory gene polymorphisms play a significant role as possible risk factors for the development of inhibitors in severe haemophilia A patients.

  10. Milk fat globule epidermal growth factor-factor VIII is down-regulated in sepsis via the lipopolysaccharide-CD14 pathway.

    PubMed

    Komura, Hidefumi; Miksa, Michael; Wu, Rongqian; Goyert, Sanna M; Wang, Ping

    2009-01-01

    Phagocytosis prevents the release of potentially harmful or immunogenic materials from dying cells. Milk fat globule epidermal growth factor (EGF)-factor VIII (MFG-E8) mediates the clearance of apoptotic cells. We have previously shown that the administration of MFG-E8-rich exosomes from immature dendritic cells promotes the phagocytosis of apoptotic cells and improves survival in sepsis. Because endotoxin is elevated in polymicrobial sepsis, we hypothesized that down-regulation of MFG-E8 is mediated via the LPS-CD14 pathway, eventually leading to the accruement of apoptotic cells. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) in CD14-deficient (CD14(-/-)), TLR4-mutated and wild-type (WT) mice. In addition, endotoxemia was elicited by i.p. injection of LPS. LPS was also neutralized by pretreating CLP-induced WT mice with polymyxin B. Splenic MFG-E8 expression, phagocytic activity, and apoptosis were assessed 5 and 20 h after CLP or 5 h after LPS administration. In septic WT mice, MFG-E8 mRNA and protein levels were suppressed by 49 and 33%, respectively. Endotoxemia reduced MFG-E8 mRNA expression in a dose dependent manner and the down-regulation of MFG-E8 mRNA expression in CLP-induced sepsis was attenuated by polymyxin B. This CLP-induced suppression was not observed in both CD14(-/-) and TLR4-mutated mice. CLP significantly decreased phagocytic activity of peritoneal macrophages in WT (by 30%), but not in CD14(-/-) mice. CLP also induced significant apoptosis in the spleen of WT (by 61%), but less in CD14(-/-) mice. Thus, MFG-E8 production is down-regulated in sepsis by LPS-CD14 dependent fashion, leading to a reduction of phagocytosis of apoptotic cells.

  11. Non-genetic risk factors in haemophilia A inhibitor management - the danger theory and the use of animal models.

    PubMed

    Lövgren, K M; Søndergaard, H; Skov, S; Wiinberg, B

    2016-09-01

    In haemophilia A (HA) management, antidrug antibodies, or inhibitors, are a serious complication that renders factor VIII (FVIII) replacement therapy ineffective, increases morbidity and reduces quality of life for affected patients. Inhibitor development aetiology is multifactorial and covers both genetic and therapy related risk factors. Many therapy-related risk factors have proven difficult to confirm due to several confounding factors and the small study populations available. However, clinical studies indicate that e.g. on-demand treatment and surgery affect inhibitor development, and explanations for this association are being investigated. A potential explanation is the danger signal effect, where the immune response is activated by endogenous or exogenous danger or damage signals present at the time and site of FVIII administration. The danger theory explains how alarm signals from stressed, injured or dying cells can activate an immune reaction, without the involvement of foreign antigens. Bleeds, trauma, surgery or concomitant infection could be events initiating danger signalling in HA patients, resulting in an immune reaction towards administered FVIII that otherwise would pass unnoticed. This role of danger in HA inhibitor formation has previously been suggested, but a thorough discussion of this subject is lacking. The present review will discuss the potential role of danger signals in haemophilia and inhibitor development, with focus on treatment related risk factors with a suspected danger signal aetiology; on-demand treatment, treatment during major bleeds or surgery, and treatment during infection or vaccination. Clinical studies as well as animal experiments addressing these factors will be reviewed.

  12. Factor 8 (F8) gene mutation profile of Turkish hemophilia A patients with inhibitors.

    PubMed

    Fidanci, Inanç D; Kavakli, Kaan; Uçar, Canan; Timur, Cetin; Meral, Adalet; Kilinç, Yurdanur; Sayilan, Hülya; Kazanci, Elif; Cağlayan, S Hande

    2008-07-01

    Factor VIII (FVIII) replacement therapy is ineffective in hemophilia A patients who develop alloantibodies (inhibitors) against FVIII. The type of factor 8 (F8) gene mutation, genes in the major histocompatibility complex loci, and also polymorphisms in IL-10 and tumor necrosis factor-alpha are the major predisposing factors for inhibitor formation. The present study was initiated to reveal the F8 gene mutation profile of 30 severely affected high-responder patients with inhibitor levels of more than 5 Bethesda U (BU)/ml and four low-responder patients with inhibitors less than 5 BU/ml. Southern blot and PCR analysis were performed to detect intron 22 and intron 1 inversions, respectively. Point mutations were screened by DNA sequence analysis of all coding regions, intron/exon boundaries, promoter and 3' UTR regions of the F8 gene. The prevalent mutation was the intron 22 inversion among the high-responder patients followed by large deletions, small deletions, and nonsense mutations. Only one missense and one splicing error mutation was seen. Among the low-responder patients, three single nucleotide deletions and one intron 22 inversion were found. All mutation types detected were in agreement with the severe hemophilia A phenotype, most likely leading to a deficiency of and predisposition to the development of alloantibodies against FVIII. It is seen that Turkish hemophilia A patients with major molecular defects have a higher possibility of developing inhibitors.

  13. Patient preference and ease of use for different coagulation factor VIII reconstitution device scenarios: a cross-sectional survey in five European countries

    PubMed Central

    Cimino, Ernesto; Linari, Silvia; Malerba, Mara; Halimeh, Susan; Biondo, Francesca; Westfeld, Martina

    2014-01-01

    Introduction Hemophilia A treatment involves replacing the deficient coagulation factor VIII. This process may involve multiple steps that might create a barrier to adherence. A new dual-chamber syringe (DCS; FuseNGo®) was recently introduced with the aim of simplifying reconstitution. Aim This study aimed to identify factors associated with adult patients’ preferences for different coagulation factor VIII reconstitution systems and to test ease of use and patient preference for the DCS. Methods A cross-sectional survey of adults with hemophilia A in five European countries was conducted; a subset of subjects also participated in a practical testing session of the DCS. Results Among the 299 survey participants, the device scenario requiring the least equipment and reconstitution steps (the DCS) received a median preference rating of 71 out of 100 (0 being “the least desirable” and 100 “the most desirable” rating). This was significantly higher than the other scenarios (the next highest achieved a median of 50 points; P<0.001). Participants would be more likely to use this device prophylactically (P<0.001). Among the 98 participants who tested the DCS, 57% preferred this device over their current device, 26% preferred their current device, and 17% had no preference. The DCS was rated as easier to use than current treatment devices (median score 9/10 versus 7/10 for current treatment, P=0.001). Conclusion The survey indicates that the prefilled DCS, FuseNGo®, requiring the least equipment and fewest reconstitution steps, was preferred by patients and was the device most likely to be used prophylactically; the practical device testing supports these results. PMID:25525348

  14. Inhibition of human factor VIIIa by anti-A2 subunit antibodies.

    PubMed Central

    Lollar, P; Parker, E T; Curtis, J E; Helgerson, S L; Hoyer, L W; Scott, M E; Scandella, D

    1994-01-01

    Human inhibitory alloantibodies and autoantibodies to Factor VIII (FVIII) are usually directed toward the A2 and/or C2 domains of the FVIII molecule. Anti-C2 antibodies block the binding of FVIII to phospholipid, but the mechanism of action of anti-A2 antibodies is not known. We investigated the properties of a patient autoantibody, RC, and a monoclonal antibody, 413, that bind to the region which contains the epitopes of all anti-A2 alloantibodies or autoantibodies studied to date. mAb 413 and RC were noncompetitive inhibitors of a model intrinsic Factor X activation complex (intrinsic FXase) consisting of Factor IXa, activated FVIII (FVIIIa), and synthetic phospholipid vesicles, since they decreased the Vmax of intrinsic FXase by > 95% at saturating concentrations without altering the Km. This indicates that RC and mAb 413 either block the binding of FVIIIa to FIXa or phospholipid or interfere with the catalytic function of fully assembled intrinsic FXase, but they do not inhibit the binding of the substrate Factor X. mAb 413 did not inhibit the increase in fluorescence anisotropy that results from the binding of Factor VIIIa to fluorescein-5-maleimidyl-D-phenylalanyl-prolyl-arginyl-FIXa (Fl-M-FPR-FIXa) on phospholipid vesicles in the absence of Factor X, indicating it does not inhibit assembly of intrinsic FXase. Addition of Factor X to Fl-M-FPR-FIXa, FVIIIa, and phospholipid vesicles produced a further increase in fluorescence anisotropy and a decrease in fluorescence intensity. This effect was blocked completely by mAb 413. We conclude that anti-A2 antibodies inhibit FVIIIa function by blocking the conversion of intrinsic FXase/FX complex to the transition state, rather than by interfering with formation of the ground state Michaelis complex. PMID:8200986

  15. Genetic factors influencing inhibitor development in a cohort of South African haemophilia A patients.

    PubMed

    Lochan, A; Macaulay, S; Chen, W C; Mahlangu, J N; Krause, A

    2014-09-01

    A critical complication of factor VIII (FVIII) replacement therapy in Haemophilia A (HA) treatment is inhibitor development. Known genetic factors predisposing to inhibitor development include FVIII (F8) gene mutations, ethnicity, a family history of inhibitors and FVIII haplotype mismatch. The aim of this study was to characterize and correlate these genetic factors in a cohort of South African HA patients. This was a retrospective study that included 229 patients and involved the analysis of patient files, HA molecular and clinical databases and molecular analysis of the F8 gene haplotype. Of the 229 patients, 51% were of black ethnicity, 49% were white, 5% had mild HA, 4% were moderate and 91% were severe, 36% were int22 positive and 13% were inhibitor positive. Of the inhibitor positive patients, 72% were black patients. Inhibitors were reported in 27% of black int22 positive patients, 13% of black int22 negative patients, 9% of white int22 positive patients and 7% of white int22 negative. The H1 haplotype was more common in whites (75%) and H2 was more common in blacks (74%). H3 and H5 were only found in black patients and had a higher frequency of inhibitor development than H1 and H2. In this small HA cohort, black patients had a significantly higher frequency of inhibitor development and the results were indicative of an association between inhibitor development, ethnicity and haplotype.

  16. Small ncRNA Expression-Profiling of Blood from Hemophilia A Patients Identifies miR-1246 as a Potential Regulator of Factor 8 Gene

    PubMed Central

    Sarachana, Tewarit; Dahiya, Neetu; Simhadri, Vijaya L.; Pandey, Gouri Shankar; Saini, Surbhi; Guelcher, Christine; Guerrera, Michael F.; Kimchi-Sarfaty, Chava; Sauna, Zuben E.; Atreya, Chintamani D.

    2015-01-01

    Hemophilia A (HA) is a bleeding disorder caused by deficiency of functional plasma clotting factor VIII (FVIII). Genetic mutations in the gene encoding FVIII (F8) have been extensively studied. Over a thousand different mutations have been reported in the F8 gene. These span a diverse range of mutation types, namely, missense, splice-site, deletions of single and multiple exons, inversions, etc. There is nonetheless evidence that other molecular mechanisms, in addition to mutations in the gene encoding the FVIII protein, may be involved in the pathobiology of HA. In this study, global small ncRNA expression profiling analysis of whole blood from HA patients, and controls, was performed using high-throughput ncRNA microarrays. Patients were further sub-divided into those that developed neutralizing-anti-FVIII antibodies (inhibitors) and those that did not. Selected differentially expressed ncRNAs were validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. We identified several ncRNAs, and among them hsa-miR-1246 was significantly up-regulated in HA patients. In addition, miR-1246 showed a six-fold higher expression in HA patients without inhibitors. We have identified an miR-1246 target site in the noncoding region of F8 mRNA and were able to confirm the suppressory role of hsa-miR-1246 on F8 expression in a stable lymphoblastoid cell line expressing FVIII. These findings suggest several testable hypotheses vis-à-vis the role of nc-RNAs in the regulation of F8 expression. These hypotheses have not been exhaustively tested in this study as they require carefully curated clinical samples. PMID:26176629

  17. Von Willebrand factor in patients on mechanical circulatory support - a double-edged sword between bleeding and thrombosis.

    PubMed

    Hudzik, Bartosz; Kaczmarski, Jacek; Pacholewicz, Jerzy; Zakliczynski, Michal; Gasior, Mariusz; Zembala, Marian

    2015-09-01

    Mechanical circulatory support (MCS) is an umbrella term describing the various technologies used in both short- and long-term management of patients with either end-stage chronic heart failure (HF) or acute HF. Most often, MCS has emerged as a bridge to transplantation, but more recently it is also used as a destination therapy. Mechanical circulatory support includes left ventricular assist device (LVAD) or bi-ventricular assist device (Bi-VAD). Currently, 2- to 3-year survival in carefully selected patients is much better than with medical therapy. However, MCS therapy is hampered by sometimes life-threatening complications including bleeding and device thrombosis. Von Willebrand factor (vWF) has two major functions in haemostasis. First, it plays a crucial role in platelet-subendothelium adhesion and platelet-platelet interactions (aggregation). Second, it is the carrier of factor VIII (FVIII) in plasma. Von Willebrand factor prolongs FVIII half-time by protecting it from proteolytic degradation. It delivers FVIII to the site of vascular injury thus enhancing haemostatic process. On one hand, high plasma levels of vWF have been associated with an increased risk of thrombosis. On the other, defects or deficiencies of vWF underlie the inherited von Willebrand disease or acquired von Willebrand syndrome. Here we review the pathophysiology of thrombosis and bleeding associated with vWF.

  18. Females with FVIII and FIX deficiency have reduced joint range of motion

    PubMed Central

    Sidonio, Robert F.; Mili, Fatima D.; Li, Tengguo; Miller, Connie H.; Hooper, William C.; DeBaun, Michael R.; Soucie, Michael

    2015-01-01

    Little is known about rates of joint bleeding among females with FVIII/FIX deficiency or hemophilia carriers. In a cross-sectional study, we tested the hypothesis that females with FVIII or FIX deficiency enrolled in the Universal Data Collection (UDC) project had a reduced mean overall joint range of motion (ROM) compared with historic controls from the Normal Joint Study. Demographics, clinical characteristics, and joint ROM measurements on 303 females without a bleeding disorder and 148 females with FVIII and FIX deficiency, respectively, between the ages of 2–69 years and a body mass index (BMI) ≤35 were compared. Multivariate linear regression was performed with the overall joint ROM (sum of the right and left ROM measurements of five joints) as the dependent variable and FVIII or FIX activity as the independent variable adjusting for age, race, BMI, and number of joint bleeds reported over the last 6 months. As FVIII and FIX activity decreased, the mean overall joint ROM became reduced and in most cases was significantly lower than that of the controls regardless of age and clinical hemophilia severity. Further investigation of reduced joint ROM as evidence of subclinical joint bleeding in females with FVIII and FIX deficiency is warranted. PMID:24838518

  19. Elective surgery on factor VIII inhibitor patients using continuous infusion of recombinant activated factor VII: plasma factor VII activity of 10 IU/ml is associated with an increased incidence of bleeding.

    PubMed

    Smith, M P; Ludlam, C A; Collins, P W; Hay, C R; Wilde, J T; Grigeri, A; Melsen, T; Savidge, G F

    2001-10-01

    We examined recombinant activated factor VII (rVIIa) administered by continuous infusion to eight patients with inhibitors to factor VIII, undergoing elective surgery. rVIIa was infused at a fixed rate of 16.5 microg/kg/h for a median of 13.5 days (range 1-26). There was effective haemostasis at this infusion rate in only one of two minor procedures and two of six major operations. Three patients experienced excessive bleeding despite plasma factor VII activity around 10 IU/ml. Serious bleeding occurred in two other patients caused by procedural errors unrelated to rVIIa and required re-operation. The median rVIIa clearance on day 1 was 57 ml/h/kg (range 18-100) and on day 3 was 100 ml/h/kg (range 61-200). Clearance on the final infusion day was not significantly different from day 3. The infusion did not induce pathological activation of the coagulation mechanism. The only thrombotic adverse events were two episodes of superficial thrombophlebitis of the infused vein in one subject. In conclusion, the 16.5 microg/kg/h infusion rate reliably achieves plasma factor VII activity levels of 10 IU/ml, but this level does not provide reliable haemostasis.

  20. Pharmacological concentrations of rFVIIa restore hemostasis independent of tissue factor in antibody-induced hemophilia mice

    PubMed Central

    KESHAVA, S.; SUNDARAM, J.; RAJULAPATI, A.; PENDURTHI, U.R.; RAO, L.V.M.

    2016-01-01

    Summary Background Recombinant factor VIIa (rFVIIa) has been used widely for treating hemophilia patients with inhibitory autoantibodies against factor VIII or IX. Its mechanism of action is not entirely known. A majority of in vitro studies suggested that pharmacological concentrations of rFVIIa restore hemostasis in hemophilia in a phospholipid-dependent mechanism, independent of tissue factor (TF). However, a few studies suggested that a TF-dependent mechanism plays a primary role in rFVIIa correction of bleeding in hemophilia patients. Here, we investigated the potential contribution of TF in rFVIIa-induced hemostasis in hemophilia employing a model system of FVIII antibody-induced hemophilia in TF transgenic mice. Methods Mice expressing low levels of human TF (LTF mice), relatively high levels of human TF (HTF mice) or wild-type mice (WT mice) were administered with neutralizing anti-FVIII antibodies to induce hemophilia in these mice. The mice were then treated with varying concentrations of rFVIIa. rFVIIa-induced hemostasis was evaluated with the saphenous vein bleeding model. Results Administration of FVIII inhibitory antibodies induced the hemophilic bleeding phenotype in all three genotypes. rFVIIa administration rescued the bleeding phenotype in all three genotypes. No significant differences were observed in rFVIIa-induced correction in the bleeding of LTF and HTF mice administered with FVIII antibodies. Conclusions Our results provide strong evidence supporting that the hemostatic effect of pharmacological doses of rFVIIa stems from a TF-independent mechanism. PMID:26727350

  1. Photoionization of Ar VIII

    NASA Astrophysics Data System (ADS)

    Liang, Liang; Jiang, Wen-xian; Zhou, Chao

    2017-01-01

    The photoionization cross section, energy levels and widths of 22 Rydberg series (in the autoionization region) for Na-like Ar VIII were investigated by using of R-matrix method. The relativistic distorted-wave method is used to calculate the radial functions, and QB method of Quigly-Berrington [Quigley et al. 1998] is employed to calculate the resonance levels and widths. We have identified the formant in the figure of the photoionization cross section.

  2. Primary and rescue immune tolerance induction in children and adults: a multicentre international study with a VWF-containing plasma-derived FVIII concentrate.

    PubMed

    Oldenburg, J; Jiménez-Yuste, V; Peiró-Jordán, R; Aledort, L M; Santagostino, E

    2014-01-01

    Most studies on immune tolerance induction (ITI) therapy in haemophilia A patients are focused on primary ITI in children. Here we report on the ITI outcome in a large retrospective cohort, including adults and patients with rescue ITI, treated with a pdFVIII/VWF concentrate. Retrospective data from haemophilic patients (FVIII< 2%) with inhibitors from 22 centres in Spain, Italy and Germany, who underwent primary or rescue ITI with pdFVIII/VWF concentrate, were collected. Complete success (CS), partial success (PS) and failure were defined based on the criteria of the consensus recommendations of the 2006 International ITI Workshop. A total of 41 cases of primary ITI (32 children and 9 adults) and 19 cases of rescue ITI (17 children and 2 adults) were evaluated. Success (CS+PS) rate of 87% was achieved in primary ITI and 74% in the higher risk profile of rescue ITI. Eight of nine (85%) patients with poorest prognosis (three or more of the known risk factors of poor response to ITI) achieved success (CS+PS). CS of 100% was observed in eight primary ITI patients with titre at start of ITI ≤2.5 BU and inhibitor peak ≤25 BU. The favourable response rates in primary and rescue ITI in children and in adult patients, even in the presence of poor prognostic factors, should be encouraged for broadening the indication of immune tolerance therapy in haemophilia A patients with inhibitors.

  3. Joint health scores in a haemophilia A cohort from Pakistan with minimal or no access to factor VIII concentrate: correlation with thrombin generation and underlying mutation.

    PubMed

    Khanum, F; Bowen, D J; Kerr, B C; Collins, P W

    2014-05-01

    Haemophilia A is associated with recurrent joint bleeding which leads to synovitis and debilitating arthropathy. Coagulation factor VIII level is an important determinant of bleed number and development of arthropathy . The aim of this study was to compare the haemophilia joint health score (HJHS) and Gilbert score with severity, age, thrombin generation (TG) and underlying mutation in a haemophilia A cohort which had minimal access to haemostatic replacement therapy. Ninety-two haemophilia A individuals were recruited from Pakistan. Age, age at first bleed, target joints, haemophilic arthropathy joints, HJHS and Gilbert score were recorded. A strong correlation was found between HJHS and Gilbert score (r = 0.98), both were significantly higher in severe (n = 59) compared with non-severe (n = 29) individuals before the age of 12 years (P ≤ 0.01) but not thereafter. When individuals were divided according to developmental age (<12 years, 12-16 years and >16 years), both HJHS and Gilbert score were significantly lower in the youngest group (P ≤ 0.001), there was no difference between 12-16 years and >16 years. In severe individuals there was no correlation between in vitro TG and joint score, whereas in non-severe individuals there was a weak negative correlation. In the severe group, no significant difference was observed for either joint score according to the underlying mutation type (inversion, missense, nonsense, frameshift). In this cohort of haemophilia A individuals with minimal access to haemostatic treatment, haemophilic arthropathy correlated with severity and age; among severe individuals, joint health scores did not relate to either the underlying mutation or in vitro TG.

  4. von Willebrand factor and von Willebrand disease.

    PubMed

    Matsui, Taei; Hamako, Jiharu

    von Willebrand factor (VWF) has two major roles in hemostasis, as a form of molecular glue which functions in platelet plug formation and as a protective transporter for coagulation factor VIII (FVIII). VWF shows a multimeric chain structure composed of 270 kDa subunits containing binding domains for FVIII, platelet and collagens. Biosynthesis, storage, secretion of VWF and the cleavage process by ADAMTS13 (a disintegrin-like and metalloproteinase with thrombospondin type 1 repeats 13) regulating VWF activity have been elucidated. von Willebrand disease (VWD) is an autosomal bleeding disorder, first documented in 1926, caused by quantitative or qualitative deficiency of VWF. The classification and molecular pathogenesis of VWD have been developed during a 90-year period based on clinical laboratory analysis of VWF, and structure-function analysis of mutant VWF with amino acid substitutions (genetic changes). VWF is a unique and very large multifunctional plasma protein, the hemostatic activity of which is dynamically regulated by physiological shear stress in the blood stream.

  5. Directed engineering of a high-expression chimeric transgene as a strategy for gene therapy of hemophilia A.

    PubMed

    Doering, Christopher B; Denning, Gabriela; Dooriss, Kerry; Gangadharan, Bagirath; Johnston, Jennifer M; Kerstann, Keith W; McCarty, David A; Spencer, H Trent

    2009-07-01

    Human coagulation factor VIII (fVIII) is inefficiently biosynthesized in vitro and has proven difficult to express at therapeutic levels using available clinical gene-transfer technologies. Recently, we showed that a porcine and certain hybrid human/porcine fVIII transgenes demonstrate up to 100-fold greater expression than human fVIII. In this study, we extend these results to describe the use of a humanized, high-expression, hybrid human/porcine fVIII transgene that is 89% identical to human fVIII and was delivered by lentiviral vectors (LVs) to hematopoietic stem cells for gene therapy of hemophilia A. Recombinant human immunodeficiency virus-based vectors encoding the fVIII chimera efficiently transduced human embryonic kidney (HEK)-293T cells. Cells transduced with hybrid human/porcine fVIII encoding vectors expressed fVIII at levels 6- to 100-fold greater than cells transduced with vectors encoding human fVIII. Transplantation of transduced hematopoietic stem and progenitor cells into hemophilia A mice resulted in long-term fVIII expression at therapeutic levels despite <5% genetically modified blood mononuclear cells. Furthermore, the simian immunodeficiency virus (SIV) -derived vector effectively transduced the human hematopoietic cell lines K562, EU1, U.937, and Jurkat as well as the nonhematopoietic cell lines, HEK-293T and HeLa. All cell lines expressed hybrid human/porcine fVIII, albeit at varying levels with the K562 cells expressing the highest level of the hematopoietic cell lines. From these studies, we conclude that humanized high-expression hybrid fVIII transgenes can be utilized in gene therapy applications for hemophilia A to significantly increase fVIII expression levels compared to what has been previously achieved.

  6. Characterization of the factor VIII defect in 147 patients with sporadic hemophilia A: Family studies indicate a mutation type-dependent sex ratio of mutation frequencies

    SciTech Connect

    Becker, J.; Schmidt, W.; Olek, K.

    1996-04-01

    The clinical manifestation of hemophilia A is caused by a wide range of different mutations. In this study the factor VIII genes of 147 severe hemophilia A patients-all exclusively from sporadic families-were screened for mutations by use of the complete panel of modern DNA techniques. The pathogenous defect could be characterized in 126 patients (85.7%). Fifty-five patients (37.4%) showed a F8A-gene inversion, 47 (32.0%) a point mutation, 14 (9.5%) a small deletion, 8 (5.4%) a large deletion, and 2 (1.4%) a small insertion. Further, four (2.7%) mutations were localized but could not be sequenced yet. No mutation could be identified in 17 patients (11.6%). Sixteen (10.9%) of the P identified mutations occurred in the B domain. Four of these were located in an adenosine nucleotide stretch at codon 1192, indicating a mutation hotspot. Somatic mosaicisms were detected in 3 (3.9%) of 76 patients` mothers, comprising 3 of 16 de novo mutations in the patients` mothers. Investigation of family relatives allowed detection of a de novo mutation in 16 of 76 two-generation and 28 of 34 three-generation families. On the basis of these data, the male:female ratio of mutation frequencies (k) was estimated as k = 3.6. By use of the quotients of mutation origin in maternal grandfather to patient`s mother or to maternal grandmother, k was directly estimated as k = 15 and k = 7.5, respectively. Considering each mutation type separately, we revealed a mutation type-specific sex ratio of mutation frequencies. Point mutations showed a 5-to-10-fold-higher and inversions a >10-fold- higher mutation rate in male germ cells, whereas deletions showed a >5-fold-higher mutation rate in female germ cells. Consequently, and in accordance with the data of other diseases like Duchenne muscular dystrophy, our results indicate that at least for X-chromosomal disorders the male:female mutation rate of a disease is determined by its proportion of the different mutation types. 68 refs., 1 fig., 5 tabs.

  7. Acquired haemophilia A in a woman with autoimmune hepatitis and systemic lupus erythematosus: review of literature.

    PubMed

    Rezaieyazdi, Zahra; Sharifi-Doloui, Davood; Hashemzadeh, Kamila; Shirdel, Abbas; Mansouritorghabeh, Hassan

    2011-12-01

    Acquired haemophilia A, secondary to systemic lupus erythematosus (SLE), is a rare bleeding diathesis. Here we report a 37-year-old woman with autoimmune hepatitis who developed SLE and acquired haemophilia caused by factor VIII (fVIII) inhibitors. She presented with spontaneous ecchymosis and haematuria. There were a prolongation of the activated partial thromboplastin time, reduced fVIII activity and a high titre of fVIII inhibitors. Therapeutic regimen was started with intravenous methylprednisolone pulse, continued with prednisolone, intravenous pulse cyclophosphamide and fresh frozen plasma. After 8 weeks, fVIII inhibitor assay was negative.

  8. Physical activity in individuals with haemophilia and experience with recombinant factor VIII Fc fusion protein and recombinant factor IX Fc fusion protein for the treatment of active patients: a literature review and case reports

    PubMed Central

    Wang, Michael; Álvarez-Román, María Teresa; Chowdary, Pratima; Quon, Doris V.; Schafer, Kim

    2016-01-01

    The World Federation of Hemophilia and the National Hemophilia Foundation encourage people with haemophilia (PWH) to participate in routine physical activity. The benefits of physical activity for PWH include improvements in joint, bone, and muscle health. Accordingly, a number of studies suggest that levels of physical activity among PWH are similar to those of their healthy peers, especially among individuals who began prophylaxis at an early age (≤3 years). Importantly, several studies found either no increased risk or only a transient increase in risk of bleeding with more intensive physical activity compared with less intensive physical activity. Data on optimal prophylaxis regimens for PWH who participate in physical/sporting activities; however, remain sparse. Long-acting recombinant factor VIII Fc fusion protein (rFVIIIFc) and recombinant factor IX Fc fusion protein (rFIXFc) demonstrated efficacy for the prevention and treatment of bleeding episodes in Phase 3 clinical trials of participants with haemophilia A and B, respectively, with most individuals able to maintain or increase their physical activities. This manuscript reviews the current literature that describes physical activity in PWH. Additionally, case studies are presented to provide supplemental information to clinicians illustrating the use of rFVIIIFc and rFIXFc in physically active patients with haemophilia A and B, respectively. These case reports demonstrate that it is possible for patients to be physically active and maintain good control of their haemophilia with extended interval prophylactic dosing using rFVIIIFc or rFIXFc. PMID:27116081

  9. High-affinity, noninhibitory pathogenic C1 domain antibodies are present in patients with hemophilia A and inhibitors

    PubMed Central

    Batsuli, Glaivy; Deng, Wei; Healey, John F.; Parker, Ernest T.; Baldwin, W. Hunter; Cox, Courtney; Nguyen, Brenda; Kahle, Joerg; Königs, Christoph; Li, Renhao; Lollar, Pete

    2016-01-01

    Inhibitor formation in hemophilia A is the most feared treatment-related complication of factor VIII (fVIII) therapy. Most inhibitor patients with hemophilia A develop antibodies against the fVIII A2 and C2 domains. Recent evidence demonstrates that the C1 domain contributes to the inhibitor response. Inhibitory anti-C1 monoclonal antibodies (mAbs) have been identified that bind to putative phospholipid and von Willebrand factor (VWF) binding epitopes and block endocytosis of fVIII by antigen presenting cells. We now demonstrate by competitive enzyme-linked immunosorbent assay and hydrogen-deuterium exchange mass spectrometry that 7 of 9 anti-human C1 mAbs tested recognize an epitope distinct from the C1 phospholipid binding site. These mAbs, designated group A, display high binding affinities for fVIII, weakly inhibit fVIII procoagulant activity, poorly inhibit fVIII binding to phospholipid, and exhibit heterogeneity with respect to blocking fVIII binding to VWF. Another mAb, designated group B, inhibits fVIII procoagulant activity, fVIII binding to VWF and phospholipid, fVIIIa incorporation into the intrinsic Xase complex, thrombin generation in plasma, and fVIII uptake by dendritic cells. Group A and B epitopes are distinct from the epitope recognized by the canonical, human-derived inhibitory anti-C1 mAb, KM33, whose epitope overlaps both groups A and B. Antibodies recognizing group A and B epitopes are present in inhibitor plasmas from patients with hemophilia A. Additionally, group A and B mAbs increase fVIII clearance and are pathogenic in a hemophilia A mouse tail snip bleeding model. Group A anti-C1 mAbs represent the first identification of pathogenic, weakly inhibitory antibodies that increase fVIII clearance. PMID:27381905

  10. The acute effect of moderate intensity aquatic exercise on coagulation factors in haemophiliacs.

    PubMed

    Beltrame, Luis Gustavo Normanton; Abreu, Laurinda; Almeida, Jussara; Boullosa, Daniel Alexandre

    2015-05-01

    The objective of this cross-sectional study was to analyse the acute effect of aquatic exercise on haemostasis in persons with haemophilia. Ten adult haemophiliacs (8 type A, 2 type B) familiarized with aquatic training performed a 20-min exercise session in a swimming pool at an intensity of ~70% maximum heart rate (HR). Blood samples were collected immediately after the training session. The haemostatic parameters selected for analyses were factor VIII (FVIII), prothrombin time (PT), activated partial thromboplastin time (APTT) and fibrinogen. There were unclear effects of the exercise bout on FVIII and APTT, with a possibly beneficial effect on PT (-11·4%; 90% confidence interval: -26·1;3·3%), and a trivial change on fibrinogen levels. It was found an association between the mean rise in HR during exercise and the decrement in PT after exercise (r = 0·729; P = 0·026). The greater changes were observed in the patients diagnosed with a moderate level of haemophilia. It is concluded that a short bout of moderate intensity of aquatic exercise may have a positive influence on PT in adults with haemophilia with greater changes in those individuals exhibiting a greater rise in HR during exercise. This may be an important issue to the haemostatic control of haemophiliacs in clinical settings. Further studies are warranted for testing the influence of different aquatic exercise intensities on haemostasis.

  11. Safety, efficacy and pharmacokinetics of rVIII-SingleChain in children with severe hemophilia A: results of a multicenter clinical trial.

    PubMed

    Stasyshyn, O; Djambas Khayat, C; Iosava, G; Ong, J; Abdul Karim, F; Fischer, K; Veldman, A; Blackman, N; St Ledger, K; Pabinger, I

    2017-04-01

    Essentials rVIII-SingleChain is a novel recombinant factor VIII with covalently bonded heavy and light chains. Efficacy, safety and pharmacokinetics were studied in pediatric patients with severe hemophilia A. Across all prophylaxis regimens, the median annualized spontaneous bleeding rate was 0.00. rVIII-SingleChain showed excellent hemostatic efficacy and a favorable safety profile.

  12. Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap

    PubMed Central

    Ayalew, Lisanework E.; Patel, Amrutlal K.; Gaba, Amit; Islam, Azharul; Tikoo, Suresh K.

    2016-01-01

    Earlier, targeting of DDX3 by few viral proteins has defined its role in mRNA transport and induction of interferon production. This study was conducted to investigate the function of bovine adenovirus (BAdV)-3 pVIII during virus infection. Here, we provided evidence regarding involvement of DDX3 in cap dependent cellular mRNA translation and demonstrated that targeting of DDX3 by adenovirus protein VIII interfered with cap-dependent mRNA translation function of DDX3 in virus infected cells. Adenovirus late protein pVIII interacted with DDX3 in transfected and BAdV-3 infected cells. pVIII inhibited capped mRNA translation in vitro and in vivo by limiting the amount of DDX3 and eIF3. Diminished amount of DDX3 and eIFs including eIF3, eIF4E, eIF4G, and PABP were present in cap binding complex in BAdV-3 infected or pVIII transfected cells with no trace of pVIII in cap binding complex. The total amount of eIFs appeared similar in uninfected or infected cells as BAdV-3 did not appear to degrade eIFs. The co-immunoprecipitation experiments indicated the absence of direct interaction between pVIII and eIF3, eIF4E, or PABP. These data indicate that interaction of pVIII with DDX3 interferes with the binding of eIF3, eIF4E and PABP to the 5′ Cap. We conclude that DDX3 promotes cap-dependent cellular mRNA translation and BAdV-3 pVIII inhibits translation of capped cellular mRNA possibly by interfering with the recruitment of eIFs to the capped cellular mRNA. PMID:28082972

  13. Small FVIII gene rearrangements in 18 hemophilia A patients: five novel mutations.

    PubMed

    Bicocchi, Maria Patrizia; Pasino, Mirella; Lanza, Tiziana; Bottini, Federico; Molinari, Angelo Claudio; Caprino, Daniela; Rosano, Camillo; Acquila, Maura

    2005-02-01

    Hemophilia A (HA) is a disorder caused by mutations of the FVIII gene, which is located on the tip of the long arm of the X chromosome. In a cohort of 18 unrelated Italian patients affected with HA of varying severity, we performed mutational screening of the gene by denaturing high-performance liquid chromatography (DHPLC) and direct sequencing of abnormal peaks. We identified five novel mutations and 9 previously reported DNA alterations. Two of the 9 previously reported alterations were each common to 3 unrelated patients. Six different mutations were characterized as missense alterations, while 8 were non-missense mutations. Among the new gene alterations, one created a stop codon, one consisted of an out-of frame deletion, and one was a splice-site mutation. The last two were missense alterations. In an attempt to better understand the causative effect of the mutations and the clinical variability of the patients, we investigated the consequences of each missense mutation and visualized the effect of the amino acid change on structural FVIII models.

  14. Membrane interaction of the factor VIIIa discoidin domains in atomistic detail

    PubMed Central

    Madsen, Jesper J.; Ohkubo, Y. Zenmei; Peters, Günther H.; Faber, Johan H.; Tajkhorshid, Emad; Olsen, Ole H.

    2016-01-01

    A recently developed membrane-mimetic model was applied to study membrane interaction and binding of the two anchoring C2-like discoidin domains of human coagulation factor (F)VIIIa, the C1 and C2 domains. Both individual domains, FVIII C1 and FVIII C2, were observed to bind the phospholipid membrane by partial or full insertion of their extruding loops (the spikes). However, the two domains adopted different molecular orientations in their membrane-bound states; FVIII C2 roughly positioned normal to the membrane plane, while FVIII C1 displayed a multitude of tilted orientations. The results indicate that FVIII C1 may be important in modulating the orientation of the FVIIIa molecule to optimize the interaction with FIXa, which is anchored to the membrane via its γ-carboxyglutamic acid-rich (Gla)-domain. Additionally, a structural change was observed in FVIII C1 in the coiled main chain leading the first spike. A tight interaction with one lipid per domain, similar to what has been suggested for the homologous FVa C2, is characterized. Finally, we rationalize known FVIII antibody epitopes and the scarcity of documented hemophilic missense mutations related to improper membrane binding of FVIIIa, based on the prevalent non-specificity of ionic interactions in the simulated membrane-bound states of FVIII C1 and FVIII C2. PMID:26346528

  15. Incidence and risk factors for inhibitor development in previously untreated severe haemophilia A patients born between 2005 and 2010.

    PubMed

    Vézina, C; Carcao, M; Infante-Rivard, C; Lillicrap, D; Stain, A M; Paradis, E; Teitel, J; Rivard, G E

    2014-11-01

    The objective of this study was to evaluate the inhibitor development (ID) in previously untreated patients (PUPs) with severe haemophilia A (FVIII ≤ 0.01 IU mL(-1) ). All Canadian Haemophilia Treatment Centres completed a questionnaire on patients born between September 2005 and August 2010 and followed for up to 7 years. Eligible patients had at least 20 exposure days (ED) or had developed an inhibitor. The odds ratio (OR) and 95% confidence intervals (95% CI) for risk factors to develop an inhibitor were estimated using unconditional logistic regression. A total of 99 haemophilia A PUPs were studied. Thirty-four (34%) developed an inhibitor (24/34 of high titre). Inhibitors developed in 25/63 (40%) patients with a high-risk mutation. ID was most frequent in Aboriginals (86%). Dose intensity (IU kg(-1)  day(-1) X number of ED) at first exposure to factor VIII (FVIII) was associated with a crude OR increase of 1.10 (95% CI: 0.99-1.23) with each increase of 100 dose-intensity units. Haemarthrosis and intracranial bleeding as the indication for first exposure to FVIII concentrate were associated with a crude OR for ID of 7.63 (95% CI: 2.14-27.17) and 5.08 (95% CI: 1.11-23.31) respectively. ID according to FVIII concentrate used was: Advate (®) 18/50 (36%), Kogenate FS(®) or Helixate FS(®) 15/36 (42%), Wilate(®) 0/11 and Xyntha(®) 1/2. In multivariate analysis, Aboriginal ethnicity (OR = 11.69; 95% CI: 1.11-122.86) and haemarthrosis (OR = 4.49; 95% CI: 1.08-18.61) were statistically significant. The cumulative incidence of ID in severe haemophilia A PUPs was 34% and varied according to ethnicity, type of bleeding at first ED, type of FVIII product and dose intensity at first exposure.

  16. Clearance of von Willebrand factor.

    PubMed

    Denis, Cécile V; Christophe, Olivier D; Oortwijn, Beatrijs D; Lenting, Peter J

    2008-02-01

    The life cycle of von Willebrand factor (VWF) comprises a number of distinct steps, ranging from the controlled expression of the VWF gene in endothelial cells and megakaryocytes to the removal of VWF from the circulation. The various aspects of VWF clearance have been the objects of intense research in the last few years, stimulated by observations that VWF clearance is a relatively common component of the pathogenesis of type 1 von Willebrand disease (VWD). Moreover, improving the survival of VWF is now considered as a viable therapeutic strategy to prolong the half-life of factor VIII in order to optimise treatment of haemophilia A. The present review aims to provide an overview of recent findings with regard to the molecular basis of VWF clearance. A number of parameters have been identified that influence VWF clearance, including its glycosylation profile and a number of VWF missense mutations. In addition, in-vivo studies have been used to identify cells that contribute to the catabolism of VWF, providing a starting point for the identification of receptors that mediate the cellular uptake of VWF. Finally, we discuss recent data describing chemically modification of VWF as an approach to prolong the half-life of the VWF/FVIII complex.

  17. Comparison of inhibitory and binding characteristics of an antibody causing acquired von Willebrand syndrome: an assay for von Willebrand factor binding by antibody.

    PubMed

    Fricke, W A; Brinkhous, K M; Garris, J B; Roberts, H R

    1985-09-01

    An acquired inhibitor of von Willebrand factor (vWF) activity occurring in a patient with benign gammopathy and von Willebrand syndrome (vWS) has been partially characterized. The inhibitor-induced syndrome resulted in low to undetectable plasma levels of vWF/ristocetin, vWF/botrocetin, FVIIIR:Ag, and FVIII:C with a normal to slightly prolonged bleeding time. Platelet vWF was normal. Intensive and continuous infusion of a heat-treated factor VIII concentrate (Hemofil-T, Hyland, Glendale, Calif) elevated the FVIII:C plasma levels to about 100%, with an increase in FVIIIR:Ag levels to about 340% and vWF/ristocetin levels to about 40%, much lower than expected based on the dose of Hemofil-T and its content of vWF and FVIII:C activities. The inhibitor bound to staphylococcal protein A (SpA) with high affinity, indicating an IgG antibody (Ab). An assay for the vWF-binding capacity was developed on the basis of absorption of the Ab from serially diluted plasma by SpA and removal of vWF and FVIII:C activities from normal plasma by the SpA-Ab complex. The Ab-binding site was on the vWF component of the factor VIII complex. The Ab was unable to bind isolated FVIII:C. The combined use of the new vWF-binding assay and a battery of tests for inhibition of vWF-dependent platelet aggregation with ristocetin (which detects high molecular weight vWF), with botrocetin (which detects high and low molecular weight vWF), and with platelet-aggregating factor (which detects high molecular weight vWF) provided a means of analysis of Ab effect on in vitro vWF function. Using these tests, a comparison was made of the effects of the vWS Ab with those of an Ab inhibitor occurring in homozygous von Willebrand's disease. The Ab of the vWS patient had weak inhibitory action on vWF/ristocetin without having an effect on vWF/botrocetin and platelet-aggregating factor, a high titer vWF-binding capacity, and no anamnestic response following concentrate therapy. These findings contrasted with those

  18. Functional Role of Milk Fat Globule-Epidermal Growth Factor VIII in Macrophage-Mediated Inflammatory Responses and Inflammatory/Autoimmune Diseases

    PubMed Central

    2016-01-01

    Inflammation involves a series of complex biological processes mediated by innate immunity for host defense against pathogen infection. Chronic inflammation is considered to be one of the major causes of serious diseases, including a number of autoimmune/inflammatory diseases, cancers, cardiovascular diseases, and neurological diseases. Milk fat globule-epidermal growth factor 8 (MFG-E8) is a secreted protein found in vertebrates and was initially discovered as a critical component of the milk fat globule. Previously, a number of studies have reported that MFG-E8 contributes to various biological functions including the phagocytic removal of damaged and apoptotic cells from tissues, the induction of VEGF-mediated neovascularization, the maintenance of intestinal epithelial homeostasis, and the promotion of mucosal healing. Recently, emerging studies have reported that MFG-E8 plays a role in inflammatory responses and inflammatory/autoimmune diseases. This review describes the characteristics of MFG-E8-mediated signaling pathways, summarizes recent findings supporting the roles of MFG-E8 in inflammatory responses and inflammatory/autoimmune diseases, and discusses MFG-E8 targeting as a potential therapeutic strategy for the development of anti-inflammatory/autoimmune disease drugs. PMID:27429513

  19. Functional Role of Milk Fat Globule-Epidermal Growth Factor VIII in Macrophage-Mediated Inflammatory Responses and Inflammatory/Autoimmune Diseases.

    PubMed

    Yi, Young-Su

    2016-01-01

    Inflammation involves a series of complex biological processes mediated by innate immunity for host defense against pathogen infection. Chronic inflammation is considered to be one of the major causes of serious diseases, including a number of autoimmune/inflammatory diseases, cancers, cardiovascular diseases, and neurological diseases. Milk fat globule-epidermal growth factor 8 (MFG-E8) is a secreted protein found in vertebrates and was initially discovered as a critical component of the milk fat globule. Previously, a number of studies have reported that MFG-E8 contributes to various biological functions including the phagocytic removal of damaged and apoptotic cells from tissues, the induction of VEGF-mediated neovascularization, the maintenance of intestinal epithelial homeostasis, and the promotion of mucosal healing. Recently, emerging studies have reported that MFG-E8 plays a role in inflammatory responses and inflammatory/autoimmune diseases. This review describes the characteristics of MFG-E8-mediated signaling pathways, summarizes recent findings supporting the roles of MFG-E8 in inflammatory responses and inflammatory/autoimmune diseases, and discusses MFG-E8 targeting as a potential therapeutic strategy for the development of anti-inflammatory/autoimmune disease drugs.

  20. Surgical experience with rFVIIa (NovoSeven) in congenital haemophilia A and B patients with inhibitors to factors VIII or IX.

    PubMed

    Valentino, L A; Cooper, D L; Goldstein, B

    2011-07-01

    Patients with congenital haemophilia with inhibitors are at risk of peri-operative bleeding complications, since replacement of the missing coagulation factor is ineffective, presenting a therapeutic challenge in elective or emergency surgery. Therefore, the management of peri-operative bleeding requires the use of bypassing agents, such as recombinant activated FVII (rFVIIa, NovoSeven(®) ). This article presents an updated evaluation of the safety and effectiveness of rFVIIa in the treatment of peri-operative bleeding in this patient population. Surgical and other medical procedures managed with rFVIIa from two randomized clinical trials, the Hemophilia Research Society/Hemophilia and Thrombosis Research Society (HRS/HTRS) registry databases and the medical literature were analysed. There were 395 rFVIIa-treated procedures (261 surgical, 89 dental and 45 other medical procedures) reported for 263 congenital haemophilia patients with inhibitors. In trials, initial rFVIIa dosing was 35-90 mcg kg(-1) bolus injection or 50 mcg kg(-1) h(-1) continuous infusion. Dosing in the registries and literature was more variable. Recombinant FVIIa effectiveness was comparable across data sources, with an overall rate of 84% (333/395). The incidence of thrombotic events was very low (0.4% of patients and 0.025% of procedures). Prior to the US approval of rFVIIa in 1999, surgical procedures in congenital haemophilia patients with inhibitors were often considered too risky. Recombinant FVIIa has consistently demonstrated effectiveness in treatment of bleeding in these patients during such procedures. Thrombotic events were rare. This analysis confirms the value of corroborating clinical trial results with post-marketing surveillance registries to assess small patient populations with clinically challenging management decisions.

  1. 12 CFR 611.1137 - Title VIII service corporations.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 12 Banks and Banking 7 2012-01-01 2012-01-01 false Title VIII service corporations. 611.1137 Section 611.1137 Banks and Banking FARM CREDIT ADMINISTRATION FARM CREDIT SYSTEM ORGANIZATION Service Organizations § 611.1137 Title VIII service corporations. (a) What is a title VIII service corporation? A title VIII service corporation is a...

  2. 12 CFR 611.1137 - Title VIII service corporations.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 12 Banks and Banking 6 2010-01-01 2010-01-01 false Title VIII service corporations. 611.1137... Organizations § 611.1137 Title VIII service corporations. (a) What is a title VIII service corporation? A title VIII service corporation is a service corporation organized for the purpose of exercising...

  3. Absorption signatures of warm-hot gas at low redshift: Ne VIII

    NASA Astrophysics Data System (ADS)

    Tepper-García, Thor; Richter, Philipp; Schaye, Joop

    2013-12-01

    At z < 1 a large fraction of the baryons is thought to reside in diffuse gas that has been shock-heated to high temperatures (105-106 K). Absorption by the 770.41, 780.32 Å doublet of Ne VIII in quasar spectra represents a unique tool to study this elusive warm-hot phase. We have developed an analytic model for the properties of Ne VIII absorbers that allows for an inhomogeneous metal distribution. Our model agrees with the predictions of a simulation from the OverWhelmingly Large Simulations project indicating that the average line-of-sight metal-filling fraction within the absorbing gas is low (cL ˜ 0.1). Most of the Ne VIII in our model is produced in low-density, collisionally ionized gas (nH = 10-6-10-4 cm-3, T = 105-106 K). Strong Ne VIII absorbers (log10(NNeVIII/cm-2)≳14), like those recently detected by Hubble Space Telescope/Cosmic Origins Spectrograph, are found to arise in higher density gas (nH ≳ 10-4 cm-3, T ≈ 5 × 105 K). Ne VIII cloudlets harbour only 1 per cent of the cosmic baryon budget. The baryon content of the surrounding gas (which has similar densities and temperatures as the Ne VIII cloudlets) is a factor c_L^{-1} higher. We conclude that Ne VIII absorbers are robust probes of shock-heated diffuse gas, but that spectra with signal-to-noise ratios S/N > 100 would be required to detect the bulk of the baryons in warm-hot gas.

  4. Prophylaxis vs. on-demand treatment with BAY 81-8973, a full-length plasma protein-free recombinant factor VIII product: results from a randomized trial (LEOPOLD II)

    PubMed Central

    Kavakli, K; Yang, R; Rusen, L; Beckmann, H; Tseneklidou-Stoeter, D; Maas Enriquez, M

    2015-01-01

    Background BAY 81-8973 is a new full-length human recombinant factor VIII product manufactured with technologies to improve consistency in glycosylation and expression to optimize clinical performance. Objectives To demonstrate superiority of prophylaxis vs. on-demand therapy with BAY 81-8973 in patients with severe hemophilia A. Patients/Methods In this multinational, randomized, open-label crossover study (LEOPOLD II; ClinicalTrials.gov identifier: NCT01233258), males aged 12–65 years with severe hemophilia A were randomized to twice-weekly prophylaxis (20–30 IU kg−1), 3-times-weekly prophylaxis (30–40 IU kg−1), or on-demand treatment with BAY 81-8973. Potency labeling for BAY 81-8973 was based on the chromogenic substrate assay or adjusted to the one-stage assay. Primary efficacy endpoint was annualized number of all bleeds (ABR). Adverse events (AEs) and immunogenicity were also assessed. Results Eighty patients (on demand, n = 21; twice-weekly prophylaxis, n = 28; 3-times-weekly prophylaxis, n = 31) were treated and analyzed. Mean ± SD ABR was significantly lower with prophylaxis (twice-weekly, 5.7 ± 7.2; 3-times-weekly, 4.3 ± 6.5; combined, 4.9 ± 6.8) vs. on-demand treatment (57.7 ± 24.6; P < 0.0001, anova). Median ABR was reduced by 97% with prophylaxis (twice-weekly, 4.0; 3-times-weekly, 2.0; combined, 2.0) vs. on-demand treatment (60.0). Median ABR was higher with twice-weekly vs. 3-times-weekly prophylaxis during the first 6-month treatment period (4.1 vs. 2.0) but was comparable in the second 6-month period (1.1 vs. 2.0). Few patients reported treatment-related AEs (4%); no treatment-related serious AEs or inhibitors were reported. Conclusions Twice-weekly or 3-times-weekly prophylaxis with BAY 81-8973 reduced median ABR by 97% compared with on-demand therapy, confirming the superiority of prophylaxis. Treatment with BAY 81-8973 was well tolerated. PMID:25546368

  5. Postpartum von Willebrand factor levels in women with and without von Willebrand disease and implications for prophylaxis.

    PubMed

    James, A H; Konkle, B A; Kouides, P; Ragni, M V; Thames, B; Gupta, S; Sood, S; Fletcher, S K; Philipp, C S

    2015-01-01

    The aim of this study was to elucidate the fall in von Willebrand factor (VWF) and factor VIII activity (FVIII) after childbirth in women with and without von Willebrand disease (VWD). VWF:RCo, VWF:Ag, and FVIII were obtained in the third trimester of pregnancy, on admission for childbirth, and 10 times postpartum. Specimens were processed within 4 h and analysed centrally. Means were calculated at each time point. Forty women (40 pregnancies) without VWD and 32 women (35 pregnancies) with VWD were enrolled. 15/32 with VWD were treated (30% of those with type 1 and all of those with type 2) in 17 pregnancies. Treatments prior to delivery consisted of desmopressin (2/17), VWF concentrate (15/17) and after delivery VWF concentrate (16/17). Duration of treatment was 0-21 days (median 6). VWF levels peaked at 250% of baseline--4 h postpartum in women with VWD and 12 h postpartum in women without VWD. Thereafter, VWF levels fell rapidly, approached baseline at 1 week and reached baseline at 3 weeks. Except immediately postpartum, when the levels among treated cases were higher, levels among women with VWD appeared to parallel, but were lower than those among women without VWD. Levels were lowest among those who received treatment. VWF levels fall rapidly after childbirth. Except immediately postpartum, current treatment strategies do not raise VWF levels to the levels of women without VWD or even to the levels of women with milder, untreated VWD. Consequently, women with VWD may be at risk of postpartum haemorrhage despite treatment.

  6. O-phospho-l-serine mediates hyporesponsiveness toward FVIII in hemophilia A-murine model by inducing tolerogenic properties in dendritic cells.

    PubMed

    Fathallah, Anas M; Ramakrishnan, Radha; Balu-Iyer, Sathy V

    2014-11-01

    The clinical use of therapeutic proteins can be complicated by the development of anti-product antibodies. We have previously observed that O-phospho-l-serine (OPLS) reduced antibody response to FVIII in Hemophilia-A (HA) mice. However, the mechanism underlying this observation is not clear. We hypothesize that OPLS reduces immunogenicity by inducing tolerogenic properties in dendritic cells (DCs). We tested this hypothesis using in vivo, in vitro, and ex vivo methods. Naive HA mice that were pre-exposed to FVIII in the presence of OPLS showed substantially lower antibody response following rechallenge with OPLS free FVIII as compared with dexamethasone-pretreated mice. Exposure of OPLS to bone-marrow-derived dendritic cells (BMDCs) in culturing conditions resulted in an increase in the regulatory cytokine TGF-β and a decrease in proinflammatory cytokines TNF-α and IL12p70. This was accompanied by a significant reduction in upregulation of costimulatory marker CD40, as measured by flow cytometry. Furthermore, ex vivo matured BMDCs in the presence of FVIII and OPLS failed to elicit a robust immune response in HA mice compared with FVIII-treated BMDCs. Our data suggest that OPLS modulates the immune response by altering the function and maturation of DCs, resulting in the induction of tolerogenic properties. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:3457-3463, 2014.

  7. Tissue Factor Pathway Inhibitor-1 Is a Valuable Marker for the Prediction of Deep Venous Thrombosis and Tumor Metastasis in Patients with Lung Cancer

    PubMed Central

    Yuan, Wufeng

    2017-01-01

    Activation of blood coagulation contributes to cancer progression. Tissue factor pathway inhibitor-1 (TFPI-1) is the main inhibitor of extrinsic coagulation pathway. The aim of this study is to assess the predicting significance of TFPI-1 for thrombotic complication and metastasis in lung cancer patients. Total of 188 non-small cell lung cancer (NSCLC) patients were included in this study. Plasma TFPI-1, D-dimer (D-D), antithrombin (AT), Fibrinogen (Fbg), and coagulating factor VIII activity (FVIII:C) were measured. In NSCLC patients, significantly decreased TFPI-1 and AT and increased D-D, Fbg, and FVIII:C levels were observed, and there was a significant correlation between TFPI-1 and other hemostatic parameters (P < 0.001, resp.). NSCLC patients with deep venous thrombosis (DVT) or metastasis had significantly lower TFPI-1 levels than those without DVT or metastasis (P < 0.01, resp.). Multivariate regression revealed that TFPI-1 acted as a predictor for DVT or tumor metastasis in NSCLC patients [OR: 4.15 or 3.28, P < 0.05, resp.]. The area under ROC curve of TFPI-1 was 0.905 (95% CI, 0.842~0.967) or 0.828 (95% CI, 0.742~0.915) for predicting DVT or metastasis (P < 0.001, resp.). The optimal point of TFPI-1 was 57.7 or 54.3 ng/mL for predicting DVT or metastasis, respectively. Combination of TFPI-1 and D-D measurements can improve the predicting power for DVT or metastasis in NSCLC patients. Our findings suggested that TFPI-1 was a valuable predictor of DVT and tumor metastasis in NSCLC patients. PMID:28246607

  8. Plasma factor and inhibitor composition contributes to thrombin generation dynamics in patients with acute or previous cerebrovascular events

    PubMed Central

    Gissel, Matthew; Undas, Anetta; Slowik, Agnieszka; Mann, Kenneth G.; Brummel-Ziedins, Kathleen E.

    2010-01-01

    Introduction More than 80% of cerebrovascular events are ischemic and largely thromboembolic by nature. We evaluated whether plasma factor composition and thrombin generation dynamics might be a contributor to the thrombotic phenotype of ischemic cerebrovascular events. Materials and Methods We studied (1) 100 patients with acute ischemic stroke (n=50) or transient ischemic attack (TIA) (n=50) within the first 24 hours from symptom onset, and (2) 100 individuals 1 to 4 years following ischemic stroke (n=50) or TIA (n=50). The tissue factor pathway to thrombin generation was simulated with a mathematical model using plasma levels of clotting factors (F)II, V, VII, VIII, IX, X, antithrombin and free tissue factor pathway inhibitor (TFPI). Results The plasma levels of free TFPI, FII, FVIII, and FX were higher, while antithrombin was lower, in the acute patients compared to the previous event group (all p≤0.02). Thrombin generation during acute events was enhanced, with an 11% faster maximum rate, a 15% higher maximum level and a 26% larger total production (all p<0.01). The increased thrombin generation in acute patients was determined by higher FII and lower antithrombin, while increased free TFPI mediated this effect. When the groups are classified by etiology, all stroke sub-types except cardioembolic have increased TFPI and decreased AT and total thrombin produced. Conclusion Augmented thrombin generation in acute stroke/TIA is to some extent determined by altered plasma levels of coagulation factors. PMID:20709367

  9. Anti-hemophilic factor (recombinant), plasma/albumin-free method (octocog-alpha; ADVATE) in the management of hemophilia A.

    PubMed

    Shapiro, Amy D

    2007-01-01

    Removal of blood-based additives from recombinant clotting factor concentrates continues to be advocated by the hemophilia community due to the history of infectious disease transmission with previous blood-derived clotting factor concentrates. In 2003, octocog-alpha, antihemophilic factor (recombinant), plasma/albumin-free method (ADVATE) was introduced, providing the first third-generation recombinant factor VIII (rFVIII) concentrate. Completed clinical trials have demonstrated ADVATE to be safe and effective in adult and pediatric subjects utilizing both prophylactic and on-demand treatment regimens, and for perioperative hemostatic coverage. In the five completed studies involving more than 200 previously treated patients (PTPs), a single incidence of low-titer, non-persistent inhibitor was reported. Active post authorization safety surveillance (PASS) is being performed to expand the efficacy and safety profile of ADVATE in routine clinical practice. Laboratory studies have documented the storage and post-reconstitution stability of ADVATE, conferring the desired versatility for home treatment. The evolving real-world experience and ongoing studies will provide further insight into ADVATE pharmacokinetics, alternative prophylactic dosing regimens, methods for perioperative hemostatic management, and utility in immune tolerance induction. Experience with ADVATE, and its place in today's treatment paradigm, is the focus of this article.

  10. Recombinant von Willebrand factor: a first-of-its-kind product for von Willebrand disease.

    PubMed

    Singal, M; Kouides, P A

    2016-12-01

    von Willebrand disease (VWD) is caused by quantitative or qualitative defects in von Willebrand factor (VWF). The mainstay of therapy is desmopressin, which is, however, not useful in certain forms of VWD notwithstanding adverse events. For these patients, plasma-derived factor VIII (pdFVIII)/VWF concentrates have been available for close to three decades but have a theoretical risk of disease transmission, hypersensitivity/allergic reactions, inhibitors and thrombosis. A recombinant VWF (vonicog alfa, Vonvendi™; manufactured by Baxalta, now part of Shire) was approved by the U.S. Food and Drug Administration (FDA) in December 2015. This review will survey the literature based on a MEDLINE review on the safety, efficacy and pharmacokinetics of Vonvendi. It will also summarize the ongoing studies on Vonvendi available in the public domain. Vonvendi may have an important role in the management of VWD. However, more studies are needed, especially in special populations such as surgical patients, patients with major gastrointestinal bleeding from arteriovenous malformations and pregnant women and children, who are most likely to benefit from it.

  11. Comparing effects of low and high-flow anesthesia on hemorheology and coagulation factors

    PubMed Central

    Binici, Orhan; Kati, Ismail; Goktas, Ugur; Soyaral, Lokman; Aytekin, Osman Cagatay

    2015-01-01

    Objectives: In the current study, we compared the effects of low- and high-flow anesthesia techniques on hemorheology and coagulation parameters in patients who received sevofluran. Methods: Forty patients classified as Risk Group I–II according to American Society of Anesthesiologists’ (ASA) guidelines who were scheduled to undergo general anesthesia were randomly assigned to one of two groups. Low-flow anesthesia was administered to the first group, and high-flow anesthesia was used in the second group. Blood samples were obtained in the preoperative and peroperative periods (at 60 and 120 min) for determination of blood and plasma viscosity, plasma oncotic pressure, international normalized ratio (INR), phorotrombin time (PT), activated partial phorotrombin time (aPTT) and fibrinogen. Blood was also drawn for analysis of factor VIII (FVIII) activity, which was measured in the preoperative period and at postoperative six hour. Results: The peroperative plasma viscosity was significantly low in Group 1 relative to Group 2. aPTT was significantly elevated at 60 minutes in Group 1 relative to Group 2, but the increase at 120 minutes was not significant. Conclusion: The effects of low-flow anesthesia on hemorheology were greater than those of high-flow anesthesia. PMID:26150868

  12. A DNA fragment from Xq21 replaces a deleted region containing the entire FVIII gene in a severe hemophilia A patient

    SciTech Connect

    Murru, S.; Casula, L.; Moi, P.

    1994-09-15

    In this paper the authors report the molecular characterization of a large deletion that removes the entire Factor VIII gene in a severe hemophilia A patient. Accurate DNA analysis of the breakpoint region revealed that a large DNA fragment replaced the 300-kb one, which was removed by the deletion. Pulsed-field gel electrophoresis analysis revealed that the size of the inserted fragment is about 550 kb. In situ hybridization demonstrated that part of the inserted region normally maps to Xq21 and to the tip of the short arm of the Y chromosome (Yp). In this patient this locus is present both in Xq21 and in Xq28, in addition to the Yp, being thus duplicated in the X chromosome. Sequence analysis of the 3` breakpoint suggested that an illegitimate recombination is probably the cause of this complex rearrangement. 52 refs., 7 figs.

  13. 32 CFR 2003.8 - Records (Article VIII).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 32 National Defense 6 2014-07-01 2014-07-01 false Records (Article VIII). 2003.8 Section 2003.8 National Defense Other Regulations Relating to National Defense INFORMATION SECURITY OVERSIGHT OFFICE...) BYLAWS, RULES, AND APPEAL PROCEDURES Bylaws § 2003.8 Records (Article VIII). (a) Integrity of...

  14. NE VIII lambda 774 and time variable associated absorption in the QSO UM 675

    NASA Technical Reports Server (NTRS)

    Hamann, Fred; Barlow, Thomas A.; Beaver, E. A.; Burbidge, E. M.; Cohen, Ross D.; Junkkarinen, Vesa; Lyons, R.

    1995-01-01

    We discuss measurements of Ne VIII lambda 774 absorption and the time variability of other lines in the z(sub a) approximately equal z(sub e) absorption system of the z(sub e) = 2.15 QSO UM 675 (0150-203). The C IV lambda 1549 and N V 1240 doublets at z(sub a) = 2.1340 (shifted approximately 1500 km/s from z(sub e) strengthened by a factor of approximately 3 between observations by Sargent, Boksenberg and Steidel (1981 November) and our earliest measurements (1990 November and December). We have no information on changes in other z(sub a) approximately equal z(sub e) absorption lines. Continued monitoring since 1990 November shows no clear changes in any of the absorptions between approximately 1100 and 1640 A rest. The short timescale of the variability (less than or approximately equal to 2.9 yr rest) strongly suggests that the clouds are dense, compact, close to the QSO, and photoionized by the QSO continuum. If the line variability is caused by changes in the ionization, the timescale requires densities greater than approximately 4000/cu cm. Photoionization calculations place the absorbing clouds within approximately 200 pc of the continuum source. The full range of line ionizations (from Ne VIII lambda 774 to C III lambda 977) in optically thin gas (no Lyman limit) implies that the absorbing regions span a factor of more than approximately 10 in distance or approximately 100 in density. Across these regions, the total hydrogen (H I + H II) column ranges from a few times 10(exp 18)/sq cm in the low-ionization gas to approximately 10(exp 20)/sq cm where the Ne VIII doublet forms. The metallicity is roughly solar or higher, with nitrogen possibly more enhanced by factors of a few. The clouds might contribute significant line emission if they nearly envelop the QSO. The presence of highly ionized Ne VIII lambda 774 absorption near the QSO supports recent studies that link z(sub a) approximately equal to z(sub e) systems with X-ray 'wamr absorbers. We show that the

  15. NE VIII lambda 774 and time variable associated absorption in the QSO UM 675

    NASA Astrophysics Data System (ADS)

    Hamann, Fred; Barlow, Thomas A.; Beaver, E. A.; Burbidge, E. M.; Cohen, Ross D.; Junkkarinen, Vesa; Lyons, R.

    1995-04-01

    We discuss measurements of Ne VIII lambda 774 absorption and the time variability of other lines in the za approximately equal ze absorption system of the ze = 2.15 QSO UM 675 (0150-203). The C IV lambda 1549 and N V 1240 doublets at za = 2.1340 (shifted approximately 1500 km/s from ze strengthened by a factor of approximately 3 between observations by Sargent, Boksenberg and Steidel (1981 November) and our earliest measurements (1990 November and December). We have no information on changes in other za approximately equal ze absorption lines. Continued monitoring since 1990 November shows no clear changes in any of the absorptions between approximately 1100 and 1640 A rest. The short timescale of the variability (less than or approximately equal to 2.9 yr rest) strongly suggests that the clouds are dense, compact, close to the QSO, and photoionized by the QSO continuum. If the line variability is caused by changes in the ionization, the timescale requires densities greater than approximately 4000/cu cm. Photoionization calculations place the absorbing clouds within approximately 200 pc of the continuum source. The full range of line ionizations (from Ne VIII lambda 774 to C III lambda 977) in optically thin gas (no Lyman limit) implies that the absorbing regions span a factor of more than approximately 10 in distance or approximately 100 in density. Across these regions, the total hydrogen (H I + H II) column ranges from a few times 1018/sq cm in the low-ionization gas to approximately 1020/sq cm where the Ne VIII doublet forms. The metallicity is roughly solar or higher, with nitrogen possibly more enhanced by factors of a few. The clouds might contribute significant line emission if they nearly envelop the QSO. The presence of highly ionized Ne VIII lambda 774 absorption near the QSO supports recent studies that link za approximately equal to ze systems with X-ray 'wamr absorbers. We show that the Ne VIII absorbing gas would itself produce measurable warm

  16. 40 CFR Appendix Viii to Part 268 - LDR Effective Dates of Injected Prohibited Hazardous Wastes

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... Prohibited Hazardous Wastes VIII Appendix VIII to Part 268 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) SOLID WASTES (CONTINUED) LAND DISPOSAL RESTRICTIONS Pt. 268, App. VIII Appendix VIII to Part 268—LDR Effective Dates of Injected Prohibited Hazardous Wastes National Capacity...

  17. 40 CFR Appendix Viii to Part 268 - LDR Effective Dates of Injected Prohibited Hazardous Wastes

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... Prohibited Hazardous Wastes VIII Appendix VIII to Part 268 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) SOLID WASTES (CONTINUED) LAND DISPOSAL RESTRICTIONS Pt. 268, App. VIII Appendix VIII to Part 268—LDR Effective Dates of Injected Prohibited Hazardous Wastes National Capacity...

  18. 40 CFR Appendix Viii to Part 268 - LDR Effective Dates of Injected Prohibited Hazardous Wastes

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Prohibited Hazardous Wastes VIII Appendix VIII to Part 268 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) SOLID WASTES (CONTINUED) LAND DISPOSAL RESTRICTIONS Pt. 268, App. VIII Appendix VIII to Part 268—LDR Effective Dates of Injected Prohibited Hazardous Wastes National Capacity...

  19. Immune tolerance induction in patients with hemophilia A.

    PubMed

    Astermark, Jan

    2011-01-01

    Replacement therapy with factor VIII (FVIII) concentrates has become the mainstay of treatment for hemophilia, but about 30% of patients with a severe disease develop neutralizing antibodies against FVIII, which can lead to treatment resistance and an increased risk of bleeding. Immune tolerance induction (ITI) overcomes the immune response to FVIII concentrates in the majority of patients. Several factors may influence the efficacy of ITI, including disease-related factors (e.g. peak inhibitor titer and pre-ITI titer), and genetic factors (e.g. type of mutation). Treatment-related factors, such as the type of FVIII concentrate used in ITI will also potentially influence the outcome. Specifically, higher success rates with von Willebrand factor (VWF)-containing factor VIII concentrates than with high-purity FVIII concentrates have been reported, but further studies are needed. Potential mechanisms involved include steric hindrance, inhibition of FVIII degradation, or immunomodulatory effects. However, the exact mechanism by which immune tolerance is induced remains unclear. High-dose FVIII ITI appears to induce immune tolerance more rapidly than low-dose protocols and with a reduced risk of bleeding episodes. The addition of immunosuppressive therapy, such as rituximab, to ITI may improve outcomes, although the optimal approach to combined ITI/immunosuppression has not been established. Ongoing studies are likely to provide further insight into the role of genetic features and the type of FVIII concentrate on the success rate of ITI.

  20. Anisotropic Bianchi types VIII and IX locally rotationally symmetric cosmologies

    SciTech Connect

    Assad, M.J.D.; Soares, I.D.

    1983-10-15

    We present a class of exact cosmological solutions of Einstein-Maxwell equations, which are anisotropic and spatially homogeneous of Bianchi types VIII and IX, and class IIIb in the Stewart-Ellis classification of locally rotationally symmetric models. If we take the electromagnetic field equal to zero, a class of Bianchi types VIII/IX spatially homogeneous anisotropic cosmological solutions with perfect fluid is obtained.

  1. Genetic map of Salmonella typhimurium, edition VIII.

    PubMed Central

    Sanderson, K E; Hessel, A; Rudd, K E

    1995-01-01

    We present edition VIII of the genetic map of Salmonella typhimurium LT2. We list a total of 1,159 genes, 1,080 of which have been located on the circular chromosome and 29 of which are on pSLT, the 90-kb plasmid usually found in LT2 lines. The remaining 50 genes are not yet mapped. The coordinate system used in this edition is neither minutes of transfer time in conjugation crosses nor units representing "phage lengths" of DNA of the transducing phage P22, as used in earlier editions, but centisomes and kilobases based on physical analysis of the lengths of DNA segments between genes. Some of these lengths have been determined by digestion of DNA by rare-cutting endonucleases and separation of fragments by pulsed-field gel electrophoresis. Other lengths have been determined by analysis of DNA sequences in GenBank. We have constructed StySeq1, which incorporates all Salmonella DNA sequence data known to us. StySeq1 comprises over 548 kb of nonredundant chromosomal genomic sequences, representing 11.4% of the chromosome, which is estimated to be just over 4,800 kb in length. Most of these sequences were assigned locations on the chromosome, in some cases by analogy with mapped Escherichia coli sequences. PMID:7603411

  2. Realization of Simple Antenna System Using ETS-VIII Satellite for Land Vehicle Communications

    NASA Astrophysics Data System (ADS)

    Basari; E. Purnomo, M. Fauzan; Saito, Kazuyuki; Takahashi, Masaharu; Ito, Koichi

    This paper presents a realization of a simple antenna system for land vehicle satellite communication that is tested in experiments conducted on the Engineering Test Satellite-VIII (ETS-VIII). The developed antenna system which was mounted onto a vehicle roof is compact, light weight with simple satellite-tracking operation. In order to realize compact antennas, an onboard-power divider and switching circuit for antenna feeding control are mounted under the array antenna. A Global Positioning System (GPS) module is used to provide accurate information on the vehicle's position and bearing during travelling. A personal computer (PC) is used as the control unit and data logger, which was specifically designed for this application, allow the switching circuit control as well as the retrieving of the received power levels and error rate. The field tests reported in this paper mainly address the tracking performance of the proposed antenna system. Satisfactory results were obtained. Good received power levels and bit error rate (BER) for tracking the ETS-VIII satellite were confirmed. Furthermore, in order to grasp the environmental factors that impact the quality of land vehicle communications, we carefully captured data at obstacles such as buildings, foliages, utility poles and highway overpasses. The results showed blockage and shadowing was confirmed. Additionally, when the antenna was tested at the inclined-road for simple propagation characteristics in elevation direction, stable reception of the satellite signals was realized.

  3. First analysis of 10-year trends in national factor concentrates usage in haemophilia: data from CHARMS, the Canadian Hemophilia Assessment and Resource Management System.

    PubMed

    Traore, A N; Chan, A K C; Webert, K E; Heddle, N; Ritchie, B; St-Louis, J; Teitel, J; Lillicrap, D; Iorio, A; Walker, I

    2014-07-01

    The Canadian Hemophilia Assessment and Resource Management System (CHARMS) tracks factor concentrates (FC) from the sole suppliers, Canadian Blood Services (CBS) and Hema-Quebec (HQ), to hospitals and to patients' homes. Patients FC infusion data are entered into CHARMS at Canadian Hemophilia Treatment Centres (HTCs) then exported to the national database (CentrePoint). From 2000 to 2009, 2260 registered haemophilia A or B patients received FVIII (1,009,097,765 IU) and FIX (272,406,859 IU). Over 91% of FVIII and over 84% of FIX was infused at home. Utilization of FVIII progressively increased; this was accounted for by an increase in the number of patients treated (r = 0.97; P < 0.001), there being a linear relationship between the increase in utilization and the increase in number of patients treated (P < 0.001). There was also a correlation with the annual amount used per patient (r = 0.95; P < 0.001). Utilization of FIX did not increase over time. The highest proportional utilization of both FVIII and FIX was for prophylaxis, and this proportion progressively increased being, in year 10 (2009), 77% and 66% for FVIII and FIX respectively. The proportion used for bleeding remained steady; in year 10 that proportion was 14% for FVIII and 26% for FIX, the use per patient for bleeding decreasing. The HTC-based CHARMS tracking system is essential, in Canada, for analysing indications for infusion, for predicting utilization and planning for future needs.

  4. Designed ankyrin repeat proteins: a new approach to mimic complex antigens for diagnostic purposes?

    PubMed

    Hausammann, Stefanie; Vogel, Monique; Kremer Hovinga, Johanna A; Lacroix-Desmazes, Sebastien; Stadler, Beda M; Horn, Michael P

    2013-01-01

    Inhibitory antibodies directed against coagulation factor VIII (FVIII) can be found in patients with acquired and congenital hemophilia A. Such FVIII-inhibiting antibodies are routinely detected by the functional Bethesda Assay. However, this assay has a low sensitivity and shows a high inter-laboratory variability. Another method to detect antibodies recognizing FVIII is ELISA, but this test does not allow the distinction between inhibitory and non-inhibitory antibodies. Therefore, we aimed at replacing the intricate antigen FVIII by Designed Ankyrin Repeat Proteins (DARPins) mimicking the epitopes of FVIII inhibitors. As a model we used the well-described inhibitory human monoclonal anti-FVIII antibody, Bo2C11, for the selection on DARPin libraries. Two DARPins were selected binding to the antigen-binding site of Bo2C11, which mimic thus a functional epitope on FVIII. These DARPins inhibited the binding of the antibody to its antigen and restored FVIII activity as determined in the Bethesda assay. Furthermore, the specific DARPins were able to recognize the target antibody in human plasma and could therefore be used to test for the presence of Bo2C11-like antibodies in a large set of hemophilia A patients. These data suggest, that our approach might be used to isolate epitopes from different sets of anti-FVIII antibodies in order to develop an ELISA-based screening assay allowing the distinction of inhibitory and non-inhibitory anti-FVIII antibodies according to their antibody signatures.

  5. Efficacy and safety of long-term prophylaxis in severe hemophilia A dogs following liver gene therapy using AAV vectors.

    PubMed

    Sabatino, Denise E; Lange, Amy M; Altynova, Ekaterina S; Sarkar, Rita; Zhou, Shangzhen; Merricks, Elizabeth P; Franck, Helen G; Nichols, Timothy C; Arruda, Valder R; Kazazian, Haig H

    2011-03-01

    Developing adeno-associated viral (AAV)-mediated gene therapy for hemophilia A (HA) has been challenging due to the large size of the factor VIII (FVIII) complementary DNA and the concern for the development of inhibitory antibodies to FVIII in HA patients. Here, we perform a systematic study in HA dogs by delivering a canine FVIII (cFVIII) transgene either as a single chain or two chains in an AAV vector. An optimized cFVIII single chain delivered using AAV serotype 8 (AAV8) by peripheral vein injection resulted in a dose-response with sustained expression of FVIII up to 7% (n = 4). Five HA dogs administered two-chain delivery using either AAV8 or AAV9 via the portal vein expressed long-term, vector dose-dependent levels of FVIII activity (up to 10%). In the two-chain approach, circulating cFVIII antigen levels were more than fivefold higher than activity. Notably, no long-term immune response to FVIII was observed in any of the dogs (1/9 dogs had a transient inhibitor). Long-term follow-up of the dogs showed a remarkable reduction (>90%) of bleeding episodes in a combined total of 24 years of observation. These data demonstrate that both approaches are safe and achieve dose-dependent therapeutic levels of FVIII expression, which supports translational studies of AAV-mediated delivery for HA.

  6. Design and operational characteristics of electrostatic charge measurement onboard Intelsat VIII & VIII-A communication satellites

    NASA Astrophysics Data System (ADS)

    Ozkul, A.; Scali, F.; Liu, D.; Bowman, C.

    1996-12-01

    Six INTELSAT VIII/VIIIA satellites currently under construction at Lockheed Martin Astrospace facilities in Princeton, New Jersey, USA, will be equipped with electrostatic charge sensors and associated equipment in order to monitor external and internal charging via normal spacecraft telemetry measurement. This paper will describe the design, implementation, measurement strategy, deployments plans associated with the two types of sensors to be installed on these satellites. The discharge threat model which will use the sensor telemetry data as input will also be described. The planned utilization of near real time charge distribution predictions as determined from this model versus the detection and mitigation of the internal and surface electrostatic discharge threat will be discussed. The first INTELSAT VIII satellite equipped with these sensors is planned to be launched in early 1997 and the following five satellites are planned to be launched four months apart following the first. Two types of sensors are planned to be installed on each INTELSAT VIII/VIIIA satellite. The first type of sensor is called "Surface Charge Monitor" which consists of a 2x2 inch alluminum plate painted with Chemglaze dielectric paint and bonded to the spacecraft structure with a non-conductive adhesive. It is designed to measure charging by magnetospheric plasma electrons with energies from 5 keV to 20 keV and with worst case current density from 0.1 to 1 nA/cm2. This range was chosen because it has the probability of occuring of 99%. The charge monitor will indicate when such an environment is present at the spacecraft's orbital location. Two of these units, one on the anti-earth panel another on either the north or south transponder panel, are being mounted onboard each INTELSAT VIII/VIIIA satellite. The second type of sensor is called "Dosimeter/Internal Charge Monitor" consisting of a sensor housing plate equipped with four identical radiation sensitive p-FET device each shielded

  7. The AfaR small RNA controls expression of the AfaD-VIII invasin in pathogenic Escherichia coli strains

    PubMed Central

    Pichon, Christophe; du Merle, Laurence; Lequeutre, Isabelle; Le Bouguénec, Chantal

    2013-01-01

    Pathogenic Escherichia coli strains carrying the afa-8 gene cluster are frequently associated with extra-intestinal infections in humans and animals. The afa-8 A to E genes determine the formation of an afimbrial adhesive sheath consisting of the AfaD-VIII invasin and the AfaE-VIII adhesin at the bacterial cell surface. This structure is thought to be required for host colonization. We characterized a new gene encoding the small RNA AfaR, which is transcribed in cis from the complementary strand of the 3′ untranslated region of the afaD messenger RNA, within the afaD–afaE intercistronic region. AfaR is a trans-acting Hfq-dependent antisense small RNA that binds the 5′ untranslated region of the afaD messenger RNA, initiating several ribonuclease E-dependent cleavages, thereby downregulating production of the AfaD-VIII invasin. AfaR transcription is dependent on σE, a member of the stress response family of extracytoplasmic alternative sigma factors. We found that the AfaR-dependent regulatory pathway was controlled by temperature, allowing the production of the AfaD-VIII invasin at temperatures above 37°C. Our findings suggest that the entry of afa-8-positive pathogenic E. coli strains into epithelial cells is tightly regulated by the AfaR small RNA. PMID:23563153

  8. Chromosome VIII disomy influences the nonsense suppression efficiency and transition metal tolerance of the yeast Saccharomyces cerevisiae.

    PubMed

    Zadorsky, S P; Sopova, Y V; Andreichuk, D Y; Startsev, V A; Medvedeva, V P; Inge-Vechtomov, S G

    2015-06-01

    The SUP35 gene of the yeast Saccharomyces cerevisiae encodes the translation termination factor eRF3. Mutations in this gene lead to the suppression of nonsense mutations and a number of other pleiotropic phenotypes, one of which is impaired chromosome segregation during cell division. Similar effects result from replacing the S. cerevisiae SUP35 gene with its orthologues. A number of genetic and epigenetic changes that occur in the sup35 background result in partial compensation for this suppressor effect. In this study we showed that in S. cerevisiae strains in which the SUP35 orthologue from the yeast Pichia methanolica replaces the S. cerevisiae SUP35 gene, chromosome VIII disomy results in decreased efficiency of nonsense suppression. This antisuppressor effect is not associated with decreased stop codon read-through. We identified SBP1, a gene that localizes to chromosome VIII, as a dosage-dependent antisuppressor that strongly contributes to the overall antisuppressor effect of chromosome VIII disomy. Disomy of chromosome VIII also leads to a change in the yeast strains' tolerance of a number of transition metal salts.

  9. Improved coagulation and hemostasis in hemophilia with inhibitors by combinations of superFactor Va and Factor VIIa

    PubMed Central

    Bhat, Vikas; von Drygalski, Annette; Gale, Andrew J.; Griffin, John H.; Mosnier, Laurent O.

    2015-01-01

    Bypassing inhibitors in hemophilia patients is limited to activated (a) Factor(F)VII products. We introduced “FVa activity augmentation” as another bypassing strategy and studied effects of an engineered FVa variant designated superFVa. Procoagulant and clot stabilizing properties of superFVa and recombinant human (rh)FVIa, either alone or in combination, were studied in thrombin generation and clot lysis assays in normal human plasma (NHP) with or without anti-FVIII inhibitors, in hemophilia plasma, and in FVIII-deficient mice or in wild-type mice with anti-FVIII inhibitors. superFVa was as effective as rhFVIIa to improve thrombin generation or clot lysis. Furthermore, procoagulant effects were significantly enhanced when these compounds were combined. RhFVIIa at 40 nM (a therapeutic concentration) improved thrombin generation mildly, but markedly improved thrombin generation when combined with a low concentration (e.g., 3 nM) of superFVa. In clot lysis studies, the concentration of rhFVIIa to normalize clot lysis times could be reduced by 100-fold (e.g., from 40 nM to 0.4 nM) when combined with a low concentration (0.37 nM) of superFVa. In hemostasis studies of FVIII-deficient mice, blood loss was dose-dependently reduced by either superFVa or rhFVIIa. SuperFVa (200 U/kg) corrected mean blood loss indistinguishably from rhFVIII. Blood loss correction by rhFVIIa was greatly improved when combined with superFVa. Similar blood loss correction results were observed for therapies in wild-type mice after infusion with anti-FVIII inhibitors. Thus, superFVa may be an effective procoagulant agent in the setting of hemophilia with inhibitors and it merits further evaluation for new bypassing strategies. PMID:26466980

  10. 19 CFR Annex Viii-A to Part 351 - Schedule for 90-Day Sunset Reviews

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 19 Customs Duties 3 2014-04-01 2014-04-01 false Schedule for 90-Day Sunset Reviews VIII Annex VIII... AND COUNTERVAILING DUTIES Pt. 351, Annex VIII-A Annex VIII-A to Part 351—Schedule for 90-Day Sunset Reviews Day 1 Event Regulation 0 Initiation § 351.218(c) 15 Filing of Notice of Intent to Participate...

  11. 19 CFR Annex Viii-A to Part 351 - Schedule for 90-Day Sunset Reviews

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 19 Customs Duties 3 2012-04-01 2012-04-01 false Schedule for 90-Day Sunset Reviews VIII Annex VIII... AND COUNTERVAILING DUTIES Pt. 351, Annex VIII-A Annex VIII-A to Part 351—Schedule for 90-Day Sunset Reviews Day 1 Event Regulation 0 Initiation § 351.218(c) 15 Filing of Notice of Intent to Participate...

  12. 19 CFR Annex Viii-A to Part 351 - Schedule for 90-Day Sunset Reviews

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 19 Customs Duties 3 2013-04-01 2013-04-01 false Schedule for 90-Day Sunset Reviews VIII Annex VIII... AND COUNTERVAILING DUTIES Pt. 351, Annex VIII-A Annex VIII-A to Part 351—Schedule for 90-Day Sunset Reviews Day 1 Event Regulation 0 Initiation § 351.218(c) 15 Filing of Notice of Intent to Participate...

  13. 40 CFR Appendix Viii to Part 600 - Fuel Economy Label Formats

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 30 2011-07-01 2011-07-01 false Fuel Economy Label Formats VIII... POLICY FUEL ECONOMY AND CARBON-RELATED EXHAUST EMISSIONS OF MOTOR VEHICLES Pt. 600, App. VIII Appendix VIII to Part 600—Fuel Economy Label Formats EC01MY92.117 EC01MY92.118 EC01MY92.119...

  14. 40 CFR Appendix Viii to Part 268 - LDR Effective Dates of Injected Prohibited Hazardous Wastes

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 28 2013-07-01 2013-07-01 false LDR Effective Dates of Injected Prohibited Hazardous Wastes VIII Appendix VIII to Part 268 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) SOLID WASTES (CONTINUED) LAND DISPOSAL RESTRICTIONS Pt. 268, App. VIII Appendix...

  15. 40 CFR Appendix Viii to Part 600 - Fuel Economy Label Formats

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 29 2010-07-01 2010-07-01 false Fuel Economy Label Formats VIII... POLICY FUEL ECONOMY AND CARBON-RELATED EXHAUST EMISSIONS OF MOTOR VEHICLES Pt. 600, App. VIII Appendix VIII to Part 600—Fuel Economy Label Formats EC01MY92.117 EC01MY92.118 EC01MY92.119...

  16. National Environmental/Energy Workforce Assessment for Region VIII.

    ERIC Educational Resources Information Center

    National Field Research Center Inc., Iowa City, IA.

    This report represents a detailed summation of existing workforce levels, training programs, career potential, and staffing level projections through 1981 for EPA Region VIII. This region serves the Rocky Mountain - Prairie Region and includes the states of Colorado, Montana, Wyoming, Utah, North Dakota, and South Dakota. The specific pollution…

  17. Volunteer Voice, Volume VIII. Tacoma Community House Training Project.

    ERIC Educational Resources Information Center

    Volunteer Voice, 1991

    1991-01-01

    This document consists of the five issues of "Volunteer Voice," the newsletter of a Tacoma, Washington, English-as-a-Second-Language (ESL) training project, issued as volume VIII, during the period June 1990-February 1991. The first issue provides a bibliography of recommended books for ESL tutors and teachers on the following topics: general…

  18. 77 FR 64401 - Order of Succession for HUD Region VIII

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-10-19

    .... Director, Denver Single Family Homeownership Center. 2. Salt Lake City Field Office Order of Succession a... URBAN DEVELOPMENT Order of Succession for HUD Region VIII AGENCY: Office of Field Policy and Management, HUD. ACTION: Notice of Order of Succession. SUMMARY: In this notice, the Assistant Deputy...

  19. Probing the WHIM Gas Through Ne VIII Absorption

    SciTech Connect

    Narayanan, Anand; Wakker, Bart P.; Savage, Blair D.

    2009-05-24

    We report on the second ever {>=}3{sigma} detection of a Ne VIII intergalactic absorption system. The system is identified in the FUSE spectrum of 3C 263 at a redshift of z = 0.32566. The presence of intermediate ionization species such as O III, O IV, N IV and C IV at the same velocity as Ne VIII, confirms that the absorption system is tracing gas at multiple ionization conditions. Unlike the intermediate ions, we find that the Ne VIII in this absorber is inconsistent with having an origin in a cool photoionization gas phase. The more likely source is collisional ionization in a warm shock-heated phase, in which case the absorber is tracing the WHIM phase of the IGM. Further constraints from H I and O IV are needed to better understand the physical conditions in the warm collisionally ionized gas. The properties of this absorber bear resemblance to the previous detection of Ne VIII reported by Savage et al.(2005)

  20. Efficacy and safety of prophylaxis with once-weekly BAY 79-4980 compared with thrice-weekly rFVIII-FS in haemophilia A patients. A randomised, active-controlled, double-blind study.

    PubMed

    Powell, Jerry; Martinowitz, Uri; Windyga, Jerzy; Di Minno, Giovanni; Hellmann, Andrzej; Pabinger, Ingrid; Maas Enriquez, Monika; Schwartz, Lawrence; Ingerslev, Jørgen

    2012-11-01

    The benefits of prophylaxis of haemophilia A patients regarding joint health and quality-of-life are well established. However, adherence to an up to every-other-day infusion regimen is a barrier to widespread adoption of prophylaxis. BAY 79-4980 is an investigational drug consisting of rFVIII-FS (sucrose-formulated recombinant FVIII) reconstituted with liposome solvent. Previous clinical studies showed extended protection from bleeding after a single injection of BAY 79-4980 (13.3 ± 6.2 days) compared with rFVIII-FS (7.2 ± 1.7 days). The effect of once-a-week prophylaxis with BAY 79-4980 (35 IU/kg) compared with three times-per-week rFVIII-FS (25 IU/kg) in previously treated, severe haemophilia A patients was evaluated in a 52-week, double-blind, two-arm, randomised, controlled study. The primary and secondary endpoints were protection from total bleeds and joint bleeds, respectively. Short- and long-term safety and tolerability of BAY 79-4980 including effects on lipid levels were assessed. A total of 139 and 131 subjects were evaluable for safety and efficacy analyses, respectively. A large difference in efficacy between treatment groups was observed with 72.1% (49/68) in the rFVIII-FS control group demonstrating <9 bleeds/year compared with 38.1% (24/63) of BAY 79-4980-treated subjects. A similar difference was seen in annualised joint bleeds, with 43 subjects (63.2%) in the control group demonstrating <5 joint bleeds/year compared with 24 subjects (38.1%) treated with BAY 79-4980. The distribution of bleeds seven days post-prophylactic treatment with BAY 79-4980 showed that 61% of bleeds occurred after day 4 post dosing. There were no safety concerns identified. The investigational treatment arm was prematurely discontinued due to failure to achieve the primary endpoint.

  1. Immunomodulation for inhibitors in hemophilia A: the important role of Treg cells.

    PubMed

    Miao, Carol H

    2010-08-01

    Approximately 25-30% of the hemophilia A patients develop inhibitory antibodies against Factor VIII (FVIII) following protein-replacement therapy. This problem is also thought to occur following gene-replacement therapy. Recently, many approaches have been investigated to modulate FVIII-specific immune responses in either protein-replacement or gene therapy hemophilia A mouse models. Several promising protocols have been demonstrated to successfully prevent or modulate the formation of anti-FVIII antibodies, including methods to manipulate antigen presentation, development of less immunogenic FVIII proteins, or formulations or gene therapy protocols to evade immune responses, as well as immunomodulation strategies to target either T- and/or B-cell responses. Most of these successful protocols involve the induction of activated Treg cells to create a regulatory immune environment during tolerance induction. Innovative strategies to overcome pre-existing anti-FVIII immune responses and induce long-term tolerance in primed subjects still need to be developed.

  2. Assessment of recombinant factor VIIa as an antidote for bleeding induced in the rabbit by low molecular weight heparin.

    PubMed

    Chan, S; Kong, M; Minning, D M; Hedner, U; Marder, V J

    2003-04-01

    While protamine sulfate reverses the anticoagulant effect of standard heparin, there currently is no effective antidote for low molecular weight heparin (LMWH)-induced bleeding. Recently, recombinant activated factor VII (rFVIIa) was approved by the FDA for use in hemophilia patients with factor (F)VIII or FIX inhibitors. However, this new pro-hemostatic agent has potential utility in other clinical scenarios. In this study, we utilized a well-characterized rabbit ear puncture model to test the efficacy of rFVIIa to reverse LMWH-induced prolonged bleeding. Animals were first treated with bolus intravenous LMWH (1800 anti-FXa U kg(-1)) which increased the primary bleeding time approximately fourfold and raised the plasma anti-FXa activity immediately and continuously throughout the 90-min experiment. In a randomized and blinded fashion, animals then received either rFVIIa (400 microg kg(-1)) or placebo by bolus intravenous injection, following which the ear puncture bleeding times were measured, along with blood levels of heparin (anti-FXa activity) and FVII. FVII activity increased 5.3-fold over baseline in treated animals, decreasing by only 24% over the full observation period. The rFVIIa-treated animals showed a slight decrease in bleeding time immediately after injection, but there was no statistically significant difference in bleeding after rFVIIa or placebo administration. In this study using a rabbit ear bleeding model, rFVIIa was not an effective antidote to LMWH-induced bleeding. However, the bolus injection of LMWH produced a very high blood anti-FXa level, which may have precluded rFVIIa effectiveness.

  3. Inhibitors – cellular aspects and novel approaches for tolerance

    PubMed Central

    SCOTT, D. W.

    2014-01-01

    Summary The immune response against therapeutic clotting factors VIII and IX (FVIII and FIX) is a major adverse event that can effectively thwart their effectiveness in correcting bleeding disorders. Thus, a significant number of haemophilia patients form antibodies, called inhibitors, which neutralize the procoagulant functions of therapeutic cofactors FVIII (haemophilia A) or FIX (haemophilia B). Understanding the cellular and molecular aspects of inhibitor formation is critical to designing tolerogenic therapies for clinical use. This review will focus on the basis of the immune response to FVIII, in particular, and will discuss emerging efforts to not only reduce immunogenicity but also to prevent and/or reverse inhibitor formation. PMID:24762281

  4. Zwitterionic Group VIII transition metal initiators supported by olefin ligands

    DOEpatents

    Bazan, Guillermo C.; Chen, Yaofeng

    2011-10-25

    A zwitterionic Group VIII transition metal complex containing the simple and relatively small 3-(arylimino)-but-1-en-2-olato ligand that catalyzes the formation of polypropylene and high molecular weight polyethylene. A novel feature of this catalyst is that the active species is stabilized by a chelated olefin adduct. The present invention also provides methods of polymerizing olefin monomers using zwitterionic catalysts, particularly polypropylene and high molecular weight polyethylene.

  5. Water excitation MPRAGE MRI of VII and VIII cranial nerves

    SciTech Connect

    Litt, A.W.; Licata, P.; Knopp, E.A.; Thomasson, D.M.

    1996-03-01

    Our goal was to compare magnetization prepared rapid gradient echo-water excitation (MPR-AGE-WE) with conventional spin echo (CSE) in the evaluation of the VII and VIII cranial nerves. One hundred three consecutive patients with symptoms referable to the VII/VIII nerves were studied with CSE T1 and MPRAGE-WE following intravenous gadolinium, contrast agent. Each right and left nerve pair was independently evaluated for the presence of an enhancing mass and for visualization of the nerves. On the CSE images, 26 definite and 2 possible lesions were identified, whereas 28 definite and 2 possible abnormalities were seen on the MPRAGE-WE. Four cases were better identified on the MPRAGE-WE and one better seen on the CSE. This difference was not statistically significant (p = 0. 19). CSE demonstrated the nerves partially in 23 instances and completely in 6; MPRAGE-WE showed the nerves partially in 35 and completely in 73. This was highly significant (p < 0.001). With equivalent or slightly improved lesion detection and better visualization of the nerves, MPRAGE-WE may replace CSE in studying the VII/VIII nerves. 14 refs., 7 figs., 3 tabs.

  6. Ehlers-Danlos Syndrome Type VIII: A Rare Cause of Leg Ulcers in Young Patients

    PubMed Central

    Lucas, Antoine; Piérard, Gérald E.; Hermanns-Lê, Trinh; De Paepe, Anne; Dupuy, Alain

    2013-01-01

    Ehlers-Danlos syndrome type VIII (EDS-VIII) is a very rare autosomal dominant disease characterized by early-onset periodontitis associated with features of Ehlers-Danlos syndrome. We report a 32-year-old man whose chronic leg ulcer led to the diagnosis of EDS-VIII. He had severe periodontitis with complete loss of permanent teeth and skin fragility with thin skin, atrophic scars, and brownish atrophic pretibial plaques. Leg ulcer is not a prominent feature of EDS-VIII. We suggest adding EDS-VIII to the list of rare diseases accounting for chronic leg ulcers, if this case report prompts others to report leg ulcers associated with EDS-VIII. PMID:24198978

  7. Positive selection during the evolution of the blood coagulation factors in the context of their disease-causing mutations.

    PubMed

    Rallapalli, Pavithra M; Orengo, Christine A; Studer, Romain A; Perkins, Stephen J

    2014-11-01

    Blood coagulation occurs through a cascade of enzymes and cofactors that produces a fibrin clot, while otherwise maintaining hemostasis. The 11 human coagulation factors (FG, FII-FXIII) have been identified across all vertebrates, suggesting that they emerged with the first vertebrates around 500 Ma. Human FVIII, FIX, and FXI are associated with thousands of disease-causing mutations. Here, we evaluated the strength of selective pressures on the 14 genes coding for the 11 factors during vertebrate evolution, and compared these with human mutations in FVIII, FIX, and FXI. Positive selection was identified for fibrinogen (FG), FIII, FVIII, FIX, and FX in the mammalian Primates and Laurasiatheria and the Sauropsida (reptiles and birds). This showed that the coagulation system in vertebrates was under strong selective pressures, perhaps to adapt against blood-invading pathogens. The comparison of these results with disease-causing mutations reported in FVIII, FIX, and FXI showed that the number of disease-causing mutations, and the probability of positive selection were inversely related to each other. It was concluded that when a site was under positive selection, it was less likely to be associated with disease-causing mutations. In contrast, sites under negative selection were more likely to be associated with disease-causing mutations and be destabilizing. A residue-by-residue comparison of the FVIII, FIX, and FXI sequence alignments confirmed this. This improved understanding of evolutionary changes in FVIII, FIX, and FXI provided greater insight into disease-causing mutations, and better assessments of the codon sites that may be mutated in applications of gene therapy.

  8. Positive Selection during the Evolution of the Blood Coagulation Factors in the Context of Their Disease-Causing Mutations

    PubMed Central

    Rallapalli, Pavithra M.; Orengo, Christine A.; Studer, Romain A.; Perkins, Stephen J.

    2014-01-01

    Blood coagulation occurs through a cascade of enzymes and cofactors that produces a fibrin clot, while otherwise maintaining hemostasis. The 11 human coagulation factors (FG, FII–FXIII) have been identified across all vertebrates, suggesting that they emerged with the first vertebrates around 500 Ma. Human FVIII, FIX, and FXI are associated with thousands of disease-causing mutations. Here, we evaluated the strength of selective pressures on the 14 genes coding for the 11 factors during vertebrate evolution, and compared these with human mutations in FVIII, FIX, and FXI. Positive selection was identified for fibrinogen (FG), FIII, FVIII, FIX, and FX in the mammalian Primates and Laurasiatheria and the Sauropsida (reptiles and birds). This showed that the coagulation system in vertebrates was under strong selective pressures, perhaps to adapt against blood-invading pathogens. The comparison of these results with disease-causing mutations reported in FVIII, FIX, and FXI showed that the number of disease-causing mutations, and the probability of positive selection were inversely related to each other. It was concluded that when a site was under positive selection, it was less likely to be associated with disease-causing mutations. In contrast, sites under negative selection were more likely to be associated with disease-causing mutations and be destabilizing. A residue-by-residue comparison of the FVIII, FIX, and FXI sequence alignments confirmed this. This improved understanding of evolutionary changes in FVIII, FIX, and FXI provided greater insight into disease-causing mutations, and better assessments of the codon sites that may be mutated in applications of gene therapy. PMID:25158795

  9. An update on the von Willebrand factor collagen binding assay: 21 years of age and beyond adolescence but not yet a mature adult.

    PubMed

    Favaloro, Emmanuel J

    2007-11-01

    von Willebrand disease (VWD) is considered to be the most common inherited bleeding disorder. It is diagnosed after a clinical and physical review, with personal and familial evidence of (primarily mucocutaneous) bleeding, and confirmed by laboratory testing. The latter typically entails initial plasma testing of factor VIII coagulant (FVIII:C), von Willebrand factor (VWF) protein (antigen; VWF:Ag), and VWF function, which has classically been assessed using the ristocetin cofactor (VWF:RCo) assay. More recent attention has focused on another functional VWF assay, the collagen binding (VWF:CB) assay, as a possible replacement for the VWF:RCo assay or as a supplementary test of VWF adhesive "activity." Additional laboratory testing can comprise a battery of confirmatory and VWD subtype assisting assays, including assessment of VWF:multimers. This review updates our knowledge of VWD diagnostics with a particular emphasis on the VWF:CB assay. There is good evidence now in place that an optimized VWF:CB assay can significantly reduce the diagnostic error rate otherwise arising from the use of a test panel restricted to including the VWF:RCo assay as the sole functional VWF assay. Nevertheless, the VWF:CB assay should not be used to wholly replace the VWF:RCo assay in phenotypic testing but rather as a supplementary assay. However, with some thought and justification, the VWF:CB assay can be used to partly replace the VWF:RCo assay in some "screening" applications and can also be used to abrogate the need to perform routine VWF:multimers in most test cases.

  10. Risk of inhibitor development in mild haemophilia A increases with age.

    PubMed

    Mauser-Bunschoten, E P; Den Uijl, I E M; Schutgens, R E G; Roosendaal, G; Fischer, K

    2012-03-01

    Mild haemophilia A is a rare disease with a relatively mild phenotype. Treatment with factor VIII (FVIII) is indicated after trauma or for surgery only. FVIII infusion may result in the development of inhibiting antibodies against FVIII. This study describes the relation between age and other risk factors for inhibitor development in mild haemophilia. A retrospective cohort study was conducted among all patients with mild haemophilia (FVIII 0.05-0.40 IU mL(-1)) registered at the van Creveldkliniek, University Medical Centre Utrecht, The Netherlands. Data on peak treatment with FVIII, gene mutation and history of inhibitor development were obtained from patient files from the period between 1st January 1970 and 31st December 2009. A total of 231 out of 297 (78%) patients had at least one exposure to FVIII, of whom 14 (6.1%) developed an inhibitor to FVIII at a median age of 66 years after a median of 50 exposure days (ED). Age at first exposure, age at peak treatment, number of peak treatments and Arg593Cys mutation were significantly associated with the development of an inhibitor, while continuous infusion with FVIII was not. Although the incidence of inhibitors in mild haemophilia is low, it increases with age and peak treatments. With increasing age patients with mild haemophilia will suffer from co-morbidity more frequently, requiring surgical interventions and exposing them to an increased risk of inhibitor development. Especially patients with a change of arginine in cysteine at 593 are at risk for inhibitor development.

  11. Genotyping of Intron Inversions and Point Mutations in Exon 14 of the FVIII Gene in Iranian Azeri Turkish Families with Hemophilia A.

    PubMed

    Shekari Khaniani, Mahmoud; Ebrahimi, Abdollah; Daraei, Setareh; Derakhshan, Sima Mansoori

    2016-12-01

    Hemophilia A (HA) is an inherited X-linked bleeding disorder caused by a variety of mutations that are distributed throughout the large FVIII gene (F8). The most common mutations in studied populations with severe HA are introns 22 and 1 inversions, gross exon deletions and point mutations in exon 14. The aim of this study was to define the frequency of these common mutations in Iranian population of Azeri Turkish in North West of Iran. Fifty patients with severe HA and forty-three female potential carriers were genotyped by inverse shifting polymerase chain reaction (IS-PCR), long-range PCR, multiplex PCR, and sequencing methods for the detection of Intron 22 and 1 inversions, gross exon deletions, and exon 14 point mutations, respectively. F8 intron 22 inversion was detected in 22 (44 %) out of 50 patients. Moreover, we detected one intron 1 inversion (2 %), and one point mutation in exon 14 (2 %). In this population, 52 % of the patients with hemophilia A did not show to carry a mutation in the analyzed regions by three mentioned methods. F8 intron 22 inversion was the major causative mutation in nearly 50 % of severe HA cases in an Azerbaijani Turkish population, which is similar to the incidence of other populations. IS-PCR is a robust, rapid, efficient, and cost-effective method for the genetic analysis of patients with severe HA and for HA carrier detection, especially in developing countries.

  12. Catalytic reforming using group VIII noble metal high silica faujasites

    SciTech Connect

    Vaughan, D.E.; Ghosh, A.K

    1989-05-23

    A process is described for reforming a naphtha feed stock utilizing a catalytic faujasite zeolite composition, which comprises contacting the feed stock at reforming conditions and in the presence of hydrogen with the catalytic zeolite having a faujasite structure and having a SiO/sub 2//Al/sub 2/O/sub 3/ ratio between about 10 and 80 and containing a Group VIII noble metal dispersed therein so as to have a dispersion of hydrogen to metal of about 0.1 to 1, as measured by hydrogen chemisorption.

  13. 40 CFR Appendix Viii to Part 85 - Vehicle and Engine Parameters and Specifications

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...) AIR PROGRAMS (CONTINUED) CONTROL OF AIR POLLUTION FROM MOBILE SOURCES Pt. 85, App. VIII Appendix VIII.... Air Inlet System. 1. Temperature control system calibration. IV. Fuel System. 1. General. a. Engine idle speed. b. Engine idle mixture. 2. Carburetion. a. Air-fuel flow calibration. b....

  14. 40 CFR Appendix Viii to Part 85 - Vehicle and Engine Parameters and Specifications

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...) AIR PROGRAMS (CONTINUED) CONTROL OF AIR POLLUTION FROM MOBILE SOURCES Pt. 85, App. VIII Appendix VIII.... Air Inlet System. 1. Temperature control system calibration. IV. Fuel System. 1. General. a. Engine idle speed. b. Engine idle mixture. 2. Carburetion. a. Air-fuel flow calibration. b....

  15. 40 CFR Appendix Viii to Part 85 - Vehicle and Engine Parameters and Specifications

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...) AIR PROGRAMS (CONTINUED) CONTROL OF AIR POLLUTION FROM MOBILE SOURCES Pt. 85, App. VIII Appendix VIII.... Air Inlet System. 1. Temperature control system calibration. IV. Fuel System. 1. General. a. Engine idle speed. b. Engine idle mixture. 2. Carburetion. a. Air-fuel flow calibration. b....

  16. 40 CFR Appendix Viii to Part 85 - Vehicle and Engine Parameters and Specifications

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...) AIR PROGRAMS (CONTINUED) CONTROL OF AIR POLLUTION FROM MOBILE SOURCES Pt. 85, App. VIII Appendix VIII.... Air Inlet System. 1. Temperature control system calibration. IV. Fuel System. 1. General. a. Engine idle speed. b. Engine idle mixture. 2. Carburetion. a. Air-fuel flow calibration. b....

  17. 40 CFR Appendix Viii to Part 85 - Vehicle and Engine Parameters and Specifications

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...) AIR PROGRAMS (CONTINUED) CONTROL OF AIR POLLUTION FROM MOBILE SOURCES Pt. 85, App. VIII Appendix VIII.... Air Inlet System. 1. Temperature control system calibration. IV. Fuel System. 1. General. a. Engine idle speed. b. Engine idle mixture. 2. Carburetion. a. Air-fuel flow calibration. b....

  18. Effectiveness of Mind Mapping in English Teaching among VIII Standard Students

    ERIC Educational Resources Information Center

    Hallen, D.; Sangeetha, N.

    2015-01-01

    The aim of the study is to find out the effectiveness of mind mapping technique over conventional method in teaching English at high school level (VIII), in terms of Control and Experimental group. The sample of the study comprised, 60 VIII Standard students in Tiruchendur Taluk. Mind Maps and Achievement Test (Pretest & Posttest) were…

  19. 40 CFR Appendix Viii to Part 266 - Organic Compounds for Which Residues Must Be Analyzed

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 27 2011-07-01 2011-07-01 false Organic Compounds for Which Residues Must Be Analyzed VIII Appendix VIII to Part 266 Protection of Environment ENVIRONMENTAL PROTECTION...—Organic Compounds for Which Residues Must Be Analyzed Volatiles Semivolatiles Benzene...

  20. 40 CFR Appendix Viii to Part 266 - Organic Compounds for Which Residues Must Be Analyzed

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 27 2014-07-01 2014-07-01 false Organic Compounds for Which Residues Must Be Analyzed VIII Appendix VIII to Part 266 Protection of Environment ENVIRONMENTAL PROTECTION...—Organic Compounds for Which Residues Must Be Analyzed Volatiles Semivolatiles Benzene...

  1. 40 CFR Appendix Viii to Part 266 - Organic Compounds for Which Residues Must Be Analyzed

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 26 2010-07-01 2010-07-01 false Organic Compounds for Which Residues Must Be Analyzed VIII Appendix VIII to Part 266 Protection of Environment ENVIRONMENTAL PROTECTION...—Organic Compounds for Which Residues Must Be Analyzed Volatiles Semivolatiles Benzene...

  2. 40 CFR Appendix Viii to Part 266 - Organic Compounds for Which Residues Must Be Analyzed

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 28 2012-07-01 2012-07-01 false Organic Compounds for Which Residues Must Be Analyzed VIII Appendix VIII to Part 266 Protection of Environment ENVIRONMENTAL PROTECTION...—Organic Compounds for Which Residues Must Be Analyzed Volatiles Semivolatiles Benzene...

  3. 7 CFR 42.113 - Defects of label, marking, or code; Table VIII.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Defects of label, marking, or code; Table VIII. 42.113... CONTAINER REGULATIONS STANDARDS FOR CONDITION OF FOOD CONTAINERS Procedures for Stationary Lot Sampling and Inspection § 42.113 Defects of label, marking, or code; Table VIII. Table VIII—Label, Marking, or...

  4. 75 FR 1333 - Notice of New Fee Site; Federal Lands Recreation Enhancement Act (Title VIII, Pub. L. 108-447)

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-01-11

    ... (Title VIII, Pub. L. 108-447) AGENCY: Forest Service, USDA. ACTION: Notice of new fee site and.... SUPPLEMENTARY INFORMATION: The Federal Lands Recreation Enhancement Act (Title VIII, Pub. L. 108-447)...

  5. Design of the INHIBIT trial: preventing inhibitors by avoiding ‘danger’, prolonging half-life and promoting tolerance

    PubMed Central

    Ragni, Margaret V; Malec, Lynn M

    2015-01-01

    Inhibitor formation is among the most serious complications of hemophilia treatment. With the US FDA licensure of the novel long-lasting recombinant factor VIII (FVIII) Fc fusion protein, Eloctate, which prolongs FVIII half-life, we propose an innovative approach to prevent inhibitor formation. In this paper, we describe a multicenter, Phase II, single-arm, 48-week trial, the INHIBIT trial, to determine if Eloctate, begun before a bleed and continued as once weekly prophylaxis, will reduce inhibitor formation in children with hemophilia A. We hypothesize that avoiding ‘danger,’ that is, immune activation by a bleed at first factor exposure and prolonging FVIII half-life will prevent inhibitors and promote FVIII-specific T-cell tolerance. If successful, this approach will suggest a new paradigm in clinical practice. PMID:25374055

  6. Detection of Ne VIII in an Intervening Multiphase Absorption System Toward 3C 263

    NASA Astrophysics Data System (ADS)

    Narayanan, Anand; Wakker, Bart P.; Savage, Blair D.

    2009-09-01

    We report the detection of Ne VIII in an intervening multiphase absorption line system at z = 0.32566 in the Far Ultraviolet Spectroscopic Explorer spectrum of the quasar 3C 263 (zem = 0.646). The Ne VIII λ770 Å detection has a 3.9σ significance. At the same velocity, we also find absorption lines from C IV, O III, O IV, and N IV. The line parameter measurements yield log [N(Ne VIII) cm-2] = 13.98+0.10 -0.13 and b = 49.8 ± 5.5 km s-1. We find that the ionization mechanism in the gas phase giving rise to the Ne VIII absorption is inconsistent with photoionization. The absorber has a multiphase structure, with the intermediate ions produced in cool photoionized gas and the Ne VIII most likely in a warm collisionally ionized medium in the temperature range (0.5-1.0) × 106 K. This is the second ever detection of an intervening Ne VIII absorption system. Its properties resemble the previous Ne VIII absorber reported by Savage and colleagues. Direct observations of H I and O VI are needed to better constrain the physical conditions in the collisionally ionized gas phase of this absorber. Based on observations with the NASA-CNES-CSA Far Ultraviolet Spectroscopic Explorer operated by Johns Hopkins University, supported by NASA contract NAS5-32985.

  7. Stimulated Raman scattering from ice-VIII by shock-induced compression in liquid water

    NASA Astrophysics Data System (ADS)

    Men, Zhiwei; Li, Zuowei; Zhou, Mi; Lu, Guohui; Zou, Bo; Li, Zhanlong; Sun, Chenglin

    2012-03-01

    Stimulated Raman scattering (SRS) from ice-VIII was investigated using shock-induced compression (SIC) generated by laser-induced breakdown in liquid water. Three backward SRS peaks of OH stretching vibrations and one backward SRS characteristic peak of lattice translation were observed. The SRS spectra indicated that the ice-VIII structure is formed by SIC, as the trajectory of the SIC passes through the stable pressure-temperature range of ice-VIII. The static electric field generated by electron jets protects proton-ordered structure. The laser-induced shock wave mechanism is also discussed.

  8. Factors affecting the quality of cryoprecipitate

    PubMed Central

    Subramaniyan, Rajeswari; Marwaha, Neelam; Jain, Ashish; Ahluwalia, Jasmina

    2017-01-01

    BACKGROUND: Many variables affect the quality of cryoprecipitate (CRYO). We investigated the effect of freezing techniques and ABO blood groups on the quality of CRYO with respect to factor VIII: C and fibrinogen levels. MATERIALS AND METHODS: Ninety-six whole blood units each collected from in-house (Group I) and blood donation camps outside the hospital premises (Group II) were processed for CRYO preparation. Within each group, half the number of plasma units was frozen using blast freezer and another half using the conventional freezer. The CRYOs from blood groups A, B, and O were equally distributed, i.e. 32 within each of the Groups I and II. The fibrinogen and factor VIII: C levels in CRYO were analyzed using single-stage clotting assay. RESULTS: In Group I, the mean ± standard deviation percentage recovery of factor VIII levels in CRYO prepared using the conventional freezer and blast freezer were 58.5% ±16.2% and 66.7% ±16.4%, respectively, and in Group II, it was 55.3% ±17.6% and 70.4% ±13.4%, respectively. Recovery of factor VIII was higher in CRYO prepared using blast freezer than that of CRYO prepared using conventional freezer (P < 0.000). In Group II, CRYOs prepared using blast freezer had higher percent recovery of fibrinogen than that of Group I. In both the groups, the mean factor VIII levels in blood group A were higher than that of factor VIII levels in the blood group O CRYO. CONCLUSION: The factor VIII recovery in CRYO improves significantly with higher baseline factor VIII: C levels, blood group A donor, and rapid freezing using blast freezer. Rapid freezing also increases the fibrinogen yield. PMID:28316438

  9. Ulex europaeus I lectin as a marker for tumors derived from endothelial cells.

    PubMed

    Miettinen, M; Holthofer, H; Lehto, V P; Miettinen, A; Virtanen, I

    1983-01-01

    Some skin and soft tumors, which generally are assumed to be derived from endothelial cells or blood vessels, were characterized with fluorochrome-labeled Ulex europaeus I agglutinin (UEA I), recently shown to bind specifically to endothelial cells in various normal human tissues. The staining pattern was compared with that obtained with immunostaining using antibodies against factor-VIII-related antigen (FVIII-RAG), a known marker for endothelial cells. The results showed that UEA-I is a specific and a more sensitive marker for the endothelial cells in benign vascular lesions as compared with anti-FVIII-RAG. UEA-I also stained many neoplastic cells of endothelial sarcomas, which generally were negative for FVIII-RAG. Melanomas, anaplastic carcinomas, and other types of sarcomas were negative for both UEA-I and FVIII-RAG. The results suggest that UEA-I lectin is a specific and sensitive adjunct tool in demonstrating endothelial cells and endothelial derivation of human tumors.

  10. Emerging Issues in Diagnosis, Biology, and Inhibitor Risk in Mild Hemophilia A.

    PubMed

    Castaman, Giancarlo; Eckhardt, Corien; van Velzen, Alice; Linari, Silvia; Fijnvandraat, Karin

    2016-07-01

    Mild hemophilia A (MHA) is an X-linked bleeding disorder defined by factor VIII (FVIII) levels between 5 and 40 U/dL. Diagnosis occurs later in life compared with severe or moderate disease. Although bleeding episodes are especially posttraumatic, their unexpected occurrence may be potentially life threatening if diagnosis is missed or delayed. Desmopressin is the treatment of choice for MHA since it is cheap and safe, but a significant proportion of cases do not attain FVIII postinfusion greater than 50 U/dL, which is considered a safe level for major surgery. Thus, replacement therapy may be needed and is usually successful in MHA, but recent data indicate that this can be associated with the occurrence of inhibitors against FVIII, as for severe hemophilia A. However, in contrast to severe or moderate hemophilia A, patients with MHA have a lifelong risk of inhibitor formation. Inhibitors may change the clinical phenotype dramatically, as the inhibitor frequently cross-reacts with the patient's endogenous FVIII, reducing the endogenous FVIII plasma levels below 1 U/dL. Specific F8 missense mutations predispose to inhibitor development. Inhibitors are frequently provoked by intensive treatment with therapeutic FVIII concentrates (more than 5 consecutive exposure days). Bleeding in inhibitor patients may be treated with desmopressin, high doses of FVIII concentrate or FVIII bypassing agents. Many inhibitors disappear over time when no FVIII concentrate is administered. However, this does not imply that a patient is tolerant and an anamnestic reaction may occur when treatment with FVIII concentrate is again necessary. To eradicate, an inhibitor different strategies may be used: watchful waiting, immunosuppression, or immune tolerance induction regimen.

  11. Acquired haemophilia A in a woman with autoimmune hepatitis and systemic lupus erythematosus; review of literature.

    PubMed

    Rezaieyazdi, Zahra; Sharifi-Doloui, Davood; Hashemzadeh, Kamila; Shirdel, Abbas; Mansouritorghabeh, Hassan

    2012-01-01

    Acquired haemophilia A, secondary to systemic lupus erythematosus (SLE) is a rare bleeding diathesis. Here we report a 37-year-old woman with autoimmune hepatitis who developed SLE and acquired haemophilia caused by factor VIII inhibitors. She presented with spontaneous ecchymosis and haematuria. There were a prolongation of the activated partial thromboplastin time, reduced factor VIII activity and a high titer of FVIII inhibitors. Therapeutic regimen was started with intravenous methylprednisolone pulse, continued with prednisolone, intravenous pulse cyclophosphamide and fresh frozen plasma. After 8 weeks, factor VIII inhibitor assay was negative.

  12. Progress and challenges in the development of a cell-based therapy for hemophilia A.

    PubMed

    Fomin, M E; Togarrati, P P; Muench, M O

    2014-12-01

    Hemophilia A results from an insufficiency of factor VIII (FVIII). Although replacement therapy with plasma-derived or recombinant FVIII is a life-saving therapy for hemophilia A patients, such therapy is a life-long treatment rather than a cure for the disease. In this review, we discuss the possibilities, progress, and challenges that remain in the development of a cell-based cure for hemophilia A. The success of cell therapy depends on the type and availability of donor cells, the age of the host and method of transplantation, and the levels of engraftment and production of FVIII by the graft. Early therapy, possibly even prenatal transplantation, may yield the highest levels of engraftment by avoiding immunological rejection of the graft. Potential cell sources of FVIII include a specialized subset of endothelial cells known as liver sinusoidal endothelial cells (LSECs) present in the adult and fetal liver, or patient-specific endothelial cells derived from induced pluripotent stem cells that have undergone gene editing to produce FVIII. Achieving sufficient engraftment of transplanted LSECs is one of the obstacles to successful cell therapy for hemophilia A. We discuss recent results from transplants performed in animals that show production of functional and clinically relevant levels of FVIII obtained from donor LSECs. Hence, the possibility of treating hemophilia A can be envisioned through persistent production of FVIII from transplanted donor cells derived from a number of potential cell sources or through creation of donor endothelial cells from patient-specific induced pluripotent stem cells.

  13. AAV-based neonatal gene therapy for hemophilia A: long-term correction and avoidance of immune responses in mice.

    PubMed

    Hu, C; Lipshutz, G S

    2012-12-01

    Hemophilia A gene therapy has been hampered by immune responses to vector-associated antigens and by neutralizing antibodies or inhibitors against the factor VIII (FVIII) protein; these 'inhibitors' more commonly affect hemophilia A patients than those with hemophilia B. A gene replacement strategy beginning in the neonatal period may avoid the development of these immune responses and lead to prolonged expression with correction of phenotype, thereby avoiding long-term consequences. A serotype rh10 adeno-associated virus (AAV) was developed splitting the FVIII coding sequence into heavy and light chains with the chicken β-actin promoter/CMV enhancer for dual recombinant adeno-associated viral vector delivery. Virions of each FVIII chain were co-injected intravenously into mice on the second day of life. Mice express sustained levels of FVIII antigen ≥5% up to 22 months of life without development of antibodies against FVIII. Phenotypic correction was manifest in all AAV-FVIII-treated mice as demonstrated by functional assay and reduction in bleeding time. This study demonstrates the use of AAV in a gene replacement strategy in neonatal mice that establishes both long-term phenotypic correction of hemophilia A and lack of antibody development against FVIII in this disease model where AAV is administered shortly after birth. These studies support the consideration of gene replacement therapy for diseases that are diagnosed in utero or in the early neonatal period.

  14. Genetic analysis of maintenance of pEC156, a naturally occurring Escherichia coli plasmid that carries genes of the EcoVIII restriction-modification system.

    PubMed

    Werbowy, Olesia; Boratynski, Robert; Dekowska, Agnieszka; Kaczorowski, Tadeusz

    2015-01-01

    In the present study the role of the mechanisms responsible for maintenance of a natural plasmid pEC156, that carries genes of the EcoVIII restriction-modification system was investigated. Analysis of this plasmid's genetic content revealed the presence of genetic determinants suggesting two such mechanisms. The first of them relies on site specific recombination utilizing the Xer/cer molecular machinery, while the second involves a restriction-modification system as an addiction module. Our analysis indicated that three factors affect the maintenance of pEC156: (i) a cis-acting cer site involved in resolution of plasmid multimers, (ii) a gene coding for EcoVIII endonuclease, and (iii) plasmid copy number control. The lowest stability was observed with pEC156 derivatives deprived of the cer site. Decreased stability of pEC156 derivatives was also observed in E.coli strains deficient in genes coding for proteins involved in plasmid multimer resolution (XerC, XerD, ArgR and PepA). A similar effect, but to a much lesser extent was observed for the pEC156 derivative without a functional gene coding for EcoVIII endonuclease. Our results indicate that the presence of the cer site is more important for pEC156 stable maintenance than the presence of a functional gene coding for EcoVIII endonuclease. In our work we also tested maintenance of pEC156 possessing a ColE1-type replicon in bacteria belonging to Enterobacteriaceae family. We have found that pEC156 was most stably maintained in Enterobacter cloacae and Klebsiella oxytoca representing coli-type enterobacteria. We have found that in all enterobacteria tested pEC156 derivatives deficient in the cer site were significantly less stably maintained than cer(+) variants.

  15. Implications of an updated ultraviolet background for the ionization mechanisms of intervening Ne VIII absorbers

    NASA Astrophysics Data System (ADS)

    Hussain, Tanvir; Khaire, Vikram; Srianand, Raghunathan; Muzahid, Sowgat; Pathak, Amit

    2017-04-01

    Ne VIII absorbers seen in QSO spectra are useful tracers of warm ionized gas, when collisional ionization is the dominant ionization process. While photoionization by the ultraviolet background (UVB) is a viable option, it tends to predict large line-of-sight thickness for the absorbing gas. Here, we study the implications of the recently updated UVB at low z to understand the ionization mechanisms of intervening Ne VIII absorbers. With the updated UVB, one typically needs higher density and metallicity to reproduce the observed ionic column densities under photoionization. Both reduce the inferred line-of-sight thicknesses of the absorbers. We find a critical density of ≥5 × 10-5 cm-3 above which the observed N({Ne VIII})/N({O VI}) can be reproduced by pure collisional processes. If the gas is of near solar metallicity (as measured for the low ions) then the cooling time-scales will be small (<108 yrs). Therefore, a continuous injection of heat is required in order to enhance the detectability of the collisionally ionized gas. Using photoionization models we find that in almost all Ne VIII systems the inferred low ion metallicity is near solar or supersolar. If we assume the Ne VIII phase to have similar metallicities then photoionization can reproduce the observed N({Ne VIII})/N({O VI}) without the line-of-sight thickness being unreasonably large and avoids cooling issues related to the collisional ionization at these metallicities. However, the indication of broad Lyα absorption in a couple of systems, if true, suggests that the Ne VIII phase is distinct from the low ion phase having much lower metallicity.

  16. A novel role for Escherichia coli endonuclease VIII in prevention of spontaneous G-->T transversions.

    PubMed

    Blaisdell, J O; Hatahet, Z; Wallace, S S

    1999-10-01

    In the bacterium Escherichia coli, oxidized pyrimidines are removed by two DNA glycosylases, endonuclease III and endonuclease VIII (endo VIII), encoded by the nth and nei genes, respectively. Double mutants lacking both of these activities exhibit a high spontaneous mutation frequency, and here we show that all of the mutations observed in the double mutants were G:C-->A:T transitions; no thymine mutations were found. These findings are in agreement with the preponderance of C-->T transitions in the oxidative and spontaneous mutational databases. The major oxidized purine lesion in DNA, 7,8-dihydro-8-oxoguanine (8-oxoG), is processed by two DNA glycosylases, formamidopyrimidine DNA glycosylase (Fpg), which removes 8-oxoG opposite C, and MutY DNA glycosylase, which removes misincorporated A opposite 8-oxoG. The high spontaneous mutation frequency previously observed in fpg mutY double mutants was significantly enhanced by the addition of the nei mutation, suggesting an overlap in the substrate specificities between endo VIII and Fpg/MutY. When the mutational specificity was examined, all of the mutations observed were G:C-->T:A transversions, indicating that in the absence of Fpg and MutY, endo VIII serves as a backup activity to remove 8-oxoG. This was confirmed by showing that, indeed, endo VIII can recognize 8-oxoG in vitro.

  17. Spectrophotometric determination of hydrogen peroxide with osmium(VIII) and m-carboxyphenylfluorone

    NASA Astrophysics Data System (ADS)

    Hoshino, Mitsuru; Kamino, Shinichiro; Doi, Mitsunobu; Takada, Shingo; Mitani, Shota; Yanagihara, Rika; Asano, Mamiko; Yamaguchi, Takako; Fujita, Yoshikazu

    2014-01-01

    Spectrophotometric determination of hydrogen peroxide was accomplished with osmium(VIII) and m-carboxyphenylfluorone (MCPF) in the presence of cetyltrimethylammonium chloride (CTAC). In the determination of hydrogen peroxide based on the fading of the color of osmium(VIII)-MCPF complex, Beer's law was obeyed in the range 20-406 ng mL-1, with an effective molar absorption coefficient (at 580 nm) of 5.21 × 104 L mol-1 cm-1 and a relative standard deviation of 0.33% (n = 6). Further, we performed the characterization of MCPF and obtained the crystal structure.

  18. Spectrophotometric determination of hydrogen peroxide with osmium(VIII) and m-carboxyphenylfluorone.

    PubMed

    Hoshino, Mitsuru; Kamino, Shinichiro; Doi, Mitsunobu; Takada, Shingo; Mitani, Shota; Yanagihara, Rika; Asano, Mamiko; Yamaguchi, Takako; Fujita, Yoshikazu

    2014-01-03

    Spectrophotometric determination of hydrogen peroxide was accomplished with osmium(VIII) and m-carboxyphenylfluorone (MCPF) in the presence of cetyltrimethylammonium chloride (CTAC). In the determination of hydrogen peroxide based on the fading of the color of osmium(VIII)-MCPF complex, Beer's law was obeyed in the range 20-406 ng mL(-1), with an effective molar absorption coefficient (at 580 nm) of 5.21×10(4) L mol(-1) cm(-1) and a relative standard deviation of 0.33% (n=6). Further, we performed the characterization of MCPF and obtained the crystal structure.

  19. 78 FR 47315 - Solar Partners VIII, LLC; Supplemental Notice That Initial Market-Based Rate Filing Includes...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-08-05

    ... Energy Regulatory Commission Solar Partners VIII, LLC; Supplemental Notice That Initial Market-Based Rate...-referenced proceeding, of Solar Partners VIII, LLC's application for market-based rate authority, with an... proceeding(s) are accessible in the Commission's eLibrary system by clicking on the appropriate link in...

  20. 77 FR 41782 - American River Power VIII, LLC; Notice of Preliminary Permit Application Accepted for Filing and...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-07-16

    ... Hydroelectric Water Power Project No. 14391, to be located at the existing Upper Appleton Dam on the Fox River... Energy Regulatory Commission American River Power VIII, LLC; Notice of Preliminary Permit Application..., 2012, American River Power VIII, LLC filed an application for a preliminary permit under section...

  1. Similarity in Joint Function Limitation in Type 3 Von Willebrand Disease and Moderate Haemophilia A

    PubMed Central

    Sood, S.L.; Cuker, A.; Wang, C.; Metjian, A.D.; Chiang, E.Y.; Soucie, J.M.; Konkle, B.A.

    2013-01-01

    Introduction Type 3 von Willebrand Disease (VWD) is a rare bleeding diathesis with complete or near complete deficiency of von Willebrand factor (VWF) and low factor VIII (FVIII) levels. In contrast, only FVIII is decreased in haemophilia A (HA). Both disorders are complicated by arthropathy. Aim The purpose of this study was to further clarify the roles of FVIII and VWF: Antigen (VWF:Ag) in joint range of motion (ROM) loss over time. Methods We compared joint ROM loss and other bleeding manifestations in 100 Type 3 VWD subjects (FVIII≤5%) and 1814 moderate HA subjects (FVIII 1–5%) within the U.S. Universal Data Collection (UDC) database. Results High rates of bleeding were reported at baseline. During follow up, moderate HA patients reported a joint (46% vs. 34%, p<0.0001) or muscle bleed (27% vs. 16%, p< 0.0001) in a higher proportion of visits than VWD patients. Other bleeds, including mucosal, were reported in a greater proportion of visits among patients with Type 3 VWD than among those with HA (49% vs. 32%, p<0.0001). Multivariate analysis revealed no difference in joint ROM loss over time in the Type 3 VWD versus moderate HA populations. A higher FVIII level was protective in both VWD and HA (p<0.001). Conclusion Our findings support the hypothesis of primacy of the FVIII level in determining risk of joint hemorrhage, and may help target therapy in Type 3 VWD and moderate HA to prevent joint disability. PMID:23534856

  2. Electron impact excitation of Mg VIII . Collision strengths, transition probabilities and theoretical EUV and soft X-ray line intensities for Mg VIII

    NASA Astrophysics Data System (ADS)

    Grieve, M. F. R.; Ramsbottom, C. A.; Keenan, F. P.

    2013-08-01

    Context. Mg viii emission lines are observed in a range of astronomical objects such as the Sun, other cool stars and in the coronal line region of Seyfert galaxies. Under coronal conditions Mg viii emits strongly in the extreme ultraviolet (EUV) and soft X-ray spectral regions which makes it an ideal ion for plasma diagnostics. Aims: Two theoretical atomic models, consisting of 125 fine structure levels, are developed for the Mg viii ion. The 125 levels arise from the 2s22p, 2s2p2, 2p3, 2s23s, 2s23p, 2s23d, 2s2p3s, 2s2p3p, 2s2p3d, 2p23s, 2p23p and 2p23d configurations. Electron impact excitation collision strengths and radiative transition probabilities are calculated for both Mg viii models, compared with existing data, and the best model selected to generate a set of theoretical emission line intensities. The EUV lines, covering 312-790 Å, are compared with existing solar spectra (SERTS-89 and SUMER), while the soft X-ray transitions (69-97 Å) are examined for potential density diagnostic line ratios and also compared with the limited available solar and stellar observational data. Methods: The R-matrix codes Breit-Pauli RMATRXI and RMATRXII are utilised, along with the PSTGF code, to calculate the collision strengths for two Mg viii models. Collision strengths are averaged over a Maxwellian distribution to produce the corresponding effective collision strengths for use in astrophysical applications. Transition probabilities are also calculated using the CIV3 atomic structure code. The best data are then incorporated into the modelling code CLOUDY and line intensities generated for a range of electron temperatures and densities appropriate to solar and stellar coronal plasmas. Results: The present effective collision strengths are compared with two previous calculations. Good levels of agreement are found with the most recent, but there are large differences with the other for forbidden transitions. The resulting line intensities compare favourably with the

  3. Information Technology: Making It All Fit. Track VIII: Academic Computing Strategy.

    ERIC Educational Resources Information Center

    CAUSE, Boulder, CO.

    Six papers from the 1988 CAUSE conference's Track VIII, Academic Computing Strategy, are presented. They include: "Achieving Institution-Wide Computer Fluency: A Five-Year Retrospective" (Paul J. Plourde); "A Methodology and a Policy for Building and Implementing a Strategic Computer Plan" (Frank B. Thomas); "Aligning…

  4. 7 CFR 1901.203 - Title VIII of the Civil Rights Act of 1968.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... made by the Federal Government and, therefore, to all RH borrowers. (2) Any person in the business of... transactions in the past year. (b) Discrimination acts prohibited. Title VIII prohibits FmHA or its successor... buyers. (2) Requiring applicants for services to meet different terms or conditions because of their...

  5. 7 CFR 1901.203 - Title VIII of the Civil Rights Act of 1968.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... made by the Federal Government and, therefore, to all RH borrowers. (2) Any person in the business of... transactions in the past year. (b) Discrimination acts prohibited. Title VIII prohibits FmHA or its successor... buyers. (2) Requiring applicants for services to meet different terms or conditions because of their...

  6. 7 CFR 1901.203 - Title VIII of the Civil Rights Act of 1968.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... made by the Federal Government and, therefore, to all RH borrowers. (2) Any person in the business of... transactions in the past year. (b) Discrimination acts prohibited. Title VIII prohibits FmHA or its successor... buyers. (2) Requiring applicants for services to meet different terms or conditions because of their...

  7. 7 CFR 1901.203 - Title VIII of the Civil Rights Act of 1968.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... made by the Federal Government and, therefore, to all RH borrowers. (2) Any person in the business of... transactions in the past year. (b) Discrimination acts prohibited. Title VIII prohibits FmHA or its successor... buyers. (2) Requiring applicants for services to meet different terms or conditions because of their...

  8. 7 CFR 1901.203 - Title VIII of the Civil Rights Act of 1968.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... made by the Federal Government and, therefore, to all RH borrowers. (2) Any person in the business of... transactions in the past year. (b) Discrimination acts prohibited. Title VIII prohibits FmHA or its successor... buyers. (2) Requiring applicants for services to meet different terms or conditions because of their...

  9. Photoelectric polarimetry of the tail of comet Ikey-Seki (1975 VIII)

    NASA Technical Reports Server (NTRS)

    Weinberg, J. L.; Beeson, D. E.

    1976-01-01

    Post-perihelion measurements of Comet 1965 VIII made on four nights in October-November 1965 using a Fabry photometer atop 3,052 m Mt. Haleakala, Hawaii are described. Detailed results of observations at 5300A on October 29, 1965 are presented.

  10. OATYC Journal, Vol. VIII, Nos. 1-2, Fall 1982-Spring 1983.

    ERIC Educational Resources Information Center

    Fullen, James, Ed.

    1983-01-01

    "OATYC Journal," which is published by the Ohio Association of Two-Year Colleges, is designed as a forum for the exchange of concepts, methods, and findings relevant to the two-year college classroom. Along with commentaries and letters of reaction from the readership, the two issues of volume VIII include the following articles: (1)…

  11. Visualizing the Life and Legacy of Henry VIII: Guiding Students with Eight Types of Graphic Organizers

    ERIC Educational Resources Information Center

    Gallavan, Nancy P.; Kottler, Ellen

    2010-01-01

    Delving into the life and legacy of Henry VIII is both complex and captivating. People seem compelled to learn more abut his critical contributions and controversial conduct that range from the significant to the scandalous. Reflecting on the history of the world would be incomplete without investigating the events and escapades associated with…

  12. Thermal management for LLNL/UC/SSRL bending magnet beamline VIII at Stanford Synchrotron Radiation Laboratory

    SciTech Connect

    Berglin, E.J.; Younger, F.C.

    1986-05-01

    All the important heat loads on the elements of Beamline VIII are cataloged. The principal elements are identified and their heat loads tabulated for various loading scenarios. The expected heat loads are those from normal operations including the anticipated performance improvements planned for the SPEAR ring and from abnormal operations due to positional perturbations of the electron beam. (LEW)

  13. Challenges and Opportunities of Information Technology in the 90s. Track VIII: Managing Distributed Computing Services.

    ERIC Educational Resources Information Center

    CAUSE, Boulder, CO.

    Six papers from the 1990 CAUSE Conference Track VIII: Managing Distributed Computing are presented. Authors discuss the challenges and opportunities involved in providing user managers with direct access to institutional databases to support their decision making and planning activities. Papers and their authors are as follows: "Rendering an…

  14. Tolerance induction in hemophilia A animal models: battling inhibitors with antigen-specific immunotherapies.

    PubMed

    Adair, Patrick; Su, Yan; Scott, David W

    2013-05-01

    Hemophilia A is an X-linked recessive bleeding disorder due to either a lack of or greatly reduced activity in the blood coagulation protein factor VIII (FVIII), due to mutations in the F8 gene. This poses significant challenges for FVIII replacement therapy since hemophilic patients are not immunologically tolerant to the protein. Thus, a proportion of patients who receive plasma-derived or recombinant FVIII replacement therapy develop anti FVIII neutralizing antibodies, known as "inhibitors." These patients require long-term regimens of high dose FVIII administration, which has varying success rates and prohibitive costs. Therefore, therapeutics for tolerance induction in such patients with inhibitors are desired. In this review, we address the current progress of immunotherapies for inducing FVIII specific tolerance in animal models of hemophilia A. Specifically we discuss the beneficial effects of B-cell depletion on immune tolerance induction (ITI), B-cell mediated gene therapy, antigen-coupled lymphocyte therapy, and regulatory T-cell epitopes (Tregitopes).

  15. VIII Olimpíada Brasileira de Astronomia e Astronáutica

    NASA Astrophysics Data System (ADS)

    Garcia Canalle, João Batista; Villas da Rocha, Jaime Fernando; Wuensche de Souza, Carlos Alexandre; Pereira Ortiz, Roberto; Aguilera, Nuricel Villalonga; Padilha, Maria De Fátima Catta Preta; Pessoa Filho, José Bezerra; Soares Rodrigues, Ivette Maria

    2007-07-01

    Neste trabalho apresentamos as motivações pelas quais organizamos, em conjunto, pela primeira vez, a Olimpíada Brasileira de Astronomia incluindo a Astronáutica, em colaboração com a Agência Espacial Brasileira. Esta ampliação contribuiu para atrair ainda mais alunos, professores, escolas e patrocinadores para participarem desta Olimpíada. Em 2005 participaram da VIII Olimpíada Brasileira de Astronomia e Astronáutica (VIII OBA) 187.726 alunos distribuídos por 3.229 escolas, pertencentes a todos os estados brasileiros, incluindo o Distrito Federal. O crescimento em número de alunos participantes foi 52,4% maior do que em 2004. Em abril de 2005 organizamos, em Itapecerica da Serra, SP, um curso para os 50 alunos previamente selecionados e participantes da VII OBA e ao final selecionamos, dentre eles, uma equipe de 5 alunos, os quais representaram o Brasil na X Olimpíada Internacional de Astronomia, na China, em outubro de 2005. Ganhamos, pela primeira vez, uma medalha de ouro naquele evento. Em Agosto de 2005, organizamos a VIII Escola de Agosto para 50 alunos e respectivos professores, em Águas de Lindóia, SP, juntamente com a XXXI reunião anual da Sociedade Astronômica Brasileira (SAB). Em novembro de 2005 realizamos a I Jornada Espacial, em São José dos Campos, com 22 alunos e 22 professores selecionados dentre os participantes que melhores resultados obtiveram nas questões de Astronáutica da VIII OBA. Neste trabalho detalhamos os resultados da VIII OBA bem como as ações subseqüentes.

  16. Spectrum of mutations in CRM-positive and CRM-reduced hemophilia A

    SciTech Connect

    McGinniss, M.J.; Kazazian, H.H. Jr.; Bi, L.; Antonarakis, S.E. ); Hoyer, L.W. ); Inaba, H. )

    1993-02-01

    Hemophilia A is due to the functional deficiency of factor VIII (FVIII, gene locus F8C). Although half the patients have no detectable FVIII protein in their plasma, the more rare patients ([approximately]5%) have normal levels of a dysfunctional FVIII and are termed cross-reacting material (CRM)-positive. More commonly ([approximately]45%), patients have plasma FVIII protein reduced to an extent roughly comparable to the level of FVIII activity and are designated CRM-reduced. We used denaturing gradient gel electrophoresis to screen for mutations within the F8C gene of 11 patients (6CRM-positive, 5 CRM-reduced) and identified 9 different mutations in 9 patients after analyses of all 26 exons, the promoter region, and the polyadenylation site. Six mutations have not been described previously. Five weree missense (Ser289Leu, Ser558Phe, Val634Ala, Val634Met, Asn1441Lys), and the sixth was a 3-bp deletion ([Delta]Phe652). A review of the literature and the assay of FVIII antigen in 5 hemophilia A patients with previously identified missense mutations from this laboratory yielded a total of 20 other unique CRM-reduced and CRM-positive mutations. Almost all CRM-positive/reduced mutations (24/26) were missense, and many (12/26) occurred at CpG dinucleotides. We examined 19 missense mutation for evolutionary conservation using the portions of the porcine and murine F8C sequences that are known, and 18/19 amino acid residue altered by mutation in these patients wer conserved. Almost 50% of mutations (11/26) clustered in the A2 domain, suggesting that this region is critical for the function of FVIII. The results indicate a nonrandom distribution of mutations and suggest that mutations in a limited number of FVIII regions may cause CRM-positive and CRM-reduced heomphilia A. 48 refs., 1 fig., 1 tab.

  17. 75 FR 26196 - Notice of Proposed New Recreation Fee Site; Federal Lands Recreation Enhancement Act, (Title VIII...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-11

    ..., (Title VIII, Pub. L. 108-447) AGENCY: National Forests in Mississippi, Forest Service, USDA. ACTION... 39269. SUPPLEMENTARY INFORMATION: The Federal Recreation Lands Enhancement Act (Title VII, Pub. L....

  18. Structure and electrical characterization of gallium arsenide nanowires with different V/III ratio growth parameters

    SciTech Connect

    Muhammad, R.; Ahamad, R.; Ibrahim, Z.; Othaman, Z.

    2014-03-05

    Gallium arsenide (GaAs) nanowires were grown vertically on GaAs(111)B substrate by gold-assisted using metal-organic chemical vapour deposition. Field-emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM) and conductivity atomic force microscopy (CAFM) analysis were carried out to investigate the effects of V/III ratio on structural properties and current-voltage changes in the wires. Results show that GaAs NWs grow preferably in the wurtzite crystal structure than zinc blende crystal structure with increasing V/III ratio. Additionally, CAFM studies have revealed that zincblende nanowires indicate ohmic characteristic compared to oscillation current occurred for wurtzite structures. The GaAs NWs with high quality structures are needed in solar cells technology for trapping energy that directly converts of sunlight into electricity with maximum capacity.

  19. Reforming with a catalyst containing a group VIII noble metal a group VIII non-noble metal and gallium on separate support particles

    SciTech Connect

    Sorrentino, C. M.; Bertolacini, R. J.; Pellet, R. J.

    1984-11-13

    The catalyst comprises a physical particle-form mixture of a Componet A, a Component B, and a Component C, said Component A comprising at least one Group VIII noble metal, preferably platinum, deposed on a solid catalyst support material providing acidic catalytic sites, said Component B comprising a small amount of a non-noble metal of Group VIII selected from cobalt, nickel, and mixtures thereof, preferably cobalt, on a solid catalyst support material providing acidic catalytic sites, said Component C comprising a small amount of gallium deposed on a solid catalyst support material providing acidic catalytic sites, and said catalyst having been prepared by thoroughly and intimately blending finely-divided particles of said Component A, B, and C to provide a thoroughly-blended composite. The catalyst can be employed suitably in a hydrocarbon conversion process. In particular, the catalyst can be employed in a process for the reforming of a hydrocarbon stream, which process comprises contacting said stream in a reforming zone under reforming conditions and in the presence of hydrogen with said catalyst. The process can be used advantageously to reform a hydrocarbon stream that contains up to 80 ppm sulfur.

  20. Ionospheric Results with Sounding Rockets and the Explorer VIII Satellite (1960 )

    NASA Technical Reports Server (NTRS)

    Bourdeau, R. E.

    1961-01-01

    A review is made of ionospheric data reported since the IGY from rocket and satellite-borne ionospheric experiments. These include rocket results on electron density (RF impedance probe), D-region conductivity (Gerdien condenser), and electron temperature (Langmuir probe). Also included are data in the 1000 kilometer region on ion concentration (ion current monitor) and electron temperature from the Explorer VIII Satellite (1960 xi). The review includes suggestions for second generation experiments and combinations thereof particularly suited for small sounding rockets.

  1. LIMITS ON THE EMISSION FROM FE VIII-XII IN THE HOT LOCAL INTERSTELLAR MEDIUM.

    SciTech Connect

    Bloch, J. J.; Roussel-Dupre, D.; Theiler, J. P.; Johnson, E. M.

    2001-01-01

    The majority of the emitted power from an optically thin million degree plasma (such as the one thought to produce the soft xray background) originates in the EUV band at wavelengths longer than 170 degrees A. Lines from Fe VIII - Fe XII dominate the emission in this wavelength region. This paper describes work in progress on the analysis of three years of data from the ALEXIS mission in search of this flux.

  2. FUNDAMENTALS OF AMHARIC (REVISED EDITION), UNITS VIII-IX. DIALOGUES FOR CONVERSATION, AMHARIC SCRIPT.

    ERIC Educational Resources Information Center

    BARTON, DONALD K.; AND OTHERS

    THIS IS THE THIRD AND FINAL VOLUME OF A NINE-UNIT COURSE IN BASIC AMHARIC. VOLUMES ONE AND TWO DEAL WITH THE PROBLEMS OF PRONUNCIATION AND THE ORAL-AURAL MASTERY OF BASIC GRAMMATICAL CONSTRUCTIONS. AT THE END OF EACH VOLUME ARE APPENDED ENGLISH-AMHARIC AND AMHARIC-ENGLISH GLOSSARIES. VOLUME THREE (UNITS VIII-IX) IS DIVIDED INTO TWO PARTS. PART I…

  3. Hydrogen-bond potential for ice VIII-X phase transition

    PubMed Central

    Zhang, Xi; Chen, Shun; Li, Jichen

    2016-01-01

    Repulsive force between the O-H bonding electrons and the O:H nonbonding pair within hydrogen bond (O-H:O) is an often overlooked interaction which dictates the extraordinary recoverability and sensitivity of water and ice. Here, we present a potential model for this hidden force opposing ice compression of ice VIII-X phase transition based on the density functional theory (DFT) and neutron scattering observations. We consider the H-O bond covalent force, the O:H nonbond dispersion force, and the hidden force to approach equilibrium under compression. Due to the charge polarization within the O:H-O bond, the curvatures of the H-O bond and the O:H nonbond potentials show opposite sign before transition, resulting in the asymmetric relaxation of H-O and O:H (O:H contraction and H-O elongation) and the H+ proton centralization towards phase X. When cross the VIII-X phase boundary, both H-O and O:H contract slightly. The potential model reproduces the VIII-X phase transition as observed in experiment. Development of the potential model may provide a choice for further calculations of water anomalies. PMID:27841335

  4. Hydrogen-bond potential for ice VIII-X phase transition.

    PubMed

    Zhang, Xi; Chen, Shun; Li, Jichen

    2016-11-14

    Repulsive force between the O-H bonding electrons and the O:H nonbonding pair within hydrogen bond (O-H:O) is an often overlooked interaction which dictates the extraordinary recoverability and sensitivity of water and ice. Here, we present a potential model for this hidden force opposing ice compression of ice VIII-X phase transition based on the density functional theory (DFT) and neutron scattering observations. We consider the H-O bond covalent force, the O:H nonbond dispersion force, and the hidden force to approach equilibrium under compression. Due to the charge polarization within the O:H-O bond, the curvatures of the H-O bond and the O:H nonbond potentials show opposite sign before transition, resulting in the asymmetric relaxation of H-O and O:H (O:H contraction and H-O elongation) and the H(+) proton centralization towards phase X. When cross the VIII-X phase boundary, both H-O and O:H contract slightly. The potential model reproduces the VIII-X phase transition as observed in experiment. Development of the potential model may provide a choice for further calculations of water anomalies.

  5. The treatment of hemophilia A: from protein replacement to AAV-mediated gene therapy.

    PubMed

    Youjin, Shen; Jun, Yin

    2009-03-01

    Factor VIII (FVIII) is an essential component in blood coagulation, a deficiency of which causes the serious bleeding disorder hemophilia A. Recently, with the development of purification level and recombinant techniques, protein replacement treatment to hemophiliacs is relatively safe and can prolong their life expectancy. However, because of the possibility of unknown contaminants in plasma-derived FVIII and recombinant FVIII, and high cost for hemophiliacs to use these products, gene therapy for hemophilia A is an attractive alternative to protein replacement therapy. Thus far, the adeno-associated virus (AAV) is a promising vector for gene therapy. Further improvement of the virus for clinical application depends on better understanding of the molecular structure and fate of the vector genome. It is likely that hemophilia will be the first genetic disease to be cured by somatic cell gene therapy.

  6. Alternative pathways of thromboplastin-dependent activation of human factor X in plasma

    SciTech Connect

    Marlar, R.A.; Griffin, J.H.

    1981-01-01

    To determine the interrelationships of the major coagulation pathways, the activation of 3H-labeled factor X in normal and various deficient human plasmas was evaluated when clotting was triggered by dilute rabbit or human thromboplastin. Various dilutions of thromboplastin and calcium were added to plasma samples containing 3H-factor X, and the time course of factor X activation was determined. At a 1/250 dilution of rabbit brain thromboplastin, the rate of factor X activation in plasmas deficient in factor VIII or factor IX was 10% of the activation rate of normal plasma or of factor XI deficient plasma. Reconstitution of the deficient plasmas with factors VIII or IX, respectively, reconstituted normal factor X activation. Similar results were obtained when various dilutions of human thromboplastin replaced the rabbit thromboplastin. From these plasma experiments, it is inferred that the dilute thromboplastin-dependent activation of factor X requires factors VII, IX, and VIII. An alternative extrinsic pathway that involves factors IX and VIII may be the physiologic extrinsic pathway and hence help to explain the consistent clinical observations of bleeding diatheses in patients deficient in factors IX or VIII.

  7. Targeting of the human F8 at the multicopy rDNA locus in Hemophilia A patient-derived iPSCs using TALENickases.

    PubMed

    Pang, Jialun; Wu, Yong; Li, Zhuo; Hu, Zhiqing; Wang, Xiaolin; Hu, Xuyun; Wang, Xiaoyan; Liu, Xionghao; Zhou, Miaojin; Liu, Bo; Wang, Yanchi; Feng, Mai; Liang, Desheng

    2016-03-25

    Hemophilia A (HA) is a monogenic disease due to lack of the clotting factor VIII (FVIII). This deficiency may lead to spontaneous joint hemorrhages or life-threatening bleeding but there is no cure for HA until very recently. In this study, we derived induced pluripotent stem cells (iPSCs) from patients with severe HA and used transcription activator-like effector nickases (TALENickases) to target the factor VIII gene (F8) at the multicopy ribosomal DNA (rDNA) locus in HA-iPSCs, aiming to rescue the shortage of FVIII protein. The results revealed that more than one copy of the exogenous F8 could be integrated into the rDNA locus. Importantly, we detected exogenous F8 mRNA and FVIII protein in targeted HA-iPSCs. After they were differentiated into endothelial cells (ECs), the exogenous FVIII protein was still detectable. Thus, it is showed that the multicopy rDNA locus could be utilized as an effective target site in patient-derived iPSCs for gene therapy. This strategy provides a novel iPSCs-based therapeutic option for HA and other monogenic diseases.

  8. The Herschel Virgo Cluster Survey - VIII. The Bright Galaxy Sample

    NASA Astrophysics Data System (ADS)

    Davies, J. I.; Bianchi, S.; Cortese, L.; Auld, R.; Baes, M.; Bendo, G. J.; Boselli, A.; Ciesla, L.; Clemens, M.; Corbelli, E.; De Looze, I.; di Serego Alighieri, S.; Fritz, J.; Gavazzi, G.; Pappalardo, C.; Grossi, M.; Hunt, L. K.; Madden, S.; Magrini, L.; Pohlen, M.; Smith, M. W. L.; Verstappen, J.; Vlahakis, C.

    2012-02-01

    We describe the Herschel Virgo Cluster Survey and the first data that cover the complete survey area (four 4 × 4 deg2 regions). We use these data to measure and compare the global far-infrared properties of 78 optically bright galaxies that are selected at 500 μm and detected in all five far-infrared bands. We show that our measurements and calibration are broadly consistent with previous data obtained by the IRAS, ISO, Spitzer and Planck. We use SPIRE and PACS photometry data to produce 100-, 160-, 250-, 350- and 500-μm cluster luminosity distributions. These luminosity distributions are not power laws, but 'peaked', with small numbers of both faint and bright galaxies. We measure a cluster 100-500 μm far-infrared luminosity density of 1.6(7.0) ± 0.2 × 109 L⊙ Mpc-3. This compares to a cluster 0.4-2.5 μm optical luminosity density of 5.0(20.0) × 109 L⊙ Mpc-3, some 3.2(2.9) times larger than the far-infrared. A 'typical' photon originates from an optical depth of 0.4 ± 0.1. Most of our sample galaxies are well fitted by a single modified blackbody (β= 2), leading to a mean dust mass of log MDust= 7.31 M⊙ and temperature of 20.0 K. We also derive both stellar and atomic hydrogen masses from which we calculate mean values for the star-to-gas (atomic) and gas (atomic)-to-dust mass ratios of 15.1 and 58.2, respectively. Using our derived dust, atomic gas and stellar masses, we estimate cluster mass densities of 8.6(27.8) × 106, 4.6(13.9) × 108 and 7.8(29.7) × 109 M⊙ Mpc-3 for dust, atomic gas and stars, respectively. These values are higher than those derived for field galaxies by factors of 39(126), 6(18) and 34(129), respectively. In the above, the luminosity/mass densities are given using the whole sample with the values in brackets using just those galaxies that lie between 17 and 23 Mpc. We provide a data table of flux densities in all the Herschel bands for all 78 bright Virgo Cluster galaxies. Herschel is an ESA space observatory with science

  9. The Carina Project. VIII. The α-element abundances

    NASA Astrophysics Data System (ADS)

    Fabrizio, M.; Nonino, M.; Bono, G.; Primas, F.; Thévenin, F.; Stetson, P. B.; Cassisi, S.; Buonanno, R.; Coppola, G.; da Silva, R. O.; Dall'Ora, M.; Ferraro, I.; Genovali, K.; Gilmozzi, R.; Iannicola, G.; Marconi, M.; Monelli, M.; Romaniello, M.; Walker, A. R.

    2015-08-01

    We have performed a new abundance analysis of Carina red giant (RG) stars from spectroscopic data collected with UVES (high spectral resolution) and FLAMES/GIRAFFE (high and medium resolution) at ESO/VLT. The former sample includes 44 RGs, while the latter consists of 65 (high-resolution) and ~800 (medium-resolution) RGs, covering a significant fraction of the galaxy's RG branch, and red clump stars. To improve the abundance analysis at the faint magnitude limit, the FLAMES/GIRAFFE data were divided into ten surface gravity and effective temperature bins. The spectra of the stars belonging to the same gravity and temperature bin were stacked. This approach allowed us to increase the signal-to-noise ratio in the faint magnitude limit (V≥ 20.5 mag) by at least a factor of five. We took advantage of the new photometry index cU,B,I introduced recently as an age and probably a metallicity indicator to split stars along the red giant branch. These two stellar populations display distinct [Fe/H] and [Mg/H] distributions: their mean iron abundances are -2.15 ± 0.06 dex (σ = 0.28), and -1.75 ± 0.03 dex (σ = 0.21), respectively. The two iron distributions differ at the 75% level. This supports preliminary results. Moreover, we found that the old and intermediate-age stellar populations have mean [Mg/H] abundances of -1.91 ± 0.05 dex (σ = 0.22) and -1.35 ± 0.03 dex (σ = 0.22); these differ at the 83% level. Carina's α-element abundances agree, within 1σ, with similar abundances for field halo stars and for cluster (Galactic and Magellanic) stars. The same outcome applies to nearby dwarf spheroidals and ultra-faint dwarf galaxies in the iron range covered by Carina stars. Finally, we found evidence of a clear correlation between Na and O abundances, thus suggesting that Carina's chemical enrichment history is quite different from that in the globular clusters. Based on spectra retrieved from the ESO/ST-ECF Science Archive Facility and collected either with UVES at

  10. A Search for Intergalactic Ne VIII in the COS Spectrum of PKS0405-123

    NASA Astrophysics Data System (ADS)

    Narayanan, Anand; Savage, B.; Wakker, B.; Yao, Y.; Danforth, C.; Froning, C.; Green, J.; Keeney, B.; Sembach, K.; Shull, M.

    2010-01-01

    The high S/N COS spectrum of the UV bright quasar PKS0405-123 provides the sensitivity required for a search of Ne VIII in QSO metal line absorption systems at z > 0.47. The EUV lines of Ne VIII 770,780 A are ideal tracers of hot collisionally ionized gas at T (0.5 - 1) x 106 K. However, detecting these weak lines require spectra of good S/N in which the effects of detector fixed pattern noise are well understood. The z = 0.49510 O VI multiphase metal line absorption system along this sight line is ideally suited for a search for Ne VIII. Chen & Mulchaey (2009) have identified an emission line galaxy of extended morphology at a projected physical separation of 77 kpc from this absorber. The COS spectrum provides coverage of O II 834, O III 833, O IV 788, C III 977 and higher order Lyman series lines associated with this absorber at a much higher S/N than the FUSE and STIS observations for this target. The improved line measurement based on the COS spectrum, combined with the information on O VI from STIS is used to derive detailed physical conditions in the multiple gas phases of this absorber. Evidence for the presence of shock-heated gas would imply that the absorber is tracing either transition temperature gas in the galaxy's halo or an intergalactic WHIM filament connecting galaxies as part of the large scale structure. This research has been supported by the NASA Cosmic Origins Spectrograph Program through a sub-contract to the University of Wisconsin from the University of Colorado.

  11. On the 1s24d Fine Structures of B III and Ne VIII

    NASA Astrophysics Data System (ADS)

    Wang, Zhiwen; Z, W. Wang; Kwong, T. Chung; Zhu, Xiaowei

    1995-01-01

    The fine structure of lithium-like 1s24d states in the literature behaves irregularly as a function of Z. The fine structures of the B III and Ne VIII fall well below the isoelectronic curve. The term energies of these two systems in the data tables also give worse agreement with the theoretical prediction. In this work, we show that the reason for this unusual situation is caused by a misidentification in the original spectra. When the correct identifications are made, the fine structures of both systems fall on the isoelectronic curve and the agreement between theory and experiment is excellent.

  12. CONSTRAINING THE MILKY WAY'S HOT GAS HALO WITH O VII AND O VIII EMISSION LINES

    SciTech Connect

    Miller, Matthew J.; Bregman, Joel N. E-mail: jbregman@umich.edu

    2015-02-10

    The Milky Way hosts a hot (≈2 × 10{sup 6} K), diffuse, gaseous halo based on detections of z = 0 O VII and O VIII absorption lines in quasar spectra and emission lines in blank-sky spectra. Here we improve constraints on the structure of the hot gas halo by fitting a radial model to a much larger sample of O VII and O VIII emission line measurements from XMM-Newton/EPIC-MOS spectra compared to previous studies (≈650 sightlines). We assume a modified β-model for the halo density distribution and a constant-density Local Bubble from which we calculate emission to compare with the observations. We find an acceptable fit to the O VIII emission line observations with χ{sub red}{sup 2} (dof) = 1.08 (644) for best-fit parameters of n{sub o}r{sub c}{sup 3β}=1.35±0.24 cm{sup –3} kpc{sup 3β} and β = 0.50 ± 0.03 for the hot gas halo and negligible Local Bubble contribution. The O VII observations yield an unacceptable χ{sub red}{sup 2} (dof) = 4.69 (645) for similar best-fit parameters, which is likely due to temperature or density variations in the Local Bubble. The O VIII fitting results imply hot gas masses of M(<50 kpc) = 3.8{sub −0.3}{sup +0.3}×10{sup 9} M{sub ⊙} and M(<250 kpc) = 4.3{sub −0.8}{sup +0.9}×10{sup 10} M{sub ⊙}, accounting for ≲50% of the Milky Way's missing baryons. We also explore our results in the context of optical depth effects in the halo gas, the halo gas cooling properties, temperature and entropy gradients in the halo gas, and the gas metallicity distribution. The combination of absorption and emission line analyses implies a sub-solar gas metallicity that decreases with radius, but that also must be ≥0.3 Z {sub ☉} to be consistent with the pulsar dispersion measure toward the Large Magellanic Cloud.

  13. Measurements of Sheath Currents and Equilibrium Potential on the Explorer VIII Satellite (1960 xi)

    NASA Technical Reports Server (NTRS)

    Bourdeau, R. E.; Donley, J. L.; Serbu, G. P.; Whipple, E. C., Jr.

    1961-01-01

    Experimental data were obtained from the Explorer VIII satellite on five parameters pertinent to the problem of the interaction of space vehicles with an ionized atmosphere. The five parameters are: photoemission current due to electrons emitted from the satellite surfaces as a result of solar radiation; electron and positive ion currents due to the diffusion of charged particles from the medium to the spacecraft; the vehicle potential relative to the medium, and the ambient electron temperature. Included in the experimental data is the aspect dependence of the photoemission and diffusion currents. On the basis of the observations, certain characteristics of the satellite's plasma sheath are postulated.

  14. Precise Calculations of Astrophysically Important Allowed and Forbidden Transitions of Xe VIII

    NASA Astrophysics Data System (ADS)

    Bhowmik, Anal; Nath Dutta, Narendra; Roy, Sourav

    2017-02-01

    The present work reports transition line parameters for Xe viii, which are potentially important for astrophysics in view of recent observations of multiply ionized xenon in hot white dwarfs. The relativistic coupled-cluster method is employed here to calculate the E1, E2, and M1 transition line parameters with high accuracy. The E1 oscillator strengths and probabilities of E2 and M1 transitions are determined using theoretical amplitudes and experimental energy values. The calculated branching ratios and the lifetimes are supplemented to the transition parameters. The accurate presentation of these calculated data is crucial for density estimation in several stellar and interstellar media.

  15. Underground Test Area Subproject Phase I Data Analysis Task. Volume VIII - Risk Assessment Documentation Package

    SciTech Connect

    1996-12-01

    Volume VIII of the documentation for the Phase I Data Analysis Task performed in support of the current Regional Flow Model, Transport Model, and Risk Assessment for the Nevada Test Site Underground Test Area Subproject contains the risk assessment documentation. Because of the size and complexity of the model area, a considerable quantity of data was collected and analyzed in support of the modeling efforts. The data analysis task was consequently broken into eight subtasks, and descriptions of each subtask's activities are contained in one of the eight volumes that comprise the Phase I Data Analysis Documentation.

  16. Similarity in joint and mucous bleeding syndromes in type 2N von Willebrand disease and severe hemophilia A coexisting with type 1 von Willebrand disease in two Chinese pedigrees.

    PubMed

    Qin, H H; Xing, Z F; Wang, X F; Ding, Q L; Xi, X D; Wang, H L

    2014-04-01

    In this study, we investigated the molecular basis of two unrelated Chinese patients with hemostatic disorders. The proband of the first family had severe hemophilia A (HA) coexisting with type 1 von Willebrand disease (VWD) and the proband of the second family had type 2N VWD. Both probands had similar phenotypes, which included joint and mucosal bleeding, very low factor VIII (FVIII) activity (FVIII:C), and moderate reductions in VWF antigen (VWF:Ag) and VWF ristocetin cofactor activity (VWF:Rco), as well as a normal multimeric pattern. One FVIII mutation and three VWF mutations were identified: FVIII p.R446* and VWF heterozygous p.E216K mutations were detected in proband 1 and compound heterozygosity of VWF mutations (p.R816W and c.1911delC) in proband 2. Transient expression studies in HEK293T cells proved that R816W mutation abolished the binding of FVIII to VWF and slightly impaired protein synthesis and secretion; 1911delC mutation mainly impaired VWF protein synthesis and secretion. These results provided insight into the possible pathogenic mechanism of type 2N VWD in Chinese patients carrying these mutations.

  17. Second-order many-body perturbation and coupled-cluster singles and doubles study of ice VIII

    SciTech Connect

    Gilliard, Kandis; Sode, Olaseni; Hirata, So

    2014-05-07

    The structure, equation of state, IR, Raman, and inelastic neutron scattering (INS) spectra of high-pressure, proton-ordered phase VIII of ice are calculated by the second-order many-body perturbation and coupled-cluster singles and doubles methods. Nearly all the observed features of the pressure-dependence of the structures and spectra are reproduced computationally up to 60 GPa insofar as the anharmonic effects can be neglected. The calculations display no sign of the hypothetical isostructural transition in 2–3 GPa to phase VIII{sup ′}, the existence of which has been a matter of controversy for over a decade, while they do not contradict the interpretation of the spectral anomaly at 10–14 GPa as a precursor of the VIII-X phase transition. The calculated INS spectra correct a systematic error in the peak positions of the observed spectra.

  18. Therapeutic approaches for treating hemophilia A using embryonic stem cells.

    PubMed

    Kasuda, Shogo; Tatsumi, Kohei; Sakurai, Yoshihiko; Shima, Midori; Hatake, Katsuhiko

    2016-06-01

    Hemophilia A is an X-linked rescessive bleeding disorder that results from F8 gene aberrations. Previously, we established embryonic stem (ES) cells (tet-226aa/N6-Ainv18) that secrete human factor VIII (hFVIII) by introducing the human F8 gene in mouse Ainv18 ES cells. Here, we explored the potential of cell transplantation therapy for hemophilia A using the ES cells. Transplant tet-226aa/N6-Ainv18 ES cells were injected into the spleens of severe combined immunodeficiency (SCID) mice, carbon tetrachloride (CCl4)-pretreated wild-type mice, and CCl4-pretreated hemophilia A mice. F8 expression was induced by doxycycline in drinking water, and hFVIII-antigen production was assessed in all cell transplantation experiments. Injecting the ES cells into SCID mice resulted in an enhanced expression of the hFVIII antigen; however, teratoma generation was confirmed in the spleen. Transplantation of ES cells into wild-type mice after CCl4-induced liver injury facilitated survival and engraftment of transplanted cells without teratoma formation, resulting in hFVIII production in the plasma. Although CCl4 was lethal to most hemophilia A mice, therapeutic levels of FVIII activity, as well as the hFVIII antigen, were detected in surviving hemophilia A mice after cell transplantation. Immunolocalization results for hFVIII suggested that transplanted ES cells might be engrafted at the periportal area in the liver. Although the development of a safer induction method for liver regeneration is required, our results suggested the potential for developing an effective ES-cell transplantation therapeutic model for treating hemophilia A in the future.

  19. [Acquired coagulant factor inhibitors].

    PubMed

    Nogami, Keiji

    2015-02-01

    Acquired coagulation factor inhibitors are an autoimmune disease causing bleeding symptoms due to decreases in the corresponding factor (s) which result from the appearance of autoantibodies against coagulation factors (inhibitor). This disease is quite different from congenital coagulation factor deficiencies based on genetic abnormalities. In recent years, cases with this disease have been increasing, and most have anti-factor VIII autoantibodies. The breakdown of the immune control mechanism is speculated to cause this disease since it is common in the elderly, but the pathology and pathogenesis are presently unclear. We herein describe the pathology and pathogenesis of factor VIII and factor V inhibitors. Characterization of these inhibitors leads to further analysis of the coagulation process and the activation mechanisms of clotting factors. In the future, with the development of new clotting examination method (s), we anticipate that further novel findings will be obtained in this field through inhibitor analysis. In addition, detailed elucidation of the coagulation inhibitory mechanism possibly leading to hemostatic treatment strategies for acquired coagulation factor disorders will be developed.

  20. Technology status of the 13 m aperture deployment antenna reflectors for Engineering Test Satellite VIII

    NASA Astrophysics Data System (ADS)

    Meguro, Akira; Tsujihata, Akio; Hamamoto, Naokazu; Homma, Masanori

    2000-07-01

    Large deployable antenna reflectors for Engineering Test Satellite VIII (ETS-VIII) are now stated in the critical design phase. The Fourteen 4.8m modules, which construct a 19.2 m × 16.7 m (13m aperture) antenna reflector, have been fabricated as Engineering Models. Ground testing for the fourteen modules will be performed until next spring. This paper describes results of critical design for the antenna reflectors and their validation plans. Each module consists of a gold-plated molybdenum mesh surface, spacially determined cable network, and a deployable truss structure as a supporting structure. Stowed size is 1 m (diameter) × 4 m (height). In stowed configuration, the lowest eigen frequencies of the antenna reflector are 47 Hz (longitudinal) and 69 Hz (lateral) respectively. The lowest eigen frequency is 0.14 Hz. Solar ray transparency of the reflector structure is designed to be more than 85% to avoid excessive solar pressure torque. Weight of each reflector is expected to be less than 100 kg. In addition, we will perform a piggyback deployment experiment in transfer orbit using the second stage of the first flight H-II A vehicle in 2000. Half scale seven modules antenna reflector will be used to validate its deployment reliability. Design, analysis and test results of LDR-P are also introduced in this paper.

  1. Characterization of Amm VIII from Androctonus mauretanicus mauretanicus: a new scorpion toxin that discriminates between neuronal and skeletal sodium channels.

    PubMed Central

    Alami, Meriem; Vacher, Hélène; Bosmans, Frank; Devaux, Christiane; Rosso, Jean-Pierre; Bougis, Pierre E; Tytgat, Jan; Darbon, Hervé; Martin-Eauclaire, Marie-France

    2003-01-01

    The venom of the scorpion Androctonus mauretanicus mauretanicus was screened by use of a specific serum directed against AaH II, the scorpion alpha-toxin of reference, with the aim of identifying new analogues. This led to the isolation of Amm VIII (7382.57 Da), which gave a highly positive response in ELISA, but was totally devoid of toxicity when injected subcutaneously into mice. In voltage-clamp experiments with rat brain type II Na+ channel rNa(v)1.2 or rat skeletal muscle Na+ channel rNa(v)1.4, expressed in Xenopus oocytes, the EC50 values of the toxin-induced slowing of inactivation were: 29+/-5 and 416+/-14 nM respectively for AmmVIII and 2.6+/-0.3 nM and 2.2+/-0.2 nM, respectively, for AaH II interactions. Accordingly, Amm VIII clearly discriminates neuronal versus muscular Na+ channel. The Amm VIII cDNA was amplified from a venom gland cDNA library and its oligonucleotide sequence determined. It shows 87% sequence homology with AaH II, but carries an unusual extension at its C-terminal end, consisting of an additional Asp due to a point mutation in the cDNA penultimate codon. We hypothesized that this extra amino acid residue could induce steric hindrance and dramatically reduce recognition of the target by Amm VIII. We constructed a model of Amm VIII based on the X-ray structure of AaH II to clarify this point. Molecular modelling showed that this C-terminal extension does not lead to an overall conformational change in Amm VIII, but drastically modifies the charge repartition and, consequently, the electrostatic dipole moment of the molecule. At last, liquid-phase radioimmunassays with poly- and monoclonal anti-(AaH II) antibodies showed the loss of conformational epitopes between AaH II and Amm VIII. PMID:12911331

  2. 46 CFR 54.01-2 - Adoption of division 1 of section VIII of the ASME Boiler and Pressure Vessel Code.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... Boiler and Pressure Vessel Code. 54.01-2 Section 54.01-2 Shipping COAST GUARD, DEPARTMENT OF HOMELAND... division 1 of section VIII of the ASME Boiler and Pressure Vessel Code. (a) Pressure vessels shall be designed, constructed, and inspected in accordance with section VIII of the ASME Boiler and Pressure...

  3. 46 CFR 54.01-2 - Adoption of division 1 of section VIII of the ASME Boiler and Pressure Vessel Code.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... Boiler and Pressure Vessel Code. 54.01-2 Section 54.01-2 Shipping COAST GUARD, DEPARTMENT OF HOMELAND... division 1 of section VIII of the ASME Boiler and Pressure Vessel Code. (a) Pressure vessels shall be designed, constructed, and inspected in accordance with section VIII of the ASME Boiler and Pressure...

  4. 46 CFR 54.01-2 - Adoption of division 1 of section VIII of the ASME Boiler and Pressure Vessel Code.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... Boiler and Pressure Vessel Code. 54.01-2 Section 54.01-2 Shipping COAST GUARD, DEPARTMENT OF HOMELAND... division 1 of section VIII of the ASME Boiler and Pressure Vessel Code. (a) Pressure vessels shall be designed, constructed, and inspected in accordance with section VIII of the ASME Boiler and Pressure...

  5. 46 CFR 54.01-2 - Adoption of division 1 of section VIII of the ASME Boiler and Pressure Vessel Code.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... Boiler and Pressure Vessel Code. 54.01-2 Section 54.01-2 Shipping COAST GUARD, DEPARTMENT OF HOMELAND... division 1 of section VIII of the ASME Boiler and Pressure Vessel Code. (a) Pressure vessels shall be designed, constructed, and inspected in accordance with section VIII of the ASME Boiler and Pressure...

  6. 46 CFR 54.01-2 - Adoption of division 1 of section VIII of the ASME Boiler and Pressure Vessel Code.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... Boiler and Pressure Vessel Code. 54.01-2 Section 54.01-2 Shipping COAST GUARD, DEPARTMENT OF HOMELAND... division 1 of section VIII of the ASME Boiler and Pressure Vessel Code. (a) Pressure vessels shall be designed, constructed, and inspected in accordance with section VIII of the ASME Boiler and Pressure...

  7. Breit-Pauli oscillator strengths and electron excitation collision strengths for Si VIII

    NASA Astrophysics Data System (ADS)

    Tayal, S. S.

    2012-05-01

    Aims: Oscillator strengths and electron impact excitation collision strengths for transitions between the 68 fine-structure levels of the 2s22p3, 2s2p4, 2p5, 2s22p23s, 2s22p23p, 2s22p23d and 2s2p33s configurations in Si VIII are calculated. Thermally averaged collision strengths are presented as a function of electron temperature for application to solar and other astrophysical plasmas. Methods: The collision strengths have been calculated using the B-splineBreit-Pauli R-matrixmethod for allowed and forbidden transitions in Si VIII. The relativistic effects have been incorporated through mass, Darwin and spin-orbit one-body operators in the Breit-Pauli Hamiltonian in the scattering calculation, while in the calculation of oscillator strengths the one-body and two-body relativistic operators are included. Flexible non-orthogonal sets of spectroscopic and correlation radial functions are used to obtain accurate description of Si VIII levels and to represent the scattering functions. The 68 fine-structure levels of the 2s22p3, 2s2p4, 2p5, 2s22p23s, 2s22p23p, 2s22p23d and 2s2p33s configurations have been considered in both the radiative and scattering calculations. The present scattering calculations are more extensive than previous ones, leading to a total 2278 transitions between fine-structure levels. Results: The calculated excitation energies are in excellent agreement with experiment and represent an improvement over the previous calculations. The present collision strengths show reasonable agreement with the previously available R-matrix and distorted-wave calculations. The oscillator strengths for E1 transitions normally compare very well with previous calculations. The effective collision strengths are obtained by integrating total resonant and non-resonant collision strengths over a Maxwellian distribution of electron energies and these are presented over a wide temperature range from 104 to 4.0 × 106 K. Tables 1-4 are only available in electronic form at

  8. Effectiveness of Peer Tutoring in Learning English among Tutors and Tutees of Class VIII Students in Kancheepuram DT

    ERIC Educational Resources Information Center

    Marieswari, M.; Prema, N.

    2016-01-01

    The peer who teaches to their mates is peer tutoring. It is a common instructional strategy used in classrooms. The aim of this study is to know whether there is any improvement in achievement marks of tutors and tutees after the process of peer tutoring. Class VIII students were selected as the sample for the present experimental study. The…

  9. 75 FR 21583 - Notice of Proposed New Fee Site; Federal Lands Recreation Enhancement Act (Title VIII, Pub. L...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-26

    ... Forest Service Notice of Proposed New Fee Site; Federal Lands Recreation Enhancement Act (Title VIII, Pub. L. 108-447) AGENCY: Manti-La Sal National Forest, USDA Forest Service. ACTION: Notice of proposed new fee site. SUMMARY: The Manti-La Sal National Forest is proposing to charge fees at the ]...

  10. 77 FR 10740 - Lock+ Hydro Friends Fund VIII, FFP Project 92, LLC, Riverbank Hydro No. 24, LLC; Notice...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-23

    ... Energy Regulatory Commission Lock+ Hydro Friends Fund VIII, FFP Project 92, LLC, Riverbank Hydro No. 24, LLC; Notice Announcing Preliminary Permit Drawing The Commission has received three preliminary permit applications deemed filed on September 1, 2011, at 8:30 a.m.,\\1\\ for proposed projects to be located at the...

  11. Comparing Population Change in Societies, Episode VIII. Resource Material Development: Population Dynamics in Eighth Grade American History.

    ERIC Educational Resources Information Center

    Massialas, Byron G.; And Others

    This is the eighth unit in a series that introduces population concepts into the eighth grade American history curriculum. (See SO 013 782 for an overview of the guide.) In Episode VIII, the history topic is population growth and trends in the United States and developing countries. Objectives are to help the student (1) examine how fertility,…

  12. 75 FR 26711 - Notice of New Recreation Fee Site; Federal Lands Recreation Enhancement Act, (Title VIII, Pub. L...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-12

    ... No: 2010-11040] DEPARTMENT OF AGRICULTURE Forest Service Notice of New Recreation Fee Site; Federal Lands Recreation Enhancement Act, (Title VIII, Pub. L. 108-447) AGENCY: National Forests in Mississippi, USDA Forest Service. ACTION: Notice of New Recreation Fee Site. SUMMARY: Bethel Motorcycle and...

  13. 75 FR 5759 - Notice of New Recreation Fee Site; Federal Lands Recreation Enhancement Act, (Title VIII, Pub. L...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-02-04

    ... Forest Service Notice of New Recreation Fee Site; Federal Lands Recreation Enhancement Act, (Title VIII... Recreation Fee Sites. SUMMARY: The Soda Springs Ranger District of the Caribou-Targhee National Forest is... appreciate and enjoy the availability of developed recreation campground and picnicking facilities....

  14. 75 FR 26714 - Notice of Proposed New Recreation Fee Site; Federal Lands Recreation Enhancement Act, (Title VIII...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-12

    ... No: 2010-11042] DEPARTMENT OF AGRICULTURE Forest Service Notice of Proposed New Recreation Fee Site; Federal Lands Recreation Enhancement Act, (Title VIII, Pub. L. 108-447) AGENCY: National Forests in Mississippi, USDA Forest Service. ACTION: Notice of Proposed New Recreation Fee Site. SUMMARY: Rattlesnake...

  15. 75 FR 1749 - Notice of Meeting; Federal Lands Recreation Enhancement Act (Title VIII, Pub. L. 108-447)

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-01-13

    ... From the Federal Register Online via the Government Publishing Office ] DEPARTMENT OF AGRICULTURE Forest Service Notice of Meeting; Federal Lands Recreation Enhancement Act (Title VIII, Pub. L. 108-447) AGENCY: Pacific Northwest Region, Forest Service, USDA. ACTION: Notice of Meeting. SUMMARY: The...

  16. 77 FR 42696 - Notice of New Fee Sites; Federal Lands Recreation Enhancement Act, (Title VIII, Pub. L. 108-447)

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-07-20

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF AGRICULTURE Forest Service Notice of New Fee Sites; Federal Lands Recreation Enhancement Act, (Title VIII, Pub. L... Enhancement Act (Title VII, Pub. L. 108-447) directed the Secretary of Agriculture to publish a six...

  17. 75 FR 30366 - Notice of Meeting; Federal Lands Recreation Enhancement Act (Title VIII, Pub. L. 108-447)

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-01

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF AGRICULTURE Forest Service Notice of Meeting; Federal Lands Recreation Enhancement Act (Title VIII, Pub. L. 108-447) AGENCY: Pacific Southwest Region, Forest Service, U.S. Department of Agriculture. ACTION: Notice...

  18. 75 FR 80789 - Notice of New Fee Site; Federal Lands Recreation Enhancement Act, (Title VIII, Pub. L. 108-447)

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-12-23

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF AGRICULTURE Forest Service Notice of New Fee Site; Federal Lands Recreation Enhancement Act, (Title VIII, Pub. L. 108... Federal Recreation Lands Enhancement Act (Title VII, Pub. L. 108-447) directed the Secretary...

  19. Expression of von Willebrand factor Normandy: An autosomal mutation that mimics hemophilia A

    SciTech Connect

    Tuley, E.A.; Worrall, N.K.; Sadler, J.E. ); Gaucher, C.; Jorieux, S.; Mazurier, C. )

    1991-07-15

    von Willebrand disease Normandy (vWD Normandy) is a recently described phenotype in which a mutant von Willebrand factor (VWF) appears structurally and functionally normal except that is does not bind to blood coagulation factor VIII. This interaction is required for normal survival of factor VIII in the circulation; consequently, vWD Normandy can present as apparent hemophilia A but with autosomal recessive rather than X chromosome-linked inheritance. AvWF missense mutation, Thr{sup 28} {r arrow} Met, was identified in the propositus in or near the factor VIII binding site. The corresponding mutant recombinant vWF(T28M) formed normal multimers and had normal ristocetin cofactor activity. However, vWF(T28M) exhibited the same defect in factor VIII binding as natural vWF Normandy, confirming that this mutation causes the vWD Normandy phenotype. The distinction between hemophilia A and vWD Normandy is clinically important and should be considered in families affected by apparent mild hemophilia A that fail to show strict X chromosome-linked inheritance and, particularly, in potential female carriers with low factor VIII levels attributed to extreme lyonization.

  20. The personality and health of King Henry VIII (1491-1547).

    PubMed

    Keynes, Milo

    2005-08-01

    The projection of Henry VIII in the first half of his reign, which began in 1509, is of a magnificent and accomplished 'imperial prince', the possessor of superb physical health. In 1528, when aged 37, he showed a marked change in personality due, it is here argued, to depressive illness, from which he recovered by the mid-1530s. Such ill health has not been recognized previously and it engenders a need for a reassessment of his character and actions during these years of illness. He did not suffer from syphilis and the well-known leg ulcers were less incapacitating than has been described in the past. This truly enormous and overweight man was 55 years old when he died in 1547 in chronic heart failure.

  1. Congenital combined deficiency of coagulation factors: a study of seven patients.

    PubMed

    Naderi, Majid; Tabibian, Shadi; Hosseini, Maryam Sadat; Alizadeh, Shaban; Hosseini, Soudabeh; Shamsizadeh, Morteza; Dorgalaleh, Akbar

    2015-01-01

    Combined deficiency of coagulation factors is considered as an extremely rare bleeding disorder (RBD) inherited in an autosomal recessive pattern. This disorder is more likely to occur in regions with a high rate of consanguineous marriages or in restricted communities. Sistan and Baluchistan, a province in southeast of Iran with a high rate of consanguinity, is a clear model of such regions with a very high prevalence of recessively inherited disorders. The aim of this study was to report the frequency of combined factor deficiency in this province. This descriptive study was conducted on 358 patients with RBD. Demographic information and medical history of each patient were recorded, and the patients were examined by a physician. Routine screening tests were carried out for all patients, and further coagulation tests including coagulation factor activity and antigen assays were subsequently performed for all suspected patients. Among 358 patients, four were found to be affected with combined factor (F)V and FVIII deficiency (F5F8D). In addition, one patient with combined deficiency of FVII-FXIII, one with combined FVII-FX and one with combined FVIII-FIX deficiency were identified. In Sistan and Baluchistan Province, coinheritance of recessively inherited disorders like combined coagulation factor deficiencies was surprisingly higher than expected.

  2. Human endonuclease VIII-like (NEIL) proteins in the giant DNA Mimivirus

    PubMed Central

    Bandaru, Viswanath; Zhao, Xiaobei; Newton, Michael R.; Burrows, Cynthia J.; Wallace, Susan S.

    2007-01-01

    Endonuclease VIII (Nei), which recognizes and repairs oxidized pyrimidines in the Base Excision Repair (BER) pathway, is sparsely distributed among both the prokaryotes and eukaryotes. Recently, we and others identified three homologs of E. coli endonuclease VIII-like (NEIL) proteins in humans. Here, we report identification of human NEIL homologs in Mimivirus, a giant DNA virus that infects Acanthamoeba. Characterization of the two mimiviral homologs, MvNei1 and MvNei2, showed that they share not only sequence homology but also substrate specificity to the human NEIL proteins, that is, they recognize oxidized pyrimidines in duplex DNA and in bubble substrates and as well show 5′2-deoxyribose-5-phosphate lyase (dRP lyase) activity. However, unlike MvNei1 and the human NEIL proteins, MvNei2 preferentially cleaves oxidized pyrimidines in single stranded DNA forming products with a different end chemistry. Interestingly, opposite base specificity of MvNei1 resembles human NEIL proteins for pyrimidine base damages whereas it resembles E. coli formamidopyrimidine DNA glycosylase (Fpg) for guanidinohydantoin (Gh), an oxidation product of 8-oxoguanine. Finally, a conserved arginine residue in the “zincless finger” motif, previously identified in human NEIL1, is required for the DNA glycosylase activity of MvNeil. Thus, Mimivirus represents the first example of a virus to carry oxidative DNA glycosylases with substrate specificities that resemble human NEIL proteins. Based on the sequence homology to the human NEIL homologs and novel bacterial NEIL homologs identified here, we predict that Mimivirus may have acquired the DNA glycosylases through the host-mediated lateral transfer from either a bacterium or from vertebrates. PMID:17627905

  3. Tissue Factor Activity in Lymphocyte Cultures from Normal Individuals and Patients with Hemophilia A

    PubMed Central

    Rickles, Frederick R.; Hardin, John A.; Pitlick, Frances A.; Hoyer, Leon W.; Conrad, Marcel E.

    1973-01-01

    The procoagulant material of lymphocytes has been characterized as tissue factor. Lymphocytes stimulated with phytohemagglutinin or the purified protein derivative of the tubercle bacillus developed procoagulant activity with incubation in tissue culture. While this material corrected the prolonged clotting time of factor VIII (AHF) deficient plasma, we have shown, utilizing a sensitive radioimmunoassay, that no AHF antigen was present in the cell cultures. Further, we have demonstrated this material to be tissue factor by coagulation techniques and immunological cross-reactivity. The published data regarding factor VIII synthesis is reviewed in light of these observations and comments are made regarding the role of the lymphocyte procoagulant. PMID:4634046

  4. Use of monoclonal antibody and colloidal gold in E.M. localization of von Willebrand factor in megakaryocytes and platelets.

    PubMed

    Jeanneau, C; Avner, P; Sultan, Y

    1984-10-01

    The subcellular localization of Factor VIII/von Willebrand protein (VIII R:Ag) is studied with monoclonal antibody and gold immunocytochemical technique. Monoclonal antibody against purified VIII R:Ag is brightly fluorescent on megakaryocytes and platelets. In E.M., gold immunolabeling is performed on thin cell sections of human megakaryocytes and platelets. Different embedding materials are used to preserve the antigenicity : Epon embedded megakaryocytes show a high concentration of VIII R:Ag in alpha-granules using 4F9 monoclonal antibody. In comparison, lowicryl K4M embedded material does not improve the same specificity, only a few platelets granules were stained. This subcellular localization, in full agreement with biochemical results appears visualized for the first time in E.M.

  5. Defect-free zinc-blende structured InAs nanowires realized by in situ two V/III ratio growth in molecular beam epitaxy.

    PubMed

    Zhang, Zhi; Lu, Zhen-Yu; Chen, Ping-Ping; Lu, Wei; Zou, Jin

    2015-08-07

    In this study, we devised a two-V/III-ratio procedure to control the Au-assisted growth of defect-free InAs nanowires in molecular beam epitaxy. The demonstrated two V/III ratio procedure consists of a first high V/III ratio growth step to prepare the nanowire foundation on the substrate surface, followed by a low V/III ratio step to induce the nanowire growth. By manipulating the V/III ratios in different steps, we have achieved the controlled growth of pure defect-free zinc-blende structured InAs nanowires on the GaAs {1̄1̄1̄} substrates. This study provides an approach to control not only the crystal structure of semiconductor nanowires, but also their structural qualities.

  6. Structure and stability of hexa-aqua V(III) cations in vanadium redox flow battery electrolytes.

    PubMed

    Vijayakumar, M; Li, Liyu; Nie, Zimin; Yang, Zhenguo; Hu, JianZhi

    2012-08-07

    The vanadium(III) cation structure in mixed acid based electrolyte solution from vanadium redox flow batteries is studied by (17)O and (35/37)Cl nuclear magnetic resonance (NMR) spectroscopy, electronic spectroscopy and density functional theory (DFT) based computational modelling. Both computational and experimental results reveal that the V(III) species can complex with counter anions (sulfate/chlorine) depending on the composition of its solvation sphere. By analyzing the powder precipitate it was found that the formation of sulfate complexed V(III) species is the crucial process in the precipitation reaction. The precipitation occurs through nucleation of neutral species formed through deprotonation and ion-pair formation process. However, the powder precipitate shows a multiphase nature which warrants multiple reaction pathways for precipitation reaction.

  7. V/III ratio effects on high quality InAlAs for quantum cascade laser structures

    NASA Astrophysics Data System (ADS)

    Demir, Ilkay; Elagoz, Sezai

    2017-04-01

    In this study we report the V/III ratio effects on growth, structural, optical and doping characteristics of low growth rate (∼1 Å/s) heteroepitaxial Metal Organic Chemical Vapor Deposition (MOCVD) grown InxAl1-xAs layers, a part of Quantum Cascade Laser (QCL) structures, on InP substrate. Especially photoluminescence (PL) properties of InAlAs-InP interface show strong dependence on AsH3 overpressure. We have shown that the V/III ratio with fixed metalorganic precursor flow is a crucial parameter on InxAl1-xAs layers to have a good material quality in terms of crystallinity, optical and electrical characteristics with and without doping.

  8. MX Siting Investigation. Geotechnical Evaluation. Volume VIII. Nevada - Utah Verification Studies, FY 79. Geotechnical Data Big Smoky CDP, Nevada.

    DTIC Science & Technology

    1979-08-24

    Angular - particles have sharp edges and relatively plane sides with unpolished surfaces. wfl . AT#UNL, EWE. FN-TR-27-VIII 6-5 Subangular - particles... GEM ~GIC UNIT My ’All .’j GP T-3 C-33 St FACE ELEV TION: 492j * 1500m) SM jSIRFICIAL GE LOGIC UNIT: AMy A3 SW-SM F -6 C-34 SURFACE ELEVATION: 4938

  9. The Funnel Geometry of Open Flux Tubes in the Low Solar Corona Constrained by O VI and Ne VIII Outflow

    NASA Technical Reports Server (NTRS)

    Byhring, Hanne S.; Esser, Ruth; Lie-Svendsen, Oystein

    2008-01-01

    Model calculations show that observed outflow velocities of order 7-10 km/s of C IV and O VI ions, and 15-20 km/s of Ne VIII ions, are not only consistent with models of the solar wind from coronas holes, but also place unique constraints on the degree of flow tube expansion as well as the location of the expansion in the transition region/lower corona.

  10. Contributions to the Mosquito Fauna of Southeast Asia. VIII. Genus Aedes, Subgenus Mucidus Theobald in Southeast Asia

    DTIC Science & Technology

    1970-01-01

    answered before the origin and routes of distribution of the groups can be hy- pothesized. BIOLOGY AND MEDICAL IMPORTANCE. Although not shown to be a...the size and shape of the trumpets. terial. The preceding descriptions were based solely on paratypic ma- BIOLOGY . The type series was collected...stellate, and from scatophugoides in the lack of noticeable secondary branching in hair g-VIII. BIOLOGY . Material from the Philippines was collected

  11. Assay discrepancy in mild haemophilia A: entire population study in a National Haemophilia Centre.

    PubMed

    Poulsen, A L; Pedersen, L H; Hvas, A-M; Poulsen, L H; Thykjaer, H; Ingerslev, J

    2009-01-01

    Assay discrepancy in mild haemophilia, here defined by a significantly higher factor VIII (FVIII):C response by the one-stage procoagulant assay as compared with a two-stage enzymatic method, has repeatedly been reported in literature. The purpose of this study was to determine the overall prevalence of this phenomenon amongst mild haemophilia families from a population of 2.95 million inhabitants in the Western Danish region. Information was collected retrospectively through a thorough search of archives of the National Haemophilia Centre in Aarhus. We identified 109 patients with mild haemophilia A amongst whom 92 were eligible to enter the study. These represent a total of 53 unrelated families. Our data illustrate that this assay discrepancy pattern is found quite frequently amongst our mild haemophilia A families. While the ratio of FVIII:C chromogenic/FVIII:C clot values was quite consistent amongst patients belonging to same family pattern, ratios in the entire cohort of families ranged from 0.18 to 1.00. Selecting a cut-off level for the FVIII:C chromogenic/FVIII:C clot ratios at 0.7, 0.6 and 0.5, respectively, we found that 38 (72%), 27 (51%) and 19 (36%) of families, respectively, displayed this assay discrepancy. In 10 patients, the FVIII:C chromogenic level was inside the category of moderate haemophilia at >0.01-<0.05 IU mL(-1), pointing to a class-shift in the biochemical phenotype. In conclusion, our data illustrate a substantial prevalence of the assay discrepancy phenomenon amongst mild haemophilia A patients in our geographical area.

  12. Mesenchymal stem cell treatment for hemophilia: a review of current knowledge.

    PubMed

    Sokal, E M; Lombard, C; Mazza, G

    2015-06-01

    Hemophilia remains a non-curative disease, and patients are constrained to undergo repeated injections of clotting factors. In contrast, the sustained production of endogenous factors VIII (FVIII) or IX (FIX) by the patient's own cells could represent a curative treatment. Gene therapy has thus provided new hope for these patients. However, the issues surrounding the durability of expression and immune responses against gene transfer vectors remain. Cell therapy, involving stem cells expanded in vitro, can provide de novo protein synthesis and, if implanted successfully, could induce a steady-state production of low quantities of factors, which may keep the patient above the level required to prevent spontaneous bleeding. Liver-derived stem cells are already being assessed in clinical trials for inborn errors of metabolism and, in view of their capacity to produce FVIII and FIX in cell culture, they are now also being considered for clinical application in hemophilia patients.

  13. Achievements, challenges and unmet needs for haemophilia patients with inhibitors: Report from a symposium in Paris, France on 20 November 2014.

    PubMed

    Dargaud, Y; Pavlova, A; Lacroix-Desmazes, S; Fischer, K; Soucie, M; Claeyssens, S; Scott, D W; d'Oiron, R; Lavigne-Lissalde, G; Kenet, G; Escuriola Ettingshausen, C; Borel-Derlon, A; Lambert, T; Pasta, G; Négrier, C

    2016-01-01

    Over the past 20 years, there have been many advances in haemophilia treatment that have allowed patients to take greater control of their disease. However, the development of factor VIII (FVIII) inhibitors is the greatest complication of the disease and a challenge in the treatment of haemophilia making management of bleeding episodes difficult and surgical procedures very challenging. A meeting to discuss the unmet needs of haemophilia patients with inhibitors was held in Paris on 20 November 2014. Topics discussed were genetic and non-genetic risk factors for the development of inhibitors, immunological aspects of inhibitor development, FVIII products and inhibitor development, generation and functional properties of engineered antigen-specific T regulatory cells, suppression of immune responses to FVIII, prophylaxis in haemophilia patients with inhibitors, epitope mapping of FVIII inhibitors, current controversies in immune tolerance induction therapy, surgery in haemophilia patients with inhibitors and future perspectives for the treatment of haemophilia patients with inhibitors. A summary of the key points discussed is presented in this paper.

  14. Achievements, challenges and unmet needs for haemophilia patients with inhibitors

    PubMed Central

    DARGAUD, Y.; PAVLOVA, A.; LACROIX-DESMAZES, S.; FISCHER, K.; SOUCIE, M.; CLAEYSSENS, S.; SCOTT, D.W.; d’OIRON, R.; LAVIGNE-LISSALDE, G.; KENET, G.; ETTINGSHAUSEN, C. ESCURIOLA; BOREL-DERLON, A.; LAMBERT, T.; PASTA, G.; NÉGRIER, C.

    2016-01-01

    Summary Over the past 20 years, there have been many advances in haemophilia treatment that have allowed patients to take greater control of their disease. However, the development of factor VIII (FVIII) inhibitors is the greatest complication of the disease and a challenge in the treatment of haemophilia making management of bleeding episodes difficult and surgical procedures very challenging. A meeting to discuss the unmet needs of haemophilia patients with inhibitors was held in Paris on 20 November 2014. Topics discussed were genetic and non-genetic risk factors for the development of inhibitors, immunological aspects of inhibitor development, FVIII products and inhibitor development, generation and functional properties of engineered antigen-specific T regulatory cells, suppression of immune responses to FVIII, prophylaxis in haemophilia patients with inhibitors, epitope mapping of FVIII inhibitors, current controversies in immune tolerance induction therapy, surgery in haemophilia patients with inhibitors and future perspectives for the treatment of haemophilia patients with inhibitors. A summary of the key points discussed is presented in this paper. PMID:26728503

  15. Towards personalised therapy for von Willebrand disease: a future role for recombinant products

    PubMed Central

    Favaloro, Emmanuel J.

    2016-01-01

    von Willebrand disease (VWD) is reportedly the most common bleeding disorder and is caused by deficiencies and/or defects in the adhesive plasma protein von Willebrand factor (VWF). Functionally, normal VWF prevents bleeding by promoting both primary and secondary haemostasis. In respect to primary haemostasis, VWF binds to both platelets and sub-endothelial matrix components, especially collagen, to anchor platelets to damaged vascular tissue and promote thrombus formation. VWF also stabilises and protects factor VIII in the circulation, delivering FVIII to the site of injury, which then facilitates secondary haemostasis and fibrin formation/thrombus stabilisation. As a result of this, patients with VWD suffer a bleeding diathesis reflective of a primary defect caused by defective/deficient VWF, which in some patients is compounded by a reduction in FVIII. Management of VWD, therefore, chiefly entails replacement of VWF, and sometimes also FVIII, to protect against bleeding. The current report principally focuses on the future potential for “personalised” management of VWD, given the emerging options in recombinant therapies. Recombinant VWF has been developed and is undergoing clinical trials, and this promising therapy may soon change the way in which VWD is managed. In particular, we can envisage a personalised treatment approach using recombinant VWF, with or without recombinant FVIII, depending on the type of VWD, the extent of deficiencies, and the period and duration of treatment. PMID:27136426

  16. Treatment of von Willebrand Disease.

    PubMed

    Curnow, Jennifer; Pasalic, Leonardo; Favaloro, Emmanuel J

    2016-03-01

    Congenital von Willebrand disease (VWD) and acquired von Willebrand syndrome (AVWS) reflect conditions caused by von Willebrand factor (VWF) deficiency and/or defects. VWD is the most common inherited bleeding disorder and AVWS arises from a variety of causes. Since VWF stabilizes and protects factor VIII (FVIII) in the circulation, this is also reduced in many patients with VWD. The treatment of VWD and AVWS therefore primarily entails replacement of VWF, and sometimes FVIII, to protect against bleeding. This may entail the use of VWF concentrates (currently plasma-derived) and/or FVIII concentrates (currently plasma-derived or more increasingly recombinant forms), and/or desmopressin to release endogenous VWF in subgroups of patients. For AVWS additional treatment of the underlying condition is also required. Adjunct therapies include antifibrinolytics. Globally, various formulations exist for both VWF and FVIII concentrates and are differentially available based on manufacturer marketing or regulatory approvals/clearances in different geographies. Also, guidelines for treatment of VWD vary for different localities and recombinant VWF is undergoing clinical trials. The current review provides an overview of the treatment of VWD as currently practiced in developed countries, and also provides a glimpse towards the future.

  17. State of the art: gene therapy of haemophilia.

    PubMed

    Spencer, H T; Riley, B E; Doering, C B

    2016-07-01

    Clinical gene therapy has been practiced for more than a quarter century and the first products are finally gaining regulatory/marketing approval. As of 2016, there have been 11 haemophilia gene therapy clinical trials of which six are currently open. Each of the ongoing phase 1/2 trials is testing a variation of a liver-directed adeno-associated viral (AAV) vector encoding either factor VIII (FVIII) or factor IX (FIX) . As summarized herein, the clinical results to date have been mixed with some perceived success and a clear recognition of the immune response to AAV as an obstacle to therapeutic success. We also attempt to highlight promising late-stage preclinical activities for AAV-FVIII where, due to inherent challenges with manufacture, delivery and transgene product biosynthesis, more technological development has been necessary to achieve results comparable to what has been observed previously for AAV-FIX. Finally, we describe the development of a stem cell-based lentiviral vector gene therapy product that has the potential to provide lifelong production of FVIII and provide a functional 'cure' for haemophilia A. Integral to this program has been the incorporation of a blood cell-specific gene expression element driving the production of a bioengineered FVIII designed for optimal efficiency. As clearly outlined herein, haemophilia remains at the forefront of the rapidly advancing clinical gene therapy field where there exists a shared expectation that transformational advances are on the horizon.

  18. Evidence-based recommendations on the treatment of von Willebrand disease in Italy

    PubMed Central

    Mannucci, Pier Mannuccio; Franchini, Massimo; Castaman, Giancarlo; Federici, Augusto B.

    2009-01-01

    Background von Willebrand disease (VWD) is the most common hereditary bleeding disorder affecting both males and females. It arises from quantitative or qualitative defects of von Willebrand factor (VWF) and causes bleeding of mucous membranes and soft tissues. The aim of treatment is to correct the dual defect of haemostasis caused by the abnormal/reduced VWF and the concomitant deficiency of factor VIII (FVIII). Material and methods This document contains evidence-based recommendations for the management of VWD compiled by AICE (the Italian Association of Haemophilia Centres). All the evidence supporting these recommendations are based on non-randomised comparative studies or case series, because randomised controlled clinical trials or meta-analyses are not available for this disease. Results and conclusions Desmopressin (DDAVP) is the treatment of choice for patients with type 1 VWD with FVIII and VWF levels of 10 U/dL or more, while VWF/FVIII concentrates are indicated for those who are unresponsive or insufficiently responsive to DDAVP (severe type 1, type 2 and 3 VWD). VWF concentrates devoid of FVIII, not yet licensed in Italy, may be considered for short-term prophylaxis in elective surgery or for long-term secondary prophylaxis. PMID:19503633

  19. Reassessment of ABO blood group, sex, and age on laboratory parameters used to diagnose von Willebrand disorder: potential influence on the diagnosis vs the potential association with risk of thrombosis.

    PubMed

    Favaloro, Emmanuel J; Soltani, Soma; McDonald, Jane; Grezchnik, Ella; Easton, Leanne; Favaloro, James W C

    2005-12-01

    We reassessed the influence of ABO blood group, sex, and age on plasma levels of von Willebrand factor (vWF) antigen, vWF:ristocetin cofactor, vWF:collagen binding assay, and factor VIII coagulant (FVIII:C). Data show that levels of vWF and FVIII:C increase with increasing age (P < .001 for all parameters) and that the ABO blood group influences plasma levels such that O group levels are significantly less than non-O group levels. There was no significant association with sex and Rh status. The selection of normal ranges based on ABO blood groups may influence the clinical diagnosis of von Willebrand disease (vWD) but might not be clinically relevant or help identify people at increased risk of bleeding. Differences in ABO-related ranges were more extensive at the high end of the ranges. This is of particular interest because high levels of vWF and FVIII are associated with thrombosis risk, and an ABO relationship also has been described. O group individuals may or may not be at greater risk for bleeding (they have lowe