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Sample records for factor viii fviii

  1. Phosphatidylserine containing liposomes reduce immunogenicity of recombinant human factor VIII (rFVIII) in a murine model of hemophilia A.

    PubMed

    Ramani, Karthik; Miclea, Razvan D; Purohit, Vivek S; Mager, Donald E; Straubinger, Robert M; Balu-Iyer, Sathy V

    2008-04-01

    Factor VIII (FVIII) is a multidomain protein that is deficient in hemophilia A, a clinically important bleeding disorder. Replacement therapy using recombinant human FVIII (rFVIII) is the main therapy. However, approximately 15-30% of patients develop inhibitory antibodies that neutralize rFVIII activity. Antibodies to epitopes in C2 domain, which is involved in FVIII binding to phospholipids, are highly prevalent. Here, we investigated the effect of phosphatidylserine (PS)-containing liposomes, which bind to C2 domain with high affinity and specificity, upon the immunogenicity of rFVIII. Circular dichroism studies showed that PS-containing liposomes interfered with aggregation of rFVIII. Immunogenicity of free- versus liposomal-rFVIII was evaluated in a murine model of hemophilia A. Animals treated with s.c. injections of liposomal-rFVIII had lower total- and inhibitory titers, compared to animals treated with rFVIII alone. Antigen processing by proteolytic enzymes was reduced in the presence of liposomes. Animals treated with s.c. injections of liposomal-rFVIII showed a significant increase in rFVIII plasma concentration compared to animals that received rFVIII alone. Based on these studies, we hypothesize that specific molecular interactions between PS-containing bilayers and rFVIII may provide a basis for designing lipidic complexes that improve the stability, reduce the immunogenicity of rFVIII formulations, and permit administration by s.c. route. PMID:17705286

  2. FcRn Rescues Recombinant Factor VIII Fc Fusion Protein from a VWF Independent FVIII Clearance Pathway in Mouse Hepatocytes

    PubMed Central

    van der Flier, Arjan; Liu, Zhan; Tan, Siyuan; Chen, Kai; Drager, Douglas; Liu, Tongyao; Patarroyo-White, Susannah; Jiang, Haiyan; Light, David R.

    2015-01-01

    We recently developed a longer lasting recombinant factor VIII-Fc fusion protein, rFVIIIFc, to extend the half-life of replacement FVIII for the treatment of people with hemophilia A. In order to elucidate the biological mechanism for the elongated half-life of rFVIIIFc at a cellular level we delineated the roles of VWF and the tissue-specific expression of the neonatal Fc receptor (FcRn) in the biodistribution, clearance and cycling of rFVIIIFc. We find the tissue biodistribution is similar for rFVIIIFc and rFVIII and that liver is the major clearance organ for both molecules. VWF reduces the clearance and the initial liver uptake of rFVIIIFc. Pharmacokinetic studies in FcRn chimeric mice show that FcRn expressed in somatic cells (hepatocytes or liver sinusoidal endothelial cells) mediates the decreased clearance of rFVIIIFc, but FcRn in hematopoietic cells (Kupffer cells) does not affect clearance. Immunohistochemical studies show that when rFVIII or rFVIIIFc is in dynamic equilibrium binding with VWF, they mostly co localize with VWF in Kupffer cells and macrophages, confirming a major role for liver macrophages in the internalization and clearance of the VWF-FVIII complex. In the absence of VWF a clear difference in cellular localization of VWF-free rFVIII and rFVIIIFc is observed and neither molecule is detected in Kupffer cells. Instead, rFVIII is observed in hepatocytes, indicating that free rFVIII is cleared by hepatocytes, while rFVIIIFc is observed as a diffuse liver sinusoidal staining, suggesting recycling of free-rFVIIIFc out of hepatocytes. These studies reveal two parallel linked clearance pathways, with a dominant pathway in which both rFVIIIFc and rFVIII complexed with VWF are cleared mainly by Kupffer cells without FcRn cycling. In contrast, the free fraction of rFVIII or rFVIIIFc unbound by VWF enters hepatocytes, where FcRn reduces the degradation and clearance of rFVIIIFc relative to rFVIII by cycling rFVIIIFc back to the liver sinusoid and

  3. Structural Characterization of Carbohydrate Binding by LMAN1 Protein Provides New Insight into the Endoplasmic Reticulum Export of Factors V (FV) and VIII (FVIII)*

    PubMed Central

    Zheng, Chunlei; Page, Richard C.; Das, Vaijayanti; Nix, Jay C.; Wigren, Edvard; Misra, Saurav; Zhang, Bin

    2013-01-01

    LMAN1 (ERGIC-53) is a key mammalian cargo receptor responsible for the export of a subset of glycoproteins from the endoplasmic reticulum. Together with its soluble coreceptor MCFD2, LMAN1 transports coagulation factors V (FV) and VIII (FVIII). Mutations in LMAN1 or MCFD2 cause the genetic bleeding disorder combined deficiency of FV and FVIII (F5F8D). The LMAN1 carbohydrate recognition domain (CRD) binds to both glycoprotein cargo and MCFD2 in a Ca2+-dependent manner. To understand the biochemical basis and regulation of LMAN1 binding to glycoprotein cargo, we solved crystal structures of the LMAN1-CRD bound to Man-α-1,2-Man, the terminal carbohydrate moiety of high mannose glycans. Our structural data, combined with mutagenesis and in vitro binding assays, define the central mannose-binding site on LMAN1 and pinpoint histidine 178 and glycines 251/252 as critical residues for FV/FVIII binding. We also show that mannobiose binding is relatively independent of pH in the range relevant for endoplasmic reticulum-to-Golgi traffic, but is sensitive to lowered Ca2+ concentrations. The distinct LMAN1/MCFD2 interaction is maintained at these lowered Ca2+ concentrations. Our results suggest that compartmental changes in Ca2+ concentration regulate glycoprotein cargo binding and release from the LMAN1·MCFD2 complex in the early secretory pathway. PMID:23709226

  4. A major determinant of the immunogenicity of factor VIII in a murine model is independent of its procoagulant function

    PubMed Central

    Meeks, Shannon L.; Cox, Courtney L.; Healey, John F.; Parker, Ernest T.; Doshi, Bhavya S.; Gangadharan, Bagirath; Barrow, Rachel T.

    2012-01-01

    A main complication of treatment of patients with hemophilia A is the development of anti–factor VIII (fVIII) antibodies. The immunogenicity of fVIII potentially is a function of its procoagulant activity, which may result in danger signals that drive the immune response. Alternatively, intrinsic structural elements in fVIII may be particularly immunogenic. Finally, VWF, the carrier protein for fVIII in plasma, may play a role in immune recognition. We compared the immunogenicity of wild-type (wt) B domain–deleted fVIII and 2 inactive fVIII molecules, R372A/R1689A fVIII and V634M fVIII in fVIII−/− and fVIII−/−/VWF−/− mice. R372A/R1689A fVIII lacks proteolytic recognition sites and is not released from VWF. In contrast, V634M fVIII undergoes proteolytic cleavage and dissociation from VWF. No significant difference was observed in the immunogenicity of wt fVIII and V634M fVIII. R372A/R1689A fVIII was slightly less immunogenic in a subset of immunization regimens tested. High doses of wt fVIII were required to produce an immune response in fVIII−/−/VWF−/− mice. Our results indicate that a main component of the immune response to fVIII is independent of its procoagulant function, is both positively and negatively affected by its association with VWF, and may involve intrinsic elements of fVIII structure. PMID:22855607

  5. Hunting down factor VIII in the immunopeptidome.

    PubMed

    Hartholt, Robin B; Peyron, Ivan; Voorberg, Jan

    2016-03-01

    Major histocompatibility complex class II (MHCII)-restricted peptide presentation is crucial for the selection and subsequent proliferation of antigen specific CD4+ T cells. While selection of antigen-specific CD4+ T cells is beneficial in the context of vaccination, emergence of antigen CD4+ T cells following administration of therapeutic proteins like factor VIII (FVIII) is not desirable. The mechanism of uptake, processing and presentation of FVIII by antigen-presenting cells (APCs) has been the subject of intense study over the past 10 years. Multiple receptors have been implicated in the uptake of FVIII by APCs. A crucial determinant directing its entry in APCs resides in the C1 domain of FVIII. Until recently, our knowledge on the repertoire of FVIII derived presented on MHCII was limited. Peptide sequences on FVIII recognized by CD4+ T cells have been identified using MHCII tetramers as well as by directly monitoring peptide-induced proliferation of CD4+ T cells. More recently, the repertoire of naturally presented peptides derived from FVIII has been identified by pulsing of immature dendritic cells with FVIII. In a complementary approach HLA-DRB1(∗)15 transgenic mice were used to identify HLA-DRB1(∗)15 restricted CD4+ T cells reactive towards human FVIII. In this review we summarize our current knowledge on FVIII derived peptides that are presented on MHCII and discuss the relevance of these findings for the etiology of inhibitor development in patients with hemophilia A. PMID:26610639

  6. Transgenic pigs produce functional human factor VIII in milk.

    PubMed

    Paleyanda, R K; Velander, W H; Lee, T K; Scandella, D H; Gwazdauskas, F C; Knight, J W; Hoyer, L W; Drohan, W N; Lubon, H

    1997-10-01

    Deficiency or abnormality of coagulation factor VIII (FVIII) causes a bleeding disorder called hemophilia A. Treatment involves FVIII concentrates prepared from pooled human plasma or recombinant FVIII (rFVIII) prepared from mammalian cell culture. The cost of highly purified FVIII or rFVIII is a major factor in hemophilia therapy and restricts prophylaxis. We have sought to generate a new source of rFVIII by targeting expression of the human FVIII cDNA to the mammary gland of transgenic pigs using the regulatory sequences of the mouse whey acidic protein gene. The identity of processed heterodimeric rFVIII was confirmed using specific antibodies, by thrombin digestion and activity assays. The secretion of as much as 2.7 micrograms/ml of rFVIII in milk was over tenfold higher than in normal plasma. Up to 0.62 U/ml of rFVIII was detected in an assay in which rFVIII restored normal clotting activity to FVIII-deficient human plasma. PMID:9335047

  7. 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.

  8. 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. PMID:26739399

  9. Domain Organization of Membrane-Bound Factor VIII

    PubMed Central

    Stoilova-McPhie, Svetla; Lynch, Gillian C.; Ludtke, Steven; Pettitt, B. Montgomery

    2014-01-01

    Factor VIII (FVIII) is the blood coagulation protein which when defective or deficient causes for hemophilia A, a severe hereditary bleeding disorder. Activated FVIII (FVIIIa) is the cofactor to the serine protease factor IXa (FIXa) within the membrane-bound Tenase complex, responsible for amplifying its proteolytic activity more than 100,000 times, necessary for normal clot formation. FVIII is composed of two noncovalently linked peptide chains: a light chain (LC) holding the membrane interaction sites and a heavy chain (HC) holding the main FIXa interaction sites. The interplay between the light and heavy chains (HCs) in the membrane-bound state is critical for the biological efficiency of FVIII. Here, we present our cryo-electron microscopy (EM) and structure analysis studies of human FVIII-LC, when helically assembled onto negatively charged single lipid bilayer nanotubes. The resolved FVIII-LC membrane-bound structure supports aspects of our previously proposed FVIII structure from membrane-bound two-dimensional (2D) crystals, such as only the C2 domain interacts directly with the membrane. The LC is oriented differently in the FVIII membrane-bound helical and 2D crystal structures based on EM data, and the existing X-ray structures. This flexibility of the FVIII-LC domain organization in different states is discussed in the light of the FVIIIa-FIXa complex assembly and function. PMID:23616213

  10. Factor VIII and glomerulonephritis.

    PubMed

    Ekberg, M; Nilsson, I M

    1975-05-17

    To find out if determination of factor VIII,which most probably is synthetised in the intima of blood-vessesls, is of value for predicting the severity of vessel damge in glomerulonephritis, factor-VIII activity, factor-VIII-related antigen, and glomerular filtration-ratewere esto,ated om 85 patients with early glomerulonephritis on admission, and in 70 of these at follow-up for up to 4 years. The levels of factor-VIII activity and factor-VIII-related antigen on admission were normal in those patients who recovered. Where renal function was impaired on admission or becaome so during follow-up, factor VIII was high. Determination of factor VIII might thus be of prognostic value in early glomerulonephritis. PMID:49471

  11. A study of reported factor VIII use around the world.

    PubMed

    Stonebraker, J S; Brooker, M; Amand, R E; Farrugia, A; Srivastava, A

    2010-01-01

    The effect of replacement therapy has significantly improved the morbidity and mortality of people with haemophilia A in high income countries, a recent socio-economic development as the availability of safe concentrates has been matched by a willingness for their provision through reimbursement. In the developing world, however, this state has not been achieved, primarily because of the low visibility of haemophilia coupled with its expense, leading to inadequate treatment with its sequelae of severe pain, joint deformities, arthropathy, disabilities, and even death in childhood or early adult life. The objective of this paper was to study the reported factor VIII (FVIII) use on a country-by-country basis. Data on the reported FVIII use for 104 countries were obtained from the Marketing Research Bureau, Inc. and the World Federation of Hemophilia. The results show that FVIII use varies considerably among countries, even among the wealthiest of countries. The use of FVIII concentrate increases as economic capacity increases; in addition, consumption of FVIII has been increasing at a greater rate in high income countries. Given these trends, there probably will be a global increase in FVIII concentrates usage. Such information is critical for national healthcare agencies to determine realistic budget priorities in planning for an increased allocation of resources required to improve the treatment of patients with haemophilia A. This information is also important for pharmaceutical manufacturers to adequately plan for increased production of FVIII concentrates. PMID:19845774

  12. Unexpected postpartum hemorrhage due to an acquired factor VIII inhibitor.

    PubMed

    Paidas, Michael J; Hossain, Nazli

    2014-09-01

    Unexplained postpartum hemorrhage (PPH) refractory to standard hemostatic measures should trigger a heightened clinical suspicion of an acquired bleeding disorder. When hemostatic medical interventions and surgical procedures fail to control the bleeding, then significant postoperative blood loss, debilitating morbidity, loss of fertility, and death may occur. In the setting of an autoantibody inhibitor to factor VIII (FVIII), control of life-threatening PPH and avoidance of subsequent bleeding episodes depends on a timely and accurate diagnosis, prompt hemostatic treatment and eradication of FVIII inhibitors, and appropriate long-term patient care and management. Acquired postpartum hemophilia due to a FVIII inhibitor is a rare cause of PPH; however, delayed treatment can lead to increased maternal morbidity and mortality. Acquired FVIII inhibitors also pose an emerging bleeding threat to the neonate as a result of possible transplacental transfer of FVIII autoantibodies to the fetus during the last trimester of pregnancy. The purpose of this review is to increase awareness among hematologists and obstetricians/gynecologists regarding the occurrence of FVIII neutralizing autoantibodies as a cause of PPH, and emphasize the importance of collaboration between obstetrician/gynecologists and hematology specialists to optimize the diagnostic evaluation, treatment, and long-term management of women who experience PPH due to an acquired FVIII inhibitor. PMID:24338123

  13. A Model for Predicting Persistent Elevation of Factor VIII among Patients with Acute Ischemic Stroke

    PubMed Central

    Samai, Alyana A.; Boehme, Amelia K.; Shaban, Amir; George, Alexander J.; Dowell, Lauren; Monlezun, Dominique J.; Leissinger, Cindy; Schluter, Laurie; El Khoury, Ramy; Martin-Schild, Sheryl

    2016-01-01

    Background and Purpose Elevated levels of coagulation factor VIII (FVIII) may persist independent of the acute-phase response; however, this relationship has not been investigated relative to acute ischemic stroke (AIS). We examined the frequency and predictors of persistently elevated FVIII in AIS patients. Methods AIS patients admitted between July 2008 and May 2014 with elevated baseline FVIII levels and repeat FVIII levels drawn for more than 7 days postdischarge were included. The patients were dichotomized by repeat FVIII level for univariate analysis at 150% and 200% activity thresholds. An adjusted model was developed to predict the likelihood of persistently elevated FVIII levels. Results Among 1616 AIS cases, 98 patients with elevated baseline FVIII had repeat FVIII levels. Persistent FVIII elevation was found in more than 75% of patients. At the 150% threshold, the prediction score ranged from 0 to 7 and included black race, female sex, prior stroke, hyperlipidemia, smoking, baseline FVIII > 200%, and baseline von Willebrand factor (vWF) level greater than 200%. At the 200% threshold, the prediction score ranged from 0–5 and included female sex, prior stroke, diabetes mellitus, baseline FVIII level greater 200%, and baseline vWF level greater than 200%. For each 1-point increase in score, the odds of persistent FVIII at both the 150% threshold (odds ratio [OR] = 10.4, 95% confidence interval [CI] 1.63–66.9, P = .0134) and 200% threshold (OR = 10.2, 95% CI 1.82–57.5, P = .0083) increased 10 times. Conclusion Because an elevated FVIII level confers increased stroke risk, our model for anticipating a persistently elevated FVIII level may identify patients at high risk for recurrent stroke. FVIII may be a target for secondary stroke prevention. PMID:26777556

  14. Neutralisation of factor VIII inhibitors by anti-idiotypes isolated from phage-displayed libraries.

    PubMed

    Schmidt, Anja; Brettschneider, Kerstin; Kahle, Jörg; Orlowski, Aleksander; Becker-Peters, Karin; Stichel, Diana; Schulze, Jörg; Braner, Markus; Tampé, Robert; Schwabe, Dirk; Königs, Christoph

    2016-07-01

    Following replacement therapy with coagulation factor VIII (FVIII), up to 30 % of haemophilia A patients develop FVIII-specific inhibitory antibodies (FVIII inhibitors). Immune tolerance induction (ITI) is not always successful, resulting in a need for alternative treatments for FVIII inhibitor-positive patients. As tolerance induction in the course of ITI appears to involve the formation of anti-idiotypes specific for anti-FVIII antibodies, such anti-idiotypes might be used to restore haemostasis in haemophilia A patients with FVIII inhibitors. We isolated anti-idiotypic antibody fragments (scFvs) binding to murine FVIII inhibitors 2-76 and 2-77 from phage-displayed libraries. FVIII inhibitor/anti-idiotype interactions were very specific as no cross-reactivity with other FVIII inhibitors or isotype controls was observed. ScFvs blocked binding of FVIII inhibitors to FVIII and neutralised their cognate inhibitors in vitro and a monoclonal mouse model. In addition, scFv JkH5 specific for FVIII inhibitor 2-76 stained 2-76-producing hybridoma cells. JkH5 residues R52 and Y226, located in complementary determining regions, were identified as crucial for the JkH5/2-76 interaction using JkH5 alanine mutants. SPR spectroscopy revealed that JkH5 interacts with FVIII inhibitor 2-76 with nanomolar affinity. Thus, FVIII inhibitor-specific, high-affinity anti-idiotypes can be isolated from phage-displayed libraries and neutralise their respective inhibitors. Furthermore, we show that anti-idiotypic scFvs might be utilised to specifically target inhibitor-specific B cells. Hence, a pool of anti-idiotypes could enable the reestablishment of haemostasis in the presence of FVIII inhibitors in patients or even allow the depletion of inhibitors by targeting inhibitor-specific B cell populations. PMID:27009573

  15. Bispecific antibody mimicking factor VIII.

    PubMed

    Nogami, Keiji

    2016-05-01

    There are some issues in the current factor (F)VIII replacement therapy for severe hemophilia A. One is mental and physical burden for the multiple intravenous infusions, and the other is difficulty in the hemostatic treatment for the patients with FVIII inhibitor. The development of novel drug with fully hemostatic effect, simply procedure, and long-acting reaction has been expected. Recently, FVIIIa-mimicking humanized recombinant bispecific antibody (ACE910) against FIXa and FX was developed. In the non-human clinical study, primate model of acquired hemophilia A demonstrated that the ACE910 was effective on both on-going and spontaneous bleedings. A phase I clinical study was conducted in healthy adults by single subcutaneous infusion of ACE910, followed by the patients' part study, Japanese patients with severe hemophilia A without or with inhibitor were treated with once-weekly subcutaneous injection of ACE910 at three dose levels for 12 successive weeks. There was no significant adverse event related to ACE910 in the clinical and laboratorial findings, and t1/2 of ACE910 was ∼30 days. The median annual bleeding rates were reduced very markedly dose-dependently, independently of inhibitor. Furthermore, among the patients with dose escalation, bleeding rate was decreased as ACE910 dose was increased. In conclusion, ACE910 would have a number of promising features: its high subcutaneous bioavailability and long half-life make the patients possible to be injected subcutaneously with a once-a-week or less frequency. In addition, ACE910 would provide the bleeding prophylactic efficacy, independently of inhibitor. PMID:27207420

  16. 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. PMID:25472812

  17. Soy Phosphatidylinositol-Containing Lipid Nanoparticle Prolongs the Plasma Survival and Hemostatic Efficacy of B-domain-Deleted Recombinant Canine Factor VIII in Hemophilia A Dogs.

    PubMed

    Shetty, Krithika A; Merricks, Elizabeth P; Raymer, Robin; Rigsbee, Natalie; Nichols, Timothy C; Balu-Iyer, Sathy V

    2016-08-01

    Soy phosphatidylinositol (PI)-containing lipid nanoparticles prolong plasma survival, improve hemostatic efficacy, and decrease immunogenicity of human B-domain-deleted factor VIII (BDD FVIII) in hemophilia A (HA) mice. We hypothesize that PI-associated BDD FVIII is more potent than the free protein and, using mathematical modeling, have projected that PI-associated BDD FVIII could be used for once-weekly prophylactic dosing in patients. To facilitate translation to the clinic, comparative plasma survival and ex vivo efficacy of PI-associated recombinant canine FVIII (PI-rcFVIII) were evaluated in HA dogs. Two HA dogs were administered a 50-U/kg intravenous dose of free or PI-rcFVIII. rcFVIII activity measurements and ex vivo efficacy analyses such as whole blood clotting time and thromboelastography were conducted on recovered plasma and whole blood samples. PI association decreased clearance (∼25%) and increased plasma exposure (∼1.4-fold) of rcFVIII. PI-rcFVIII-treated animals had prolonged improvements in whole blood clotting time and thromboelastography parameters compared to free rcFVIII-treated animals. Because rcFVIII is a BDD form of FVIII, these studies provide proof of principle that observations with human BDD FVIII in mice translate to higher animal species. In addition, PI-rcFVIII has potential applications in canine HA management and as a bypass therapy in inhibitor-positive HA patients. PMID:27372547

  18. Immunogenicity of long-lasting recombinant factor VIII products.

    PubMed

    Ing, Mathieu; Gupta, Nimesh; Teyssandier, Maud; Maillère, Bernard; Pallardy, Marc; Delignat, Sandrine; Lacroix-Desmazes, Sébastien

    2016-03-01

    Replacement therapy for patients with hemophilia A using plasma-derived or recombinant factor VIII (FVIII) is complicated by the short half-life of the FVIII products and by the occurrence of neutralizing antibodies in a substantial number of patients. In the recent years, enormous efforts have been invested to develop new generations of coagulation factors with extended half-lives. Presumably, the use of long-lasting FVIII products should reduce the frequency of administration to the patients and drastically improve their quality of life. The question of their immunogenicity remains however unanswered as yet. The present review proposes a summary of the different strategies developed to enhance the half-life of FVIII, including fusion of FVIII to the Fc fragment of the human IgG1 or to human serum albumin, or attachment of polyethylene glycol. Based on the available literature, we hypothesize on the potential benefits or risks associated with each of the latter strategies in terms of immunogenicity of the newly derived hemostatic drugs. PMID:26723503

  19. 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. PMID:27126696

  20. The role of plasma-derived factor VIII/von Willebrand factor concentrates in the treatment of hemophilia A patients.

    PubMed

    Auerswald, Günter; Spranger, Torsten; Brackmann, Hans-Hermann

    2003-06-01

    Besides preventing bleeding episodes, common goals of the treatment of hemophilia include integrating of patients into a normal social life and optimizing their quality of life. Sufficient amounts of factor VIII (FVIII) concentrates, whether recombinant or plasma-derived, are continuously needed. Guidelines for quality assurance of treatment will be a cornerstone to maintain optimal clinical management of patients especially considering financial aspects. Advances in manufacturing technologies have made possible general availability of modern concentrates for the management of hemophilia A patients. Safety, cost and continuous supply of concentrates must be considered when deciding on a product for replacement therapy. As todays' products have reached an excellent margin of safety with regard to virus transmission, the development and treatment of inhibitors is currently the main concern for physicians and patients. The incidence of inhibitors is influenced by various patient-related factors such as mutation type or severity of the disease. Plasma-derived FVIII concentrates containing von Willebrand factor (VWF) may have clinical advantages over pure FVIII concentrates with regard to inhibitor development and inhibitor eradication. Clinical trials comparing FVIII/VWF concentrates with pure FVIII concentrates are lacking, thus a lower inhibitor incidence has not yet been proven. Data from Germany on immune tolerance induction with FVIII/VWF concentrates indicate higher success rates with these than with pure FVIII concentrates. In addition FVIII/VWF concentrates are the therapy of choice when immune tolerance therapy with pure FVIII products is not successful. PMID:12826531

  1. Analysis of factor VIII mediated suppression of lentiviral vector titres.

    PubMed

    Radcliffe, P A; Sion, C J M; Wilkes, F J; Custard, E J; Beard, G L; Kingsman, S M; Mitrophanous, K A

    2008-02-01

    Effective gene therapy for haemophilia A necessitates a vector system that is not subject to a pre-existing immune response, has adequate coding capacity, gives long-term expression and preferably can target non-dividing cells. Vector systems based on lentiviruses such as equine infectious anaemia virus (EIAV) fulfil these criteria for the delivery of factor VIII (FVIII). We have found that B domain-deleted (BDD) FVIII protein inhibits functional viral particle production when co-expressed with the EIAV vector system. Although particle numbers (as measured by reverse transcriptase activity) are near normal, RNA genome levels are reduced and measurement of integrated copies revealed the virus is severely defective in its ability to transduce target cells. This is due to the absence of sufficient vesicular stomatitis virus glycoprotein (VSV-G) envelope on viral particles derived from cells expressing FVIII. By using an internal tissue-specific promoter, that has low activity in the producer cells, to drive expression of FVIII we have overcome this inhibitory effect allowing us to generate titres approaching those obtained with vector genomes encoding reporter genes. Furthermore, we report that codon optimization of the full-length FVIII gene increased vector titres approximately 10-fold in addition to substantially improving expression per integrated vector copy. PMID:18046428

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

    PubMed

    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

    2016-03-01

    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

  3. 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

  4. Factor VIII efficient and specific non-covalent binding to PEGylated liposomes enables prolongation of its circulation time and haemostatic efficacy.

    PubMed

    Baru, Moshe; Carmel-Goren, Lea; Barenholz, Yechezkel; Dayan, Inbal; Ostropolets, Savely; Slepoy, Igal; Gvirtzer, Nira; Fukson, Vladimir; Spira, Jack

    2005-06-01

    Haemophilia A is a bleeding disorder caused by the lack of factor VIII (FVIII). We report the prolongation of exogenous FVIII circulation time and haemostatic efficacy by its formulation with PEGylated liposomes (PEGLip). FVIII binds non-covalently but with high affinity in a specific mode with the external surface of PEGLip neither losing its activity nor its binding to von Willebrand Factor. Experiments in haemophilic and non-haemophilic mice indicate that the circulation time and clotting efficacy of PEGLip-formulated exogenous FVIII (PEGLip-FVIII) are significantly enhanced over those of free FVIII. The data support the feasibility of using PEGLip-FVIII to extend the duration of haemostatic efficacy in the treatment of haemophilia A. PMID:15968389

  5. 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. PMID:15566360

  6. A subset of high-titer anti–factor VIII A2 domain antibodies is responsive to treatment with factor VIII

    PubMed Central

    Eubanks, Joshua; Baldwin, W. Hunter; Markovitz, Rebecca; Parker, Ernest T.; Cox, Courtney; Kempton, Christine L.

    2016-01-01

    The primary B-cell epitopes of factor VIII (fVIII) are in the A2 and C2 domains. Within the C2 domain, antibody epitope and kinetics are more important than inhibitor titer in predicting pathogenicity in a murine bleeding model. To investigate this within the A2 domain, the pathogenicity of a diverse panel of antihuman fVIII A2 domain monoclonal antibodies (MAbs) was tested in the murine model. MAbs were injected into hemophilia A mice, followed by injection of human B domain-deleted fVIII. Blood loss after a 4-mm tail snip was measured. The following anti-A2 MAbs were tested: high-titer type 1 inhibitors 4A4, 2-76, and 1D4; 2-54, a high-titer type 2 inhibitor; B94, a type 2 inhibitor; and noninhibitory MAbs GMA-012, 4C7, and B25. All high-titer type 1 MAbs produced blood loss that was significantly greater than control mice, whereas all non-inhibitory MAbs produced blood loss that was similar to control. The type 2 MAbs were not pathogenic despite 2-54 having an inhibitor titer of 34 000 BU/mg immunoglobulin G. In addition, a patient with a high-titer type 2 anti-A2 inhibitor who is responsive to fVIII is reported. The discrepancy between inhibitor titer and bleeding phenotype combined with similar findings in the C2 domain stress the importance of inhibitor properties not detected in the standard Bethesda assay in predicting response to fVIII therapy. PMID:26825708

  7. Stable Expression of Recombinant Factor VIII in CHO Cells Using Methotrexate-Driven Transgene Amplification.

    PubMed

    Orlova, N A; Kovnir, S V; Vorobiev, I I; Yuriev, A S; Gabibov, A G; Vorobiev, A I

    2012-01-01

    Prophylaxis and treatment of inherited clotting disorder hemophilia A requires regular administration of factor VIII. Recombinant factor VIII, which is produced in CHO or BHK cells, is equivalent to the plasma-derived one and is prevalent in current clinical practice in developed countries. Development of a biosimilar recombinant FVIII requires the creation of a highly productive clonal cell line and generation of monoclonal antibodies suitable for affinity purification of the product. Methotrexate-driven transgene amplification of genetic cassettes that code full-length and truncated variants of FVIII under the control of the CMV promoter was studied. It was shown that the expression level of the truncated variant of FVIII is 6.5 times higher than that of the full-length molecule. The transgene amplification procedure was sufficient for a twofold increase of the expression level in the transfected cells pool and subsequent selection of the clonal line, stably producing truncated FVIII at the level of 0.52 IU/ml during cultivation in a chemically defined protein-free culture medium. Four generated mouse monoclonal antibodies toward the heavy chain of FVIII were found suitable for binding the truncated variant of FVIII directly from the conditioned medium and elution of the FVIII with a more than 85% yield and normal pro-coagulant activity. The producer cell line and monoclonal antibodies obtained are sufficient for the development of upstream and downstream processes of biosimilar FVIII production. Generation of more productive cell lines by the use of stronger, nonviral promoters and shorter cDNA of FVIII will be the subject of further studies. PMID:22708069

  8. 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

  9. Factor VIII and von Willebrand factor are ligands for the carbohydrate-receptor Siglec-5

    PubMed Central

    Pegon, Julie N.; Kurdi, Mohamad; Casari, Caterina; Odouard, Soline; Denis, Cécile V.; Christophe, Olivier D.; Lenting, Peter J.

    2012-01-01

    Background Factor VIII (FVIII) and von Willebrand factor (VWF) circulate in plasma in a tight non-covalent complex, being critical to hemostasis. Although structurally unrelated, both share the presence of sialylated glycan-structures, making them potential ligands for sialic-acid-binding-immunoglobulin-like-lectins (Siglecs). Design and Methods We explored the potential interaction between FVIII/VWF and Siglec-5, a receptor expressed in macrophages using various experimental approaches, including binding experiments with purified proteins and cell-binding studies with Siglec-5 expressing cells. Finally, Siglec-5 was overexpressed in mice via hydrodynamic gene transfer. Results In different systems using purified proteins, saturable, dose-dependent and reversible interactions between a soluble Siglec-5 fragment and both hemostatic proteins were found. Sialidase treatment of VWF resulted in a complete lack of Siglec-5 binding. In contrast, sialidase treatment left interactions between FVIII and Siglec-5 unaffected. FVIII and VWF also bound to cellsurface exposed Siglec-5, as was visualized by classical immunostaining as well as by Duolinkproximity ligation assays. Co-localization of FVIII and VWF with early endosomal markers further suggested that binding to Siglec-5 is followed by endocytosis of the proteins. Finally, overexpression of human Siglec-5 in murine hepatocytes following hydrodynamic gene transfer resulted in a significant decrease in plasma levels of FVIII and VWF in these mice. Conclusions Our data indicate that FVIII and VWF may act as a ligand for Siglec-5, and that Siglec-5 may contribute to the regulation of plasma levels of the FVIII/VWF complex. PMID:22733016

  10. 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

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

    PubMed

    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

    2015-10-01

    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

  12. Intraosseous Delivery of Lentiviral Vectors Targeting Factor VIII Expression in Platelets Corrects Murine Hemophilia A

    PubMed Central

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

    2015-01-01

    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. PMID:25655313

  13. The first recombinant human coagulation factor VIII of human origin: human cell line and manufacturing characteristics

    PubMed Central

    Casademunt, Elisabeth; Martinelle, Kristina; Jernberg, Mats; Winge, Stefan; Tiemeyer, Maya; Biesert, Lothar; Knaub, Sigurd; Walter, Olaf; Schröder, Carola

    2012-01-01

    Introduction Since the early 1990s, recombinant human clotting factor VIII (rhFVIII) produced in hamster cells has been available for haemophilia A treatment. However, the post-translational modifications of these proteins are not identical to those of native human FVIII, which may lead to immunogenic reactions and the development of inhibitors against rhFVIII. For the first time, rhFVIII produced in a human host cell line is available. Aim We describe here the establishment of the first human production cell line for rhFVIII and the manufacturing process of this novel product. Methods and results A human cell line expressing rhFVIII was derived from human embryonic kidney (HEK) 293 F cells transfected with an FVIII expression plasmid. No virus or virus-like particles could be detected following extensive testing. The stringently controlled production process is completely free from added materials of animal or human origin. Multistep purification employing a combination of filtration and chromatography steps ensures the efficient removal of impurities. Solvent/detergent treatment and a 20 nm pore size nanofiltration step, used for the first time in rhFVIII manufacturing, efficiently eliminate any hypothetically present viruses. In contrast to hamster cell-derived products, this rhFVIII product does not contain hamster-like epitopes, which might be expected to be immunogenic. Conclusions HEK 293 F cells, whose parental cell line HEK 293 has been used by researchers for decades, are a suitable production cell line for rhFVIII and will help avoid immunogenic epitopes. A modern manufacturing process has been developed to ensure the highest level of purity and pathogen safety. PMID:22690791

  14. 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. PMID:25927594

  15. Abnormal coagulation factor VIII transcript in a Tennessee Walking Horse colt with hemophilia A.

    PubMed

    Norton, Elaine M; Wooldridge, Anne A; Stewart, Allison J; Cusimano, Layla; Schwartz, Dean D; Johnson, Calvin M; Boudreaux, Mary K; Christopherson, Pete W

    2016-03-01

    Hemophilia A is an X-chromosome-linked disorder caused by a deficiency in factor VIII (FVIII). Although foals have been diagnosed with hemophilia A based on deficiency in FVIII activity, causative gene mutations have not been identified. The genomic DNA and cDNA encoding FVIII of a Tennesee Walking Horse colt affected with hemophilia A and the genomic DNA of his dam and a normal unrelated horse were analyzed with no splice site or coding sequence abnormalities identified in any of the horses. Polymerase chain reactions (PCR) were then performed on hepatic cDNA from the affected colt and an unrelated normal horse, and no product was obtained for the sequence between and including exon 1 and exon 2 in the affected colt. Based on these results, suspected mutations were identified in the noncoding region of FVIII (intron 1), and genomic sequencing of intron 1 in the dam and the affected colt suggested maternal inheritance. PMID:26765501

  16. Cryo-electron microscopy of coagulation Factor VIII bound to lipid nanotubes

    SciTech Connect

    Parmenter, Christopher D.J.; Cane, Matthew C.; Zhang Rui; Stoilova-McPhie, Svetla

    2008-02-08

    Factor VIII (FVIII) is a key protein in blood coagulation, deficiency or malfunction of which causes Haemophilia A. The sole cure for this condition is intravenous administration of FVIII, whose membrane-bound structure we have studied by Cryo-electron microscopy and image analysis. Self-assembled lipid nanotubes were optimised to bind FVIII at close to native conditions. The tubes diameter was constant at 30 nm and the lipid bilayer resolved. The FVIII molecules were well defined, forming an 8.5 nm thick outer layer, and appeared to reach the hydrophobic core of the bilayer. The two known FVIII atomic models were superimposed with the averaged 2D protein densities. The insertion of the FVIII within the membrane was evaluated, reaffirming that the membrane-binding C2 or C1-C2 domain(s) fully penetrate the outer leaflet of the lipid layer. The presented results lay the basis for new models of the FVIII overall orientation and membrane-binding mechanism.

  17. The diversity of the immune response to the A2 domain of human factor VIII

    PubMed Central

    Markovitz, Rebecca C.; Healey, John F.; Parker, Ernest T.; Meeks, Shannon L.

    2013-01-01

    Approximately 30% of patients with severe hemophilia A develop inhibitory anti–factor VIII (fVIII) antibodies (Abs). We characterized 29 anti-human A2 monoclonal Abs (mAbs) produced in a murine hemophilia A model. A basis set of nonoverlapping mAbs was defined by competition enzyme-linked immunosorbent assay, producing 5 major groups. The overlapping epitopes covered nearly the entire A2 surface when mapped by homolog-scanning mutagenesis. Most group A mAbs recognized a previously described epitope bounded by Arg484-Ile508 in the N-terminal A2 subdomain, resulting in binding to activated fVIII and noncompetitive inhibition of the intrinsic fXase complex. Group B and C mAbs displayed little or no inhibitory activity. Group D and E mAbs recognized epitopes in the C-terminal A2 subdomain. A subset of group D mAbs inhibited the activation of fVIII by interfering with thrombin-catalyzed cleavage at Arg372 at the A1-A2 domain junction. Other group D mAbs displayed indeterminate or no inhibitory activity despite inhibiting cleavage at Arg740 at the A2-B domain junction. Group E mAbs inhibited fVIII light-chain cleavage at Arg1689. Inhibition of cleavages at Arg372 and Arg1689 represent novel mechanisms of inhibitor function and, along with the extensive epitope spectrum identified in this study, reveal hitherto unrecognized complexity in the immune response to fVIII. PMID:23349389

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

    PubMed Central

    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. PMID:26909355

  19. Mapping the interaction between factor VIII and von Willebrand factor by electron microscopy and mass spectrometry

    PubMed Central

    Chiu, Po-Lin; Bou-Assaf, George M.; Chhabra, Ekta Seth; Chambers, Melissa G.; Peters, Robert T.; Kulman, John D.

    2015-01-01

    Association with the D′D3 domain of von Willebrand factor (VWF) stabilizes factor VIII (FVIII) in the circulation and maintains it at a level sufficient to prevent spontaneous bleeding. We used negative-stain electron microscopy (EM) to visualize complexes of FVIII with dimeric and monomeric forms of the D′D3 domain. The EM averages show that FVIII interacts with the D′D3 domain primarily through its C1 domain, with the C2 domain providing a secondary attachment site. Hydrogen-deuterium exchange mass spectrometry corroborated the importance of the C1 domain in D′D3 binding and implicates additional surface regions on FVIII in the interaction. Together, our results establish that the C1 domain is the major binding site on FVIII for VWF, reiterate the importance of the a3 acidic peptide in VWF binding, and suggest that the A3 and C2 domains play ancillary roles in this interaction. PMID:26065652

  20. Safety and prolonged activity of recombinant factor VIII Fc fusion protein in hemophilia A patients

    PubMed Central

    Josephson, Neil C.; Quon, Doris; Ragni, Margaret V.; Cheng, Gregory; Li, Ella; Jiang, Haiyan; Li, Lian; Dumont, Jennifer A.; Goyal, Jaya; Zhang, Xin; Sommer, Jurg; McCue, Justin; Barbetti, Margaret; Luk, Alvin

    2012-01-01

    Current factor VIII (FVIII) products display a half-life (t1/2) of ∼ 8-12 hours, requiring frequent intravenous injections for prophylaxis and treatment of patients with hemophilia A. rFVIIIFc is a recombinant fusion protein composed of a single molecule of FVIII covalently linked to the Fc domain of human IgG1 to extend circulating rFVIII t1/2. This first-in-human study in previously treated subjects with severe hemophilia A investigated safety and pharmacokinetics of rFVIIIFc. Sixteen subjects received a single dose of rFVIII at 25 or 65 IU/kg followed by an equal dose of rFVIIIFc. Most adverse events were unrelated to study drug. None of the study subjects developed anti-rFVIIIFc antibodies or inhibitors. Across dose levels, compared with rFVIII, rFVIIIFc showed 1.54- to 1.70-fold longer elimination t1/2, 1.49- to 1.56-fold lower clearance, and 1.48- to 1.56-fold higher total systemic exposure. rFVIII and rFVIIIFc had comparable dose-dependent peak plasma concentrations and recoveries. Time to 1% FVIII activity above baseline was ∼ 1.53- to 1.68-fold longer than rFVIII across dose levels. Each subject showed prolonged exposure to rFVIIIFc relative to rFVIII. Thus, rFVIIIFc may offer a viable therapeutic approach to achieve prolonged hemostatic protection and less frequent dosing in patients with hemophilia A. This trial was registered at www.clinicaltrials.gov as NCT01027377. PMID:22223821

  1. Dimeric Organization of Blood Coagulation Factor VIII bound to Lipid Nanotubes

    PubMed Central

    Dalm, Daniela; Galaz-Montoya, Jesus G.; Miller, Jaimy L.; Grushin, Kirill; Villalobos, Alex; Koyfman, Alexey Y.; Schmid, Michael F.; Stoilova-McPhie, Svetla

    2015-01-01

    Membrane-bound Factor VIII (FVIII) has a critical function in blood coagulation as the pro-cofactor to the serine-protease Factor IXa (FIXa) in the FVIIIa-FIXa complex assembled on the activated platelet membrane. Defects or deficiency of FVIII cause Hemophilia A, a mild to severe bleeding disorder. Despite existing crystal structures for FVIII, its membrane-bound organization has not been resolved. Here we present the dimeric FVIII membrane-bound structure when bound to lipid nanotubes, as determined by cryo-electron microscopy. By combining the structural information obtained from helical reconstruction and single particle subtomogram averaging at intermediate resolution (15-20 Å), we show unambiguously that FVIII forms dimers on lipid nanotubes. We also demonstrate that the organization of the FVIII membrane-bound domains is consistently different from the crystal structure in solution. The presented results are a critical step towards understanding the mechanism of the FVIIIa-FIXa complex assembly on the activated platelet surface in the propagation phase of blood coagulation. PMID:26082135

  2. von Willebrand factor and factor VIII are independently required to form stable occlusive thrombi in injured veins

    PubMed Central

    Chauhan, Anil K.; Kisucka, Janka; Lamb, Colin B.; Bergmeier, Wolfgang

    2007-01-01

    von Willebrand factor (VWF) protects factor VIII (FVIII) from proteolysis and mediates the initial contact of platelets with the injured vessel wall, thus playing an important role in hemostasis and thrombosis. VWF is crucial for the formation of occlusive thrombi at arterial shear rates. However, with only a few conflicting studies published, the role of VWF in venous thrombosis is still unclear. Using gene-targeted mice, we show that in ferric chloride–injured veins platelet adhesion to subendothelium is decreased and thrombus growth is impaired in VWF−/− mice when compared with wild type (WT). We also observed increased embolization in the VWF−/− mice, which was due to lower FVIII levels in these mice as recombinant factor VIII (r-FVIII) restored thrombus stability. Despite normalization of blood clotting time and thrombus stability after r-FVIII infusion, the VWF−/− venules did not occlude. Transgenic platelets lacking the VWF receptor GPIbα extracellular domain showed decreased adhesion to injured veins. But, after a delay, all the injured venules occluded in these transgenic mice. Thus, VWF likely uses other adhesion receptors besides GPIbα in thrombus growth under venous shear conditions. Our studies document crucial roles for VWF and FVIII in experimental thrombosis under venous flow conditions in vivo. PMID:17119108

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

    PubMed

    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; Jiang, Haiyan

    2012-03-29

    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

  4. Concurrent acquired inhibitors to factor VIII and IX, a laboratory artifact: a case report

    PubMed Central

    Doma, Saša Anžej; Hillarp, Andreas; Pajič, Tadej; Andoljšek, Dušan; Černelč, Peter; Preldžnik Zupan, Irena

    2016-01-01

    Acquired inhibitors to coagulation factors other than factor VIII are extremely rare. We describe a case of a 59-year-old woman with abnormal bleeding, diagnosed with concurrent inhibitor antibodies to factor VIII and IX by Bethesda testing. We demonstrate that anti-FVIII antibodies of a very high titre are capable of disturbing the aPTT-based Bethesda assay, resulting in falsely-positive antibodies to factor IX. The case also illustrates the usefulness of the immunological assay (ELISA) in complementing the inhibitor diagnosis. PMID:27346976

  5. Half-life extended factor VIII for the treatment of hemophilia A.

    PubMed

    Tiede, A

    2015-06-01

    Prophylactic infusion of factor VIII (FVIII) prevents joint bleeding and other hemorrhages in patients with hemophilia A. Conventional FVIII concentrates have a short half-life, with an average of about 12 h in adults, ranging in individual patients between 6 and 24 h, and even shorter in younger children. Therefore, effective prophylaxis requires frequent intravenous injection, usually three times per week or every other day. Several technologies are currently under investigation to extend the half-life of FVIII, including Fc fusion (Eloctate, Elocta, efmoroctocog alfa), addition of polyethylene glycol (turoctocog alfa pegol [N8-GP], BAY 94-9027, BAX 855), and a single-chain construct (CSL627). This review summarizes characteristics of products in clinical development and discusses their potential benefits. PMID:26149020

  6. SK-HEP cells and lentiviral vector for production of human recombinant factor VIII.

    PubMed

    da Rosa, Nathalia Gonsales; Swiech, Kamilla; Picanço-Castro, Virgínia; Russo-Carbolante, Elisa Maria de Sousa; Soares Neto, Mario Abreu; de Castilho-Fernandes, Andrielle; Faça, Vitor Marcel; Fontes, Aparecida Maria; Covas, Dimas Tadeu

    2012-08-01

    Hemophilia A is caused by a deficiency in coagulation factor VIII. Recombinant factor VIII can be used as an alternative although it is unavailable for most patients. Here, we describe the production of a human recombinant B-domain-deleted FVIII (rBDDFVIII) by the human cell line SK-HEP-1, modified by a lentiviral vector rBDDFVIII was produced by recombinant SK-HEP cells (rSK-HEP) at 1.5-2.1 IU/10(6) in 24 h. The recombinant factor had increased in vitro stability when compared to commercial pdFVIII. The functionality of rBDDFVIII was shown by its biological activity and by tail-clip challenge in hemophilia A mice. The rSK-HEP cells grew in a scalable system and produced active rBDDFVIII, indicating that this platform production can be optimized to meet the commercial production scale needs. PMID:22488441

  7. To clear or to fear: An innate perspective on factor VIII immunity.

    PubMed

    Lai, Jesse Derek; Georgescu, Maria Teofana; Hough, Christine; Lillicrap, David

    2016-03-01

    The enigma that is factor VIII immunogenicity remains ever pertinent in the treatment of hemophilia A. Development of neutralizing antibodies against the therapeutic protein in 25-30% of patients likely depends on the appropriate activation of the innate immune response shortly following antigen encounter. Our understanding of this important immunological synapse remains ill-defined. In this review, we examine the three distinct factors contributing to the fate of factor VIII almost immediately after infusion: the characteristics of the protein, the cell, and the microenvironment. We propose a continuum between clearance and antigen presentation that facilitates removal of FVIII from circulation leading to either tolerance or immunity. PMID:26547364

  8. Acquired coagulant factor VIII deficiency induced by Bacillus anthracis lethal toxin in mice

    PubMed Central

    Sun, Der-Shan; Lee, Po-Chien; Kau, Jyh-Hwa; Shih, Yung-Luen; Huang, Hsin-Hsien; Li, Chen-Ru; Lee, Chin-Cheng; Wu, Yu-Ping; Chen, Kuo-Ching; Chang, Hsin-Hou

    2015-01-01

    Mice treated with anthrax lethal toxin (LT) exhibit hemorrhage caused by unknown mechanisms. Moreover, LT treatment in mice induced liver damage. In this study, we hypothesized that a suppressed coagulation function may be associated with liver damage, because the liver is the major producing source of coagulation factors. The hepatic expression of coagulant factors and the survival rates were analyzed after cultured cells or mice were exposed to LT. In agreement with our hypothesis, LT induces cytotoxicity against hepatic cells in vitro. In addition, suppressed expression of coagulation factor VIII (FVIII) in the liver is associated with a prolonged plasma clotting time in LT-treated mice, suggesting a suppressive role of LT in coagulation. Accordingly, we further hypothesized that a loss-of-function approach involving treatments of an anticoagulant should exacerbate LT-induced abnormalities, whereas a gain-of-function approach involving injections of recombinant FVIII to complement the coagulation deficiency should ameliorate the pathogenesis. As expected, a sublethal dose of LT caused mortality in the mice that were non-lethally pretreated with an anticoagulant (warfarin). By contrast, treatments of recombinant FVIII reduced the mortality from a lethal dose of LT in mice. Our results indicated that LT-induced deficiency of FVIII is involved in LT-mediated pathogenesis. Using recombinant FVIII to correct the coagulant defect may enable developing a new strategy to treat anthrax. PMID:25906166

  9. Acquired coagulant factor VIII deficiency induced by Bacillus anthracis lethal toxin in mice.

    PubMed

    Sun, Der-Shan; Lee, Po-Chien; Kau, Jyh-Hwa; Shih, Yung-Luen; Huang, Hsin-Hsien; Li, Chen-Ru; Lee, Chin-Cheng; Wu, Yu-Ping; Chen, Kuo-Ching; Chang, Hsin-Hou

    2015-01-01

    Mice treated with anthrax lethal toxin (LT) exhibit hemorrhage caused by unknown mechanisms. Moreover, LT treatment in mice induced liver damage. In this study, we hypothesized that a suppressed coagulation function may be associated with liver damage, because the liver is the major producing source of coagulation factors. The hepatic expression of coagulant factors and the survival rates were analyzed after cultured cells or mice were exposed to LT. In agreement with our hypothesis, LT induces cytotoxicity against hepatic cells in vitro. In addition, suppressed expression of coagulation factor VIII (FVIII) in the liver is associated with a prolonged plasma clotting time in LT-treated mice, suggesting a suppressive role of LT in coagulation. Accordingly, we further hypothesized that a loss-of-function approach involving treatments of an anticoagulant should exacerbate LT-induced abnormalities, whereas a gain-of-function approach involving injections of recombinant FVIII to complement the coagulation deficiency should ameliorate the pathogenesis. As expected, a sublethal dose of LT caused mortality in the mice that were non-lethally pretreated with an anticoagulant (warfarin). By contrast, treatments of recombinant FVIII reduced the mortality from a lethal dose of LT in mice. Our results indicated that LT-induced deficiency of FVIII is involved in LT-mediated pathogenesis. Using recombinant FVIII to correct the coagulant defect may enable developing a new strategy to treat anthrax. PMID:25906166

  10. Production of human factor VIII-FL in 293T cells using the bicistronic MGMT(P140K)-retroviral vector.

    PubMed

    Fontes, A M; Melo, F U F; Greene, L J; Faça, V M; Lin, Y; Gerson, S L; Covas, D T

    2012-01-01

    Hemophilia A is the most common X-linked bleeding disorder; it is caused by deficiency of coagulation factor VIII (FVIII). Replacement therapy with rFVIII produced from human cell line is a major goal for treating hemophilia patients. We prepared a full-length recombinant FVIII (FVIII-FL), using the pMFG-P140K retroviral vector. The IRES DNA fragment was cloned upstream to the P140K gene, providing a 9.34-kb bicistronic vector. FVIII-FL cDNA was then cloned upstream to IRES, resulting in a 16.6-kb construct. In parallel, an eGFP control vector was generated, resulting in a 10.1- kb construct. The 293T cells were transfected with these constructs, generating the 293T-FVIII-FL/P140K and 293T-eGFP/P140K cell lines. In 293T-FVIII-FL/P140K cells, FVIII and P140K mRNAs levels were 4,410 (±931.7)- and 295,400 (±75,769)-fold higher than in virgin cells. In 293T-eGFP/P140K cells, the eGFP and P140K mRNAs levels were 1,501,000 (±493,700)- and 308,000 (±139,300)-fold higher than in virgin cells. The amount of FVIII-FL was 0.2 IU/mL and 45 ng/mL FVIII cells or 4.4 IU/μg protein. These data demonstrate the efficacy of the bicistronic retroviral vector expressing FVIII-FL and MGMT(P140K), showing that it could be used for producing the FVIII-FL protein in a human cell line. PMID:22576836

  11. 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

  12. 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. PMID:27445481

  13. Evidence that a secondary binding and protecting site for factor VIII on von Willebrand factor is highly unlikely.

    PubMed Central

    Layet, S; Girma, J P; Obert, B; Peynaud-Debayle, E; Bihoreau, N; Meyer, D

    1992-01-01

    A binding domain for Factor VIII (F.VIII) has been previously identified on the N-terminal portion of human von Willebrand Factor (vWF) subunit [amino acids (AA) 1-272]. In order to characterize other possible structures of vWF involved in its capacity to bind and to protect F.VIII against human activated protein C (APC), we used a series of purified vWF fragments overlapping the whole sequence of the subunit. Among those were fragments SpIII (dimer; AA 1-1365), SpII (dimer; AA 1366-2050) and SpI (monomer; AA 911-1365) generated by Staphylococcus aureus V8 proteinase, a P34 species (monomer; AA 1-272) obtained with plasmin, a monomeric 39/34 kDa dispase fragment (AA 480-718) and a tetrameric III-T2 fragment (AA 273-511/674-728) produced from SpIII by trypsin. Three other fragments without precise extremities were located using selected monoclonal antibodies to vWF. Two C-terminal fragments of 270 and 260 kDa, overlapping SpI and SpII, were respectively generated from vWF with trypsin and protease 1 from Crotalus atrox venom. An N-terminal 120 kDa fragment, overlapping P34 and 39/34 kDa fragments, was produced by protease 1. Our results show that vWF bound to F.VIII and protected it from degradation by APC in a dose-dependent way. Among the C-terminal and central vWF fragments (SpII, tryptic 270 kDa, 260 kDa, SpI, 39/34 kDa and III-T2), none had the capacity to bind or to protect F.VIII, even at high concentrations. The three N-terminal fragments (SpIII, 120 kDa and P34) bound to F.VIII in a dose-dependent and saturable fashion. SpIII and the 120 kDa fragment had the capacity to protect F.VIII in a dose-dependent way. In contrast, the P34 species did not significantly protect F.VIII, even when using high concentrations of the fragment. In conclusion, the N-terminal end of vWF subunit (AA 1-272) plays a crucial role in binding to F.VIII, but requires additional structures of the 120 kDa fragment to protect it against APC. In addition, the presence of a secondary

  14. Anti-factor VIII IgA as a potential marker of poor prognosis in acquired hemophilia A: results from the GTH-AH 01/2010 study.

    PubMed

    Tiede, Andreas; Hofbauer, Christoph J; Werwitzke, Sonja; Knöbl, Paul; Gottstein, Saskia; Scharf, Rüdiger E; Heinz, Jürgen; Groß, Jürgen; Holstein, Katharina; Dobbelstein, Christiane; Scheiflinger, Fritz; Koch, Armin; Reipert, Birgit M

    2016-05-12

    Neutralizing autoantibodies against factor VIII (FVIII), also called FVIII inhibitors, are the cause of acquired hemophilia A (AHA). They are quantified in the Bethesda assay or Nijmegen-modified Bethesda assay by their ability to neutralize FVIII in normal human plasma. However, FVIII inhibitors do not represent the whole spectrum of anti-FVIII autoantibodies. Here, we studied isotypes, immunoglobulin G subclasses, and apparent affinities of anti-FVIII autoantibodies to assess their prognostic value for the outcome in AHA. We analyzed baseline samples from patients enrolled in the prospective GTH-AH 01/2010 study. Our data suggest that anti-FVIII immunoglobulin A (IgA) autoantibodies are predictors of poor outcome in AHA. Anti-FVIII IgA-positive patients achieved partial remission similar to anti-FVIII IgA-negative patients but had a higher risk of subsequent recurrence. Consequently, IgA-positive patients achieved complete remission less frequently (adjusted hazard ratio [aHR], 0.35; 95% confidence interval [CI], 0.18-0.68; P < .01) and had a higher risk of death (aHR, 2.62; 95% CI, 1.11-6.22; P < .05). Anti-FVIII IgA was the strongest negative predictor of recurrence-free survival after achieving partial remission and remained significant after adjustment for baseline demographic and clinical characteristics. In conclusion, anti-FVIII IgA represents a potential novel biomarker that could be useful to predict prognosis and tailor immunosuppressive treatment of AHA. PMID:26912467

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

    PubMed Central

    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. PMID:26473492

  16. Analysis of factor VIII gene inversion mutations in 166 unrelated haemophilia A families: frequency and utility in genetic counselling.

    PubMed

    Vnencak-Jones, C L; Iii, J A; Janco, R L; Cohen, M P; Dupont, W D; Kazazian, H H; Rossiter, J P

    1996-01-01

    Haemophilia A is an X-linked recessive bleeding disorder of variable severity that is caused by a deficiency of coagulation factor VIII (FVIII). The disease results from mutations in the FVIII gene which are heterogenous both in type and position within the gene. Recently, however, inversion mutations were found to be common to patients with severe disease (Lakich et al., 1993). These mutations result from intrachromosomal recombinations between DNA sequences in the A gene (located in intron 22 of the FVIII gene) and one of two A genes upstream to the FVIII gene. To determine the frequency of these inversions we performed Southern blot analysis on banked DNA from 166 consecutive, unrelated haemophilia A families previously referred for carrier or prenatal testing. In 57/166 (34%) families an inversion or other unique mutation was detected. The distal and proximal A genes lying upstream to the FVIII gene were involved in 79% and 18% of the mutations, respectively, but in 3% of the families the sequences involved in the mutation have not been identified. In 20/38 (53%) families with severe disease a mutation was detected. Interestingly, the relative risk of developing inhibitors in patients with FVIII gene inversions or other 3° mutations detected by this assay, as compared to patients with no detectable mutation by this assay, was 3.8. In families for which a mutation is detected, direct DNA testing is an accurate and inexpensive alternative to linkage analysis for prenatal or haemophilia A carrier testing. PMID:27213900

  17. The impact of minimally oversized adeno-associated viral vectors encoding human factor VIII on vector potency in vivo

    PubMed Central

    Kyostio-Moore, Sirkka; Berthelette, Patricia; Piraino, Susan; Sookdeo, Cathleen; Nambiar, Bindu; Jackson, Robert; Burnham, Brenda; O’Riordan, Catherine R; Cheng, Seng H; Armentano, Donna

    2016-01-01

    Recombinant adeno-associated viral (rAAV) vectors containing oversized genomes provide transgene expression despite low efficiency packaging of complete genomes. Here, we characterized the properties of oversized rAAV2/8 vectors (up to 5.4 kb) encoding human factor VIII (FVIII) under the transcriptional control of three liver promoters. All vectors provided sustained production of active FVIII in mice for 7 months and contained comparable levels of vector genomes and complete expression cassettes in liver. Therefore, for the 5.4 kb genome size range, a strong expression cassette was more important for FVIII production than the vector genome size. To evaluate the potency of slightly oversized vectors, a 5.1 kb AAVrh8R/FVIII vector was compared to a 4.6 kb (wild-type size) vector with an identical expression cassette (but containing a smaller C1-domain deleted FVIII) for 3 months in mice. The 5.1 kb vector had twofold to threefold lower levels of plasma FVIII protein and liver vector genomes than that obtained with the 4.6 kb vector. Vector genomes for both vectors persisted equally and existed primarily as high molecular weight concatemeric circular forms in liver. Taken together, these results indicate that the slightly oversized vectors containing heterogeneously packaged vector genomes generated a functional transgene product but exhibited a twofold to threefold lower in vivo potency. PMID:26958574

  18. Concurrent influenza vaccination reduces anti-FVIII antibody responses in murine hemophilia A.

    PubMed

    Lai, Jesse D; Moorehead, Paul C; Sponagle, Kate; Steinitz, Katharina N; Reipert, Birgit M; Hough, Christine; Lillicrap, David

    2016-06-30

    Inflammatory signals such as pathogen- and danger-associated molecular patterns have been hypothesized as risk factors for the initiation of the anti-factor VIII (FVIII) immune response seen in 25% to 30% of patients with severe hemophilia A (HA). In these young patients, vaccines may be coincidentally administered in close proximity with initial exposure to FVIII, thereby providing a source of such stimuli. Here, we investigated the effects of 3 vaccines commonly used in pediatric patients on FVIII immunogenicity in a humanized HA murine model with variable tolerance to recombinant human FVIII (rhFVIII). Mice vaccinated intramuscularly against the influenza vaccine prior to multiple infusions of rhFVIII exhibited a decreased incidence of rhFVIII-specific neutralizing and nonneutralizing antibodies. Similar findings were observed with the addition of an adjuvant. Upon exposure to media from influenza- or FVIII-stimulated lymph node or splenic lymphocytes, naïve CD4(+) lymphocytes preferentially migrated toward media from influenza-stimulated cells, indicating that antigen competition, by means of lymphocyte recruitment to the immunization site, is a potential mechanism for the observed decrease in FVIII immunogenicity. We also observed no differences in incidence or titer of rhFVIII-specific antibodies and inhibitors in mice exposed to the live-attenuated measles-mumps-rubella vaccine regardless of route of administration. Together, our results suggest that concomitant FVIII exposure and vaccination against influenza does not increase the risk of inhibitor formation and may in fact decrease anti-FVIII immune responses. PMID:27034428

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

    PubMed Central

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

    2011-01-01

    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. PMID:21245323

  20. Recombinant to modified factor VIII and factor IX - chromogenic and one-stage assays issues.

    PubMed

    Kitchen, S; Kershaw, G; Tiefenbacher, S

    2016-07-01

    The recent development of modified recombinant factor VIII (FVIII) and factor IX (FIX) therapeutic products with extended half-lives will create challenges for the haemostasis laboratory in obtaining recovery estimates of these products in clinical samples using existing assays. The new long-acting therapeutic concentrates contain molecular modifications of Fc fusion, site-specific of polyethylene glycol or albumin fusion. The optimum methods for monitoring each new product will need to be assessed individually and laboratories should select an assay which gives similar results to the assay used to assign potency to the product in question. For some extended half-life FVIII and FIX products some one stage assays are entirely unsuitable for monitoring purposes. For most products and assay reagents studied so far, and reviewed in this manuscript, chromogenic FVIII or FIX assays can be safely used with conventional plasma standards. If one stage assays are used then they should be performed using carefully selected reagents/methods which have been shown to recover activity close to the labelled potency for the specific product being monitored. PMID:27405680

  1. 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. PMID:27064790

  2. Genotype-phenotype correlation in combined deficiency of factor V and factor VIII

    PubMed Central

    Spreafico, Marta; Zheng, Chunlei; Yang, Angela; Platzer, Petra; Callaghan, Michael U.; Avci, Zekai; Ozbek, Namik; Mahlangu, Johnny; Haw, Tabitha; Kaufman, Randal J.; Marchant, Kandice; Tuddenham, Edward G. D.; Seligsohn, Uri; Peyvandi, Flora; Ginsburg, David

    2008-01-01

    Combined deficiency of factor V and factor VIII (F5F8D) is caused by mutations in one of 2 genes, either LMAN1 or MCFD2. Here we report the identification of mutations for 11 additional F5F8D families, including 4 novel mutations, 2 in MCFD2 and 2 in LMAN1. We show that a novel MCFD2 missense mutation identified here (D81Y) and 2 previously reported mutations (D89A and D122V) abolish MCFD2 binding to LMAN1. Measurement of platelet factor V (FV) levels in 7 F5F8D patients (4 with LMAN1 and 3 with MCFD2 mutations) demonstrated similar reductions to those observed for plasma FV. Combining the current data together with all previous published reports, we performed a genotype-phenotype analysis comparing patients with MCFD2 mutations with those with LMAN1 mutations. A previously unappreciated difference is observed between these 2 classes of patients in the distribution of plasma levels for FV and factor VIII (FVIII). Although there is considerable overlap, the mean levels of plasma FV and FVIII in patients with MCFD2 mutations are significantly lower than the corresponding levels in patients with LMAN1 mutations. No differences in distribution of factor levels are observed by sex. These data suggest that MCFD2 may play a primary role in the export of FV and FVIII from the ER, with the impact of LMAN1 mediated indirectly through its interaction with MCFD2. PMID:18391077

  3. Molecular structural studies of human factor VIII.

    PubMed

    McKee, P A; Andersen, J C; Switzer, M E

    1975-01-20

    Neither normal nor hemophilic factor VIII protein enters a 5% sosium dodecyl sulfate gel; on reduction, however, a single 195 000-molecular-weight peptide is observed. Hemophilic and normal factor VIII contain carbohydrate and appear identical in subunit molecular weight, electrical charge, and major antigenic determinants. Thrombin activation and inactivation of factor VIII does not detectably change the subunit molecular weight. Trypsin causes similar activity changes and obviously cleaves the factor VIII subunit. Human plasmin destroys factor VIII procoagulant activity and degrades the factor VIII subunit to 103 000-, 88 000-, and 17 000-molecular-weight peptides. Both normal and hemophilic factor VIII as well as thrombin-inactivated factor VIII support ristocetin-induced platelet aggregation. Purified factor VIII chromatographed on 4% agarose in 1.0 M sodium chloride shows no dissociation of the procoagulant activity from the void volume protein. Gel chromatography on 4% agarose in 0.25 M calcium chloride results in a procoagulant activity peak removed from the void volume protein; both peaks contain protein which does not enter a 5% SDS gel, but on reduction a 195 000-molecular-weight subunit band is observed for each. Both the void volume protein peak and the procoagulant activity peak from the 0.25 M calcium chloride-agarose gel column support ristocetin-induced platelet aggregation. After removal of calcium, a small amount of procoagulant activity is present only in the void volume peak. These data suggest that both the procoagulant and von Willebrand activities are on the same molecule. Thus our previous conclusion remains the same: human factor VIII is a large glycoprotein composed of identical 195 000-molecular-weight subunits jointed by disulfide bonds and is responsible for both antihemophilic and von Willebrand activities in human plasma. PMID:122889

  4. Factor VIII A3 domain substitution N1922S results in hemophilia A due to domain-specific misfolding and hyposecretion of functional protein

    PubMed Central

    Summers, Ryan J.; Meeks, Shannon L.; Healey, John F.; Brown, Harrison C.; Parker, Ernest T.; Kempton, Christine L.; Doering, Christopher B.

    2011-01-01

    A point mutation leading to amino acid substitution N1922S in the A3 domain of factor VIII (fVIII) results in moderate to severe hemophilia A. A heterologous expression system comparing N1922S-fVIII and wild-type fVIII (wt-fVIII) demonstrated similar specific coagulant activities but poor secretion of N1922S-fVIII. Immunocytochemical analysis revealed that intracellular levels of N1922S-fVIII were similar to those of wt-fVIII. The specific activity of intracellular N1922S-fVIII was 10% of that of wt-fVIII, indicating the presence of large amounts of a nonfunctional N1922S-fVIII–folding intermediate. wt-fVIII colocalized with both endoplasmic reticulum (ER)– and Golgi-resident proteins. In contrast, N1922S-fVIII colocalized only with ER-resident proteins, indicating a block in transit from the ER to the Golgi. A panel of conformation-dependent monoclonal antibodies was used to determine native or nonnative folding of N1922S-fVIII. Intracellular N1922S-fVIII but not secreted N1922S-fVIII displayed abnormal folding in the A3 and C1 domains, indicating that the A1, A2, and C2 domains fold independently into antigenically intact tertiary structures, but that folding is stalled in the mutant A3 and its contiguous C1 domain. In summary, the N1922S substitution results in poor secretion of a functional protein, and the domain-specific defect in folding and intracellular trafficking of N1922S-fVIII is a novel mechanism for secretion defects leading to hemophilia A. PMID:21217077

  5. Transient transfection of serum-free suspension HEK 293 cell culture for efficient production of human rFVIII

    PubMed Central

    2011-01-01

    Background Hemophilia A is a bleeding disorder caused by deficiency in coagulation factor VIII. Recombinant factor VIII (rFVIII) is an alternative to plasma-derived FVIII for the treatment of hemophilia A. However, commercial manufacturing of rFVIII products is inefficient and costly and is associated to high prices and product shortage, even in economically privileged countries. This situation may be solved by adopting more efficient production methods. Here, we evaluated the potential of transient transfection in producing rFVIII in serum-free suspension HEK 293 cell cultures and investigated the effects of different DNA concentration (0.4, 0.6 and 0.8 μg/106 cells) and repeated transfections done at 34° and 37°C. Results We observed a decrease in cell growth when high DNA concentrations were used, but no significant differences in transfection efficiency and in the biological activity of the rFVIII were noticed. The best condition for rFVIII production was obtained with repeated transfections at 34°C using 0.4 μg DNA/106 cells through which almost 50 IU of active rFVIII was produced six days post-transfection. Conclusion Serum-free suspension transient transfection is thus a viable option for high-yield-rFVIII production. Work is in progress to further optimize the process and validate its scalability. PMID:22115125

  6. Experience with a third generation recombinant factor VIII concentrate (Advate) for immune tolerance induction in patients with haemophilia A.

    PubMed

    Valentino, L A; Recht, M; Dipaola, J; Shapiro, A D; Pipe, S W; Ewing, N; Urgo, J; Bullock, T; Simmons, M; Deguzman, C

    2009-05-01

    The development of an inhibitor represents one of the most challenging complications in patients with haemophilia A. Optimal management is immune tolerance induction (ITI), typically through the administration of high doses of factor VIII (FVIII) concentrate. Among 12 patients who underwent ITI using Advate, a third-generation recombinant FVIII product that is free of animal and human protein additives, tolerance was achieved in nine (75%), including seven of 10 patients (70%) with high-titre inhibitors. ITI is ongoing in two patients and not yet successful; immune tolerance failed in the third patient. The median time to success was 4.0 months for group as a whole and for patients with high-titre inhibitors. Treatment was well tolerated, and no adverse events were observed. Advate was found to be equivalent to other FVIII products with regard to both ITI success rates and the incidence of adverse effects when used in these immune tolerance regimens. PMID:19298383

  7. Potency determination of factor VIII and factor IX for new product labelling and postinfusion testing: challenges for caregivers and regulators.

    PubMed

    Dodt, J; Hubbard, A R; Wicks, S J; Gray, E; Neugebauer, B; Charton, E; Silvester, G

    2015-07-01

    A workshop organized by the European Medicines Agency and the European Directorate for the Quality of Medicines and HealthCare was held in London, UK on November 28-29, 2013, to provide an overview of the current knowledge of the characterization of new factor VIII (FVIII) and factor IX (FIX) concentrates with respect to potency assays and testing of postinfusion material. The objective was to set the basis for regulatory authorities' discussion on the most appropriate potency assay for the individual products, and European Pharmacopoeia (Ph. Eur.) discussion on whether to propose revision of the Ph. Eur. monographs with respect to potency assays in the light of information on new FVIII and FIX concentrates. The workshop showed that for all products valid assays vs. the international concentrate standards were obtained and potency could be expressed in International Units. The Ph. Eur. chromogenic potency assay gave valid assay results which correlate with in vivo functionality of rFVIII products. For some modified rFVIII products and all modified rFIX products, one-stage clotting assay methods result in different potencies depending on the activated partial thromboplastin time reagent. As a consequence, monitoring of patients' postinfusion levels is challenging but it was pointed out that manufacturers are responsible for providing the users with appropriate information for use and laboratory testing of their product. Strategies to avoid misleading determination of patents' plasma levels, e.g. information on suitable assays, laboratory standards or correction factors were discussed. PMID:25623631

  8. In silico analyses of missense mutations in coagulation factor VIII: identification of severity determinants of haemophilia A.

    PubMed

    Sengupta, M; Sarkar, D; Ganguly, K; Sengupta, D; Bhaskar, S; Ray, K

    2015-09-01

    Factor VIII (FVIII) mutations cause haemophilia A (HA), an X-linked recessive coagulation disorder. Over 1000 missense mutations in FVIII are known and they lead to variable clinical phenotypes (severe, moderate and mild). The exact molecular basis of this phenotypic heterogeneity by FVIII missense mutations is elusive to date. In this study, we aimed to identify the severity determinants that cause phenotypic heterogeneity of HA. We compiled and curated a data set of 766 missense mutations from the repertoire of missense mutations in FVIII. We analysed these mutations by computational programs (e.g. Swiss-PdbViewer) and different mutation analysis servers (e.g. SIFT, PROVEAN, CUPSAT, PolyPhen2, MutPred); and various sequence- and structure-based parameters were assessed for any significant distribution bias among different HA phenotypes. Our analyses suggest that 'mutations in evolutionary conserved residues', 'mutations in buried residues', mutation-induced 'steric clash' and 'surface electrostatic potential alteration' act as risk factors towards severe HA. We have developed a grading system for FVIII mutations combining the severity determinants, and the grading pattern correlates with HA phenotype. This study will help to correctly associate the HA phenotype with a mutation and aid early characterization of novel variants. PMID:25854144

  9. Von Willebrand factor-containing factor VIII concentrates and inhibitors in haemophilia A. A critical literature review.

    PubMed

    Franchini, Massimo; Lippi, Giuseppe

    2010-11-01

    The development of inhibitors that neutralise the function of factor VIII (FVIII) is currently not only the most challenging complication associated with the treatment of haemophilia A but it also increases the disease-related morbidity as bleeding episodes do not respond to standard therapy. The main short-term goal of the treatment of inhibitor patients is to control bleeding episodes while the long-term one is to permanently eradicate the inhibitor by immune tolerance induction, particularly in the case of high-titer antibodies. Due to some in vitro studies and clinical observations, some investigators have suggested that FVIII concentrates containing von Willebrand factor (VWF) may be less immunogenic than high-purity or recombinant FVIII products. It has also been suggested that success rates for immune tolerance induction are higher when plasma-derived FVIII products are used. The currently available data from laboratory and clinical studies on the role of VWF in inhibitor development and eradication in haemophilia A is critically analysed in this review. As a result, we have not found definitive evidence supporting a role for product type on inhibitor incidence and inhibitor eradication in haemophilia A patients. PMID:20838738

  10. Molecular design and downstream processing of turoctocog alfa (NovoEight), a B-domain truncated factor VIII molecule.

    PubMed

    Ahmadian, Haleh; Hansen, Ernst B; Faber, Johan H; Sejergaard, Lars; Karlsson, Johan; Bolt, Gert; Hansen, Jens J; Thim, Lars

    2016-07-01

    Turoctocog alfa (NovoEight) is a third-generation recombinant factor VIII (rFVIII) with a truncated B-domain that is manufactured in Chinese hamster ovary cells. No human or animal-derived materials are used in the process. The aim of this study is to describe the molecular design and purification process for turoctocog alfa. A five-step purification process is applied to turoctocog alfa: protein capture on mixed-mode resin; immunoaffinity chromatography using a unique, recombinantly produced anti-FVIII mAb; anion exchange chromatography; nanofiltration and size exclusion chromatography. This process enabled reduction of impurities such as host cell proteins (HCPs) and high molecular weight proteins (HMWPs) to a very low level. The immunoaffinity step is very important for the removal of FVIII-related degradation products. Manufacturing scale data shown in this article confirmed the robustness of the purification process and a reliable and consistent reduction of the impurities. The contribution of each step to the final product purity is described and shown for three manufacturing batches. Turoctocog alfa, a third-generation B-domain truncated rFVIII product is manufactured in Chinese hamster ovary cells without the use of animal or human-derived proteins. The five-step purification process results in a homogenous, highly purified rFVIII product. PMID:26761578

  11. Molecular design and downstream processing of turoctocog alfa (NovoEight), a B-domain truncated factor VIII molecule

    PubMed Central

    Ahmadian, Haleh; Hansen, Ernst B.; Faber, Johan H.; Sejergaard, Lars; Karlsson, Johan; Bolt, Gert; Hansen, Jens J.; Thim, Lars

    2016-01-01

    Turoctocog alfa (NovoEight) is a third-generation recombinant factor VIII (rFVIII) with a truncated B-domain that is manufactured in Chinese hamster ovary cells. No human or animal-derived materials are used in the process. The aim of this study is to describe the molecular design and purification process for turoctocog alfa. A five-step purification process is applied to turoctocog alfa: protein capture on mixed-mode resin; immunoaffinity chromatography using a unique, recombinantly produced anti-FVIII mAb; anion exchange chromatography; nanofiltration and size exclusion chromatography. This process enabled reduction of impurities such as host cell proteins (HCPs) and high molecular weight proteins (HMWPs) to a very low level. The immunoaffinity step is very important for the removal of FVIII-related degradation products. Manufacturing scale data shown in this article confirmed the robustness of the purification process and a reliable and consistent reduction of the impurities. The contribution of each step to the final product purity is described and shown for three manufacturing batches. Turoctocog alfa, a third-generation B-domain truncated rFVIII product is manufactured in Chinese hamster ovary cells without the use of animal or human-derived proteins. The five-step purification process results in a homogenous, highly purified rFVIII product. PMID:26761578

  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. PMID:24389588

  13. Safety of Switching Factor VIII Products in the Era of Evolving Concentrates: Myths and Facts.

    PubMed

    Coppola, Antonio; Marrone, Emiliana; Conca, Paolo; Cimino, Ernesto; Mormile, Rosaria; Baldacci, Erminia; Santoro, Cristina

    2016-07-01

    Recent advances in the development of factor VIII (FVIII) concentrates offer patients with hemophilia the opportunity to switch to products considered safer or with improved properties. In some cases, product switch occurs due to side effects, convenience issues, or economic reasons affecting clinical choices. Reluctance to change FVIII concentrates is shown by patients and also by their physicians, because of concerns in particular about the risk of inhibitor development. A literature review was performed to retrieve the best evidence regarding safety issues of switching FVIII concentrate in patients with severe hemophilia A. Product switch was not associated with an increased inhibitor risk in four studies in patients during the first 50 to 75 exposure days, or in three studies reporting national switches in Canada and United Kingdom. The latter, the only available study comparing switcher and nonswitcher patients, showed an inhibitor incidence similar to that historically reported in the United Kingdom. In 16 phase III clinical trials and 6 postmarketing studies of FVIII concentrates, few de novo inhibitors were detected in previously treated patients, mostly transient and low-titer, with some additional recurrent inhibitors in patients with previous positive testing. On the whole, although rigorous controlled studies are lacking, literature data do not support increased risk of inhibitor development or other safety issues related to product switch. Therefore, in the presence of clinical needs, the advantages of switching FVIII products should not be missed because of perceived more than evidence-based challenges, in particular in this era of products with improved properties recently introduced or available in few years. Caution, however, is suggested in patients with high inhibitor risk, including in those in concomitance with surgery or intensive treatment. A careful inhibitor testing prior to and after product switch is always needed, to identify real de

  14. Coagulation factor VIII, IX and XI levels in north Indian patients with venous thromboembolism: first study from India.

    PubMed

    Chougule, Abhijit; Rajpal, Sweta; Ahluwalia, Jasmina; Bose, Sunil Kumar; Masih, Joseph; Das, Reena; Kumar, Narender; Malhotra, Pankaj; Suri, Vikas

    2016-01-01

    Studies have shown elevated levels of certain coagulation factors as risk factors for venous thromboembolism (VTE). In this study, we investigated the levels of coagulation factor VIII (FVIII), FIX and FXI in north Indian patients with VTE. A total of 123 patients with VTE were screened prospectively for FVIII, FIX and FXI levels and the conventional risk factors - deficiencies of protein C, S and antithrombin, positivity for antiphospholipid antibodies and the factor V Leiden mutation. Age-matched and sex-matched controls were included. VTE was secondary to known circumstantial and thrombophilic risk factors in 66 (53.7%) patients. In 46.3% (idiopathic VTE) patients, no cause was identified. The mean FVIII levels in idiopathic (187 IU/dl) and secondary VTE patients (185.4 IU/dl) were significantly higher compared with controls (129.6 IU/dl; P < 0.001). However, there was no statistically significant difference in the levels of FIX and FXI between patients and controls (P = 0.214 and 0.198, respectively). Patients with elevated FVIII levels had increased risk of VTE compared with controls (odds ratio: 9.4, 95% confidence interval: 4.7-18.79). On logistic regression analysis after adjusting for surgery and presence of antiphospholipid antibodies, this risk remained unchanged (odds ratio: 9.54, 95% confidence interval: 4.68-19.44). A dose-response relationship was observed with progressive increase in FVIII levels. Elevated FVIII levels constitute an independent risk factor for VTE in the north Indian population. Elevated levels of FIX and FXI were not associated with increased risk of VTE. PMID:26340461

  15. Combined deficiency of coagulation factors V and VIII: an update.

    PubMed

    Zheng, Chunlei; Zhang, Bin

    2013-09-01

    Combined deficiency of factor V (FV) and FVIII (F5F8D) is an autosomal recessive bleeding disorder characterized by simultaneous decreases of both coagulation factors. This review summarizes recent reports on the clinical presentations, treatments, and molecular mechanism of F5F8D. Genetic studies identified LMAN1 and MCFD2 as causative genes for this disorder, revealing a previously unknown intracellular transport pathway shared by the two important blood coagulation factors. LMAN1 and MCFD2 form a Ca2+-dependent cargo receptor complex that functions in the transport of FV/FVIII from the endoplasmic reticulum (ER) to the Golgi. Disrupting the LMAN1-MCFD2 receptor, complex formation is the primary molecular defect of missense mutations leading to F5F8D. The EF-hand domains of MCFD2 are necessary and sufficient for the interactions with both LMAN1 and FV/FVIII. Similarly, the carbohydrate recognition domain of LMAN1 contains distinct and separable binding sites for both MCFD2 and FV/FVIII. Therefore, FV and FVIII likely carry duel sorting signals that are separately recognized by LMAN1 and MCFD2 and necessary for the efficient ER-to-Golgi transport. FV and FVIII likely bind LMAN1 through the high-mannose N-linked glycans under the higher Ca2+ conditions in the ER and dissociate in the lower Ca2+ environment of the ER-Golgi intermediate compartment. PMID:23852824

  16. Combined Deficiency of Coagulation Factors V and VIII: An Update

    PubMed Central

    Zheng, Chunlei; Zhang, Bin

    2015-01-01

    Combined deficiency of factor V (FV) and FVIII (F5F8D) is an autosomal recessive bleeding disorder characterized by simultaneous decreases of both coagulation factors. This review summarizes recent reports on the clinical presentations, treatments, and molecular mechanism of F5F8D. Genetic studies identified LMAN1 and MCFD2 as causative genes for this disorder, revealing a previously unknown intracellular transport pathway shared by the two important blood coagulation factors. LMAN1 and MCFD2 form a Ca2+-dependent cargo receptor complex that functions in the transport of FV/FVIII from the endoplasmic reticulum (ER) to the Golgi. Disrupting the LMAN1-MCFD2 receptor, complex formation is the primary molecular defect of missense mutations leading to F5F8D. The EF-hand domains of MCFD2 are necessary and sufficient for the interactions with both LMAN1 and FV/FVIII. Similarly, the carbohydrate recognition domain of LMAN1 contains distinct and separable binding sites for both MCFD2 and FV/FVIII. Therefore, FV and FVIII likely carry duel sorting signals that are separately recognized by LMAN1 and MCFD2 and necessary for the efficient ER-to-Golgi transport. FV and FVIII likely bind LMAN1 through the high-mannose N-linked glycans under the higher Ca2+ conditions in the ER and dissociate in the lower Ca2+ environment of the ER–Golgi intermediate compartment. PMID:23852824

  17. 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.

  18. Stabilization of a human recombinant factor VIII by poloxamer 188 in relation to polysorbate 80.

    PubMed

    Clark, Jakson; Montgomery, Jade; Squires, Ryan; McGuire, Joseph

    2016-03-01

    Detection of enhanced surface tension depression by surfactant in the presence of protein was recently suggested as a basis for determining whether protein stabilization by that surfactant is owing to surfactant forming a steric barrier at interfaces or surfactant association with the protein. In particular, protein interaction with surfactant aggregates may lead to an increased concentration of monomers thus enhancing surfactant adsorption, or to formation of surfactant-protein complexes having little or no effect on adsorption. We compared the initial rates of surface tension depression by poloxamer 188 and polysorbate 80 (PS 80) in the presence and absence of a human recombinant factor VIII (rFVIII). Indirect evidence had suggested poloxamer 188 enters into stable associations with rFVIII in solution but does not form a steric barrier at the interface, while PS 80 behaves in contrary fashion. In this study, we show the presence of rFVIII caused an increase in the rate (reduction in the activation energy) of PS 80 adsorption, while no such change was recorded in the case of poloxamer 188. Thus, we provide substantiation for detection of protein-mediated acceleration of surfactant adsorption as a means to compare different surfactants in relation to their favored mechanism for protein stabilization. PMID:25471699

  19. 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.

  20. Adult severe haemophilia A patients under long-term prophylaxis with factor VIII in routine clinical practice.

    PubMed

    Mingot-Castellano, María E; González-Díaz, Laura; Tamayo-Bermejo, Rocio; Heiniger-Mazo, Anabel I

    2015-07-01

    There is no clear consensus on the best practice for long-term prophylaxis in adults with severe haemophilia A. This is a single-centre prospective case series study. We describe here the demographic data, type and reason of prophylaxis in adult patients (>18 years old) with severe (<1%) haemophilia A, treated in our centre from 2006 to 2013. Prophylaxis was tailored according to pharmacokinetic studies and posterior factor VIII (FVIII) trough level adjustment. We analysed FVIII consumption, bleeding rate, adherence and adverse events in this group of patients. In adult patients who initiated long-term prophylaxis during this period, we compared FVIII consumption and bleeding rate with the previous on-demand period. We analysed data from 18 patients. Median annual FVIII consumption was 2374.2 IU/kg/year. Among the patients receiving tertiary prophylaxis, initiated from 2006 onwards, the annual FVIII consumption was 2557.8 vs. 1696.8 IU/kg per year during the on-demand period (P = 0.312). In this group of patients, there was a decrease in annual bleeding events of 88.3% during prophylaxis compared with the on-demand therapy (P < 0.0001). A high adherence to prophylaxis was observed (84%). No cases of anaphylaxis or symptomatic thromboembolic events were recorded. In adult severe haemophilia A patients, the type of and reason to indicate long-term prophylaxis are diverse nowadays. FVIII consumption varies depending on the justification of prophylaxis. The observations reported provide further support for the efficacy of long-term prophylaxis in adult haemophilia A patients. PMID:25767895

  1. EF-hand domains of MCFD2 mediate interactions with both LMAN1 and coagulation factor V or VIII

    PubMed Central

    Zheng, Chunlei; Liu, Hui-hui; Zhou, Jiahai

    2010-01-01

    Combined deficiency of factor V and factor VIII (F5F8D) is a bleeding disorder caused by mutations in either LMAN1 or MCFD2. LMAN1 (ERGIC-53) and MCFD2 form a Ca2+-dependent cargo receptor that cycles between the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment for efficient transport of FV/FVIII from the ER to the Golgi. Here we show that the C-terminal EF-hand domains are both necessary and sufficient for MCFD2 to interact with LMAN1. MCFD2 with a deletion of the entire N-terminal non-EF hand region still retains the LMAN1-binding function. Deletions that disrupt core structure of the EF-hand domains abolish LMAN1 binding. Circular dichroism spectroscopy studies on missense mutations localized to different structural elements of the EF-hand domains suggest that Ca2+-induced folding is important for LMAN1 interaction. The EF-hand domains also mediate the interaction with FV and FVIII. However, mutations in MCFD2 that disrupt the tertiary structure and abolish LMAN1 binding still retain the FV/FVIII binding activities, suggesting that this interaction is independent of Ca2+-induced folding of the protein. Our results suggest that the EF-hand domains of MCFD2 contain separate binding sites for LMAN1 and FV/FVIII that are essential for cargo receptor formation and cargo loading in the ER. PMID:20007547

  2. Interaction of the 268-282 region of glycoprotein Ibalpha with the heparin-binding site of thrombin inhibits the enzyme activation of factor VIII.

    PubMed Central

    De Cristofaro, R; De Filippis, V

    2003-01-01

    Activation of factor VIII (FVIII) by thrombin plays a fundamental role in the amplification of the coagulation cascade and takes place through specific proteolytic cleavages at Arg(372), Arg(740) and Arg(1689). Full FVIII activation requires cleavage at Arg(372), a process involving the alpha-thrombin exosite-II; referred to as heparin-binding site (HBS). The present study was aimed at investigating the effect of glycoprotein Ibalpha (GpIbalpha; 1-282 fragment) binding to thrombin HBS on FVIII activation. Similar experiments were also performed using a synthetic peptide modelled on the 268-282 sequence of GpIbalpha, and sulphated successfully at all tyrosine residues present along its sequence, at positions 276, 278 and 279. Both GpIbalpha 1-282 and the sulphated GpIb 268-282 peptides induced a progressive decrease (up to 70%) in activated FVIII generation, assessed by coagulation and FXa-generation assays. Furthermore, SDS/PAGE and Western-blot experiments showed that the specific appearance of the 44 kDa A2 domain on cleavage of the FVIII Arg(372)-Ser(373) peptide bond was delayed significantly in the presence of either GpIbalpha 1-282 or GpIb 268-282 peptide. Moreover, the effect of the latter on thrombin-mediated hydrolysis of a peptide having the sequence 341-376 of FVIII was investigated using reverse-phase HPLC. The k (cat)/ K (m) values of the FVIII 341-376 peptide hydrolysis by thrombin decreased linearly as a function of the GpIbalpha 268-282 peptide concentration, according to a competitive inhibition effect. Taken together, these experiments suggest that the sulphated 268-282 region of GpIbalpha binds to thrombin HBS, and is responsible for the inhibition of the Arg(372)-Ser(373) bond cleavage and activation of FVIII. PMID:12689334

  3. 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. PMID:24870350

  4. A shift towards a T cell cytokine deficiency along with an anti-inflammatory/regulatory microenvironment may enable the synthesis of anti-FVIII inhibitors in haemophilia A patients

    PubMed Central

    Chaves, D G; Velloso-Rodrigues, C; Oliveira, C A; Teixeira-Carvalho, A; Santoro, M M; Martins-Filho, O A

    2010-01-01

    Despite the clinical relevance of anti-factor VIII (FVIII) antibodies (anti-FVIII inhibitors) impairing haemostatic activity of haemophilia A (HA) patients, the immunological mechanisms underlying their production are unknown. Aiming to understand more clearly the immune response in patients with [HAα-FVIII(+)] and without [HAα-FVIII(−)] anti-FVIII inhibitors, we have characterized the cytokine pattern of peripheral blood leucocytes, using an in vitro stimulation of whole blood samples with plasma-derived (pFVIII) or recombinant FVIII (rFVIII). The results highlighted decreased levels of tumour necrosis factor (TNF)-α+ neutrophils with higher interleukin (IL)-5/TNF-α ratio in HAα-FVIII(+). All HA samples displayed decreased levels of IL-10+ monocytes when compared to the blood donor (BD) samples. HAα-FVIII(+) showed lower levels of TNF-α+ monocytes and increased IL-10/TNF-α ratio. Analysis of adaptive immunity revealed increased levels of interferon (IFN)-γ+, TNF-α+ and IL-4+ T-cells, from both CD4+ and CD8+ T cells, in HAα-FVIII(−) when compared to BD. Moreover, increased frequency of IL-10+ B cells and higher levels of α-FVIII IgG1 were observed in HAα-FVIII(−). Basal levels of cytokine+ B-cells, similar to BD, and higher levels of α-FVIII IgG4 are major features in HAα-FVIII(+). The global cytokine profile demonstrated a major anti-inflammatory/regulatory pattern in HAα-FVIII(+), confirmed by the in vitro stimuli with pFVIII or rFVIII. The polarized anti-inflammatory/regulatory immune response in HAα-FVIII(+) and the mixed pattern with a bias towards an inflammatory cytokine profile, modulated by IL-4 in HAα-FVIII(−), may be the key element to drive the development of distinct subclasses of anti-FVIII antibodies. These finding have implications for the design of safe and effective therapeutic protocols to control inhibitors synthesis in HA patients. PMID:20846164

  5. Correction of murine hemophilia A following nonmyeloablative transplantation of hematopoietic stem cells engineered to encode an enhanced human factor VIII variant using a safety-augmented retroviral vector

    PubMed Central

    Ramezani, Ali

    2009-01-01

    Insertional mutagenesis by retroviral vectors is a major impediment to the clinical application of hematopoietic stem cell gene transfer for the treatment of hematologic disorders. We recently developed an insulated self-inactivating gammaretroviral vector, RMSinOFB, which uses a novel enhancer-blocking element that significantly decreases genotoxicity of retroviral integration. In this study, we used the RMSinOFB vector to evaluate the efficacy of a newly bioengineered factor VIII (fVIII) variant (efVIII)—containing a combination of A1 domain point mutations (L303E/F309S) and an extended partial B domain for improved secretion plus A2 domain mutations (R484A/R489A/P492A) for reduced immunogenicity—toward successful treatment of murine hemophilia A. In cell lines, efVIII was secreted at up to 6-fold higher levels than an L303E/F309S A1 domain–only fVIII variant (sfVIIIΔB). Most important, when compared with a conventional gammaretroviral vector expressing sfVIIIΔB, lower doses of RMSin-efVIII-OFB–transduced hematopoietic stem cells were needed to generate comparable curative fVIII levels in hemophilia A BALB/c mice after reduced-intensity total body irradiation or nonmyeloablative chemotherapy conditioning regimens. These data suggest that the safety-augmented RMSin-efVIII-OFB platform represents an encouraging step in the development of a clinically appropriate gene addition therapy for hemophilia A. PMID:19470695

  6. The role of previously untreated patient studies in understanding the development of FVIII inhibitors.

    PubMed

    Carcao, M; Re, W; Ewenstein, B

    2016-01-01

    Development of inhibitors against factor VIII (FVIII), the major complication of haemophilia A treatment today, is influenced by multiple factors. Genetic (F8 mutation, family history, ethnicity, polymorphisms in immune modulating genes) and non-genetic (intensive exposure to FVIII, presence of pro-inflammatory signals as might occur with large bleeds, infections, surgery, or other immune stimulants [e.g. vaccines]) risk factors as well as their complex inter-relationships contribute to the inhibitor risk profile of haemophilia patients, particularly in the previously untreated patient (PUP) population. Studies in PUPs have been fundamental to furthering the understanding of FVIII inhibitor development, as well as discovering previously unappreciated risk factors. The multi-factorial nature of inhibitor development makes it difficult to ascertain the contribution of FVIII products in inhibitor development through individual PUP studies. Sufficiently powered studies of large cohorts may overcome these limitations but interpretations should be conducted cautiously. Proper design and implementation of PUP safety studies will become even more important with the introduction of new molecules, such as extended half-life or human cell-line derived FVIII that propose reduced immunogenicity. Despite these difficulties, carefully performed clinical studies in PUPs may provide important insights into the natural history of the immune response to FVIII and may suggest targets for intervention to reduce immunogenicity. PMID:26315604

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

    PubMed Central

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

    2014-01-01

    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. PMID:25339360

  8. Factoring in Factor VIII With Acute Ischemic Stroke

    PubMed Central

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

    2016-01-01

    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. PMID:25669199

  9. Use of affinity-directed liquid chromatography-mass spectrometry to map the epitopes of a factor VIII inhibitor antibody fraction

    PubMed Central

    Griffiths, Amy E.; Wang, Wensheng; Hagen, Fred K.; Fay, Philip J.

    2011-01-01

    Summary Background Neutralizing factor (F) VIII antibodies develop in ~30% of individuals with hemophilia A and show specificity to multiple sites in the FVIII protein. Methods Reactive epitopes to an immobilized IgG fraction prepared from a high-titer, FVIII inhibitor plasma were determined following immuno-precipitation (IP) of tryptic and chymotryptic peptides derived from digests of the A1 and A2 subunits of FVIIIa and FVIII light chain. Peptides were detected and identified using highly sensitive liquid chromatography-mass spectrometry (LC-MS). Results Coverage maps of the A1 subunit, A2 subunit and light chain represented 79%, 69% and 90%, respectively, of the protein sequences. Dot blots indicated that the inhibitor IgG reacted with epitopes contained within each subunit of FVIIIa. IP coupled with LC-MS identified 19 peptides representing epitopes from all FVIII A and C domains. The majority of peptides (10) were derived from the A2 domain. Three peptides mapped to the C2 domain, while two mapped to the A1 and A3 domains, and single peptides mapped to the a1 segment and C1 domain. Epitopes were typically defined by peptide sequences of <12 residues. Conclusions IP coupled with LC-MS identified extensive antibody reactivity at high resolution over the entire functional FVIII molecule and yielded sequence lengths of less than 15 residues. A number of the peptides identified mapped to known sequences involved in functionally important protein-protein and protein-membrane interactions. PMID:21668738

  10. 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. PMID:26830966

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

    PubMed Central

    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. PMID:27382347

  12. Sucrose-Formulated Recombinant Factor VIII Dosing Flexibility in Prophylaxis Regimens: Experience from Postmarketing Surveillance Studies

    PubMed Central

    Humphries, Thomas J.; Rauchensteiner, Stephan; Tückmantel, Claudia; Pieper, Alexander; Maas Enriquez, Monika; Mathew, Prasad

    2015-01-01

    Objectives. Prophylaxis regimens for severe hemophilia A allowing more flexible dosing while maintaining efficacy may improve adherence and decrease the cost of prophylaxis. Here, we compared the clinical effectiveness of once- or twice-weekly versus ≥3-times-weekly prophylaxis with sucrose-formulated recombinant factor VIII (rFVIII-FS) in a “real-world” practice setting. Methods. Data from 3 postmarketing studies were pooled. Patients with severe hemophilia A receiving ≥1 prophylaxis infusion/wk of rFVIII-FS for ≥80% of a prophylaxis observation period (≥5 months) were included. Patients were categorized based on physician-assigned treatment regimens of 1-2 prophylaxis injections/wk (n = 63) or ≥3 prophylaxis injections/wk (n = 76). Descriptive statistics were determined for annualized bleeding rates (ABRs). Results. Median (quartile 1; quartile 3) ABR for all bleeds was 2.0 (0; 4.0) in the 1-2 prophylaxis injections/wk group and 3.9 (1.5; 9.3) in the ≥3 prophylaxis injections/wk group. Median ABRs for joint, spontaneous, and trauma-related bleeds were numerically lower with 1-2 prophylaxis injections/wk. As an estimate of prophylaxis success, 63% (≥3 prophylaxis injections/wk) to 84% of patients (1-2 prophylaxis injections/wk) had ≤4 annualized joint bleeds. Conclusions. Dosing flexibility and successful prophylaxis with rFVIII-FS were demonstrated. Very good bleeding control was achieved with both once-twice-weekly and ≥3-times-weekly prophylaxis dosing regimens. PMID:26356675

  13. Role of factor VIII-von Willebrand factor and fibronectin in the interaction of platelets in flowing blood with monomeric and fibrillar human collagen types I and III.

    PubMed

    Houdijk, W P; Sakariassen, K S; Nievelstein, P F; Sixma, J J

    1985-02-01

    Platelet adhesion to monomeric collagen types I and III, which were purified from human umbilical arteries, was studied in a perfusion chamber under well defined flow conditions. For this purpose, glass coverslips were coated with 20-30 micrograms/cm2 of collagen types I and III by spraying a solution of these collagens with a retouching air brush. Platelet deposition increased with the time of perfusion. Adhesion to both collagen types was similar in the first 3 min, but increased platelet deposition occurred on collagen type III after 3 min due to thrombus formation. Adhesion at a shear rate of 800 s-1 was strongly impaired with plasma of a patient with von Willebrand's disease or with fibronectin-free plasma. Addition of purified fibronectin to fibronectin-free plasma restored adhesion to the level obtained with normal plasma. Platelet deposition in normal plasma increased with increasing shear rates. Platelet deposition in VWD-plasma was normal at 490 s-1, but there was no increase at higher shear rates. Platelet deposition in fibronectin-free plasma was diminished at all shear rates studied from 490 to 1,300 s-1. Perfusion with a human albumin solution (HAS) to which purified Factor VIII-von Willebrand factor complex (FVIII-VWF) and fibronectin had been added gave similar platelet deposition as with normal plasma. Preincubation of collagen with FVIII-VWF and perfusion with HAS containing fibronectin, or, conversely, preincubation with fibronectin and perfusion with HAS containing FVIII-VWF, also resulted in adhesion similar to that observed in normal plasma. Similar adhesion was also observed after preincubation with both FVIII-VWF and fibronectin and subsequent perfusion with HAS alone. Sequential preincubations with first FVIII-VWF and then fibronectin, or with first fibronectin and then FVIII-VWF followed by perfusion with HAS, also gave a similar adhesion as observed with normal plasma. These data indicate that platelet adhesion to monomeric collagen types

  14. Role of factor VIII-von Willebrand factor and fibronectin in the interaction of platelets in flowing blood with monomeric and fibrillar human collagen types I and III.

    PubMed Central

    Houdijk, W P; Sakariassen, K S; Nievelstein, P F; Sixma, J J

    1985-01-01

    Platelet adhesion to monomeric collagen types I and III, which were purified from human umbilical arteries, was studied in a perfusion chamber under well defined flow conditions. For this purpose, glass coverslips were coated with 20-30 micrograms/cm2 of collagen types I and III by spraying a solution of these collagens with a retouching air brush. Platelet deposition increased with the time of perfusion. Adhesion to both collagen types was similar in the first 3 min, but increased platelet deposition occurred on collagen type III after 3 min due to thrombus formation. Adhesion at a shear rate of 800 s-1 was strongly impaired with plasma of a patient with von Willebrand's disease or with fibronectin-free plasma. Addition of purified fibronectin to fibronectin-free plasma restored adhesion to the level obtained with normal plasma. Platelet deposition in normal plasma increased with increasing shear rates. Platelet deposition in VWD-plasma was normal at 490 s-1, but there was no increase at higher shear rates. Platelet deposition in fibronectin-free plasma was diminished at all shear rates studied from 490 to 1,300 s-1. Perfusion with a human albumin solution (HAS) to which purified Factor VIII-von Willebrand factor complex (FVIII-VWF) and fibronectin had been added gave similar platelet deposition as with normal plasma. Preincubation of collagen with FVIII-VWF and perfusion with HAS containing fibronectin, or, conversely, preincubation with fibronectin and perfusion with HAS containing FVIII-VWF, also resulted in adhesion similar to that observed in normal plasma. Similar adhesion was also observed after preincubation with both FVIII-VWF and fibronectin and subsequent perfusion with HAS alone. Sequential preincubations with first FVIII-VWF and then fibronectin, or with first fibronectin and then FVIII-VWF followed by perfusion with HAS, also gave a similar adhesion as observed with normal plasma. These data indicate that platelet adhesion to monomeric collagen types

  15. Characteristics of rabbit transgenic mammary gland expressing recombinant human factor VIII.

    PubMed

    Chrenek, P; Makarevich, A V; Pivko, J; Massanyi, P; Lukac, N

    2009-02-01

    The objective of this research was to compare (i) the content of milk protein and recombinant human factor VIII (rhFVIII) in the milk of transgenic and non-transgenic rabbit females at three lactations and (ii) histological structure, ultrastructural morphology and occurrence of apoptosis in rabbit transgenic and non-transgenic mammary gland during third lactation and involution. Significant differences (t(0.05)) in milk protein content were found between transgenic and non-transgenic at all three lactations. The percentage of apoptotic cells was significantly higher (t(0.01)) in non-transgenic ones compared with transgenic mammary gland tissues (6.5% versus 2.4%) taken at the involution stage. Morphometrical analysis of histological preparations at the involution stage detected a significantly higher (t(0.05)) relative volume of lumen in transgenic animals compared with non-transgenic ones (60.00 versus 46.51%). Ultrastructural morphology of the transgenic mammary gland epithelium at the involution stage revealed an increased relative volume of protein globules (t(0.05)); at the lactation stage, a significantly higher volume of mitochondria (13.8%) compared with the non-transgenic (9.8%) ones was observed. These results, although revealing differences in some parameters of ultrastructure and histology, indicate no harmful effect of the mouse whey acid protein-hFVIII transgene expression on the state of mammary gland of transgenic rabbit females. PMID:19143684

  16. Method validation and clinical utility of chromogenic factor VIII assay compared to one-stage assay.

    PubMed

    Gouws, Wilmare; Botha, Elsabie; Visser, Adele

    2014-01-01

    The chromogenic FVIII assay is currently considered the gold standard for quantitation of factor VIII levels in both haemophilia A patients and as part of screening for thrombophilia. A method validation and evaluation of clinical utility within a routine diagnostic laboratory was undertaken by comparing the currently used one-stage assay to a commercially available chromogenic assay (Siemens, Johannesburg, South Africa). In total, 60 samples were included in this study to encompass the whole diagnostic range of the assay. Both low and high values showed very good correlation on linear regression analysis with correlation coeffients of 0.949 and 0.888 respectively. However, the lower detection limit of the Siemens Chromogenic assay was 1.5 IU/dL rendering it impossible to utilize in the setting of classifying a haemophilia A patient in terms of disease severity. Although the Siemens FVIII chromogenic assay shows excellent correlation to the currently used one-stage assay, the relatively high detection limit restrict implementation as a stand-alone assay in a routine diagnostic laboratory. PMID:23504571

  17. The Association Between HLA Class II Alleles and the Occurrence of Factor VIII Inhibitor in Thai Patients with Hemophilia A

    PubMed Central

    Nathalang, Oytip; Sriwanitchrak, Pramote; Sasanakul, Werasak; Chuansumrit, Ampaiwan

    2012-01-01

    Objective: This study aimed to investigate the association between HLA class II alleles and the occurrence of FVIIIinhibitor in Thai hemophilia A patients. Material and Methods: The distribution of HLA-DRB1 alleles and DQB1 alleles in 57 Thai hemophilia A patientsand 36 blood donors as controls was determined using the PCR sequence-specific primer (PCR-SSP) method, and theassociation between the occurrence of factor VIII (FVIII) inhibitor and the presence of certain HLA class II alleles wasinvestigated. Results: The frequency of HLA-DRB1*15 was higher in the hemophilia A patients with and without FVIII inhibitor,whereas that of DRB1*14, DRB1*07, and DQB1*02 was lower in the hemophilia A patients with FVIII inhibitor, ascompared to controls. Interestingly, only the frequency of DRB1*15 was significantly higher in the patients with inhibitorthan in the controls (P = 0.021). Moreover, the frequency of DRB1*15 in the patients with inhibitor was higher than inthose without inhibitor (P = 0.198). Conclusion: The study’s findings show that the DRB1*15 allele might have contributed to the occurrence of inhibitorin the Thai hemophilia A patients; however, additional research using larger samples and high-resolution DRB1 typingis warranted. PMID:24744621

  18. An Acquired Factor VIII Inhibitor in a Patient with HIV and HCV: A Case Presentation and Literature Review

    PubMed Central

    Zeichner, S. B.; Harris, A.; Turner, G.; Francavilla, M.; Lutzky, J.

    2013-01-01

    Introduction. Despite its low incidence, acquired factor VIII inhibitor is the most common autoantibody affecting the clotting cascade. The exact mechanism of acquisition remains unclear, but postpartum patients, those with autoimmune conditions or malignancies, and those with exposure to particular drugs appear most susceptible. There have been several case reports describing acquired FVIII inhibitors in patients receiving interferon alpha for HCV treatment and in patients being treated for HIV. To our knowledge, this is the first case of a patient with HCV and HIV who was not actively receiving treatment for either condition. Case Presentation. A 57-year-old Caucasian male with a history of HIV and HCV was admitted to our hospital for a several day history of progressively worsening right thigh bruising and generalized weakness. CTA of the abdominal arteries revealed large bilateral retroperitoneal hematomas. Laboratory studies revealed the presence of a high titer FVIII inhibitor. Conclusion. Our case of a very rare condition highlights the importance of recognizing and understanding the diagnosis of acquired FVIII inhibitor. Laboratory research and clinical data on the role of newer agents are needed in order to better characterize disease pathogenesis, disease associations, genetic markers, and optimal disease management. PMID:24198984

  19. 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

  20. 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. PMID:27436242

  1. 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

  2. Engineered antigen-specific human regulatory T cells: immunosuppression of FVIII-specific T- and B-cell responses

    PubMed Central

    Kim, Yong Chan; Zhang, Ai-Hong; Su, Yan; Rieder, Sadiye Amcaoglu; Rossi, Robert J.; Ettinger, Ruth A.; Pratt, Kathleen P.; Shevach, Ethan M.

    2015-01-01

    Expansion of human regulatory T cells (Tregs) for clinical applications offers great promise for the treatment of undesirable immune responses in autoimmunity, transplantation, allergy, and antidrug antibody responses, including inhibitor responses in hemophilia A patients. However, polyclonal Tregs are nonspecific and therefore could potentially cause global immunosuppression. To avoid this undesirable outcome, the generation of antigen-specific Tregs would be advantageous. Herein, we report the production and properties of engineered antigen-specific Tregs, created by transduction of a recombinant T-cell receptor obtained from a hemophilia A subject’s T-cell clone, into expanded human FoxP3+ Tregs. Such engineered factor VIII (FVIII)-specific Tregs efficiently suppressed the proliferation and cytokine production of FVIII-specific T-effector cells. Moreover, studies with an HLA-transgenic, FVIII-deficient mouse model demonstrated that antibody production from FVIII-primed spleen cells in vitro were profoundly inhibited in the presence of these FVIII-specific Tregs, suggesting potential utility to treat anti-FVIII inhibitory antibody formation in hemophilia A patients. PMID:25498909

  3. Role of enhanced half-life factor VIII and IX in the treatment of haemophilia.

    PubMed

    Mahdi, Ali J; Obaji, Samya G; Collins, Peter W

    2015-06-01

    Treatment of congenital haemophilia with factor VIII and IX concentrates often requires frequent infusions. This has obvious implications in establishing effective administration strategies and, in turn, adherence. To overcome these issues, three main technologies--polyethylene-glycol, Fc-neonatal IgG1 and albumin fusion products--have emerged into various stages of clinical development. Published data indicates an approximately 1·5- and fivefold increase in half-life of factor VIII and IX, respectively, compared to standard recombinant concentrates. Studies into efficacy and safety are starting to be published. Monitoring and optimal use of these new concentrates remains unknown. Weekly factor IX prophylaxis appears to be a feasible prophylactic regimen in haemophilia B patients. Weekly longer-acting FVIII is unlikely to provide adequate prophylaxis in most patients with haemophilia A but may reduce the frequency of infusions. Ongoing clinical trials and real life experience will help shape how these products can be used in practice and their cost effectiveness. The drive for convenience however should not overshadow the ultimate goal of prophylaxis, namely, preventing bleeding and arthropathy. PMID:25754016

  4. 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

  5. Blood Clotting Factor VIII: From Evolution to Therapy

    PubMed Central

    Orlova, N. A.; Kovnir, S. V.; Vorobiev, I. I.; Gabibov, A. G.; Vorobiev, A. I.

    2013-01-01

    Recombinant blood clotting factor VIII is one of the most complex proteins for industrial manufacturing due to the low efficiency of its gene transcription, massive intracellular loss of its proprotein during post-translational processing, and the instability of the secreted protein. Improvement in hemophilia A therapy requires a steady increase in the production of factor VIII drugs despite tightening standards of product quality and viral safety. More efficient systems for heterologous expression of factor VIII can be created on the basis of the discovered properties of its gene transcription, post-translational processing, and behavior in the bloodstream. The present review describes the deletion variants of factor VIII protein with increased secretion efficiency and the prospects for the pharmaceutical development of longer acting variants and derivatives of factor VIII. PMID:23819034

  6. [Gene mutation analysis of coagulation factor VIII from a female patient with hemophilia A].

    PubMed

    Zhou, Jing; Yan, Nai-hong; Jia, Yong-qian; Lu, Yi-lu; Yu, Jiang; Cao, Gui-qun; Chen, Qing-ying; Wang, Ling; Zhang, Fa-qiang; Xia, Oing-jie

    2006-05-01

    Hemophilia A affects male, whereas females are carriers and generally spared from this disease. However, we here reported a 65-year-old female with Hemophilia A while screening the gene mutation of coagulation factor VIII. The female went to hospital because of tripping to lead her right chest to be injured with subcutaneous hematoma. She had historically a hemorrhagic diathesis. The physical examination discovered her hip limited to bend and move, but no discrepancy length between her two legs. The initial laboratory tests showed that the activated partial thromboplastin time (APTT) was 61. 3 seconds (20-40 seconds), and the APTT corrected by mixing with normal plasma was 41.3 s, but the levels of PT, FIB and TT were normal. The plain radiographs revealed the hip joints to suffer from the acetabular dysplasia and osteoarthritis. The level of FVIII:C was 2%, F IX:C 200%, vWF:Ag 120%, vWF:Rcof 100%, vWF:CBA 128%, and the F VIII binding assay to vWF was normal. The primers for exon 14 of F VIII gene were designed according to the NM - 000132 gene sequence. DNA was abstracted from the patient blood. PCR were carried out and the DNA sequence was followed. A new mutation of 4111A-->C was discovered, which caused the amino acid sequence changed (T 1314 P). The mutation of T 1314 P may be the cause of this female patient to get the hemophilia A. This mutation was a novel one which has never been reported before. PMID:16761442

  7. Inversions in the factor VIII gene: improvement of carrier detection and prenatal diagnosis in Dutch haemophilia A families.

    PubMed Central

    Deutz-Terlouw, P P; Losekoot, M; Olmer, R; Pieneman, W C; de Vries-v d Weerd, S; Briët, E; Bakker, E

    1995-01-01

    Haemophilia A is an X linked bleeding disorder caused by a heterogeneous spectrum of mutations in the factor VIII gene. It has recently been reported that about 50% of severe haemophilia A cases are the result of an iversion in the factor VIII gene. The inversion results from homologous recombination between the A gene located in intron 22 of the FVIII gene and one of the two distal A genes, thus disrupting the coding sequence of the factor VIII gene. The inversion can be detected by conventional Southern blotting and hybridisation techniques. Here we present an analysis of 177 unrelated Dutch haemophilia A cases for the presence of an inversion. In 57% of the patients with severe disease an inversion was found and also in at least one of the 26 patients with moderately severe disease. The majority of inversions (85%) involved the most distal A gene, while in a minority (15%) the more proximal A gene was involved. We show that direct mutation detection greatly improves the assessment of carrier status and prenatal diagnosis for haemophilia A, especially in families with an isolated patient. The inversion is predominantly of grandpaternal origin. Images PMID:7643361

  8. 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. PMID:24251950

  9. Long-term anti-FVIII antibody response in Bethesda-negative haemophilia A patients receiving continuous replacement therapy.

    PubMed

    Klintman, Jenny; Hillarp, Andreas; Berntorp, Erik; Astermark, Jan

    2013-11-01

    It has previously been shown that patients with haemophilia A may develop non-neutralizing anti-factor VIII (FVIII) antibodies (NNA) that escape detection by the Bethesda assay, but are detected using immune-based assays. We and others found NNAs to be directed not only towards non-functional parts of the protein, but towards all regions of the FVIII protein. We also showed a heterogeneous antibody response towards different FVIII products. However, the clinical relevance and the natural history of NNA remain unclear. Therefore, we followed a cohort of unrelated subjects with haemophilia A for 4 years with the goal of exploring the long-term development of NNA using an enzyme-linked immunosorbent assay (ELISA). Ten of 78 subjects (12·8%) exhibited an immune response that was transient and heterogeneous, and none of the subjects developed an FVIII inhibitor. The result of the ELISA was examined in relation to clinical variables and no significant associations between a positive ELISA and age, F8 mutation, port-à-cath implantation and HCV infection were shown. Interestingly, patients with NNA had significantly fewer bleeding episodes (P = 0·048) compared with NNA-negative subjects. The results indicate that the immune response to FVIII products within an individual may vary over time. However, the clinical impact of NNA remains unclear. PMID:24032553

  10. Production of Factor VIII by Human Liver Sinusoidal Endothelial Cells Transplanted in Immunodeficient uPA Mice

    PubMed Central

    Fomin, Marina E.; Zhou, Yanchen; Beyer, Ashley I.; Publicover, Jean; Baron, Jody L.; Muench, Marcus O.

    2013-01-01

    Liver sinusoidal endothelial cells (LSECs) form a semi-permeable barrier between parenchymal hepatocytes and the blood. LSECs participate in liver metabolism, clearance of pathological agents, immunological responses, architectural maintenance of the liver and synthesis of growth factors and cytokines. LSECs also play an important role in coagulation through the synthesis of Factor VIII (FVIII). Herein, we phenotypically define human LSECs isolated from fetal liver using flow cytometry and immunofluorescence microscopy. Isolated LSECs were cultured and shown to express endothelial markers and markers specific for the LSEC lineage. LSECs were also shown to engraft the liver when human fetal liver cells were transplanted into immunodeficient mice with liver specific expression of the urokinase-type plasminogen activator (uPA) transgene (uPA-NOG mice). Engrafted cells expressed human Factor VIII at levels approaching those found in human plasma. We also demonstrate engraftment of adult LSECs, as well as hepatocytes, transplanted into uPA-NOG mice. We propose that overexpression of uPA provides beneficial conditions for LSEC engraftment due to elevated expression of the angiogenic cytokine, vascular endothelial growth factor. This work provides a detailed characterization of human midgestation LSECs, thereby providing the means for their purification and culture based on their expression of CD14 and CD32 as well as a lack of CD45 expression. The uPA-NOG mouse is shown to be a permissive host for human LSECs and adult hepatocytes, but not fetal hepatoblasts. Thus, these mice provide a useful model system to study these cell types in vivo. Demonstration of human FVIII production by transplanted LSECs encourages further pursuit of LSEC transplantation as a cellular therapy for the treatment of hemophilia A. PMID:24167566

  11. 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. PMID:26484639

  12. Innovative approach for improved rFVIII concentrate.

    PubMed

    Morfini, Massimo

    2014-11-01

    The development of a new recombinant factor VIII was designed and implemented to answer a number of unmet needs of patients affected by hemophilia A. Turoctocog alfa is bioengineered in a specific Chinese hamster ovary clone to present translational and posttranslational characteristics (sulphation, glycosylation) biosimilar to natural circulating forms of FVIII, with the aim to devoid any minimal change which may impact immunogenicity and antigenicity of recombinant protein. Both producer cell line and media are maintained free of any animal or human plasma derivative. Downstream processes of purification are performed by five steps (immunoaffinity chromatography, ion-exchange chromatography, virus inactivation by means of solvent-detergent treatment and nanofiltration, and to end with gel filtration), to provide the best possible margin of safety from known and unknown infectious agents. Large clinical trials seem to confirm the expectations placed in Turoctocog alfa in terms of high quality and safety of recombinant FVIII toward the goal of overcoming actual and future challenges of hemophilia therapy. PMID:24766411

  13. Freeze-dried activated substrate for factor VIII assays.

    PubMed

    Margolis, J

    1987-01-01

    Factor VIII-deficient plasma (natural or artificial) mixed with kaolin and phospholipid can be lyophilized to provide ready-to-use substrate which is stable for months at 4 degrees C and usable after many weeks at room temperature. Factor VIII assays are much simplified and more reproducible using this reagent and can be quantified with the aid of a programmable calculator according to the equation (formula; see text) as % of standard and X, S and B are clotting times of test, standard and blank samples respectively. The slope of the log/log function (k) is approximately--6.5. PMID:3111909

  14. Lymphocyte subset ratios and factor VIII usage in haemophilia.

    PubMed Central

    Beddall, A C; Al-Rubei, K; Williams, M D; Hill, F G

    1985-01-01

    Type and quantity of replacement treatment, together with haematological and immunological parameters were determined in 37 boys with severe haemophilia A and 41 children with other bleeding disorders. The quantity of factor VIII concentrate given to boys with severe haemophilia A (mean U/year) showed a significant inverse correlation with total white cell counts, lymphocyte counts, platelet counts, and the ratio of monoclonal antibody defined T lymphocyte subsets, T4 and T8 (T4:T8). Of the boys with severe haemophilia A, 49% had inversed T4:T8 ratios and 24% had thrombocytopenia. Treatment with high dose factor VIII concentrate (more than 25 000 U/year) was associated with low platelet counts, low lymphocyte counts, low T4:T8 ratios, and hypergammaglobulinaemia. In addition, six patients with severe haemophilia A and factor VIII inhibitors had inversed T4:T8 ratios. Patients treated exclusively with cryoprecipitate or prothrombin complex concentrates had normal T4:T8 ratios and platelet counts. The severity of the haematological and immunological abnormalities observed seems to be associated with high usage of factor VIII concentrates. Similar abnormalities have been described in patients with the acquired immune deficiency syndrome (AIDS). Prospective study of haemophiliacs is required to assess long term sequelae of factor concentrate usage, including the possible development of AIDS. PMID:3925894

  15. Molecular basis of LMAN1 in coordinating LMAN1-MCFD2 cargo receptor formation and ER-to-Golgi transport of FV/FVIII

    PubMed Central

    Zheng, Chunlei; Liu, Hui-hui; Yuan, Shuguang; Zhou, Jiahai

    2010-01-01

    The LMAN1-MCFD2 (lectin, mannose binding 1/multiple coagulation factor deficiency protein 2) cargo receptor complex transports coagulation factors V (FV) and VIII (FVIII) from the endoplasmic reticulum (ER) to the ER-Golgi intermediate compartment (ERGIC). LMAN1 (ERGIC-53) is a hexameric transmembrane protein with a carbohydrate recognition domain (CRD) on the ER luminal side. Here, we show that mutations in the first beta sheet of the CRD abolish MCFD2 binding without affecting the mannose binding, suggesting that LMAN1 interacts with MCFD2 through its N-terminal beta sheet, consistent with recently reported crystal structures of the CRD-MCFD2 complex. Mutations in the Ca2+- and sugar-binding sites of the CRD disrupt FV and FVIII interactions, without affecting MCFD2 binding. This interaction is independent of MCFD2, as LMAN1 mutants defective in MCFD2 binding can still interact with FVIII. Thus, the CRD of LMAN1 contains distinct, separable binding sites for both its partner protein (MCFD2) and the cargo proteins (FV/FVIII). Monomeric LMAN1 mutants are defective in ER exit and unable to interact with MCFD2, suggesting that the oligomerization of LMAN1 is necessary for its cargo receptor function. These results point to a central role of LMAN1 in regulating the binding in the ER and the subsequent release in the ERGIC of FV and FVIII. PMID:20817851

  16. Systematic review of the role of FVIII concentrates in inhibitor development in previously untreated patients with severe hemophilia a: a 2013 update.

    PubMed

    Franchini, Massimo; Coppola, Antonio; Rocino, Angiola; Santagostino, Elena; Tagliaferri, Annarita; Zanon, Ezio; Morfini, Massimo

    2013-10-01

    Nowadays, patients with hemophilia A receive a high standard of care; therefore, the most challenging complication of factor VIII (FVIII) replacement therapy has become the development of FVIII inhibitors, which render the concentrate infusion ineffective and expose patients to an increased risk of morbidity and mortality. Among environmental risk factors influencing inhibitor development, the type of FVIII products has always drawn the attention of investigators. Conflicting results are reported in the literature concerning rates of inhibitor development after either plasma-derived or recombinant FVIII concentrates. To help elucidate this controversial issue, we have performed a systematic review and meta-analysis of prospective studies evaluating the incidence of inhibitors in previously untreated patients with severe hemophilia A receiving plasma-derived or recombinant FVIII products. The quality of the studies was assessed using the Newcastle-Ottawa Scale (NOS), the STrenghtening the Reporting of OBservational studies in Epidemiology and an ad hoc quality score. Overall, 28 prospective studies, including 1,421 patients with hemophilia A, fulfilled our selection criteria and were included in the systematic review. No statistically significant differences were observed in the inhibitor incidence between plasma-derived and recombinant FVIII concentrates considering all (weighted means: 23%, 95% CI: 15-33% vs. 29%, 95% CI: 26-32%) and high titer (16%, 95% CI: 10-26% vs. 18%, 95% CI: 15-21%) inhibitors. Similarly, no significant differences were found in the inhibitor incidence among the different classes of recombinant products. In conclusion, the results of our meta-analysis show that the different types of FVIII products are not associated with different risks of inhibitor development. PMID:24022806

  17. Fc-fusion technology and recombinant FVIII and FIX in the management of the hemophilias

    PubMed Central

    Mancuso, Maria Elisa; Mannucci, Pier Mannuccio

    2014-01-01

    Prophylaxis with regular infusions of factor VIII (FVIII)- or factor IX (FIX)- containing products is the mainstay of modern hemophilia care. However, this therapeutic regimen is inconvenient, requiring repeated intravenous injections from childhood. Approaches meant to prolong the half-life of FVIII and FIX in plasma have been developed in order to improve the feasibility and acceptability of replacement therapy, extending protection from bleeding, reducing infusion frequency and hence the need for venous access devices in young children. Several strategies have been implemented to enhance the pharmacokinetics of clotting factors, including conjugation with polyethylene glycol and the production by genetic engineering of fusion proteins containing the coagulation factors linked to a long-lived plasma protein such as albumin or the Fc fragment of immunoglobulin (Ig)G. The latter technology is one of the most promising, since the prolongation of FVIII and FIX half-life is obtained by exploiting the physiological binding of the Fc domain to the neonatal Fc receptor. Fc fusion monomers have been obtained with both recombinant FVIII (rFVIIIFc) and FIX (rFIXFc), and data from preclinical and clinical studies showed improved pharmacokinetics for both factors, which are produced in human embryonic kidney (HEK) 293 cells, thus ensuring full human post-translational modifications. In Phase I/IIa studies, rFVIIIFc and rFIXFc showed 1.5–1.7 fold and 3.0–4.0 fold longer elimination half-life, respectively. Similar data have been obtained in the Phase III clinical studies with rFVIIIFc and rFIX-Fc published recently. Both drugs were satisfactorily safe, particularly with respect to immunogenicity, and no serious adverse event was observed. PMID:24729686

  18. 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

    2016-08-01

    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 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

  20. 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.

  1. Survival of 125iodine-labeled Factor VIII in normals and patients with classic hemophilia. Observations on the heterogeneity of human Factor VIII.

    PubMed Central

    Over, J; Sixma, J J; Bruïne, M H; Trieschnigg, M C; Vlooswijk, R A; Beeser-Visser, N H; Bouma, B N

    1978-01-01

    Radiolabeled human Factor VIII was used to study its survival in normals and patients with classic hemophilia, and to study the heterogeneity of Factor VIII; Purified Factor VIII was radiolabeled with 125iodine (125I-VIII) without loss of its structural integrity. The survival of 125I-VIII was studied in six normals and six hemophiliacs of whom four of the hemophiliacs had received transfusions with normal cryoprecipitate before the 125I-VIII infusion. No significant difference was observed between the disappearance of Factor VIII coagulant activity and radioactivity in these hemophiliacs. 125I-VIII in plasma showed a biphasic disappearance with an average t1/2 of 2.9 +/- 0.4 h (SEM) for the first phase and 18.6 +/- 0.7 h (SEM) for the second phase, respectively. The survival of 125I-VIII was similar comparing normals and hemophiliacs. The highest molecular weight forms of Factor VIII disappear more rapidly than the lower molecular weight ones. This was established by analysis of the fractions obtained by gel chromatography of plasma collected at several times after infusion and by analysis of the in vivo disappearance of three subfractions of Factor VIII. The fraction of 125I-VIII binding to platelets in the presence of ristocetin (containing the highest molecular weight forms of Factor VIII including the ristocetin cofactor) represented about 50% of the radioactivity present in plasma after infusion and showed a t 1/2 of 11.7 +/- 0.9 h (SEM) for the second phase. The fraction, which was recovered in cryoprecipitate of the recipient's plasma, represented about 90% of the initial radioactivity and showed a t 1/2 of 16.3 +/- 0.8 h (SEM) for the second phase. The fraction of 125I-VIII remaining in the cryosupernatant plasma (containing low molecular weight forms of Factor (VIII) showed a t 1/2 of 27.2 +/- 1.1 h (SEM). The first phase of the disappearance of 125I-VIII is caused in part by the disappearance of the highest molecular weight forms, which are possibly

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

    PubMed Central

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

    2015-01-01

    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. PMID:26192111

  3. Correlation between endogenous VWF:Ag and PK parameters and bleeding frequency in severe haemophilia A subjects during three-times-weekly prophylaxis with rFVIII-FS

    PubMed Central

    Lalezari, S; Martinowitz, U; Windyga, J; Enriquez, M M; Delesen, H; Schwartz, L; Scharrer, I

    2014-01-01

    Patients with severe haemophilia A experience frequent and spontaneous bleeding, causing debilitating damage to joints and decreasing quality of life. Prophylaxis with factor VIII (FVIII) reduces joint damage if initiated early. Circulating FVIII levels may be influenced by endogenous von Willebrand factor (VWF), a chaperone protein that binds and stabilizes FVIII. The aim of this study was to determine whether endogenous VWF antigen (VWF:Ag) levels are correlated with FVIII pharmacokinetic (PK) parameters and clinical outcomes in patients with severe haemophilia A. Previously treated, non-inhibitor patients in a multinational, randomized, double-blind, Ph II study received prophylaxis with once-weekly BAY 79-4980 (35 IU kg−1) or thrice-weekly recombinant sucrose-formulated FVIII (rFVIII-FS; 25 IU kg−1). PK parameters were evaluated at weeks 1 and 26. The number of bleeds per patient during the study was captured as part of the core efficacy endpoint. Spearman rank correlations assessed relationships of VWF:Ag levels with patient age, PK and annualized bleeding rate. Of 131 study patients (aged 13−64 years; BAY 79-4980, n = 63; rFVIII-FS, n = 68), 27 (21%; n = 15 and 12 respectively) were evaluable for PK assessment. Baseline VWF:Ag levels correlated with patient age (P < 0.0001). There was no significant difference in PK results between treatments; thus, PK parameters and VWF levels of all patients were analysed together. AUCnorm and T1/2 significantly increased with increased VWF:Ag (P < 0.001); clearance significantly decreased with increased VWF:Ag (P = 0.002). Annualized bleeding rate in patients treated with 3× per week rFVIII-FS significantly correlated with VWF:Ag and age (P = 0.038 and 0.021 respectively). PK parameters as well as the clinical outcome significantly correlated with endogenous VWF:Ag. The improved clinical outcome in subjects with high VWF:Ag levels may be explained by VWF:Ag influence on FVIII PK. PMID:24252058

  4. 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

  5. Challenges of the management of severe hemophilia A with inhibitors: two case reports emphasizing the potential interest of a high-purity human Factor VIII/von Willebrand factor concentrate and individually tailored prophylaxis guided by thrombin-generation test.

    PubMed

    Mathieu, Sophie; Crampe, Carine; Dargaud, Yesim; Lavigne-Lissalde, Géraldine; Escuriola-Ettingshausen, Carmen; Tardy, Brigitte; Meley, Roland; Thouvenin, Sandrine; Stephan, Jean L; Berger, Claire

    2015-12-01

    Severe hemophilia A is an X-linked bleeding disorder. Immune tolerance induction (ITI) is the best strategy of treatment when patients develop inhibitors. The objective is to illustrate the benefit of a high-purity human factor VIII/von Willebrand factor (VWF) concentrate (Octanate) in the management of ITI. We also wanted to raise the potential interest of laboratory assays such as thrombin-generation test (TGT) and epitope mapping. Two patients were treated during ITI, first with a recombinant FVIII and then with plasma-derived factor VIII without success, and, finally, with Octanate. Bypassing agents were used based on the results of TGT. Epitope mapping was performed during ITI therapy. These observations suggest the potential contribution of Octanate in the management of ITI in difficult cases. The use of bypassing agents can be necessary in prophylaxis or to treat bleedings, and may be guided by TGT results. Epitope mapping is used to describe the inhibitor. This article shows a decrease of the inhibitor directed against the C2 domain after initiation of Octanate. A high-purity human factor VIII/von Willebrand factor concentrate (Octanate) may be a valuable therapeutical option for ITI therapy. TGT and epitope mapping could be of help in the management of ITI. PMID:26517064

  6. The dataset from administration of single or combined immunomodulation agents to modulate anti-FVIII antibody responses in FVIII plasmid or protein primed hemophilia A mice.

    PubMed

    Liu, Chao Lien; Lyle, Meghan J; Shin, Simon C; Miao, Carol H

    2016-06-01

    Hemophilia A mice with pre-existing inhibitory antibodies against factor VIII (FVIII) were treated with single agents, AMD3100 and GCS-F, respectively. Inhibitor titers in treated mice and control HemA inhibitors mice were followed over time. Total B cells and plasma cells (PCs) were characterized by flow cytometry. HemA inhibitor mice were then treated with a combination regimen of IL-2/IL-2mAb complexes plus rapamycin and AMD3100. Finally, HemA inhibitor mice were treated with a new combination therapy using include IL-2/IL-2mAb complexes + Anti-CD20+AMD3100+G-CSF. The timeline of combination therapy was illustrated. Inhibitor titers following treatment in FVIII plasmid or protein induced inhibitor mice were evaluated overtime. A representative figure and gating strategies to characterize the subsets of Treg cells and B cells are presented. Please see http://dx.doi.org/10.1016/j.cellimm.2016.01.005 [1] for interpretation and discussion of these data and results. PMID:27081675

  7. The dataset from administration of single or combined immunomodulation agents to modulate anti-FVIII antibody responses in FVIII plasmid or protein primed hemophilia A mice

    PubMed Central

    Liu, Chao Lien; Lyle, Meghan J.; Shin, Simon C.; Miao, Carol H.

    2016-01-01

    Hemophilia A mice with pre-existing inhibitory antibodies against factor VIII (FVIII) were treated with single agents, AMD3100 and GCS-F, respectively. Inhibitor titers in treated mice and control HemA inhibitors mice were followed over time. Total B cells and plasma cells (PCs) were characterized by flow cytometry. HemA inhibitor mice were then treated with a combination regimen of IL-2/IL-2mAb complexes plus rapamycin and AMD3100. Finally, HemA inhibitor mice were treated with a new combination therapy using include IL-2/IL-2mAb complexes + Anti-CD20+AMD3100+G-CSF. The timeline of combination therapy was illustrated. Inhibitor titers following treatment in FVIII plasmid or protein induced inhibitor mice were evaluated overtime. A representative figure and gating strategies to characterize the subsets of Treg cells and B cells are presented. Please see http://dx.doi.org/10.1016/j.cellimm.2016.01.005 [1] for interpretation and discussion of these data and results. PMID:27081675

  8. Synthesis, processing, and secretion of recombinant human factor VIII expressed in mammalian cells

    SciTech Connect

    Kaufman, R.J.; Wasley, L.C.; Dorner, A.J.

    1988-05-05

    The synthesis, processing, and secretion of factor VIII expressed from heterologous genes introduced into Chinese hamster ovary cells has been studied. The results show factor VIII to be synthesized as a primary translation product of approximately 230 kDa that can be detected in the lumen of the endoplasmic reticulum. In this compartment, the majority of the factor VIII is in a complex with a resident protein of the endoplasmic reticulum, binding protein, and may never appear in the medium. Some factor VIII transits the endoplasmic reticulum to the Golgi apparatus, where it is cleaved to generate the mature heavy and light chains. In the absence of von Willebrand factor in the medium, the secreted heavy and light chains are unassociated and subsequently degraded. In the presence of von Willebrand factor in the medium, the heavy and light chains are secreted as a stable complex and activity accumulates linearly with time. The utilization and complexity of asparagine-linked carbohydrate present on the secreted recombinant-derived factor VIII and human plasma-derived factor VIII were compared and found to be very similar. In both cases, the asparagine-linked carbohydrate moieties on the heavy chain are primarily of the hybrid or complex-type. In contrast, the factor VIII from both sources contains a high-mannose type of asparagine-linked carbohydrate on the light chain.

  9. 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

  10. A first-in-human phase 1 study of ACE910, a novel factor VIII-mimetic bispecific antibody, in healthy subjects.

    PubMed

    Uchida, Naoki; Sambe, Takehiko; Yoneyama, Koichiro; Fukazawa, Naoki; Kawanishi, Takehiko; Kobayashi, Shinichi; Shima, Midori

    2016-03-31

    ACE910 is a recombinant humanized bispecific antibody that binds to activated factor IX and factor X and mimics the cofactor function of factor VIII (FVIII). This first-in-human study examined the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of ACE910 in healthy male adults. A total of 40 Japanese and 24 white subjects were randomized to receive a single subcutaneous injection of ACE910 (Japanese: 0.001, 0.01, 0.1, 0.3, or 1 mg/kg; white: 0.1, 0.3, or 1 mg/kg; n = 6 per dose group) or placebo (n = 2 per dose group). ACE910 exhibited a linear PK profile and had a half-life of ∼4 to 5 weeks. In FVIII-neutralized plasma, ACE910 shortened activated partial thromboplastin time and increased peak height of thrombin generation in a dose-dependent manner. All adverse events were nonserious and did not lead to any subject's withdrawal. Neither clinical findings nor laboratory abnormalities indicating hypercoagulability were observed. Two of 48 subjects receiving ACE910 (1 Japanese and 1 white) were positive for anti-ACE910 antibodies (anti-drug antibodies [ADAs]). One subject tested positive for ADAs both before and after ACE910 administration, whereas the other became ADA positive after receiving ACE910. The PK and PD profiles of ACE910 were similar in healthy Japanese and white subjects and suggest that ACE910 will be an effective and convenient prophylactic treatment of hemophilia A. This trial was registered at www.clinicaltrials.jp as #JapicCTI-121934. PMID:26626991

  11. 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.

  12. 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.

  13. Studies of the human factor VIII/von Willebrand factor protein. III. Qualitative defects in von Willebrand's disease.

    PubMed Central

    Gralnick, H R; Coller, B S; Sultan, Y

    1975-01-01

    The Factor VIII/von Willebrand factor protein was characterized in two unrelated patients with von Willebrand's disease in whom procoagulant and Factor VIII/von Willebrand factor antigen levels were normal. In both patients evidence of an abnormal protein was observed on crossed antigen-antibody electrophoresis. In one patient the Factor VIII/von Willebrand factor protein eluted from Sepharose 4B in a position and distribution identical to normal with normal levels of procoagulant activity and antigen. However, the partially purified Factor VIII/von Willebrand factor protein had markedly reduced von Willebrand factor activity in a ristocetin assay. In the second patient the peak of Factor VIII/von Willebrand factor protein, antigen, and procoagulant activity eluted from a Sepharose 4B column with an estimated molecular weight of approximately half that of normal. This protein had no von Willebrand factor activity. In both patients the reduced Factor VIII/von Willebrand factor protein subunit was indistinguishable from normal on polyacrylamide gel electrophoresis. These studies indicate that in some patients with von Willebrand's disease there is a qualitative defect of the Factor VII/von Willebrand factor protein; the total amount of protein, antigen, and procoagulant activity are normal while the von Willebrand factor activity is deficient. Images PMID:1080491

  14. 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

  15. 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

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

    PubMed Central

    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

    2015-01-01

    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. PMID:25911555

  17. Factor VIII/factor IX prophylaxis for severe hemophilia.

    PubMed

    Carcao, Manuel; Srivastava, Alok

    2016-01-01

    Experience with clotting factor concentrate (CFC) replacement products over several decades has shown that regular replacement (prophylaxis) is the only way to prevent musculoskeletal damage in hemophilia and impact the natural history of hemophilia. Yet there is a lack of data on the optimal age to start such replacement therapy and the regimens to be used. While very early administration of high doses is certainly more effective in preventing bleeding, cost and compliance are major constraints all over the world. Starting prophylaxis with even lower doses comparable to that used in episodic therapies leads to major reduction in bleeding. Recognition of the clinical heterogeneity of hemophilia even among patients with a label of severe hemophilia in terms of their spontaneous bleeding has led to efforts aimed at individualizing CFC replacement, based on clinical responses or pharmacokinetic data of the CFC. The importance of long-term outcome assessment being combined with CFC replacement therapy cannot be overemphasized. PMID:26805901

  18. 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. PMID:26105150

  19. 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

  20. 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. PMID:19358933

  1. Permanent cell line expressing human factor VIII-related antigen established by hybridization.

    PubMed Central

    Edgell, C J; McDonald, C C; Graham, J B

    1983-01-01

    A permanent human cell line, EA . hy 926, has been established that expresses at least one highly differentiated function of vascular endothelium, factor VIII-related antigen. This line was derived by fusing human umbilical vein endothelial cells with the permanent human cell line A549. Hybrid cells that survived in selective medium had more chromosomes than either progenitor cell type and included a marker chromosome from the A549 line. Factor VIII-related antigen can be identified intracellularly in the hybrids by immunofluorescence and accumulates in the culture fluid. Expression of factor VIII-related antigen by these hybrid cells has been maintained for more than 100 cumulative population doublings, including more than 50 passages and three cloning steps. This is evidence that EA . hy 926 represents a permanent line. Images PMID:6407019

  2. 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).

  3. Response of protein C and protein C inhibitor to warfarin therapy in patient with combined deficiency of Factors V and VIII.

    PubMed

    Bern, M M; Suzuki, K; Mann, K; Tracy, P; Hoyer, L; Jensen, W; Gallivan, M; Arkin, C; Davis, G

    1984-12-15

    The role of Protein C in combined factor V/VIII deficiency was examined by reducing the Protein C concentration using warfarin therapy in a patient with the combined deficiency. The factor VIII deficiency was like Hemophilia-A, with deficiency of VIII:C and VIII:C(Ag), but normal VIIIR:Ag and VIIIR:cof. The factor V deficiency was due to loss of the V antigen. During warfarin therapy the Protein C level was reduced, but concentrations of factors V and VIII did not change. Protein C Inhibitor was normal throughout. Thus combined factor V/VIII deficiency is not related to Protein C levels. PMID:6098970

  4. Direct characterization of factor VIII in plasma: detection of a mutation altering a thrombin cleavage site (arginine-372----histidine).

    PubMed Central

    Arai, M; Inaba, H; Higuchi, M; Antonarakis, S E; Kazazian, H H; Fujimaki, M; Hoyer, L W

    1989-01-01

    An immunoadsorbent method has been developed for the direct analysis of normal and variant plasma factor VIII. Using this method, the molecular defect responsible for mild hemophilia A has been identified for a patient whose plasma factor VIII activity is 0.05 unit/ml, even though the factor VIII antigen content is 3.25 units/ml. Although the variant factor VIII has an apparently normal molecular mass and chain composition, the 92-kDa heavy chain accumulates when the variant protein is incubated with thrombin and the 44-kDa heavy chain fragment cannot be detected. In contrast, thrombin cleavage of the 80-kDa light chain to the 72-kDa fragment is normal. As these data indicate a loss of factor VIII cleavage by thrombin at arginine-372, the genetic defect was determined by polymerase-chain-reaction amplification of exon 8 of the factor VIII gene and direct sequencing of the amplified product. A single-base substitution (guanine----adenine) was identified that produces an arginine to histidine substitution at amino acid residue 372. These data identify the molecular basis of an abnormal factor VIII, "factor VIII-Kumamoto," that lacks procoagulant function because of impaired thrombin activation. Images PMID:2498882

  5. Immune tolerance therapy in paediatric haemophiliacs with factor VIII inhibitors: 14 years follow-up.

    PubMed

    Kreuz, W; Ehrenforth, S; Funk, M; Auerswald, G; Mentzer, D; Joseph-Steiner, J; Beeg, T; Klarman, D; Scharrer, I; Kornhuber, B

    1995-01-01

    We report our clinical experience in the immune tolerance (IT) therapy of 21 paediatric haemophiliacs with FVIII inhibitor: high responders (16HR) received initially FVIII twice daily at a dosage of 50-300 U/kg/day, 11/16 received a concomitant treatment with activated prothrombin complex concentrate (100-200 U/kg/day). Low responders (five LR) received 20-100 FVIII U/kg every second or third day. Inhibitor elimination was achieved in 19/21 patients in a median time of 4 months in HR and 1.5 months in LR. The outcome and length of time needed to induce IT was significantly correlated with FVIII exposure between the first inhibitor detection and onset of IT therapy and to interruption of IT therapy. For a rapid elimination of FVIII inhibitors it is important to start continuous administration of high-dose FVIII (≥ 100 FVIII U/kg/day) before repeated exposure to FVIII, in order to prevent rebooster effects, prolongation of elimination time, and to reduce expense. PMID:27214218

  6. 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.

  7. [Molecular biologic study and the factor VIII gene in hemophilia A].

    PubMed

    Bock, I; Melegh, B; Nagy, A; Losonczy, H; Csete, B; Schröder, W; Kardos, M; István, L; Jager, R; Tóth, A M; Tóth, A; Falko, H; Mózsik, G

    1996-11-17

    Results of inversion in the intron 22 region of the VIII factor gene studied by Southern blot are presented. Inversion was found in 20 of 46 patients. In 14 cases (70%) distal and in 6 cases (30%) proximal type of inversion was detected. The significance of the positive result in genetic counseling and in presymptomatic diagnosis of Haemophilia A is emphasized. PMID:9005386

  8. Risk Factors for High-Titer Inhibitor Development in Children with Hemophilia A: Results of a Cohort Study

    PubMed Central

    Heller, Christine; Gutsche, Sven; Holzhauer, Susanne; Kenet, Gili; Kurnik, Karin; 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. PMID:24199202

  9. Inhibitors of propagation of coagulation (factors VIII, IX and XI): a review of current therapeutic practice

    PubMed Central

    Franchini, Massimo; Mannucci, Pier Mannuccio

    2011-01-01

    The management of patients with congenital haemophilia who develop alloantibodies against factors of the propagation phase of blood coagulation, commonly known as inhibitors, is the most important challenge facing haemophilia caregivers at present, as this complication not only compromises the efficacy of replacement therapy but also consumes an enormous amount of economic resources. Development of inhibitors further complicates the clinical course of severe haemophilia, with a prevalence of up to 30% in patients with haemophilia A (factor VIII deficiency) and up to 5% in those with haemophilia B (factor IX deficiency) and haemophilia C (factor XI deficiency). While the short-term goal of treatment of patients who develop alloantibodies is the control of bleeding, the eradication of the inhibitor is the main long-term goal. The management of severe bleeding episodes and the eradication of the autoantibody are also the mainstays of treatment of patients with acquired haemophilia, a rare but life-threatening haemorrhagic condition characterized by the development of inhibitory autoantibodies against coagulation factor VIII. The most recent options available for treating patients with congenital haemophilia complicated by inhibitors and acquired haemophilia because of autoantibodies against factor VIII are summarized in this review article. PMID:21204915

  10. A Hematological Menace: Multiple Venous Thrombosis Complicated by Acquired Factor VIII Deficiency

    PubMed Central

    Paudel, Robin; Dominguez, Luis W.; Dogra, Prerna; Suman, Saurav; Badin, Simon; Wasserman, Carrie

    2016-01-01

    Patient: Male, 21 Final Diagnosis: Acquired Factor VIII Deficiency Symptoms: Abdominal hematoma • DVT • life threatening bleeding Medication: — Clinical Procedure: Life saving medical therapy Specialty: Hematology Objective: Rare disease Background: Acquired hemophilia A (AHA) classically presents with spontaneous bleeding of mucosal sites, GI tract, and sub-cutaneous tissues, often leading to large hematomas and ecchymosis. Among documented cases, 50% are idiopathic and few have been associated with trauma or surgery. We present a case of life-threatening bleeding caused by AHA, following trauma and complicated by multiple venous thrombi. Case Report: A 21-year-old man presented with multiple injuries secondary to trauma leading to extensive life-saving surgery. Two weeks post-operatively, he developed multiple deep venous thrombi and was started on anticoagulation. Twenty-four days post-operatively, he started bleeding from multiple mucosal sites and developed an abdominal hematoma. Anticoagulation was stopped, with administration of fresh frozen plasma and vitamin K. Diagnosis of AHA was made based on low factor VIII level and presence of factor VIII inhibitors after an appropriate battery of tests ruled out other possible diagnoses. He was started on steroids and recombinant factor VIIa, leading to immediate improvement. Once stable, Rituximab infusions resulted in decreasing factor VIII inhibitor levels, with gradual normalization of PTT. Conclusions: AHA remains a diagnostic challenge because of its rarity, leading to delay in diagnosis and causing significant morbidity and mortality. Elevated PTT relative to PT/INR is a strong clue which should be followed by mixing studies. Very few cases have been associated with surgery or trauma and relatively few large, controlled trials have compared different treatment modalities for AHA. Growing evidence supports anti-CD20 (Rituximab) as an effective treatment option, as in this case. PMID:27040655

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

    NASA Astrophysics Data System (ADS)

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

    2014-11-01

    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.

  12. Prediction of giant thermoelectric power factor in type-VIII clathrate Si46.

    PubMed

    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 Si(46). 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 Si(46)-VIII clathrates is responsible for the formation of such a large number of carrier pockets. PMID:25391971

  13. 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.

  14. Intragenic integration in DLC1 sustains factor VIII expression in primary human cells without insertional oncogenicity

    PubMed Central

    Sivalingam, J; Phan, T T; Kon, O L

    2014-01-01

    Techniques enabling precise genome modifications enhance the safety of gene-based therapy. DLC1 is a hot spot for phiC31 integrase-mediated transgene integration in vitro and in vivo. Here we show that integration of a coagulation factor VIII transgene into intron 7 of DLC1 supports durable expression of factor VIII in primary human umbilical cord-lining epithelial cells. Oligoclonal cells with factor VIII transgene integrated in DLC1 did not have altered expression of DLC1 or neighbouring genes within a 1-Mb interval. Only 1.9% of all expressed genes were transcriptionally altered; most were downregulated and mapped to cell cycle and DNA repair pathways. DLC1-integrated cells were not tumourigenic in vivo and were normal by high-resolution genomic DNA copy number analysis. Our data identify DLC1 as a locus for durable transgene expression that does not incur features of insertional oncogenesis, thus expanding options for developing ex vivo cell therapy mediated by site-specific integration methods. PMID:24553346

  15. 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. PMID:23926300

  16. 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

  17. 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. PMID:18633304

  18. 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.

  19. 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.

  20. Immunoradiometric assay of factor VIII related antigen, with observations in 32 patients with von Willebrand's disease.

    PubMed

    Ruggeri, Z M; Mannucci, P M; Jeffcoate, S L; Ingram, G I

    1976-06-01

    A solid phase non-competitive immunoradiometric assay (IRMA) has been developed which allows measurement of factor VIII related antigen (VIIIR:AG) levels in normal plasma as low as 2.5 x 10(-4) U/ml. The assay is based on the extraction of VIIIR:AG from test plasma by means of polystrene tubes coated with a specific unlabelled anti-VIIIR:AG rabbit antiserum and subsequent labelling of the extracted antigen with 125I-labelled anti-VIIIR:AG rabbit IgG. PMID:1083743

  1. Pharmacokinetics and acute tolerance of a double virus inactivated plasma derived factor VIII concentrate.

    PubMed

    Saez, A; Bosh, N; Boadas, N; Arguello, A; Horvat, D; Dinapoli, G; Lisciani, R

    1999-07-01

    To further reduce the risk of possible viral disease transmission, an additional virucidal step was performed in the manufacturing process of a solvent/detergent treated factor VIII concentrate, which consisted of heating the lyophilized preparation at 100 degrees C for 30 min (Emoclot DI; ISI, Italy). Because thermal treatment may modify factor VIII bioavailability, the pharmacokinetic parameters and the acute tolerance of the single viral inactivated concentrate (preparation A) were compared with that of the double viral inactivated one (preparation B). Fifteen patients with severe haemophilia A and positive for HAV Ab were enrolled in a double-blind cross-over study and injected with 32.5 IU kg-1 of preparation A and 27 IU kg-1 of the preparation B. No significant differences between terminal half-life, area under the curve/dose, clearance/kg, volume of distribution at the steady state, in vivo recovery and acute tolerance of the two preparations was observed. The only statistical difference was restricted to Cmax. PMID:10469180

  2. FVIII inhibitors: pathogenesis and avoidance

    PubMed Central

    2015-01-01

    The pathogenesis of inhibitory antibodies has been the focus of major scientific interest over the last decades, and several studies on underlying immune mechanisms and risk factors for formation of these antibodies have been performed with the aim of improving the ability to both predict and prevent their appearance. It seems clear that the decisive factors for the immune response to the deficient factor are multiple and involve components of both a constitutional and therapy-related nature. A scientific concern and obstacle for research in the area of hemophilia is the relatively small cohorts available for studies and the resulting risk of confounded and biased results. Careful interpretation of data is recommended to avoid treatment decisions based on a weak scientific platform. This review will summarize current concepts of the underlying immunological mechanisms and risk factors for development of inhibitory antibodies in patients with hemophilia A and discuss how these findings may be interpreted and influence our clinical management of patients. PMID:25712994

  3. Pharmacokinetic studies with FVIII/von Willebrand factor concentrate can be a diagnostic tool to distinguish between subgroups of patients with acquired von Willebrand syndrome.

    PubMed

    Luboshitz, J; Lubetsky, A; Schliamser, L; Kotler, A; Tamarin, I; Inbal, A

    2001-05-01

    Acquired von Willebrand syndrome (AVWS) has been associated mainly with monoclonal gammopathy of uncertain significance (MGUS), clonal lymphoproliferative or myeloproliferative disorders and autoimmunity. In the present work we studied 6 patients with AVWS: four with MGUS IgG (lambda or kappa), one with small lymphocytic lymphoma and one with agnogenic myeloid metaplasia (AMM). All the patients underwent a pharmacokinetic analysis at presentation in order to study potential differences in recovery, clearance (CL) or terminal half-life (THL) following administration of von Willebrand factor (VWF) concentrate. In all the patients with AVWS an increase in clearance and a decrease in THL was observed as compared to these parameters in patients with hereditary type 3 von Willebrand disease (VWD). No difference in recovery was observed among the groups. The increase in clearance and the decrease in THL were significantly more pronounced in the group of MGUS patients (57.93 +/- 25.6 ml/h/kg, and 1.39 +/- 0.5 h, respectively) as compared to these parameters in the AMM (8.06 ml/h/kg, and 6.96 h, respectively) or the lymphoma (4.76 ml/h/kg, and 6.76 h. respectively) patients (p = 0.03 for clearance and 0.001 for THL). These data indicate that the pharmacokinetic analysis can be a useful tool to distinguish between MGUS-related and other causes of AVWS, and to plan an appropriate treatment accordingly. PMID:11372672

  4. 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

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

    DOE PAGESBeta

    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

  6. 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.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26598467

  8. 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

  9. 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

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

    PubMed Central

    Haji-Valizadeh, Hassa n; Modery-Pawlowski, Christa L.

    2015-01-01

    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. PMID

  11. 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.

  12. Sensitivity of the reaction time of the resonance thrombogram for factor VIII:C and factor IX deficiencies in the blood of dogs with haemophilia A or B.

    PubMed

    Mischke, R

    2000-09-01

    The sensitivity of the reaction time of the resonance thrombogram (RTG-r) was examined for two instruments based on 105 canine samples with a reduced factor VIII:C activity and 26 samples with a reduced factor IX activity. These samples were taken from dogs suffering from haemophilia A (n=18) or B (n=3) before and at different times after the substitution therapy with fresh frozen plasma. The RTG-r sensitivity was low; with reference to the total of all examined samples, it was between 0.39 and 0.65 for the factor VIII:C activity and between 0.46 and 0.62 for the factor IX activity, depending on the instrument and the testing procedure used. Low sensitivity was shown especially by the fact that even some of the samples with a factor VIII:C activity < 5% showed false negative results. Moreover, the correlation found between RTG-r and factor VIII:C activity (Spearman's rank correlation coefficient, r(S)=-0.556 to -0.700) as well as between factor IX activity and RTG-r (r(S)=-0.380 to -0.612) was only moderate. The low sensitivity of the RTG towards a reduced activity of the haemophilia factors VIII:C and IX contradicts the exclusive use of the RTG for monitoring haemophilic dogs as well as a global screening test for the haemostasis of dogs. PMID:11012705

  13. Cerebral venous thrombosis associated with thyrotoxicosis, the use of desmopressin and elevated factor VIII/von Willebrand factor.

    PubMed

    Waheed, Waqar; Aljerdi, Salman; Decker, Barbara; Cushman, Mary; Hamill, Robert W

    2016-01-01

    Cerebral venous thrombosis (CVT) is an uncommon disorder associated with diverse processes. We report a patient who, while receiving desmopressin and contraceptive pills (OCP), developed straight sinus thrombosis. Clinical assessment and laboratory investigations revealed untreated hyperthyroidism and a hypercoagulable state, characterised by high levels of von Willebrand factor, factor VIII coagulant activity and IgM cardiolipin antibody. The clinical picture improved with anticoagulation, treatment of hyperthyroidism and discontinuation of OCP and desmopressin. To the best of our knowledge, the association between the use of oral desmopressin and CVT has not been described. The multiple risk factors present in our case were probably additive in increasing the risk of CVT. Although this case represents a rare occurrence, practitioners should be alerted to the possible associations of desmopressin, oral contraceptives and Graves' disease with venous thrombosis. PMID:27503942

  14. Efficacy and safety during formulation switch of a pasteurized VWF/FVIII concentrate: results from an Italian prospective observational study in patients with von Willebrand disease.

    PubMed

    Castaman, G; Coppola, A; Zanon, E; Boeri, E; Musso, M; Siragusa, S; Federici, A B; Mancuso, G; Barillari, G; Biasoli, C; Feola, G; Franchini, M; Moratelli, S; Gamba, G; Schinco, P; Valdrè, L; Dragani, A; Mazzucconi, G; Tagliaferri, A; Morfini, M

    2013-01-01

    Von Willebrand disease (VWD) is an inherited bleeding disorder caused by the quantitative or qualitative deficiency of von Willebrand factor (VWF). Replacement therapy with plasma-derived VWF/factor VIII (FVIII) concentrates is required in patients unresponsive to desmopressin. To assess the efficacy, safety and ease of use of a new, volume-reduced (VR) formulation of VWF/FVIII concentrate Haemate(®) P in patients requiring treatment for bleeding or prophylaxis for recurrent bleeding or for invasive procedures. Pharmacoeconomic variables were also recorded. Data were analysed using descriptive statistics. This was a multicentre, prospective, observational study. Consecutively enrolled patients received Haemate(®) P VR according to their needs, and were followed for 24 months. Of the 121 patients enrolled, 25.6% had type 3 VWD and more than 40% had severe disease. All patients were followed for 2 years, for a total of 521 visits. On-demand treatment was given to 61.9% of patients, secondary long-term prophylaxis to 25.6% and prophylaxis for surgery, dental or invasive procedures to 45.5%. The response to treatment was rated as good to excellent in >93-99% of interventions. The new formulation was well tolerated by all patients with no report of drug-related adverse events. The switch to volume-reduced Haemate(®) P was easy to perform and infusion duration was decreased twofold compared with the previous formulation. Volume-reduced Haemate(®) P was at least as effective and well-tolerated as the previous formulation. PMID:22957493

  15. Functional domains on von Willebrand factor. Recognition of discrete tryptic fragments by monoclonal antibodies that inhibit interaction of von Willebrand factor with platelets and with collagen.

    PubMed Central

    Sixma, J J; Sakariassen, K S; Stel, H V; Houdijk, W P; In der Maur, D W; Hamer, R J; de Groot, P G; van Mourik, J A

    1984-01-01

    We have identified two functional domains on the von Willebrand factor (VWF) moiety of the Factor VIII-von Willebrand factor complex (FVIII-VWF), one interacting with blood platelets, and one interacting with vessel wall collagens, by means of two monoclonal antibodies directed against the VWF molecule, CLB-RAg 35 and CLB-RAg 201. The monoclonal antibody CLB-RAg 35 inhibited virtually all platelet adherence to artery subendothelium and to purified vessel wall collagen type III, at relatively high wall shear rates. CLB-RAg 35 also inhibited the ristocetin-induced platelet aggregation and the binding of FVIII-VWF to the platelet in the presence of ristocetin but did not affect the binding of FVIII-VWF to collagen. The monoclonal antibody CLB-RAg 201 inhibited the binding of FVIII-VWF to purified vessel wall collagen type I and III and all platelet adherence to collagen type III and the platelet adherence to subendothelium that was mediated by FVIII-VWF in plasma. The two functional domains on FVIII-VWF that were recognized by CLB-RAg 35 and CLB-RAg 201 were identified by means of immunoprecipitation studies of trypsin-digested FVIII-VWF. The domains resided on different polypeptide fragments, with a Mr of 48,000 for the collagen binding domain and a Mr of 116,000 for the platelet binding domain. The 116,000-mol wt fragment consisted of subunits of 52,000/56,000 mol wt and 14,000 mol wt after reduction. The 52,000/56,000-mol wt subunits possessed the epitope for CLB-RAg 35. Images PMID:6332119

  16. Detection and Characterization of Porcine Endogenous Retrovirus in Porcine Plasma and Porcine Factor VIII

    PubMed Central

    Takefman, Daniel M.; Wong, Susan; Maudru, Thomas; Peden, Keith; Wilson, Carolyn A.

    2001-01-01

    The pig genome contains porcine endogenous retroviruses (PERVs) capable of infecting human cells. Detection of infectious retrovirus in porcine peripheral blood mononuclear cells and endothelial cells suggested to us that pig plasma is likely to contain PERV. Both PERV env sequences and viral reverse transcriptase (RT) activity were detected in all plasma samples isolated from four NIH minipigs. To detect infectious virus from plasma, we performed a culture assay using three cell lines of feline, swine, and human origin that had previously been shown to be permissive for PERV. Infectious virus was successfully cultured from all four NIH minipig plasmas on the swine cell line ST-IOWA. Using RT-PCR with env-specific primers, we could detect expression of PERV class C envelope in the supernatant of ST-IOWA cells that had been exposed to each pig plasma. We next examined a pig plasma derivative, Hyate:C (porcine factor VIII), and found evidence of PERV particles, since all six lots examined were positive for PERV RNA and RT activity. However, infectious virus could not be detected in clinical lots of Hyate:C, suggesting that the manufacturing process might reduce the load of infectious virus to levels below detectable limits of the assay. Detection of infectious virus in porcine plasma confirms and extends the previous findings that certain porcine cells express PERV when manipulated in vitro and clearly demonstrates that there are porcine cells that express infectious PERV constitutively in vivo. PMID:11312325

  17. Influence of factor VIII:C and factor IX activity in plasmas of haemophilic dogs on the activated partial thromboplastin time measured with two commercial reagents.

    PubMed

    Mischke, R

    2000-05-01

    The present study is based on 145 plasma samples with a reduced activity of factor VIII:C (range: 0.009-0.62 IU mL-1) and 28 samples with a reduced factor IX activity (range: 0.035-0.55 IU mL-1). The samples were collected from dogs with haemophilia A (n=22) or haemophilia B (n=3), some of these during substitution therapy. For all samples the activated partial thromboplastin time (APTT) was measured with two commercial reagents containing kaolin as a contact activator. In each case, the deficiency of factor VIII:C or IX was reflected in abnormal results of the APTT. This was true for both reagents. A significant correlation (P < 0.001) was found between factor VIII:C activity and APTT (reagent 1, Pathromtin(R); Spearman's rank correlation coefficient, rS=-0.731, reagent 2, PTT-Reagenz; rS=-0.875) as well as between factor IX activity and APTT (reagent 1, rS=-0.819; reagent 2, rS=-0.955]. In each case, the relationship between coagulation factor activity and APTT could be proven most precisely by geometric regression. The results of this study illustrate the applicability of commercial APTT test kits as a sensitive screening test of factor VIII:C and IX deficiencies in canine plasma. PMID:10792470

  18. 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.

  19. 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

  20. 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

    Lau, A G; Sun, J; Hannah, W B; Livingston, E W; Heymann, D; Bateman, T A; Monahan, P E

    2014-09-01

    While chronic degenerative arthropathy is the main morbidity of haemophilia, a very high prevalence of low bone density is also seen in men and boys with haemophilia. This study investigates bone degradation in the knee joint of haemophilic mice resulting from haemarthrosis and the efficacy of aggressive treatment with factor VIII in the period surrounding injury to prevent bone pathology. Skeletally mature factor VIII knock-out mice were subjected to knee joint haemorrhage 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 haemorrhage. Mice were killed 2-weeks after injury and the joint morphology and loss of bone in the proximal tibia was assessed using microCT imaging. 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. Knee joint haemorrhage resulted in acute changes in 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

  1. 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.

  2. Long-acting recombinant factor VIII Fc fusion protein (rFVIIIFc) for perioperative haemostatic management in severe haemophilia A.

    PubMed

    Mahlangu, Johnny N; Ragni, Margaret; Gupta, Naresh; Rangarajan, Savita; Klamroth, Robert; Oldenburg, Johannes; Nogami, Keiji; Young, Guy; Cristiano, Lynda M; Dong, Yingwen; Allen, Geoffrey; Pierce, Glenn F; Robinson, Brian

    2016-07-01

    The Phase 3 A-LONG and Kids A-LONG studies demonstrated the prolonged half-life of rFVIIIFc compared with rFVIII, and the safety and efficacy of rFVIIIFc in subjects with severe haemophilia A. Eligible subjects from A-LONG and Kids A-LONG continued rFVIIIFc treatment by enrolling in ASPIRE, an ongoing extension study. Based on combined data from the primary studies and ASPIRE interim data, the safety and efficacy of rFVIIIFc in subjects requiring surgery were evaluated. Perioperative dosing regimens were determined by investigators with guidance based on pharmacokinetic data and recommendations from a clinical dosing committee. In addition to dosing frequency, factor consumption, blood loss, transfusions, bleeding episodes, and haemostatic response were assessed. Across studies, 21 subjects underwent 23 evaluable major surgeries, including 19 orthopaedic surgeries; 41 subjects underwent 52 minor surgeries, including 30 dental procedures. No major and 10 minor surgeries were performed in paediatric subjects. Of the major (n = 22) and minor (n = 32) surgeries assessed for haemostatic response, all were rated as excellent or good by the investigator/surgeon. During most major surgeries (95.7 %), haemostasis was maintained with one rFVIIIFc infusion. Blood loss in major surgeries was consistent with similar surgeries in subjects without haemophilia. Across studies, rFVIIIFc was well tolerated; no subject developed an inhibitor. PMID:26962852

  3. Evidence of normal functional levels of activated protein C inhibitor in combined Factor V/VIII deficiency disease.

    PubMed Central

    Canfield, W M; Kisiel, W

    1982-01-01

    Human activated protein C (APC) is a plasma serine protease that possesses amidolytic and anticoagulant activity. The rate at which the amidolytic and anticoagulant activity of APC was neutralized in normal plasma was essentially identical to that observed in plasma obtained from four individuals with combined Factor V/VIII deficiency disease. Incubation of radioiodinated APC with either normal human plasma or the combined Factor V/VIII-deficient plasmas resulted in the formation of a stable complex (Mr = 96,000) of the enzyme and a plasma protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Pretreatment of the radiolabeled APC with diisopropyl fluorophosphate prevented the formation of the enzyme-protein complex. On the basis of its ability to form a complex with radiolabeled APC, the APC-binding protein was purified to homogeneity from normal human plasma by ammonium sulfate fractionation, heparin-agarose chromatography, and QAE-Sephadex A-50 chromatography. The APC-binding protein (Mr = 54,000) is a glycoprotein, and possesses an amino-terminal sequence of Gly-Arg-Thr-Cys-Pro-Lys-Pro-Asp. The amino-terminal sequence of the APC-binding protein exhibited considerable homology with bovine colostrum inhibitor and pancreatic trypsin inhibitor, but no apparent sequence homology with the plasma serine protease inhibitors. Affinity-purified antibody against APC-binding protein immunoprecipitated a complex of radiolabeled APC and native APC-binding protein from normal human plasma. Complex formation was virtually eliminated in plasma immunodepleted of the APC-binding protein. Quantitative electroimmunoassay indicated essentially equal levels of APC-binding protein antigen in normal plasma compared with plasma from four patients with combined Factor V/VIII deficiency disease. Images PMID:6294139

  4. Phenotypic correction of hemophilia A in sheep by postnatal intraperitoneal transplantation of FVIII-expressing MSC.

    PubMed

    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

    2011-12-01

    We recently re-established a line of sheep that accurately mimics the clinical symptoms and genetics of severe hemophilia A (HA). Here, we tested a novel, nonablative transplantation therapy in two 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 human FVIII 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. Postmortem 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, nonablative MSC transplantation was straightforward, safe, and converted life-threatening, debilitating HA to a moderate phenotype in a large animal model. PMID:21906573

  5. Congenital factor V and VIII deficiency in women: a systematic review of literature and report of two new cases.

    PubMed

    Spiliopoulos, Dimitrios; Kadir, Rezan A

    2016-04-01

    Factor V and factor VIII deficiency (F5F8D) is a rare congenital bleeding disorder. There is a paucity of data in the literature about obstetric and gynaecological problems in women affected by F5F8D. The aim of this review was to examine obstetric complications and gynaecological problems in women with congenital F5F8D and present two new cases. An electronic search was performed to identify the published literature on PUBMED, MEDLINE and EMBASE databases using the following keywords 'congenital factor V and factor VIII deficiency' and 'women or pregnancy'. A total of 23 relevant articles were found and included in this systematic review: 15 case reports and 10 case series dating from 1976 to 2015. A total number of 86 women were identified. Heavy menstrual bleeding was the most common bleeding symptom in women (49%). Recurrent ovulation bleeding and haemorrhagic ovarian cyst were reported in three women. Nineteen pregnancies were reported (including our two case reports). There were no miscarriages. Postpartum bleeding occurred in six (32%) deliveries. In conclusion, data are very limited on gynaecological and obstetric problems in women with F5F8D. Heavy menstrual bleeding is a common problem. There is also an increased risk of postpartum haemorrhage. Close collaboration between haemophilia, obstetric and gynaecological teams is important to prevent and manage obstetric and gynaecological bleeding complications. PMID:26376169

  6. Quantification of perioperative changes in von Willebrand factor and factor VIII during elective orthopaedic surgery in normal individuals

    PubMed Central

    Kahlon, A.; Grabell, J.; Tuttle, A.; Engen, D.; Hopman, W.; Lillicrap, D.; James, P.

    2014-01-01

    Summary von Willebrand’s disease (VWD) patients undergoing major surgery are prophylactically treated to promote haemostasis. There is variability in perioperative clinical practice; however, most guidelines suggest replacing the deficient factor to a level of 1.0 IU mL−1 (or 100%). A review of the literature reveals a paucity of well constructed descriptive data quantifying the changes in coagulation that occur in response to surgical stress. The aim of this study was to quantify the changes in haemostatic variables occurring in response to elective orthopaedic surgery in normal individuals. Eligible subjects >18 years of age undergoing total hip or knee replacement were recruited. Blood samples were drawn at five time points: baseline, preoperatively, 30 min after surgical incision, 30 min postoperatively, postoperative day (POD) 1. Analyses included t-tests and repeated measures anova. Overall 30 patients, 21 women and 9 men, with a mean age of 65 were included in the final analysis. All von Willebrand factor (VWF) variables were seen to significantly decrease intraoperatively and increase postoperatively. VWF multimers showed a statistically significant decrease in high molecular weight multimers intraoperatively and an increase postoperatively. On subgroup analysis, age, gender and anaesthesia type were significantly correlated with changes in VWF parameters. Data presented in the current study establish a physiological baseline for VWF parameters in the normal population and demonstrate mean VWF/factor VIII levels greater than 1.0 IU mL−1 intraoperatively. As such, current management in VWD patients does not appear to mimic the normal physiological response to surgery. PMID:23711418

  7. F8 gene mutation profile in Indian hemophilia A patients: Identification of 23 novel mutations and factor VIII inhibitor risk association.

    PubMed

    Pinto, Patricia; Ghosh, Kanjaksha; Shetty, Shrimati

    2016-04-01

    'FVIII inhibitors', especially in severe hemophilia A (HA) patients, is a serious adverse effect that complicates their clinical management. Many genetic and non-genetic risk factors have been proposed for FVIII inhibitor development, diverse in different population groups. This is the first study in Indian hemophiliacs that analyzes inhibitor risk in relation to the complete F8 mutation profile, in a case-control study that included 145 Indian severe HA patients, i.e. 69 inhibitor positive (with 18 inhibitor concordant/discordant family members), and 58 inhibitor negative patients, after informed consent. While 53.54% (68/127) index cases were positive for intron 22 or intron 1 inversions, 55 causative F8 mutations were detected in the 59 inversion negative patients, of which 23 were novel mutations (in 24 patients) and 32 were reported earlier (in 35 patients). A higher incidence of mutations, in the C1 and C2 domains in inhibitor positive patients, and in the A1 domain in inhibitor negative patients was observed, though not significantly different. The study suggests that large F8 rearrangements (significantly higher in the inhibitor positive patients) pose the highest risk, while missense mutations (significantly higher in the inhibitor negative patients) pose the lowest risk of inhibitor development in Indian hemophilia A patients. PMID:26897466

  8. 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

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

    PubMed

    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

  10. Von Willebrand factor is reversibly decreased during torpor in 13-lined ground squirrels.

    PubMed

    Cooper, Scott; Sell, Shawn; Nelson, Luke; Hawes, Jennifer; Benrud, Jacob A; Kohlnhofer, Bridget M; Burmeister, Bradley R; Flood, Veronica H

    2016-01-01

    During torpor in a hibernating mammal, decreased blood flow increases the risk of blood clots such as deep vein thrombi (DVT). In other animal models platelets, neutrophils, monocytes and von Willebrand factor (VWF) have been found in DVT. Previous research has shown that hibernating mammals decrease their levels of platelets and clotting factors VIII (FVIII) and IX (FIX), increasing both bleeding time and activated partial thromboplastin time. In this study, FVIII, FIX and VWF activities and mRNA levels were measured in torpid and non-hibernating ground squirrels (Ictidomys tridecemlineatus). Here, we show that VWF high molecular weight multimers, collagen-binding activity, lung mRNA and promoter activity decrease during torpor. The VWF multimers reappear in plasma within 2 h of arousal in the spring. Similarly, FIX activity and liver mRNA both dropped threefold during torpor. In contrast, FVIII liver mRNA levels increased twofold while its activity dropped threefold, consistent with a post-transcriptional decrease in FVIII stability in the plasma due to decreased VWF levels. Finally, both neutrophils and monocytes are decreased eightfold during torpor which could slow the formation of DVT. In addition to providing insight in how blood clotting can be regulated to allow mammals to survive in extreme environments, hibernating ground squirrels provide an interesting model for studying. PMID:26481634

  11. Application of intron 9 and intron 25 dinucleotide repeats of the factor VIII gene for carrier diagnosis in haemophilia A.

    PubMed

    Venceslá, A; Baena, M; Fares Taie, L; Cornet, M; Baiget, M; Tizzano, E F

    2008-05-01

    We describe the usefulness of two dinucleotide repeats located in intron 9 and in intron 25 of the factor VIII gene for carrier diagnosis of haemophilia A. We analyzed 100 unrelated Spanish women and 34 women from haemophilia A (HA) families in whom known intragenic markers were unhelpful in determining their carrier status. The heterozygosity rate of intron 9 and intron 25 markers in the 100 control women was lower (0.28 and 0.38, respectively) than the values obtained with common markers routinely used in our laboratory. However, the application of intron 9 and intron 25 markers was effective in identifying the at-risk X chromosome in 11 of 34 (32%) of the uninformative women from HA families. The combined use of these repeats with current markers may facilitate the identification of the X chromosome in HA families for application in carrier, prenatal and pre-implantation diagnoses. PMID:18384354

  12. Successful percutaneous coronary intervention in a patient with combined deficiency of FV and FVIII due to novel compound heterozygous mutations in LMAN1.

    PubMed

    Patel, A J; Liu, H-H; Lager, R A; Malkovska, V; Zhang, B

    2013-07-01

    Percutaneous coronary intervention (PCI) in patients with congenital coagulation factor deficiencies presents a unique challenge. They are not only at increased risk of perioperative bleeding but can also suffer thrombosis of the stent as preventive anticoagulation and antiplatelet therapy is difficult. Several cases of successful PCI have been described in patients with haemophilia A and B, but there are no reports in patients with combined coagulation factor deficiencies. We used PCI to treat the coronary artery disease in a patient with the combined deficiency of factor V and factor VIII (F5F8D) and analysed the molecular basis of the disorder for this patient. A 68-year-old patient was admitted for urgent PCI with bare metal stent placement after the diagnosis of the F5F8D. Peripheral blood DNA was extracted for the sequence analysis of LMAN1 and MCFD2 genes. Mutations in LMAN1 was confirmed by molecular cloning of the PCR product and resequencing of the resulting clones. The patient underwent successful PCI with good long-term outcome. Our patient tolerated anticoagulation therapy well, with unfractionated heparin, and double antiplatelet therapy while he was initially supported with fresh frozen plasma and recombinant FVIII. Molecular analysis revealed that the patient carries unusual compound heterozygous frameshift mutations on the same microsatellite repeat region in exon 8 of LMAN1, one of which is a novel mutation (c.912delA). Our results suggest that patients with F5F8D can safely undergo PCI for coronary artery disease, with the treatment individualized to the specific patient. PMID:23557496

  13. 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.

    PubMed Central

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

    1991-01-01

    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 large gene size, its many scattered exons, and the high frequency of de novo mutations. Recently, we 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, we 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, we analyzed 99% of the coding region, 94% of the splice junctions, the promoter region, and the polyadenylylation site of the gene. We 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. Since DGGE after computer analysis appears to detect all mutations in a given fragment, the lower-than-expected yield of mutations in patients with severe disease is likely not due to failure of the detection method; it is probably due to the presence of mutations in DNA sequences outside the regions studied. Such sequences may include locus-controlling regions, other sequences within introns or outside the gene that are important for its expression, or another gene involved in factor VIII expression that is very closely linked to the factor VIII gene. Images PMID:1908096

  14. 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

  15. 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.

  16. Identification of deep intronic variants in 15 haemophilia A patients by next generation sequencing of the whole factor VIII gene.

    PubMed

    Bach, J Elisa; Wolf, Beat; Oldenburg, Johannes; Müller, Clemens R; Rost, Simone

    2015-10-01

    Current screening methods for factor VIII gene (F8) mutations can reveal the causative alteration in the vast majority of haemophilia A patients. Yet, standard diagnostic methods fail in about 2% of cases. This study aimed at analysing the entire intronic sequences of the F8 gene in 15 haemophilia A patients by next generation sequencing. All patients had a mild to moderate phenotype and no mutation in the coding sequence and splice sites of the F8 gene could be diagnosed so far. Next generation sequencing data revealed 23 deep intronic candidate variants in several F8 introns, including six recurrent variants and three variants that have been described before. One patient additionally showed a deletion of 9.2 kb in intron 1, mediated by Alu-type repeats. Several bioinformatic tools were used to score the variants in comparison to known pathogenic F8 mutations in order to predict their deleteriousness. Pedigree analyses showed a correct segregation pattern for three of the presumptive mutations. In each of the 15 patients analysed, at least one deep intronic variant in the F8 gene was identified and predicted to alter F8 mRNA splicing. Reduced F8 mRNA levels and/or stability would be well compatible with the patients' mild to moderate haemophilia A phenotypes. The next generation sequencing approach used proved an efficient method to screen the complete F8 gene and could be applied as a one-stop sequencing method for molecular diagnostics of haemophilia A. PMID:25948085

  17. 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.

  18. 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 Central

    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. PMID:25775247

  19. Factor VIII assay

    MedlinePlus

    ... B. In: Hoffman R, Benz EJ Jr, Silberstein LE, Heslop HE, Weitz JI, Anastasi, J, eds. Hematology: ... disorders. In: Hoffman R, Benz EJ Jr, Silberstein LE, Heslop HE, Weitz JI, Anastasi J, eds. Hematology: ...

  20. Factor VIII assay

    MedlinePlus

    ... LE, Heslop HE, Weitz JI, Anastasi, J, eds. Hematology: Basic Principles and Practice . 6th ed. Philadelphia, PA: ... LE, Heslop HE, Weitz JI, Anastasi J, eds. Hematology: Basic Principles and Practice . 6th ed. Philadelphia, PA: ...

  1. Local skin burn causes systemic (lung and kidney) endothelial cell injury reflected by increased circulating and decreased tissue factor VIII-related antigen.

    PubMed

    Gross, M A; Viders, D E; Brown, J M; Mulvin, D W; Miles, R H; Brentlinger, E R; Velasco, S E; Crawford, T S; Burton, L K; Repine, J E

    1989-08-01

    Inasmuch as xanthine oxidase (XO)-derived O2* metabolites may contribute to vascular endothelial injury and Factor VIII antigen (F8Ag) is a component of endothelial cells, we hypothesized that XO-derived O2* might damage and cause distant organ endothelial cells to release F8Ag in rats subjected to skin burn. We found that serum F8Ag (ELISA) increased in the blood of rats subjected to skin burn (70 degrees C water to shaved dorsal skin for 30 seconds) but not in sham control rats (30 degrees C water). Coincidentally, F8Ag levels also decreased in lung and kidney tissue sections (immunofluorescent staining) of burned rats but not sham rats. Increases in circulating F8Ag levels and decreases in tissue F8Ag levels appeared to result from XO-derived O2* metabolites: F8Ag levels did not increase in the blood and did not decrease in the tissues of rats pretreated with allopurinol (a specific XO inhibitor, 50 mg/kg) or dimethylthiourea (DMTU) (a permeable O2* metabolite scavenger, 250 mg/kg). Lung injury as assessed by permeability studies (I125-albumin leak) paralleled changes in blood F8Ag levels in sham, burn, allopurinol-, and DMTU-treated groups. We conclude that skin burn causes a systemic vascular injury that can be inhibited by allopurinol or DMTU and is reflected by increased circulating and tissue decreased Factor VIII antigen levels. Release of Factor VIII antigen may serve as a valuable marker of distant organ injury in patients with skin burn. PMID:2503901

  2. 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

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

    PubMed Central

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

    2015-01-01

    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. PMID:26702279

  4. 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.

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

    PubMed Central

    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

    2013-01-01

    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 PMID:23716558

  6. Rhesus monkeys and baboons develop FVIII inhibitors in response to porcine endothelial cells or islets

    PubMed Central

    Stewart, JM; Tarantal, AF; Hawthorne, WJ; Salvaris, EJ; O’Connell, PJ; Nottle, MB; d’Apice, AJF; Cowan, PJ; Kearns-Jonker, M

    2014-01-01

    Background Xenotransplantation of porcine organs holds promise of solving the human organ donor shortage. The use of α-1,3-galactosyltransferase knockout (GTKO) pig donors mitigates hyperacute rejection, while delayed rejection is currently precipitated by potent immune and hemostatic complications. Previous analysis by our laboratory suggests FVIII inhibitors might be elicited by the structurally restricted xenoantibody response which occurs after transplantation of either pig GTKO/hCD55/hCD59/hHT transgenic neonatal islet cell clusters or GTKO endothelial cells. Methods A recombinant xenoantibody was generated using sequences from baboons demonstrating an active xenoantibody response at day 28 after GTKO/hCD55/hCD59/hHT transgenic pig neonatal islet cell cluster transplantation. Rhesus monkeys were immunized with GTKO pig endothelial cells to stimulate an anti-nonGal xenoantibody response. Serum was collected at day 0 and 7 after immunization. A two stage chromogenic assay was used to measure FVIII cofactor activity and identify antibodies which inhibit FVIII function. Molecular modeling and molecular dynamics simulations were used to predict antibody structure and the residues which contribute to antibody-FVIII interactions. Competition ELISA was used to verify predictions at the domain structural level. Results Antibodies which inhibit recombinant human FVIII function are elicited after non-human primates are transplanted with either GTKO pig neonatal islet cell clusters or endothelial cells. There is an apparent increase of inhibitor titer by 15 Bethesda units after transplant; where an increase greater than 5 Bu can indicate pathology in humans. Furthermore, competition ELISA verifies the computer modeled prediction that the recombinant xenoantibody, H66K12, binds the C1 domain of FVIII. Conclusions The development of FVIII inhibitors is a novel illustration of the potential impact the humoral immune response can have on coagulative dysfunction in

  7. A protective effect of milk fat globule EGF factor VIII (MFG-E8) on the spontaneous fusion of milk fat globules in breast milk.

    PubMed

    Yasueda, Takehiko; Oshima, Kenzi; Nakatani, Hajime; Tabuchi, Kanji; Nadano, Daita; Matsuda, Tsukasa

    2015-07-01

    Lipid droplets synthesized in mammary epithelial cells are secreted into breast milk by the budding-off mechanism. The milk lipids, termed mik fat globules (MFGs), are surrounded with the cell plasma membrane and contain various membrane proteins, including milk fat globule epidermal growth factor (EGF)-factor VIII (MFG-E8), on their surface. We report here that the MFGs in the milk of MFG-E8-deficient mice fused each other and turned into abnormally large size of lipid droplets within ∼48 h after being secreted into mammary alveolar lumen in situ or being incubated at 37°C in vitro. This biophysical degeneration of MFGs in the MFG-E8-deficient milk was efficiently rescued in vitro by adding the milk serum of wild-type mice, isolated MFG-E8 or annexin V. Moreover, addition of ethylenediaminetetraacetic acid (30 mM) also protected the MFG fusion remarkably in vitro. In addition, bovine MFGs also fused each other when isolated from milk serum, and the fusion was inhibited by adding isolated MFG-E8 or mouse milk serum, but not the milk serum of MFG-E8-deficient mice. MFG-E8 in breast milk may mask the phosphatidylserine exposed on the surface of MFGs with time after secretion and thereby suppress the membrane fusion among MFGs resulting in the enlargement of MFGs in the breast milk. PMID:25661589

  8. Efficacy, safety and tolerability of recombinant factor VIII (REFACTO) in patients with haemophilia A: interim data from a postmarketing surveillance study in Germany and Austria.

    PubMed

    Pollmann, H; Externest, D; Ganser, A; Eifrig, B; Kreuz, W; Lenk, H; Pabinger, I; Schramm, W; Schwarz, T F; Zimmermann, R; Zavazava, N; Oldenburg, J; Klamroth, R

    2007-03-01

    An open-label, multicentre, postmarketing surveillance study conducted in Germany and Austria with recombinant factor VIII (REFACTO) has enrolled 217 patients (mean age 26.3 years) from 38 haemophilia centres during the first 4.8 years. Most patients (188/217; 86.6%) had severe to moderately severe haemophilia A, of whom 153 completed sufficient diary information for the main efficacy analysis. These 153 patients experienced a median of 6.6 (interquartile range 1.4-18.6) bleeding episodes per year. Patients treated with prophylaxis experienced a median of 4.4 (1.1-9.3) bleeds per year, while patients treated on-demand experienced a median of 22.8 (11.3-29.0) bleeds per year. Overall, most physicians (41/43 [95.3%]) were 'very satisfied' or 'satisfied' with the efficacy of REFACTO in the treatment of bleeding episodes. A total of 137 non-serious adverse events have been reported in 52/217 patients (24.0%) to date. In addition, 129 serious adverse events in 87 patients (40%) were reported, including 41 cases of 'less than expected therapeutic effect' (LETE). Of these, 39 LETE cases were reported in one centre; however, patients in this centre experienced considerably fewer bleeding episodes per year than patients outside this centre. Overall, six patients (2.8%) have developed de novo inhibitors, three of which were considered high titre. Four of these patients were at high risk (0-50 exposure days [ED]) of inhibitor formation, one was at intermediate risk (51-100 ED) and one was at low risk (>100 ED). These results emphasize the benefit of postmarketing surveillance and, overall, this study confirms the efficacy, safety and tolerability of REFACTO in the treatment of patients with haemophilia A. PMID:17286765

  9. 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. PMID:24354535

  10. Is ABO blood group truly a risk factor for thrombosis and adverse outcomes?

    PubMed Central

    Zhou, Shan; Welsby, Ian

    2014-01-01

    ABO blood type is one of the most readily available laboratory tests, and serves as a vital determinant in blood transfusion and organ transplantation. The ABO antigens are expressed not only on red blood cell membranes, determining the compatibility of transfusion, but also on the surface of other human cells, including epithelium, platelet and vascular endothelium, therefore extending the research into other involvements of cardiovascular disease and postoperative outcomes. ABO blood group has been recognized as a risk factor of venous thrombosis embolism since the 1960’s, effects now understood to be related to ABO dependent variations are procoagulant factor VIII (FVIII) and von Willebrand factor (vWF) levels. Levels of vWF, mostly genetically determined, are strongly associated with venous thromboembolism (VTE). It mediates platelet adhesion aggregation and stabilizes FVIII in plasma. Moreover, many studies have tried to identify the relationship between ABO blood types and ischemic heart disease. Unlike the clear and convincing associations between VTE and ABO blood type, the link between ABO blood type and ischemic heart disease is less consistent and may be confusing. Other than genetic factors, ischemic heart disease is strongly related to diet, race, lipid metabolism and economic status. In this review, we’ll summarize the data relating race and genetics, including ABO blood type, to VTE, ischemic heart disease and postoperative bleeding after cardiac surgery. PMID:25276299

  11. Inactivation of human immunodeficiency virus by gamma radiation and its effect on plasma and coagulation factors

    SciTech Connect

    Hiemstra, H.; Tersmette, M.; Vos, A.H.; Over, J.; van Berkel, M.P.; de Bree, H. )

    1991-01-01

    The inactivation of HIV by gamma-radiation was studied in frozen and liquid plasma; a reduction of the virus titer of 5 to 6 logs was achieved at doses of 5 to 10 Mrad at -80 degrees C and 2.5 Mrad at 15 degrees C. The effect of irradiation on the biologic activity of a number of coagulation factors in plasma and in lyophilized concentrates of factor VIII (FVIII) and prothrombin complex was examined. A recovery of 85 percent of the biologic activity of therapeutic components present in frozen plasma and in lyophilized coagulation factor concentrates was reached at radiation doses as low as 1.5 and 0.5 Mrad, respectively. As derived from the first-order radiation inactivation curves, the radiosensitive target size of HIV was estimated to be 1 to 3 MDa; the target size of FVIII was estimated to be 130 to 160 kDa. Gamma radiation must be disregarded as a method for the sterilization of plasma and plasma-derived products, because of the low reduction of virus infectivity at radiation doses that still give acceptable recovery of biologic activity of plasma components.

  12. Complex Changes in von Willebrand Factor-Associated Parameters Are Acquired during Uncomplicated Pregnancy

    PubMed Central

    Drury-Stewart, Danielle N.; Lannert, Kerry W.; Chung, Dominic W.; Teramura, Gayle T.; Zimring, James C.; Konkle, Barbara A.; Gammill, Hilary S.; Johnsen, Jill M.

    2014-01-01

    Background The coagulation protein von Willebrand Factor (VWF) is known to be elevated in pregnancy. However, the timing and nature of changes in VWF and associated parameters throughout pregnancy are not well understood. Objectives To better understand the changes in VWF provoked by pregnancy, we studied VWF-associated parameters in samples collected over the course of healthy pregnancies. Methods We measured VWF antigen (VWF:Ag), VWF propeptide (VWFpp), Factor VIII (FVIII), and ADAMTS13 activity in samples collected from 46 women during pregnancy and at non-pregnant baseline. We also characterized pregnant vs. non-pregnant VWF multimer structure in 21 pregnancies, and performed isoelectric focusing (IEF) of VWF in two pregnancies which had samples from multiple trimesters. Results VWF:Ag and FVIII levels were significantly increased during pregnancy. ADAMTS13 activity was unchanged. VWFpp levels increased much later in pregnancy than VWF:Ag, resulting in a progressive decrease in VWFpp:Ag ratios. FVIII:VWF ratios also decreased in pregnancy. Most pregnancies exhibited a clear loss of larger VWF multimers and altered VWF triplet structure. Further evidence of acquired VWF qualitative changes in pregnancy was found in progressive, reversible shifts in VWF IEF patterns over gestation. Conclusions These data support a new view of pregnancy in which VWF can acquire qualitative changes associated with advancing gestational age. Modeling supports a scenario in which both increased VWF production and doubling of the VWF half-life would account for the data observed. We propose that gestation induces a prolongation in VWF survival, which likely contributes to increased total VWF levels and altered VWF structure. PMID:25409031

  13. 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.

    PubMed Central

    Becker, J.; Schwaab, R.; Möller-Taube, A.; Schwaab, U.; Schmidt, W.; Brackmann, H. H.; Grimm, T.; Olek, K.; Oldenburg, J.

    1996-01-01

    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 percent). Fifty-five patients (37.4 percent) showed a F8A-gene inversion, 47 (32.0 percent) a point mutation, 14 (9.5 percent) a small deletion, 8 (5.4 percent) a large deletion, and 2 (1.4 percent) a small insertion. Further, four (2.7 percent) mutations were localized but could not be sequenced yet. No mutation could be identified in 17 patients (11.6 percent). Sixteen (10.9 percent) of the 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 percent) 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 patients 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

  14. 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.

  15. cDNA cloning of a mouse mammary epithelial cell surface protein reveals the existence of epidermal growth factor-like domains linked to factor VIII-like sequences

    SciTech Connect

    Stubbs, J.D.; Bui, A. San Francisco State Univ., CA ); Lekutis, C.; Singer, K.L.; Srinivasan, U.; Parry, G. ); Yuzuki, D. )

    1990-11-01

    A 2.1-kilobase cDNA coding for a surface protein of mammary epithelial cells has been isolated from a mouse mammary gland {lambda}gt11 cDNA library. Sequence analysis of this cDNA reveals an open reading frame of 1,389 base pairs that defines a protein with a molecular mass of 51.5 dKa. Structural analysis of the predicted sequence identifies two putative functional domains of the protein: (i) an N-terminal cysteine-rich region that is similar to epidermal growth factor-like domains of Drosophila Notch-1 protein and (ii) a large segment of the sequence that exhibited 54.5% identify with C-terminal domains of human coagulation factors VIII and V. These similarities in structure are used to predict the possible functions of the protein and its means of interaction with the cell surface. mRNA expression was detectable in mammary tissue from nonpregnant animals but was maximal in the lactating gland. In cultured cells, mRNA levels also correlated with the degree of cellular differentiation.

  16. Changes in von Willebrand factor level and von Willebrand activity with age in type 1 von Willebrand disease

    PubMed Central

    Rydz, Natalia; Grabell, Julie; Lillicrap, David; James, Paula D.

    2015-01-01

    In a normal population, VWF plasma levels (VWF:Ag) and VWF activity (VWF:RCo) increase by approximately 0.17 and 0.15 IU/ml per decade, but the influence of age is unknown in patients with type 1 von Willebrand disease (VWD). In a retrospective cohort study, the medical records of 31 type 1 VWD patients over the age of 30, who had been followed for ≥5 years, were reviewed for baseline clinical data and previously performed VWF:Ag, VWF:RCo and factor VIII levels (FVIII:C). VWF multimer analysis was normal in 27/31 cases performed. Mean bleeding score was 9.4 (range 2-21). Mean age at diagnosis was 33 (range 16-60 years), and duration of follow-up ranged from 5-26 years (mean 11 years). Patients had 2-10 time points of VWD testing (mean of 5.2). The mean VWF:Ag, VWF:RCo and FVIII:C at time of diagnosis were 0.44 IU/ml 0.34 IU/ml and 0.75 IU/ml. At last follow-up, the mean VWF:Ag, VWF:RCo and FVIII:C were significantly increased to 0.71 IU/L, 0.56 IU/ml and 0.90 IU/ml (p=<0.001, <0.001, and 0.0081, respectively). 18/31 patients had VWF:Ag, VWF:RCo and FVIII:C levels that increased into the normal range. The rate of change in VWF:Ag and VWF:RCo was 0.30 IU/ml (0.214-0.386, CI 95%, p<0.0001) and 0.20 IU/ml per year (0.126-0.274, CI 95%, p=0.0001). Patients with type 1 VWD experience age-related increases to VWF:Ag and VWF:RCo which can result in normalization of VWF levels. Further studies are required to determine if the bleeding phenotype resolves with the increases in VWF:Ag and VWF:RCo levels. PMID:25756206

  17. [Von Willebrand factor and ADAMTS13 balancing primary haemostasis].

    PubMed

    Schneppenheim, R; Budde, U

    2011-11-01

    Von Willebrand factor (VWF) is an adhesive, multi-functional huge multimerized protein with multiple domains harboring binding sites for collagen, platelet glycoprotein receptors and coagulation factor VIII (FVIII). The functional domains enable VWF to bind to the injured vessel wall, to recruit platelets to the site of injury by adhesion and aggregation and to bind and protect FVIII, an important cofactor of the coagulation cascade. VWF function in primary haemostasis is located in particular in the arterial and micro-circulation. This environment is exposed to high shear forces with hydrodynamic shear rates ranging over several orders of magnitude from 10⁻¹ to 10⁵ s-1 and requires particular mechanisms to enable platelet adhesion and aggregation under these variable conditions. The respective VWF function is strictly correlating with its multimer size. Lack or reduction of large VWF multimers is seen in patients with von Willebrand disease (VWD) type 2A which correlates with reduction of both VWF:platelet GPIb-binding and VWF:collagen binding and a bleeding phenotype. To prevent unlimited platelet adhesion and aggregation which is the cause of the microangiopathic disorder thrombotic thrombocytopenic purpura (TTP), VWF function is regulated by its specific protease ADAMTS13. Whereas a particular susceptibility of VWF to ADAMTS13 proteolysis is the cause of a frequent VWD type 2A phenotype, lack or dysfunction of ADAMTS13, either acquired by ADAMTS13 antibodies or by inherited ADAMTS13 deficiency (Upshaw-Schulman Syndrome), causes TTP. Therefore VWD and TTP represent the opposite manifestations of VWF related disorders, tightly linked to each other. PMID:21792464

  18. Prevalence, Incidence, and Factor Concentrate Usage Trends of Hemophiliacs in Taiwan

    PubMed Central

    Tu, Tsu-Chiang; Liou, Wen-Shyong; Chou, Tsui-Yun; Lin, Tsung-Kun; Lee, Chuan-Fang; Chen, Jye-Daa; Cham, Thau-Ming

    2013-01-01

    Purpose Hemophilia A and B (HA, HB) are the most common X-linked inherited bleeding disorders. The introduction of factor concentrates has allowed for control of the lifelong chronic disease. However, no studies have been published regarding the epidemiology of hemophilia in Taiwan. Our aim was to determine the prevalence, incidence, and mortality rate, as well as trends in the use of factor concentrates, in individuals with hemophilia in Taiwan. Materials and Methods A retrospective study was conducted using the National Health Insurance Research Database between 1997 and 2007. Results We identified 988 males with hemophilia (HA : HB ratio=5.4 : 1). The mean prevalence per 100000 males was 6.7±0.1 for HA and 1.2±0.1 for HB. The estimated mean annual incidence per live male birth was 1 in 10752 for HA and 1 in 47619 for HB. Standardized mortality ratios for males with hemophilia (all severities) or severe hemophilia were 1.3- and 2.1-fold higher than that of the general male population, respectively. Mean factor VIII (FVIII) and factor IX (FIX) usage was 1.5003±0.4029 and 0.3126±0.0904 international units (IUs) per capita, respectively. Mean FVIII and FIX usage per patient with hemophilia (all severities) or severe hemophilia was 44027±11532 and 72341±17298, respectively, and 49407±13015 and 74369±18411 IUs per person with HA or HB, respectively. Conclusion Our data revealed epidemiologic and factor concentrate usage trends in males with hemophilia in Taiwan, highlighting a need for improvements in the mandatory National Health Insurance registry. A better-designed, patient-centered registry system would enable more detailed patient information collection and analysis, improving subsequent care. PMID:23225801

  19. Human plasma-derived FVIII/VWD concentrate (Biostate): a review of experimental and clinical pharmacokinetic, efficacy and safety data

    PubMed Central

    Harper, Paul; Favaloro, Emmanuel J.; Curtin, Julie; Barnes, Chris; Dunkley, Scott

    2016-01-01

    Human plasma-derived factor VIII/von Willebrand factor complex concentrates are used to control bleeding in patients with von Willebrand disease (VWD) or haemophilia A (HA). The properties of these haemostatic factor concentrates vary widely, which can have significant clinical implications. This review provides an extensive overview of the molecular properties, in addition to pharmacokinetic, efficacy and safety data, and case studies of clinical experience of one such concentrate, Biostate. These data are discussed in the context of various therapeutic applications and compared with other factor concentrate products. Data are presented from data on file from the manufacturer; product information and published experimental and clinical pharmacokinetic, safety and efficacy study data; and example case studies of clinical experience. The data discussed herein demonstrate that Biostate has well-established efficacy profiles in the treatment of patients with VWD or HA, with the control of bleeding rated as ‘excellent’, ‘good’ or ‘moderate’ in >90% of patients. In an immune-tolerance induction setting, 73% of patients achieved a complete response following treatment with Biostate. Biostate was generally well tolerated in patients with HA or VWD, with infrequent minor adverse events reported and no reported cases of clinically relevant thrombosis. PMID:27114741

  20. T1-Weighted Dynamic Contrast-Enhanced MRI as a Noninvasive Biomarker of Epidermal Growth Factor Receptor vIII Status

    PubMed Central

    Arevalo-Perez, J.; Thomas, A.A.; Kaley, T.; Lyo, J.; Peck, K.K.; Holodny, A.I.; Mellinghoff, I.K.; Shi, W.; Zhang, Z.; Young, R.J.

    2016-01-01

    BACKGROUND AND PURPOSE Epidermal growth factor receptor variant III is a common mutation in glioblastoma, found in approximately 25% of tumors. Epidermal growth factor receptor variant III may accelerate angiogenesis in malignant gliomas. We correlated T1-weighted dynamic contrast-enhanced MR imaging perfusion parameters with epidermal growth factor receptor variant III status. MATERIALS AND METHODS Eighty-two consecutive patients with glioblastoma and known epidermal growth factor receptor variant III status who had dynamic contrast-enhanced MR imaging before surgery were evaluated. Volumes of interest were drawn around the entire enhancing tumor on contrast T1-weighted images and then were transferred onto coregistered dynamic contrast-enhanced MR imaging perfusion maps. Histogram analysis with normalization was performed to determine the relative mean, 75th percentile, and 90th percentile values for plasma volume and contrast transfer coefficient. A Wilcoxon rank sum test was applied to assess the relationship between baseline perfusion parameters and positive epidermal growth factor receptor variant III status. The receiver operating characteristic method was used to select the cutoffs of the dynamic contrast-enhanced MR imaging perfusion parameters. RESULTS Increased relative plasma volume and increased relative contrast transfer coefficient parameters were both significantly associated with positive epidermal growth factor receptor variant III status. For epidermal growth factor receptor variant III–positive tumors, relative plasma volume mean was 9.3 and relative contrast transfer coefficient mean was 6.5; for epidermal growth factor receptor variant III–negative tumors, relative plasma volume mean was 3.6 and relative contrast transfer coefficient mean was 3.7 (relative plasma volume mean, P < .001, and relative contrast transfer coefficient mean, P = .008). The predictive powers of relative plasma volume histogram metrics outperformed those of the

  1. A 1.6-Mb contig of yeast artificial chromosomes around the human factor VIII gene reveals three regions homologous to probes for the DXS115 locus and two for the DXYS64 locus.

    PubMed Central

    Freije, D; Schlessinger, D

    1992-01-01

    Two yeast artificial chromosome (YAC) libraries were screened for probes in Xq28, around the gene for coagulation factor VIII (F8). A set of 30 YACs were recovered and assembled into a contig spanning at least 1.6 Mb from the DXYS64 locus to the glucose 6-phosphate dehydrogenase gene (G6PD). Overlaps among the YACs were determined by several fingerprinting techniques and by additional probes generated from YAC inserts by using Alu-vector or ligation-mediated PCR. Analysis of more than 30 probes and sequence-tagged sites (STSs) made from the region revealed the presence of several homologous genomic segments. For example, a probe for the DXYS64 locus, which maps less than 500 kb 5' of F8, detects a similar but not identical locus between F8 and G6PD. Also, a probe for the DXS115 locus detects at least three identical copies in this region, one in intron 22 of F8 and at least two more, which are upstream of the 5' end of the gene. Comparisons of genomic and YAC DNA suggest that the multiple loci are not created artifactually during cloning but reflect the structure of uncloned human DNA. On the basis of these data, the most likely order for the loci analyzed is tel-DXYS61-DXYS64-(DXS115-3-DXS115-2)-5'F8-(D XS115-1)-3'F8-G6PD. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:1609806

  2. 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

  3. A first-in-human phase 1 study of ACE910, a novel factor VIII–mimetic bispecific antibody, in healthy subjects

    PubMed Central

    Uchida, Naoki; Sambe, Takehiko; Yoneyama, Koichiro; Fukazawa, Naoki; Kawanishi, Takehiko; Kobayashi, Shinichi

    2016-01-01

    ACE910 is a recombinant humanized bispecific antibody that binds to activated factor IX and factor X and mimics the cofactor function of factor VIII (FVIII). This first-in-human study examined the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of ACE910 in healthy male adults. A total of 40 Japanese and 24 white subjects were randomized to receive a single subcutaneous injection of ACE910 (Japanese: 0.001, 0.01, 0.1, 0.3, or 1 mg/kg; white: 0.1, 0.3, or 1 mg/kg; n = 6 per dose group) or placebo (n = 2 per dose group). ACE910 exhibited a linear PK profile and had a half-life of ∼4 to 5 weeks. In FVIII-neutralized plasma, ACE910 shortened activated partial thromboplastin time and increased peak height of thrombin generation in a dose-dependent manner. All adverse events were nonserious and did not lead to any subject’s withdrawal. Neither clinical findings nor laboratory abnormalities indicating hypercoagulability were observed. Two of 48 subjects receiving ACE910 (1 Japanese and 1 white) were positive for anti-ACE910 antibodies (anti-drug antibodies [ADAs]). One subject tested positive for ADAs both before and after ACE910 administration, whereas the other became ADA positive after receiving ACE910. The PK and PD profiles of ACE910 were similar in healthy Japanese and white subjects and suggest that ACE910 will be an effective and convenient prophylactic treatment of hemophilia A. This trial was registered at www.clinicaltrials.jp as #JapicCTI-121934. PMID:26626991

  4. Recombinant activated factor VII in patients at high risk of bleeding.

    PubMed

    Kubisz, Peter; Stasko, Ján

    2004-01-01

    Currently, recombinant activated factor VII (rFVIIa) (NovoSeven) is indicated for the treatment of spontaneous and surgical bleeding in congenital haemophilia A and B patients with inhibitors to factors VIII (FVIII) and IX (FIX) >5 Bethesda units (BU) worldwide, and in patients with acquired haemophilia, congenital FVII deficiency and Glanzmann's thrombasthenia in Europe. Until April 2003, almost three-quarters of a milion doses of rFVIIa have been administered proving its efficacy and excellent safety record. According to results from initial clinical trials and a large number of case reports, the rFVIIa may be effective not only in treating haemophilia patients but also in treatment of bleeding in patients on oral anticoagulation or heparin, patients with liver diseases, von Willebrand disease (vWD), thrombocytopenia, various platelet defects, congenital or acquired deficiency of FVII, and in subjects without any pre-existing coagulopathy with diffuse life-threatening bleeding triggered by surgery or trauma. This review will briefly summarize rFVIIa mode of action in haemostasis, the current clinical experience with rFVIIa and focus on the alternative use of rFVIIa in patients at the high risk of bleeding in both spontaneous cases and clinical trials reports. PMID:15763970

  5. 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 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...

  6. 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.

  7. The constitutive activity of epidermal growth factor receptor vIII leads to activation and differential trafficking of wild-type epidermal growth factor receptor and erbB2.

    PubMed

    Zeineldin, Reema; Ning, Yan; Hudson, Laurie G

    2010-06-01

    A constitutively active epidermal growth factor receptor (EGFR) mutant, EGFR variant III (EGFRvIII), has been detected at high frequencies in certain human cancers. This study evaluated transactivation and trafficking of erbB family members as a result of constitutive EGFR activity in a cancer cell line. Expression of EGFRvIII modulated erbB family members through different mechanisms; the erbB3 mRNA level was reduced, whereas wild-type EGFR (wtEGFR) and erbB2 protein levels were diminished, with no change in their mRNA levels, and there was no change in the erbB4 expression level. Both EGFR and erbB2 were internalized as a result of EGFRvIII's activity and redistributed to the cell surface upon addition of AG1478, an inhibitor of wtEGFR/EGFRvIII catalytic activity. Acute activation of EGFRvIII by removing AG1478 from cells increased phosphorylation of both wtEGFR and erbB2 and caused differential trafficking of EGFRvIII's activation partners; wtEGFR was directed primarily to lysosomal compartments and partially to recycling compartments, whereas erbB2 was directed primarily to recycling compartments and partially to lysosomal compartments. Our data demonstrate that the constitutive activity of EGFRvIII is sufficient to trigger endocytosis and trafficking of wtEGFR and erbB2, which may play a role in activating signaling pathways that are triggered during receptor endocytosis. PMID:20159766

  8. Pulmonary embolism in congenital bleeding disorders: intriguing discrepancies among different clotting factors deficiencies.

    PubMed

    Girolami, Antonio; Cosi, Elisabetta; Tasinato, Valentina; Peroni, Edoardo; Girolami, Bruno; Lombardi, Anna Maria

    2016-07-01

    Pulmonary embolism is a complication of deep vein thrombosis. It occurs in the population with a normal clotting mechanism, but it may also occur in patients with congenital bleeding conditions. Here, we report on all cases of pulmonary embolism in congenital hemorrhagic disorders. All reported cases of pulmonary embolism in congenital coagulation disorders have been gathered by a time-unlimited PubMed search. Cross-checking of the references listed at the end of the single papers was carried out to avoid omissions. Seventy-two patients had an objectively demonstrated pulmonary embolism. The event occurred in patients with fibrinogen, factor V, factor VIII (FVII), FVIII, FIX, and FXI deficiency, and in those with von Willebrand's disease. No embolism was reported in FII, factor X, and FXIII deficiency. Thirty were women and 28 were men, whereas in the remaining 14 cases, sex was not reported. Age varied from 6 to 81 years (mean age 34.3 years). The management varied from only supportive to the administration of unfractionated heparin, low-molecular-weight heparin, and anti-vitamin K medications, accompanied by adequate replacement therapy. Evolution was fair or good in the majority of cases, but there were 10 fatalities. Risk factors were present in 61 patients. The most frequent of these were replacement therapy (35 cases), surgery (34), and old age (13). Some patients had more than one risk factor. Eleven patients had no risk factors. There are discrepancies in the prevalence of pulmonary embolism among different clotting disorders. The conditions most frequently affected are FVII deficiency and fibrinogen defects. The significance of the findings is discussed. PMID:26829362

  9. 32 CFR 2003.8 - Records (Article VIII).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 32 National Defense 6 2013-07-01 2013-07-01 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...

  10. 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...

  11. 77 FR 64401 - Order of Succession for HUD Region VIII

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-10-19

    ... 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 Secretary... Succession for the Denver Regional Office and its Field Offices (Region VIII). This Order of...

  12. Possible Pasts: Historiography and Legitimation in "Henry VIII."

    ERIC Educational Resources Information Center

    Kamps, Ivo

    1996-01-01

    Aims to rehabilitate the reputation of Shakespeare's "Henry VIII" and emphasizes its potential usefulness in the classroom by reconsidering it in the context of Renaissance history writing. Shows how "Henry VIII" can be taught as a commentary on or seen as a continuation of incipient themes in "The Tempest" and "Henry V." (TB)

  13. 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

  14. Modified Thoracoscopic Hepatectomy For Segment VIII

    PubMed Central

    Li, Hongyu; Wei, Yonggang; Li, Bo; Peng, Bing

    2016-01-01

    Abstract Although previous studies have reported the use of total thoracoscopic hepatectomy (TH) for malignant liver tumors, it is technically impossible to perform intraoperative ultrasonography (IUSO) to exclude intrahepatic metastasis for the whole liver via a thoracic approach. Therefore, total TH may be inappropriate for these patients. We here report the first case of modified TH for a malignant liver tumor in China. The patient was a 26-year-old man with a 10-year HBV infection. Preoperative CT showed a 1.2 cm × 0.9 cm mass located in segment VIII. His alpha-fetal protein level was 444 ng/mL. Child–Pugh was Grade (A, 6), whereas an ICG-15 test yielded 2.7%. HCC was diagnosed preoperatively. The indications for TH were difficult tumor location, HCC, and a young patient with good resilience. The modified TH included 2 steps: (1) the patient was placed in a supine position. IUSO was used to locate the tumor in segment VIII and determine that intrahepatic metastasis had not occurred. The hepatoduodenal ligament was hanged over using a Pringle maneuver; (2) patient was then placed in the left-lateral position with single-lung ventilation. Three trocars were placed into the right thoracic cavity. The intra-thoracic space was observed using a regular 10-mm rigid scope. The diaphragm was transected and retracted. IUSO was used again to confirm the tumor location. Under the Pringle maneuver (once every 10 min), the superficial portion of the liver was transected by ultrasonic shears, whereas the deeper tissue was transected by LigaSure. Bipolar was used for hemostasis. The specimen was put into a retrieval bag and removed from the abdominal trocar. The diaphragm was repaired by running suturing. The operation time was 260 minutes and estimated blood loss was 30 mL. The patient was discharged on postoperative day 5 with normal liver function. No complications arose. Total TH may be inappropriate for malignant liver tumors due to the limitation of IUSO

  15. 40 CFR Appendix Viii to Part 268 - LDR Effective Dates of Injected Prohibited Hazardous Wastes

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 27 2014-07-01 2014-07-01 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 VIII to Part 268—LDR Effective Dates...

  16. 40 CFR Appendix Viii to Part 268 - LDR Effective Dates of Injected Prohibited Hazardous Wastes

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 26 2010-07-01 2010-07-01 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 VIII to Part 268—LDR Effective Dates...

  17. 19 CFR Annex Viii-B to Part 351 - Schedule for Expedited Sunset Reviews

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 19 Customs Duties 3 2011-04-01 2011-04-01 false Schedule for Expedited Sunset Reviews VIII Annex VIII-B to Part 351 Customs Duties INTERNATIONAL TRADE ADMINISTRATION, DEPARTMENT OF COMMERCE ANTIDUMPING AND COUNTERVAILING DUTIES Pt. 351, Annex VIII-B Annex VIII-B to Part 351—Schedule for...

  18. 19 CFR Annex Viii-C to Part 351 - Schedule for Full Sunset Reviews

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 19 Customs Duties 3 2011-04-01 2011-04-01 false Schedule for Full Sunset Reviews VIII Annex VIII-C to Part 351 Customs Duties INTERNATIONAL TRADE ADMINISTRATION, DEPARTMENT OF COMMERCE ANTIDUMPING AND COUNTERVAILING DUTIES Pt. 351, Annex VIII-C Annex VIII-C to Part 351—Schedule for Full Sunset Reviews Day 1...

  19. 19 CFR Annex Viii-A to Part 351 - Schedule for 90-Day Sunset Reviews

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 19 Customs Duties 3 2011-04-01 2011-04-01 false Schedule for 90-Day Sunset Reviews VIII Annex VIII-A to Part 351 Customs Duties INTERNATIONAL TRADE ADMINISTRATION, DEPARTMENT OF COMMERCE ANTIDUMPING AND COUNTERVAILING DUTIES Pt. 351, Annex VIII-A Annex VIII-A to Part 351—Schedule for 90-Day...

  20. 19 CFR Annex Viii-B to Part 351 - Schedule for Expedited Sunset Reviews

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 19 Customs Duties 3 2010-04-01 2010-04-01 false Schedule for Expedited Sunset Reviews VIII Annex VIII-B to Part 351 Customs Duties INTERNATIONAL TRADE ADMINISTRATION, DEPARTMENT OF COMMERCE ANTIDUMPING AND COUNTERVAILING DUTIES Pt. 351, Annex VIII-B Annex VIII-B to Part 351—Schedule for...

  1. 19 CFR Annex Viii-A to Part 351 - Schedule for 90-Day Sunset Reviews

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 19 Customs Duties 3 2010-04-01 2010-04-01 false Schedule for 90-Day Sunset Reviews VIII Annex VIII-A to Part 351 Customs Duties INTERNATIONAL TRADE ADMINISTRATION, DEPARTMENT OF COMMERCE ANTIDUMPING AND COUNTERVAILING DUTIES Pt. 351, Annex VIII-A Annex VIII-A to Part 351—Schedule for 90-Day...

  2. 19 CFR Annex Viii-C to Part 351 - Schedule for Full Sunset Reviews

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 19 Customs Duties 3 2010-04-01 2010-04-01 false Schedule for Full Sunset Reviews VIII Annex VIII-C to Part 351 Customs Duties INTERNATIONAL TRADE ADMINISTRATION, DEPARTMENT OF COMMERCE ANTIDUMPING AND COUNTERVAILING DUTIES Pt. 351, Annex VIII-C Annex VIII-C to Part 351—Schedule for Full Sunset Reviews Day 1...

  3. Ab initio studies on the lattice thermal conductivity of silicon clathrate frameworks II and VIII

    NASA Astrophysics Data System (ADS)

    Härkönen, Ville J.; Karttunen, Antti J.

    2016-01-01

    The lattice thermal conductivities of silicon clathrate frameworks II and VIII are investigated by using ab initio lattice dynamics and an iterative solution of the linearized Boltzmann transport equation (BTE) for phonons. Within the temperature range 100-350 K, the clathrate structures II and VIII were found to have lower lattice thermal conductivity values than the silicon diamond structure (d -Si) by factors of 1/2 and 1/3, respectively. The main reason for the lower lattice thermal conductivity of the clathrate structure II in comparison to d -Si was found to be the harmonic phonon spectra, while in the case of the clathrate structure VIII, the difference is mainly due to the harmonic phonon spectra and partly due to the shorter relaxation times of phonons. In the studied clathrate frameworks, the anharmonic effects have larger impact on the lattice thermal conductivity than the size of the unit cell. For the structure II, the predicted lattice thermal conductivity differs approximately by a factor of 20 from the previous experimental results obtained for a polycrystalline sample at room temperature.

  4. 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.

  5. DR-07TARGETING EGFR-VIII WITH DICHLOROACETATE IN TEMOZOLOMIDE RESISTANT GLIOBLASTOMA MODELS

    PubMed Central

    Velpula, Kiran Kumar; Asuthkar, Swapna; Lathia, Justin D.; Tsung, Andrew J.

    2014-01-01

    Epidermal growth factor receptor (EGFR) gene amplification and its subsequent over-expression is the most frequent genetic alteration associated with glioblastoma (GBM) approximating 40%. EGFR amplification is accompanied by a gene rearrangement that overexpresses the receptor variant III (EGFRvIII) characterized by an in-frame deletion of 801 bp of coding sequence from exons 2-7. Our in vitro preliminary studies conducted using U373 cell lines stably expressing EGFRvIII (U373vIII) conferred increased proliferation and invasiveness when compared to control. Temozolomide (TMZ) resistance in this context is a well-known concept, where high initial response is met with eventual failure and tumor propagation. We thus generated an aggressive TMZ resistance model for U373vIII cells (U373vIII-TMZ-R) by continual exposure to 150uM TMZ for 6 months. After the initial phase of cell death in the majority and prolonged lag in replication, the eventual surviving U373vIII-TMZ-R colonies showed identical morphology and proliferation potential of the parent U373vIII. This resulted in a cell line mimicking the actual growth potential of recurrent GBM. We have previously shown that dichloroacetic acid (DCA) reduces EGFR expression in GBMs. Since EGFRvIII expression displays an aggressive phenotype yet remains tumor specific, we hypothesize studying DCA alone or in combination with dasatinib, an inhibitor of EGFR subcellular translocation, in U373vIII and U373vIII-TMZ-R cell lines. By more accurately depicting the status of GBM at the time of recurrence after standard of care chemotherapy treatment, the investigation of DCA will augment and possibly redefine our current understanding of resistance mechanisms and establish novel methods of circumventing this pervasive phenomena.

  6. Tumbling and spaceflight: the Gemini VIII experience.

    PubMed

    Mohler, S R; Nicogossian, A E; McCormack, P D; Mohler, S R

    1990-01-01

    A malfunctioning orbital flight attitude thruster during the flight of Gemini VIII led to acceleration forces on astronauts Neil Armstrong (commander) and David Scott (pilot) that created the potential for derogation of oculo-vestibular and eye-hand coordination effects. The spacecraft attained an axial tumbling rotation of 50 rpm and would have exceeded this had not the commander accurately diagnosed the problem and taken immediate corrective action. By the time counter-measure controls were applied, both astronauts were experiencing vertigo and the physiological effects of the tumbling acceleration. Data from the recorders reveal that one astronaut experienced -Gy of 0.92 G-units, and the other +Gy of 0.92 for approximately 46 s. Both received a -Gz of 0.89 G-units from the waist up with a +Gz of 0.05 from the waist down. A substantial increase of time and/or an increase in rpm would ultimately have produced incapacitation of both astronauts. NASA corrected the Gemini thruster problem by changing the ignition system wiring. Future space-craft undertaking long-term missions could be equipped with unambiguous thruster fault displays and could have computer-controlled automatic cutoffs to control excessive thruster burns. PMID:2302130

  7. 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

  8. Congenital Type III von Willebrand's disease unmasked by hypothyroidism in a Shetland sheepdog.

    PubMed

    Scuderi, Margaret; Bessey, Lauren; Snead, Elisabeth; Burgess, Hilary; Carr, Anthony

    2015-09-01

    A 7-year-old, spayed female Shetland sheepdog had sudden onset of right-sided epistaxis. Diagnostic tests revealed Type III von Willebrand's disease and primary hypothyroidism leading to an acute hypothyroid crisis and acquired factor VIII (FVIII) deficiency. Levothyroxine therapy normalized the serum thyroxine and FVIII concentrations. The delayed onset of disease and the reversible FVIII deficiency that was corrected with levothyroxine therapy, support a role for hypothyroidism in the pathogenesis of this dog's sudden bleeding tendency as has been seen with hypothyroidism in humans. PMID:26347307

  9. 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

  10. Congenital Type III von Willebrand’s disease unmasked by hypothyroidism in a Shetland sheepdog

    PubMed Central

    Scuderi, Margaret; Bessey, Lauren; Snead, Elisabeth; Burgess, Hilary; Carr, Anthony

    2015-01-01

    A 7-year-old, spayed female Shetland sheepdog had sudden onset of right-sided epistaxis. Diagnostic tests revealed Type III von Willebrand’s disease and primary hypothyroidism leading to an acute hypothyroid crisis and acquired factor VIII (FVIII) deficiency. Levothyroxine therapy normalized the serum thyroxine and FVIII concentrations. The delayed onset of disease and the reversible FVIII deficiency that was corrected with levothyroxine therapy, support a role for hypothyroidism in the pathogenesis of this dog’s sudden bleeding tendency as has been seen with hypothyroidism in humans. PMID:26347307

  11. 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

  12. von Willebrand Factor Test

    MedlinePlus

    ... Platelet Count , Platelet Function Tests , Complete Blood Count , Coagulation Factor VIII , PT , PTT At a Glance Test ... a protein , one of several components of the coagulation system that work together to stop bleeding and ...

  13. 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 to Part 85—Vehicle and Engine Parameters and Specifications a. light duty vehicle parameters...

  14. 40 CFR Appendix Viii to Part 266 - Organic Compounds for Which Residues Must Be Analyzed

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... SPECIFIC TYPES OF HAZARDOUS WASTE MANAGEMENT FACILITIES Pt. 266, App. VIII Appendix VIII to Part 266... residues collected from areas downstream of the combustion chamber (e.g., ductwork, boiler tubes,...

  15. Transmed Transect VIII: from Laurussia to Gondwana

    NASA Astrophysics Data System (ADS)

    Stephenson, R. A.; Mart, Y.; Okay, A.; Robertson, A.; Stovba, S.; Khriachtchevskaia, O.

    2003-04-01

    TransMed transect VIII crosses the easternmost Mediterranean Sea. Some 2600 km long, it runs from Laurussia in the north (Ukrainian Shield) to Gondwana in the south (Arabian Shield). The Late Palaeozoic aged intracratonic Dniepr-Donets rift basin lies near the northern end of the transect and the modern intracratonic Red Sea rift lies at the southern end. Intervening tectonic units, north to south, include the Scythian Platform and Crimean Orogen in Ukraine; the Black Sea; the Pontides, Kirsehir Massif, and Taurides in Turkey; Cyprus with the Troodos complex; the Levant Basin and distal parts of the Nile Delta in the eastern Mediterranean; and the Sinai in Egypt. Deep structure, including crustal thickness and affinity, is poorly known along much of the transect. Although all available crustal scale seismic data have been considered, much of the deeper parts of the transect are inferred from the exposed geology and how this can be modelled in terms of regional paleotectonics and are, accordingly, speculative. Palaeozoic and younger sediments of the Scythian Platform overlie basement that is surmised to be younger than the adjoining Archean Ukrainian Shield. The whole of the crust of the Crimean Orogen, underlying thrust nappes emplaced during the early Mesozoic, is speculated to comprise an accretionary complex of mainly Triassic age that is correlated with the Sakarya Zone of northern Turkey. The Black Sea is a back-arc rift basin that opened in Late Cretacous-Tertiary times but the margins of the Black Sea intersected by the transect are not conjugate. The Pontide margin of the Black Sea, in northern Turkey, is formed by the Istanbul Zone, with Proterozoic-aged basement correlative with Moesian crust found in southern Romania and Bulgaria. The Kirsehir Massif is made up of Cretaceous metamorphic and granitic rocks, and represents the deeper parts of a magmatic arc. The Taurides form a south-vergent thrust-stack involving Palaeozoic to Eocene sediments. The

  16. 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 Appendix VIII to Part 600 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) ENERGY POLICY FUEL ECONOMY AND CARBON-RELATED EXHAUST EMISSIONS OF MOTOR VEHICLES Pt. 600, App. VIII...

  17. 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...

  18. 40 CFR Appendixes Vii-Viii to Part... - [Reserved

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 25 2010-07-01 2010-07-01 false VII Appendixes VII-VIII to Part 264 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) SOLID WASTES (CONTINUED) STANDARDS FOR OWNERS AND OPERATORS OF HAZARDOUS WASTE TREATMENT, STORAGE, AND DISPOSAL FACILITIES Appendixes...

  19. Academics 2000: Cycle VIII Evaluation Report, 2001-2002.

    ERIC Educational Resources Information Center

    Huskey, Barton

    This report presents evaluation findings regarding the implementation and effectiveness of the Academics 2000, Cycle VIII grant in the Austin Independent School District (AISD), Texas. The purpose of Academics 2000 was to raise the level of academic achievement of all Texas students by ensuring that each child achieves early mastery of the…

  20. 10. Historic American Buildings Survey Photocopy of Plate VIII (dated ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    10. Historic American Buildings Survey Photocopy of Plate VIII (dated 1889) in John Calvin Stevens and Albert Winslow Cobb, Examples of American Domestic Architecture, New York, William T. Comstock, 1889 PLANS AND INTERIOR DETAILS, 1889 - John Calvin Stevens House, 52 Bowdoin Street, Portland, Cumberland County, ME

  1. Henry VIII, McLeod syndrome and Jacquetta's curse.

    PubMed

    Stride, P; Lopes Floro, K

    2013-01-01

    The mental decline of King Henry VIII from being a jovial, charismatic and athletic young man into an increasingly paranoid, brutal tyrant in later life, ever more concerned at his lack of one or more male heirs, has attracted many medical diagnostic theories. Previous hypotheses have included diabetes, syphilis and hypothyroidism, among others. However, these inadequately explain Henry's failure to produce a male heir, despite multiple pairings. The latest postulated diagnoses for Henry are the coexistence of both Kell blood group antigenicity (possibly inherited from Jacquetta Woodville, Henry's maternal great grandmother) causing related impaired fertility, and McLeod syndrome, causing psychotic changes. As the mutated McLeod protein of the syndrome significantly reduces the expression, effectively inactivating the Kell antigen, we critically review this theory, examining in detail the pathophysiology of these conditions and assessing the genealogy of Henry VIII and its effect in subsequent generations. PMID:24350322

  2. Zwitterionic Group VIII transition metal initiators supported by olefin ligands

    SciTech Connect

    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.

  3. Experimental Excitation Rate Coefficients for Ne VIII Ions

    NASA Astrophysics Data System (ADS)

    Chang, C. C.; Greve, P.; Kolk, K.-H.; Kunze, H.-J.

    1984-02-01

    From the line emission of a pure neon plasma produced in a theta pinch discharge rate coefficients for the excitation of the n = 3 and 4 levels in Ne VIII ions are derived and compared with theoretical calculations and previous measurements. The general agreement between theory and all measurements is rather satisfactory for the excitation to the n = 3 levels, the measured rate coefficients to the 4p and 4d levels, however, being consistently too low.

  4. 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.

  5. Spanish Consensus Guidelines on prophylaxis with bypassing agents in patients with haemophilia and inhibitors.

    PubMed

    López-Fernández, Maria Fernanda; Altisent Roca, Carmen; Álvarez-Román, Maria Teresa; Canaro Hirnyk, Mariana Isabel; Mingot-Castellano, Maria Eva; Jiménez-Yuste, Víctor; Cid Haro, Ana Rosa; Pérez-Garrido, Rosario; Sedano Balbas, Carmen

    2016-05-01

    Prophylaxis with the blood clotting factor, factor VIII (FVIII) is ineffective for individuals with haemophilia A and high-titre inhibitors to FVIII. Prophylaxis with the FVIII bypassing agents activated prothrombin complex concentrates (aPCC; FEIBA® Baxalta) or recombinant activated factor VII (rFVIIa; Novo-Seven®, Novo Nordisk) may be an effective alternative. It was our aim to develop evidence -and expert opinion- based guidelines for prophylactic therapy for patients with high-titre inhibitors to FVIII. A panel of nine Spanish haematologists undertook a systematic review of the literature to develop consensus-based guidance. Particular consideration was given to prophylaxis in patients prior to undergoing immune tolerance induction (ITI) (a process of continued exposure to FVIII that can restore sensitivity for some patients), during the ITI period and for those not undergoing ITI or for whom ITI had failed. These guidelines offer guidance for clinicians in deciding which patients might benefit from prophylaxis with FVIII bypassing agents, the most appropriate agents in various clinical settings related to ITI, doses and dosing regimens and how best to monitor the efficacy of prophylaxis. The paper includes recommendations on when to interrupt or stop prophylaxis and special safety concerns during prophylaxis. These consensus guidelines offer the most comprehensive evaluation of the clinical evidence base to date and should be of considerable benefit to clinicians facing the challenge of managing patients with severe haemophilia A with high-titre FVIII inhibitors. PMID:26842562

  6. Crystal structure of EstSRT1, a family VIII carboxylesterase displaying hydrolytic activity toward oxyimino cephalosporins.

    PubMed

    Cha, Sun-Shin; An, Young Jun

    2016-09-16

    EstSRT1 is a family VIII carboxylesterase that hydrolyzes oxyimino third- and fourth-generation cephalosporins, first-generation cephalosporins and ester substrates. According to the crystal structure of EstSRT1 (2.0-Å resolution), this protein contains a large α/β domain and a small α-helical domain and harbors three catalytic residues (Ser71, Lys74, and Tyr160) in the cavity at the domain interface, similarly to other family VIII carboxylesterases. Comparison of the structures of EstSRT1 and EstU1, a family VIII carboxylesterase with no hydrolytic activity toward bulky oxyimino cephalosporins, revealed that EstSRT1 has a smaller active site, despite its extended substrate range. The B-factors of the active site segments that could potentially contact with the oxyimino groups and the R2 side chains of oxyimino cephalosporins are higher in EstSRT1 than in EstU1, thus suggesting the role of the active site's structural flexibility in the extension of EstSRT1's substrate spectrum. PMID:27501751

  7. Cost–consequence analysis of long-term prophylaxis in the treatment of von Willebrand disease in the Italian context

    PubMed Central

    Schinco, Piercarla; Cultrera, Dorina; Valeri, Federica; Borchiellini, Alessandra; Mantuano, Michela; Gorla, Francesca; Savarese, Alessia; Teruzzi, Cristina

    2015-01-01

    Purpose Prophylaxis with von Willebrand factor (VWF)/factor VIII (FVIII) concentrates is a potential approach for patients with severe von Willebrand disease (VWD). As far as we are aware, to date there have been no pharmacoeconomic analyses in order to assess the economic impact of treatments for severe VWD. The analysis presented here estimates the cost–benefit ratio of VWF with a low FVIII content when compared with VWF/FVIII concentrates currently used in Italy for long-term prophylaxis in patients with severe VWD. Methods A cost–consequence analysis was undertaken to assess the economic impact of the treatment of severe VWD from the perspective both of the Italian National Health Service and society. The analysis was based on four case reports of long-term prophylaxis with VWD with VWF/FVIII concentrates and VWF with a low FVIII content. The costs per patient included direct and indirect costs for each treatment. Results Considering the four case reports, health care costs (without cost of treatment) and indirect costs per patient per year were lower with VWF with a low FVIII content than VWF/FVIII concentrates. The total health care costs (without cost of treatment) and indirect costs avoided with VWF with a low FVIII content per patient per year ranged from €2,295 to €17,530 and from €1,867 to €4,978, respectively. Conclusion VWF with a low FVIII content seems to be a cost-effective treatment option for patients with severe VWD. Although the drug cost per se is higher, the use of VWF with a low FVIII content is associated with decreased consumption of hospital resources and fewer lost working days due to bleedings and consequently with an improvement of the quality of life of the patients. PMID:25565871

  8. Circulating immune complexes containing anti-VIII antibodies in multi-transfused patients with haemophilia A.

    PubMed

    Kazatchkine, M D; Sultan, Y; Burton-Kee, E J; Mowbray, J F

    1980-02-01

    Evidence for the presence of circulating immune complexes was found in thirty-four out of fifty-five samples from forty-seven patients with haemophilia A. In eleven patients the complexes, precipitated from the blood with polyethylene glycol, were digested with pepsin. The F(ab')2 antibody was tested, and found to have neutralizing activity against coagulant Factor VIII in two patients. In one of these no free antibody had ever been found in the plasma, while in the other the antibody was concentrated tenfold in the complex. In two other samples free without complexed antibody was found. In comparison, IgG-containing complexes were found in nine out of nineteen patients with von Willebrand's disease and no complexes were found in the sera from twelve multi-transfused thalassaemics. PEG precipitation is a useful technique for the preparation of concentrated immune complexes for further study such as antigen identification. PMID:6771074

  9. AAV-based Neonatal Gene Therapy for Hemophilia A: Long-Term Correction and Avoidance of Immune Responses in Mice

    PubMed Central

    Hu, Chuhong; Lipshutz, Gerald S.

    2012-01-01

    Hemophilia A gene therapy has been hampered by immune responses to vector-associated antigens and by neutralizing antibodies or inhibitors to the factor VIII (FVIII) protein; these ‘inhibitors’ more commonly effect 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. Serotype rh10 AAV was developed splitting the FVIII coding sequence into heavy and light chains with the chicken β-actin promoter/CMV enhancer for dual recombinant AAV vector delivery. Coinjection of virions of each FVIII chain intravenously to mice on the second day of life was performed. Mice express sustained FVIII antigen levels of ≥5% to 22 months of life without the development of antibodies to 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 to FVIII in this disease model where AAV is administered shortly after birth. These studies support consideration of gene replacement therapy for diseases that are diagnosed in utero or in the early neonatal period. PMID:22241178

  10. THE KINETICS OF PENETRATION : VIII. TEMPORARY ACCUMULATION.

    PubMed

    Osterhout, W J; Kamerling, S E

    1934-03-20

    solution it will tend to enter and displace the weaker acid HA (if this be carbonic it can readily escape): hence potassium may accumulate to a greater or less extent as KCl. Injury of the cell may produce a twofold effect, (1) increase of permeability, (2) lessened accumulation. The total amount of electrolyte taken up in a given time will be influenced by these factors and may be greater than normal in the injured cell or less, depending somewhat on the length of the interval of time chosen. PMID:19872797

  11. Different subcellular localizations and functions of Arabidopsis myosin VIII

    PubMed Central

    Golomb, Lior; Abu-Abied, Mohamad; Belausov, Eduard; Sadot, Einat

    2008-01-01

    Background Myosins are actin-activated ATPases that use energy to generate force and move along actin filaments, dragging with their tails different cargos. Plant myosins belong to the group of unconventional myosins and Arabidopsis myosin VIII gene family contains four members: ATM1, ATM2, myosin VIIIA and myosin VIIIB. Results In transgenic plants expressing GFP fusions with ATM1 (IQ-tail truncation, lacking the head domain), fluorescence was differentially distributed: while in epidermis cells at the root cap GFP-ATM1 equally distributed all over the cell, in epidermal cells right above this region it accumulated in dots. Further up, in cells of the elongation zone, GFP-ATM1 was preferentially positioned at the sides of transversal cell walls. Interestingly, the punctate pattern was insensitive to brefeldin A (BFA) while in some cells closer to the root cap, ATM1 was found in BFA bodies. With the use of different markers and transient expression in Nicotiana benthamiana leaves, it was found that myosin VIII co-localized to the plasmodesmata and ER, colocalized with internalized FM4-64, and partially overlapped with the endosomal markers ARA6, and rarely with ARA7 and FYVE. Motility of ARA6 labeled organelles was inhibited whenever associated with truncated ATM1 but motility of FYVE labeled organelles was inhibited only when associated with large excess of ATM1. Furthermore, GFP-ATM1 and RFP-ATM2 (IQ-tail domain) co-localized to the same spots on the plasma membrane, indicating a specific composition at these sites for myosin binding. Conclusion Taken together, our data suggest that myosin VIII functions differently in different root cells and can be involved in different steps of endocytosis, BFA-sensitive and insensitive pathways, ER tethering and plasmodesmatal activity. PMID:18179725

  12. Efmoroctocog Alfa: A Review in Haemophilia A.

    PubMed

    Frampton, James E

    2016-09-01

    Efmoroctocog alfa (Elocta(®), Eloctate(®), Eloctate™), a first-in-class recombinant factor VIII-Fc fusion protein (rFVIIIFc), has an extended half-life compared with conventional factor VIII (FVIII) preparations, including recombinant FVIII (rFVIII) products. It is approved for the treatment and prophylaxis of bleeding in patients with haemophilia A in multiple countries worldwide. Data accumulated from pivotal phase III studies (A-LONG in adults and adolescents aged ≥12 years; Kids A-LONG in children aged <12 years) and their ongoing extension study (ASPIRE) have demonstrated the long-term effectiveness of efmoroctocog alfa for the treatment of acute bleeding episodes, perioperative management and routine prophylaxis in previously treated males with severe haemophilia A. Among patients on individualized efmoroctocog alfa prophylaxis who had previously received FVIII prophylaxis, all but one of those aged ≥12 years and three-quarters of those aged <12 years reduced their injection frequency compared with their pre-study regimen. FVIII replacement therapy with efmoroctocog alfa was generally well tolerated in previously treated patients, with no evidence of increased immunogenicity. The safety and efficacy of FVIII replacement therapy with efmoroctocog alfa in previously untreated males aged <6 years with severe haemophilia A are currently being evaluated. Although there are no direct, head-to-head studies, the available clinical trial evidence indicates that efmoroctocog alfa provides an effective alternative to conventional FVIII preparations (including rFVIIIs) for the management of haemophilia A. Moreover, by reducing the frequency of injections required, it has the potential to reduce treatment burden, and hence improve adherence to prophylaxis. PMID:27487799

  13. 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…

  14. 40 CFR Appendix Viii to Part 86 - Aging Bench Equipment and Procedures

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 19 2014-07-01 2014-07-01 false Aging Bench Equipment and Procedures.... VIII Appendix VIII to Part 86—Aging Bench Equipment and Procedures This appendix provides specifications for standard aging bench equipment and aging procedures which may be used to conduct bench...

  15. 40 CFR Appendix Viii to Part 86 - Aging Bench Equipment and Procedures

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 19 2011-07-01 2011-07-01 false Aging Bench Equipment and Procedures.... 86, App. VIII Appendix VIII to Part 86—Aging Bench Equipment and Procedures This appendix provides specifications for standard aging bench equipment and aging procedures which may be used to conduct bench...

  16. 40 CFR Appendix Viii to Part 86 - Aging Bench Equipment and Procedures

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 20 2012-07-01 2012-07-01 false Aging Bench Equipment and Procedures.... 86, App. VIII Appendix VIII to Part 86—Aging Bench Equipment and Procedures This appendix provides specifications for standard aging bench equipment and aging procedures which may be used to conduct bench...

  17. 40 CFR Appendix Viii to Part 86 - Aging Bench Equipment and Procedures

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 19 2010-07-01 2010-07-01 false Aging Bench Equipment and Procedures.... 86, App. VIII Appendix VIII to Part 86—Aging Bench Equipment and Procedures This appendix provides specifications for standard aging bench equipment and aging procedures which may be used to conduct bench...

  18. 40 CFR Appendix Viii to Part 86 - Aging Bench Equipment and Procedures

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 20 2013-07-01 2013-07-01 false Aging Bench Equipment and Procedures.... 86, App. VIII Appendix VIII to Part 86—Aging Bench Equipment and Procedures This appendix provides specifications for standard aging bench equipment and aging procedures which may be used to conduct bench...

  19. 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 AGENCY (CONTINUED) SOLID WASTES (CONTINUED) STANDARDS FOR THE MANAGEMENT OF SPECIFIC HAZARDOUS WASTES AND SPECIFIC TYPES OF HAZARDOUS WASTE...

  20. Paducah-Louisville Consortium; Project VIII: Focus on Dropouts... a New Design. Final Evaluation Report.

    ERIC Educational Resources Information Center

    Paducah Public Schools, KY.

    Project VIII is an innovative behavioral science oriented educational program, funded under Title VIII Elementary Secondary Education Act, designed to attack the personal, social, and educational problems of children whose previous record of school failure and frustration has indicated high dropout potential. The program is operated as a…

  1. 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....

  2. 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....

  3. 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....

  4. 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....

  5. 40 CFR Appendix Viii to Part 266 - Organic Compounds for Which Residues Must Be Analyzed

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 28 2013-07-01 2013-07-01 false Organic Compounds for Which Residues Must Be Analyzed VIII Appendix VIII to Part 266 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) SOLID WASTES (CONTINUED) STANDARDS FOR THE MANAGEMENT OF SPECIFIC HAZARDOUS WASTES AND SPECIFIC TYPES OF HAZARDOUS WASTE...

  6. Atomic data for astrophysics: improved collision strengths for Fe viii

    NASA Astrophysics Data System (ADS)

    Del Zanna, G.; Badnell, N. R.

    2014-10-01

    We describe, and present the results of, a new large-scale R-matrix scattering calculation for the electron collisional excitation of Fe viii. We first discuss the limitations of the previous calculations, in particular concerning some strong EUV lines observed in the solar corona by the Hinode EUV Imaging Spectrometer. We then present a new target which represents an improvement over the previous ones for this particularly complex ion. We developed a new method, based on the use of term energy corrections within the intermediate coupling frame transformation method, to calculate the collision strengths. We compare predicted and observed line intensities using laboratory and solar spectra, finding excellent agreement for all the main soft X-ray and extreme ultraviolet (EUV) transitions, using the present atomic data. In particular, we show that Fe viii EUV lines observed by Hinode EIS can now be used to provide reliable electron temperatures for the solar corona. The full dataset (energies, transition probabilities and rates) is only available in electronic form at our APAP website (http://www.apap-network.org) as well as at the CDS via anonymous ftp to http://cdsarc.u-strasbg.fr (ftp://130.79.128.5) or via http://cdsarc.u-strasbg.fr/viz-bin/qcat?J/A+A/570/A56

  7. Modified Thoracoscopic Hepatectomy For Segment VIII: A Case Report.

    PubMed

    Li, Hongyu; Wei, Yonggang; Li, Bo; Peng, Bing

    2016-05-01

    Although previous studies have reported the use of total thoracoscopic hepatectomy (TH) for malignant liver tumors, it is technically impossible to perform intraoperative ultrasonography (IUSO) to exclude intrahepatic metastasis for the whole liver via a thoracic approach. Therefore, total TH may be inappropriate for these patients. We here report the first case of modified TH for a malignant liver tumor in China.The patient was a 26-year-old man with a 10-year HBV infection. Preoperative CT showed a 1.2 cm × 0.9 cm mass located in segment VIII. His alpha-fetal protein level was 444 ng/mL. Child-Pugh was Grade (A, 6), whereas an ICG-15 test yielded 2.7%. HCC was diagnosed preoperatively. The indications for TH were difficult tumor location, HCC, and a young patient with good resilience.The modified TH included 2 steps: (1) the patient was placed in a supine position. IUSO was used to locate the tumor in segment VIII and determine that intrahepatic metastasis had not occurred. The hepatoduodenal ligament was hanged over using a Pringle maneuver; (2) patient was then placed in the left-lateral position with single-lung ventilation. Three trocars were placed into the right thoracic cavity. The intra-thoracic space was observed using a regular 10-mm rigid scope. The diaphragm was transected and retracted. IUSO was used again to confirm the tumor location. Under the Pringle maneuver (once every 10 min), the superficial portion of the liver was transected by ultrasonic shears, whereas the deeper tissue was transected by LigaSure. Bipolar was used for hemostasis. The specimen was put into a retrieval bag and removed from the abdominal trocar. The diaphragm was repaired by running suturing. The operation time was 260 minutes and estimated blood loss was 30 mL. The patient was discharged on postoperative day 5 with normal liver function. No complications arose.Total TH may be inappropriate for malignant liver tumors due to the limitation of IUSO for the whole

  8. Life-threatening hemorrhage from acquired hemophilia A as a presenting manifestation of prostate cancer.

    PubMed

    Sheth, Chirag; Gill, Amandeep; Sekhon, Sumeet

    2016-01-01

    Acquired factor VIII deficiency (acquired hemophilia A) is a rare condition characterized by the acquisition of autoantibodies that affect the clotting activity of factor VIII (fVIII). The most common manifestation in affected patients is a hemorrhagic diathesis. This disorder is associated with autoimmune diseases, pregnancy, postpartum period, drugs, and malignancy. Management of this condition begins with attempts to arrest an acute bleed based on the site and severity of bleeding and inhibitor titer. The next priority is eradication of the fVIII antibodies using immunosuppressive therapies. We report the case of a 66-year-old male who presented with spontaneous right thigh hematoma with prolonged activated partial prothrombin time and normal prothrombin time. Mixing studies confirmed the presence of an inhibitor. Further investigation for the underlying etiology of acquired hemophilia A leads to diagnosis of prostate cancer. Treatment consisted of bypassing agents including activated factor VII and activated prothrombin plasma concentrate to arrest the bleeding. Steroids and cyclophosphamide were added to suppress the fVIII inhibitors. Concomitant treatment of locally advanced prostate cancer with chemotherapy confirmed the eradication of the inhibitors. To our knowledge, this is the first reported case of prostate cancer diagnosed and treated simultaneously with acquired hemophilia A resulting in favorable patient outcome. PMID:27609734

  9. Safety and efficacy of turoctocog alfa (NovoEight®) during surgery in patients with haemophilia A: results from the multinational guardian™ clinical trials

    PubMed Central

    Santagostino, E; Lentz, S R; Misgav, M; Brand, B; Chowdary, P; Savic, A; Kilinc, Y; Amit, Y; Amendola, A; Solimeno, L P; Saugstrup, T; Matytsina, I

    2015-01-01

    Recombinant factor VIII (rFVIII) products provide a safe and efficacious replacement therapy for prevention and treatment of bleeding episodes in patients with haemophilia A. The present investigations from the multinational, open-label guardian™ clinical trials assessed the haemostatic response of turoctocog alfa (NovoEight®), a rFVIII product, in patients with severe haemophilia A (FVIII ≤ 1%) undergoing surgery. All patients had a minimum of 50 exposure days to any FVIII product prior to surgery and no history of inhibitors. A total of 41 procedures (13 orthopaedic, 19 dental and 9 general) were performed in 33 patients aged 4–59 years. Of the 41 procedures, 15 were major surgeries in 13 patients and 26 were minor surgeries in 21 patients. The success rate for haemostatic response was 100% (success was defined as ‘excellent’ or ‘good’ haemostatic outcome). Turoctocog alfa consumption on the day of surgery ranged from 27 to 153 IU kg−1. The mean daily dose declined over time, while retaining adequate FVIII coverage as measured by trough levels. Overall, no safety issues were identified. No thrombotic events were observed and none of the patients developed FVIII inhibitors. In conclusion, the present results show that turoctocog alfa was effective in controlling blood loss by obtaining a sufficient haemostatic response in patients with severe haemophilia A undergoing surgery. PMID:25273984

  10. [Hypertension in rural communities. Study in the VIII Region, Chile].

    PubMed

    Fasce, E; Pérez, H; Boggiano, G; Ibáñez, P; Nieto, C

    1993-09-01

    The aim of this work was to study the prevalence of hypertension in rural populations of the VIII region of Chile. Six thousand twenty four non institutionalized subjects. 14 years old or older, coming from eight randomly chosen rural communities were studied. Blood pressure was measured by trained personnel in two occasions within 30 minutes; in those classified as hypertensive, a third measurement was performed within 24 hours, eliminating alcohol intake and medications. Results show prevalence rates in the first, second and third measurement of 38.5, 30.8 and 22.8% respectively. The elimination of alcohol intake and medications decreased the prevalence in 3.9%. All the chosen communities has similar rates (19.8 to 21.2%) except Arauco, that had a significantly higher frequency of 31.9%. Zones of extreme ruralness had higher rates than small towns (23.8 vs 21.7%). There was a secular increase in the prevalence of hypertension from 4.9 in the 15 to 24 years old group to 57.8% among people 75 years old or older. Forty three percent of hypertensives were aware of such condition, 26.1% were receiving treatment and in 8.2% it was successful; all these figures were slightly better among women. PMID:8191159

  11. Alternative therapies for the management of inhibitors.

    PubMed

    Shima, M; Lillicrap, D; Kruse-Jarres, R

    2016-07-01

    The development of inhibitors to factor VIII (FVIII) or factor IX (FIX) remains a major treatment complication encountered in the treatment of haemophilia. Not all patients with even the same severity and genotype develop inhibitors suggesting an underlying mechanism of tolerance against FVIII- or FIX-related immunity. One mechanism may be central tolerance observed in patients in whom the FVIII mutation enables some production of the protein. The other is a peripheral tolerance mechanism which may be evident in patients with null mutation. Recently, recombinant porcine FVIII (rpFVIII, Obixur, OBI-1, BAX801) has been developed for the haemostatic treatment of both congenital haemophilia with inhibitor (CHAWI) and acquired haemophilia A (AHA). In 28 subjects with AHA with life-/limb-threatening bleeding, rpFVIII reduced or stopped bleeding in all patients within 24 h. The cross-reactivity of anti-human FVIII antibodies to rpFVIII remains around 30-50%. Recently, new therapeutics based on the quite novel concepts have been developed and clinical studies are ongoing. These are humanized asymmetric antibody mimicking FVIIIa function by maintaining a suitable interaction between FIXa and FX (Emicizumab, ACE910), and small interfering RNAs (siRNA, ALN-AT3) suppress liver production of AT through post-transcriptional gene silencing and a humanized anti-TFPI monoclonal antibody (Concizumab). Their main advantages are longer half-life, subcutaneous applicability and efficacy irrespective of the presence of inhibitors which will make it easier to initiate more effective treatment especially early childhood. PMID:27405674

  12. 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.

  13. New products for the treatment of haemophilia.

    PubMed

    Laffan, Mike

    2016-01-01

    Current developments in haemophilia therapy are directed at two therapeutic targets: reduction of injection frequency and reduction or bypassing of inhibitors. A variety of new molecules addressing these aims are now completing clinical trials and are ready to enter clinical use. First amongst these are modified Factor VIII (FVIII) and Factor IX (FIX) molecules with extended half-lives. FIX modifications have achieved 5-fold prolongation of half-life whilst effects on FVIII have been more modest, at less than two-fold. We now face the problem of integrating these into clinical practice. Other approaches have generated chemically modified FVIII molecules with altered activation profiles. An alternative way of correcting the haemophilia defect is to reduce the activity of natural anticoagulants in an attempt to restore the balance of haemostasis. These methods are also giving promising results but, as with all new approaches, it will be some while before they all find their place in practice. PMID:26456702

  14. Acquired hemophilia complicated by cardiorenal syndrome type 3

    PubMed Central

    Sharma, Rakesh; Dash, Sananta Kumar; Chawla, Rajesh; Kansal, Sudha; Agrawal, Devender Kumar; Dua, Harsh

    2013-01-01

    Development of autoantibodies against coagulation factor VIII (FVIII) leads to a rare condition defined as acquired hemophilia (AH). If not diagnosed and treated early, AH may be associated with high mortality and morbidity. A 65-year-old woman presented with history of macrohematuria, acute renal failure, cardiogenic shock, and acute respiratory failure. Blood investigation revealed azotemia, prolonged activated partial thromboplastin time (aPTT), coagulation FVIII level of <1%, and presence of FVIII inhibitor. Echocardiography showed global hypokinesia and ultrasonography and computed tomography (CT) revealed bilateral hydroureteronephrosis. The final diagnosis was acquired hemophilia A, complicated by acute obstructive renal failure and cardiorenal syndrome (CRS) type 3. Patient was managed with mechanical ventilation, heparin-free hemodialysis, negative fluid balance, recombinant activated factor VII, and prednisolone. Hematuria was relieved, renal function improved, and cardiac function showed improvement on repeat echocardiography. Patient was discharged on prednisolone with subsequent follow ups. PMID:24501492

  15. Type VIII Collagen Mediates Vessel Wall Remodeling after Arterial Injury and Fibrous Cap Formation in Atherosclerosis

    PubMed Central

    Lopes, Joshua; Adiguzel, Eser; Gu, Steven; Liu, Shu-Lin; Hou, Guangpei; Heximer, Scott; Assoian, Richard K.; Bendeck, Michelle P.

    2014-01-01

    Collagens in the atherosclerotic plaque signal regulation of cell behavior and provide tensile strength to the fibrous cap. Type VIII collagen, a short-chain collagen, is up-regulated in atherosclerosis; however, little is known about its functions in vivo. We studied the response to arterial injury and the development of atherosclerosis in type VIII collagen knockout mice (Col8−/− mice). After wire injury of the femoral artery, Col8−/− mice had decreased vessel wall thickening and outward remodeling when compared with Col8+/+ mice. We discovered that apolipoprotein E (ApoE) is an endogenous repressor of the Col8a1 chain, and, therefore, in ApoE knockout mice, type VIII collagen was up-regulated. Deficiency of type VIII collagen in ApoE−/− mice (Col8−/−;ApoE−/−) resulted in development of plaques with thin fibrous caps because of decreased smooth muscle cell migration and proliferation and reduced accumulation of fibrillar type I collagen. In contrast, macrophage accumulation was not affected, and the plaques had large lipid-rich necrotic cores. We conclude that in atherosclerosis, type VIII collagen is up-regulated in the absence of ApoE and functions to increase smooth muscle cell proliferation and migration. This is an important mechanism for formation of a thick fibrous cap to protect the atherosclerotic plaque from rupture. PMID:23567639

  16. Acquired von Willebrand disease--hemostatic management of major orthopedic surgery with high-dose immunoglobulin, desmopressin, and continuous factor concentrate infusion.

    PubMed

    Frank, Rolf Dario; Kunz, Dagmar; Wirtz, Dieter Christian

    2002-05-01

    Acquired von Willebrand disease (aVWD) is a rare bleeding disorder that mimics congenital VWD in previously healthy individuals; it is most frequently associated with monoclonal gammopathy. Hemostatic therapy of aVWD is challenging due to the extremely shortened half-life of endogenous and exogenous VWF. High-dose intravenous immunoglobulin (ivIG) is recommended as the treatment of choice, usually rapidly normalizing coagulation; but in case of failure, alternative treatment options are not well explored. We report successful major orthopedic surgery in a 61-year-old woman with multiple myeloma IgG lambda and aVWD. IvIG alone failed to correct hemostasis. However, ivIG pretreatment improved the VWF ristocetin cofactor (VWF:RCo) half-life from only 1.5 hr to more than 4 hr, allowing desmopressin infusions twice daily to maintain sufficient VWF:RCo levels. Because of diminishing desmopressin effect, we attempted for the first time in aVWD a continuous VWF/FVIII infusion (Haemate HS), 2.1-2.7 FVIII U/kg/hr or 51-64 U/kg/day, respectively 4.6-6.0 VWF:RCo U/kg/hr or 110-145 U/kg/day) to reach constant factor levels. The steady-state clearance was 2.4 mL/kg/hr for FVIII:C and 13.5 mL/kg/hr for VWF:RCo. During surgery, VWF:RCo, FVIII:C, and PFA-100 closure time were normalized. Until day 5, VWF:RCo was kept above 50%, from day 6 to 10 at least 30% activity were attained. FVIII:C levels were always >70%. The clinical course was uneventful without bleeding. Two weeks after hip surgery the patient was discharged from the hospital without complaints. The therapy described can be recommended as safe and feasible for further evaluation in aVWD patients who are hyporesponsive to ivIG treatment alone. Continuous VWF/FVIII infusion can improve substitution therapy in aVWD. PMID:11994985

  17. Compound heterozygous hemophilia A in a female patient and the identification of a novel missense mutation, p.Met1093Ile.

    PubMed

    Qiao, Shu-Kai; Ren, Han-Yun; Ren, Jin-Hai; Guo, Xiao-Nan

    2014-02-01

    Hemophilia A (HA) in females is rare. Female HA cases are often misdiagnosed as acquired HA (AHA) or as von Willebrand disease type 2N (vWD-2N). Here, we report the case of a 37-year-old female HA patient with a moderate factor VIII (FVIII) deficiency. The patient had no personal or family history of bleeding disorders, but presented with heavy uterine bleeding following surgery to remove an intrauterine device. IgG inhibitory antibodies against FVIII were undetected. A compound heterozygote mutation of the FVIII gene (F8) was found in this patient. The p.Val502Asp mutation, which has been reported previously, affects A2 domain function. A novel missense point mutation, p.Met1093Ile, was identified in the B domain. The compound heterozygote mutations in F8, p.Val502Asp and p.Met1093Ile, caused HA in this female patient, with a moderate phenotype. PMID:24317041

  18. Compound heterozygous hemophilia A in a female patient and the identification of a novel missense mutation, p.Met1093Ile

    PubMed Central

    QIAO, SHU-KAI; REN, HAN-YUN; REN, JIN-HAI; GUO, XIAO-NAN

    2014-01-01

    Hemophilia A (HA) in females is rare. Female HA cases are often misdiagnosed as acquired HA (AHA) or as von Willebrand disease type 2N (vWD-2N). Here, we report the case of a 37-year-old female HA patient with a moderate factor VIII (FVIII) deficiency. The patient had no personal or family history of bleeding disorders, but presented with heavy uterine bleeding following surgery to remove an intrauterine device. IgG inhibitory antibodies against FVIII were undetected. A compound heterozygote mutation of the FVIII gene (F8) was found in this patient. The p.Val502Asp mutation, which has been reported previously, affects A2 domain function. A novel missense point mutation, p.Met1093Ile, was identified in the B domain. The compound heterozygote mutations in F8, p.Val502Asp and p.Met1093Ile, caused HA in this female patient, with a moderate phenotype. PMID:24317041

  19. 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...

  20. 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

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF ENERGY Federal Energy Regulatory Commission Lock+ Hydro Friends Fund VIII, FFP Project 92, LLC, Riverbank Hydro No. 24... Madison Counties, Kentucky. The applications were filed by Lock+ Hydro Friends Fund VIII for Project...

  1. 75 FR 27770 - Lock+ Hydro Friends Fund VIII; Notice of Preliminary Permit Application Accepted for Filing and...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-18

    ... From the Federal Register Online via the Government Publishing Office ] DEPARTMENT OF ENERGY Federal Energy Regulatory Commission Lock+ Hydro Friends Fund VIII; Notice of Preliminary Permit... 11, 2010. On April 13, 2010 Lock+ Hydro Friends Fund VIII filed an application for a...

  2. 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...

  3. 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

  4. 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. PMID:26921444

  5. 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.

  6. 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)

  7. 25 CFR 36.23 - Standard VIII-Junior high/middle school instructional program.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 25 Indians 1 2010-04-01 2010-04-01 false Standard VIII-Junior high/middle school instructional program. 36.23 Section 36.23 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR EDUCATION MINIMUM ACADEMIC STANDARDS FOR THE BASIC EDUCATION OF INDIAN CHILDREN AND NATIONAL CRITERIA FOR DORMITORY SITUATIONS Minimum Program of Instruction...

  8. Nurse Training. Title VIII--Public Health Service Act. The Complete Law.

    ERIC Educational Resources Information Center

    1972

    To keep the nursing profession and other interested groups informed of the new provisions in nurse training legislation, this publication contains the complete text of Title VIII of the Public Health Service Act, as of November 18, 1971. Major provisions of the Act cover: (1) Grants for Expansion and Improvement of Nurse Training, (2) Assistance…

  9. SURVEY OF STRAWBERRY VIRUSES IN FRAGARIA X ANNANASA AND FRAGARIA CHILOENSIS IN THE VIII REGION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In April 2004, a survey was conducted of strawberry plants with symptoms in two areas (Los Angeles and Contulmo) of Chilean Region VIII. Three types of plantings were sampled: nurseries, commercial fields of Fragaria annanasa and commercial fields of Fragaria chiloensis. Nursery plants were sample...

  10. 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…

  11. 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…

  12. Exemplary Project Region VIII Workshop (Riverton, Wyoming, July 12-14, 1972).

    ERIC Educational Resources Information Center

    Lutz, Richard

    To discuss common problems and plan for their solution, this meeting of personnel from the Office of Education, the State Department of Education in Region VIII, and career Education Projects in the Rocky Mountain region was held. Specific goals included efforts to: (1) identify new approaches and alternatives in an effort to continue examplary…

  13. III-V nanowire growth mechanism: V/III ratio and temperature effects.

    PubMed

    Dayeh, Shadi A; Yu, Edward T; Wang, Deli

    2007-08-01

    We have studied the dependence of Au-assisted InAs nanowire (NW) growth on InAs(111)B substrates as a function of substrate temperature and input V/III precursor ratio using organometallic vapor-phase epitaxy. Temperature-dependent growth was observed within certain temperature windows that are highly dependent on input V/III ratios. This dependence was found to be a direct consequence of the drop in NW nucleation and growth rate with increasing V/III ratio at a constant growth temperature due to depletion of indium at the NW growth sites. The growth rate was found to be determined by the local V/III ratio, which is dependent on the input precursor flow rates, growth temperature, and substrate decomposition. These studies advance understanding of the key processes involved in III-V NW growth, support the general validity of the vapor-liquid-solid growth mechanism for III-V NWs, and improve rational control over their growth morphology. PMID:17608541

  14. 7 CFR 1901.203 - Title VIII of the Civil Rights Act of 1968.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 12 2013-01-01 2013-01-01 false Title VIII of the Civil Rights Act of 1968. 1901.203 Section 1901.203 Agriculture Regulations of the Department of Agriculture (Continued) RURAL HOUSING SERVICE, RURAL BUSINESS-COOPERATIVE SERVICE, RURAL UTILITIES SERVICE, AND FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE PROGRAM...

  15. Evaluation of ESEA Title VIII Dropout Prevention Program, "Project KAPS," School Year 1970-71.

    ERIC Educational Resources Information Center

    Cutter, N. Craig; Jones, Earl R.

    The KAPS (or Keeping All Pupils in School) program was initiated by the Baltimore City Public Schools in 1969, using funds provided under Title VIII of the Elementary and Secondary Education Act. This evaluation deals with the academic year 1970-71, the second full year of the project's evaluation. Most important among the goals of Project KAPS is…

  16. 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.

  17. Rituximab for treatment of inhibitors in haemophilia A. A Phase II study.

    PubMed

    Leissinger, C; Josephson, C D; Granger, S; Konkle, B A; Kruse-Jarres, R; Ragni, M V; Journeycake, J M; Valentino, L; Key, N S; Gill, J C; McCrae, K R; Neufeld, E J; Manno, C; Raffini, L; Saxena, K; Torres, M; Marder, V; Bennett, C M; Assmann, S F

    2014-09-01

    The development of antibodies against infused factor VIII (FVIII) in patients with haemophilia A is a serious complication leading to poorly controlled bleeding and increased morbidity. No treatment has been proven to reduce high titre antibodies in patients who fail immune tolerance induction or are not candidates for it. The Rituximab for the Treatment of Inhibitors in Congenital Hemophilia A (RICH) study was a phase II trial to assess whether rituximab can reduce anamnestic FVIII antibody (inhibitor) titres. Male subjects with severe congenital haemophilia A and an inhibitor titre ≥5 Bethesda Units/ml (BU) following a FVIII challenge infusion received rituximab 375 mg/m² weekly for weeks 1 through 4. Post-rituximab inhibitor titres were measured monthly from week 6 through week 22 to assess treatment response. Of 16 subjects who received at least one dose of rituximab, three (18.8%) met the criteria for a major response, defined as a fall in inhibitor titre to <5 BU, persisting after FVIII re-challenge. One subject had a minor response, defined as a fall in inhibitor titre to <5 BU, increasing to 5-10 BU after FVIII re-challenge, but <50% of the original peak inhibitor titre. Rituximab is useful in lowering inhibitor levels in patients, but its effect as a solo treatment strategy is modest. Future studies are indicated to determine the role of rituximab as an adjunctive therapy in immune tolerisation strategies. PMID:24919980

  18. LRP1/CD91 is up-regulated in monocytes from patients with haemophilia A: a single-centre analysis.

    PubMed

    Franchini, M; Urbani, S; Amadei, B; Rivolta, G F; Di Perna, C; Riccardi, F; Frattini, F; Crestani, S; Bonfanti, C; Formentini, A; Quintavalla, R; Tagliaferri, A

    2013-05-01

    The low-density lipoprotein receptor-related protein 1 (LRP1) is an ubiquitously expressed endocytic receptor that, among its several functions, is involved in the catabolism of coagulation factor VIII (FVIII) and in the regulation of its plasma concentrations. Although LRP1/CD91 polymorphisms have been associated with increased FVIII levels and a consequent thrombotic risk, no data are available on LRP1/CD91 expression in patients with inherited FVIII deficiency. With the aim of elucidating this issue, 45 consecutive patients with haemophilia A (HA) (18 severe, 5 moderate and 22 mild HA) were enrolled in this cross-sectional, single-centre survey. The LRP1/CD91 mean fluorescence intensity (MFI) in monocytes from HA patients was significantly higher than that detected in 90 healthy blood donors (105 vs. 67, P < 0.001). This over-expression was independent of hepatitis C virus infection status and varied according to the severity of the haemophilia, being higher in patients with more severe FVIII deficiency. In conclusion, our study documents for the first time that LRP1/CD91 is over-expressed on monocytes from HA patients, with the intensity of expression varying according to the severity of the FVIII deficiency. Further studies are needed to assess the clinical implications of these findings. PMID:23387825

  19. Progress and challenges in the development of a cell-based therapy for hemophilia A

    PubMed Central

    Fomin, Marina E.; Togarrati, Padma Priya; Muench, Marcus O.

    2015-01-01

    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 (iPSCs) 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 iPSCs. PMID:25297648

  20. 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. PMID:27131224

  1. 75 FR 4340 - Notice of New Recreation Fee Site; Federal Lands Recreation Enhancement Act, (Title VIII, Pub. L...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-01-27

    ... Forest Supervisor. BILLING CODE 3410-11-M ... Forest Service Notice of New Recreation Fee Site; Federal Lands Recreation Enhancement Act, (Title VIII, Pub. L. 108-447) AGENCY: Caribou-Targhee National Forest, USDA Forest Service. ACTION: Notice of...

  2. 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....

  3. Complete Genome Sequence of Clostridium sp. Strain DL-VIII, a Novel Solventogenic Clostridium Species Isolated from Anaerobic Sludge

    PubMed Central

    Taghavi, Safiyh; Izquierdo, Javier A.

    2013-01-01

    We report the genome sequence of Clostridium sp. strain DL-VIII, a novel Gram-positive, endospore-forming, solventogenic bacterium isolated from activated anaerobic sludge of a wastewater treatment plant. Aside from a complete sol operon, the 6,477,357-bp genome of DL-VIII reveals genes for several unique enzymes with applications in lignocellulose degradation, including two phenolic acid decarboxylases. PMID:23929491

  4. 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.

  5. 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.

  6. 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.

  7. Search for plasma shifts of C VI, N VII, and O VIII resonance lines

    NASA Technical Reports Server (NTRS)

    Goldsmith, S.; Griem, H. R.; Cohen, L.

    1984-01-01

    The resonance lines of the hydrogenlike ions C VI, N VII, and O VIII were observed in laser-produced plasmas with electron densities in the range 10 to the 21st - 10 to the 22nd per cu cm and electron temperatures between about 70 and 100 eV. The lines of the hydrogenlike ions were recorded photographically with a 3-m grazing-incidence spectrograph. No significant plasma line shift was observed.

  8. 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…

  9. 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.

  10. Molecular Characterization and Subcellular Localization of Arabidopsis Class VIII Myosin, ATM1*

    PubMed Central

    Haraguchi, Takeshi; Tominaga, Motoki; Matsumoto, Rie; Sato, Kei; Nakano, Akihiko; Yamamoto, Keiichi; Ito, Kohji

    2014-01-01

    Land plants possess myosin classes VIII and XI. Although some information is available on the molecular properties of class XI myosins, class VIII myosins are not characterized. Here, we report the first analysis of the enzymatic properties of class VIII myosin. The motor domain of Arabidopsis class VIII myosin, ATM1 (ATM1-MD), and the motor domain plus one IQ motif (ATM1-1IQ) were expressed in a baculovirus system and characterized. ATM1-MD and ATM1-1IQ had low actin-activated Mg2+-ATPase activity (Vmax = 4 s−1), although their affinities for actin were high (Kactin = 4 μm). The actin-sliding velocities of ATM1-MD and ATM1-1IQ were 0.02 and 0.089 μm/s, respectively, from which the value for full-length ATM1 is calculated to be ∼0.2 μm/s. The results of actin co-sedimentation assay showed that the duty ratio of ATM1 was ∼90%. ADP dissociation from the actin·ATM1 complex (acto-ATM1) was extremely slow, which accounts for the low actin-sliding velocity, low actin-activated ATPase activity, and high duty ratio. The rate of ADP dissociation from acto-ATM1 was markedly biphasic with fast and slow phase rates (5.1 and 0.41 s−1, respectively). Physiological concentrations of free Mg2+ modulated actin-sliding velocity and actin-activated ATPase activity by changing the rate of ADP dissociation from acto-ATM1. GFP-fused full-length ATM1 expressed in Arabidopsis was localized to plasmodesmata, plastids, newly formed cell walls, and actin filaments at the cell cortex. Our results suggest that ATM1 functions as a tension sensor/generator at the cell cortex and other structures in Arabidopsis. PMID:24637024

  11. ITI choice for the optimal management of inhibitor patients - from a clinical and pharmacoeconomic perspective.

    PubMed

    Oldenburg, J; Austin, S K; Kessler, C M

    2014-09-01

    The development of alloantibody inhibitors against factor VIII (FVIII) represents the most significant complication of haemophilia care. Inhibitors tend to develop early in the course of treatment in about 20-30% of patients with severe haemophilia who receive on-demand or prophylactic FVIII therapy. Many factors are associated with inhibitor formation, including disease severity, major FVIII gene defects, family history and non-Caucasian race, as well as age at first treatment, intensity of early treatment, use of prophylaxis and product choice. As these latter treatment-related variables are modifiable, they provide opportunity to minimize inhibitor incidence at the clinical level. Data from the Bonn Centre in Germany have indicated an overall success rate of 78% for immune tolerance induction (ITI) therapy, with a failure rate of 15% and with some treatments either ongoing (3%) or withdrawn (4%). Similarly, data from the G-ITI study, the largest international multicentre ITI study using a single plasma-derived (pd) FVIII/von Willebrand factor (VWF) product, have demonstrated success rates (complete and partial) in primary and rescue ITI of 87% and 74%, respectively, with 85% of poor prognosis patients achieving success. Favourable clinical results based on success rates and time to tolerization continue to be reported for use of pdFVIII/VWF in ITI, with pdFVIII/VWF having a particular role in patients who require rescue ITI and those with a poor prognosis for success. Data from prospective, randomized, controlled clinical studies, such as RES.I.ST (Rescue Immune Tolerance Study), are eagerly awaited. Another factor to consider with ITI therapy is cost; preliminary data from an updated decision analytic model have provided early evidence that ITI has an economic advantage compared with on-demand or prophylactic therapy. PMID:24975701

  12. Imbalance of Pro- vs. Anti-Coagulation Factors in Chinese Patients with Budd-Chiari Syndrome and Non-Cirrhotic Portal Vein Thrombosis

    PubMed Central

    He, Chuangye; Yin, Zhanxin; Wu, Feifei; Fan, Daiming; Han, Guohong

    2015-01-01

    Background and Aim The coagulation abnormalities in non-cirrhotic Budd-Chiari syndrome (NC-BCS) and non-cirrhotic portal vein thrombosis (NC-PVT) are unclear. We conducted this case-control study to investigate the coagulation profile of NC-BCS and NC-PVT in Chinese patients. Methods We measured the levels of factors II, V, VII, VIII, IX, X, XI, XII, protein C (PC), protein S (PS) and antithrombin (AT) in blood samples from 37 NC-BCS patients, 74 NC-PVT patients, and 100 healthy controls. The levels and ratios of pro- and anti-coagulation factors were compared between patients with NC-BCS and healthy controls, between different types of NC-BCS and between NC-PVT and healthy controls. Results In patients with NC-BCS, factor VIII (P<0.001) was significantly elevated; factor V (P<0.001), VII (P<0.001), IX (P = 0.003), X (P<0.001), XI (P<0.001), XII (P<0.001), PC (P<0.001) and AT (P<0.001) were significantly decreased; and no difference was observed for factor II (P = 0.088) and PS (P = 0.199) compared with healthy controls. Factor VIII-to-PC (P = 0.008), factor VIII-to-PS (P = 0.037) and factor VIII-to-AT (P = 0.001) were significantly increased; other ratios were significantly reduced or did not show any difference. No differences were observed between different types of NC-BCS for individual pro- and anti-coagulation factors or the ratios between them. Among patients with NC-PVT, factor VIII (P<0.001) was significantly elevated and other factors were significantly decreased. Factor II-to-PC (P<0.001), factor VIII-to-PC (P<0.001), factor IX-to-PC (P<0.001), factor VIII-to-PS (P<0.001), factor II-to-AT (P<0.001), factor VIII-to-AT (P<0.001) and factor IX-to-AT (P<0.001) were significantly increased; all other ratios for NC-PVT were significantly reduced or did not show any significant difference. Conclusions NC-BCS and NC-PVT are associated with elevated levels of factor VIII and the decreased levels of PC and AT were probably the most significant features of

  13. 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.

  14. 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.

  15. WAVELENGTHS, ENERGY LEVELS, LIFETIMES, AND WEIGHTED OSCILLATOR STRENGTHS FOR THE S VIII SPECTRUM

    SciTech Connect

    Pagan, C. J. B.; Cavalcanti, G. H.; Trigueiros, A. G.; Jupen, C.

    2011-10-01

    The weighted oscillator strengths (gf) and lifetimes for S VIII presented in this work were obtained by a multiconfigurational Hartree-Fock relativistic approach. In this calculation, the electrostatic energy parameters were optimized by a least-squares procedure in order to improve the adjustment to experimental energy levels. The values for gf and lifetimes were then calculated on the basis of these adjusted parameters. New classifications are proposed for energy levels belonging to the 4s and 4d configurations and lines related to them.

  16. 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

  17. Myosin VIII associates with microtubule ends and together with actin plays a role in guiding plant cell division

    PubMed Central

    Wu, Shu-Zon; Bezanilla, Magdalena

    2014-01-01

    Plant cells divide using the phragmoplast, a microtubule-based structure that directs vesicles secretion to the nascent cell plate. The phragmoplast forms at the cell center and expands to reach a specified site at the cell periphery, tens or hundreds of microns distant. The mechanism responsible for guiding the phragmoplast remains largely unknown. Here, using both moss and tobacco, we show that myosin VIII associates with the ends of phragmoplast microtubules and together with actin plays a role in guiding phragmoplast expansion to the cortical division site. Our data lead to a model whereby myosin VIII links phragmoplast microtubules to the cortical division site via actin filaments. Myosin VIII's motor activity along actin provides a molecular mechanism for steering phragmoplast expansion. DOI: http://dx.doi.org/10.7554/eLife.03498.001 PMID:25247701

  18. Structural characterization of the novel aminoglycoside phosphotransferase AphVIII from Streptomyces rimosus with enzymatic activity modulated by phosphorylation.

    PubMed

    Boyko, Konstantin M; Gorbacheva, Marina A; Korzhenevskiy, Dmitry A; Alekseeva, Maria G; Mavletova, Dilara A; Zakharevich, Natalia V; Elizarov, Sergey M; Rudakova, Natalia N; Danilenko, Valery N; Popov, Vladimir O

    2016-09-01

    Aminoglycoside phosphotransferases represent a broad class of enzymes that promote bacterial resistance to aminoglycoside antibiotics via the phosphorylation of hydroxyl groups in the latter. Here we report the spatial structure of the 3'-aminoglycoside phosphotransferase of novel VIII class (AphVIII) solved by X-ray diffraction method with a resolution of 2.15 Å. Deep analysis of APHVIII structure and its comparison with known structures of aminoglycoside phosphotransferases of various types reveals that AphVIII has a typical two-domain fold and, however, possesses some unique characteristics that distinguish the enzyme from its known homologues. The most important difference is the presence of the activation loop with unique Ser146 residue. We demonstrate that in the apo-state of the enzyme the activation loop does not interact with other parts of the enzyme and seems to adopt catalytically competent state only after substrate binding. PMID:27338640

  19. Monte Carlo simulation of V/III flux ratio influence on GaAs island nucleation during MBE

    NASA Astrophysics Data System (ADS)

    Ageev, O. A.; Solodovnik, M. S.; Balakirev, S. V.; Mikhaylin, I. A.

    2016-02-01

    The kinetic Monte Carlo simulation of GaAs/GaAs(001) molecular beam epitaxial growth considering V/III flux ratio influence on nucleating island characteristics is presented. It is shown that the island density increases with the surface coverage increase and reaches saturation after deposition of ∼0.1 monolayer of GaAs. The increase of V/III flux ratio from 3 to 40 leads to the increase of the island density from 1.9-1012 to 2.6-1012 cm-2. At the same time the average size decreases from 4.4 to 4.1 nm. The island size distribution function narrows with V/III flux ratio increase. This is attributed to the shortage of gallium atoms in comparison with deposited arsenic molecules that prevents large island formation and leads to the dramatic growth of little island concentration. The simulation demonstrates good agreement with experimental results.

  20. QSO ABSORPTION SYSTEMS DETECTED IN Ne VIII: HIGH-METALLICITY CLOUDS WITH A LARGE EFFECTIVE CROSS SECTION

    SciTech Connect

    Meiring, J. D.; Tripp, T. M.; Werk, J. K.; Prochaska, J. X.; Howk, J. C.; Jenkins, E. B.; Lehner, N.; Sembach, K. R.

    2013-04-10

    Using high-resolution, high signal-to-noise ultraviolet spectra of the z{sub em} = 0.9754 quasar PG1148+549 obtained with the Cosmic Origins Spectrograph (COS) on the Hubble Space Telescope, we study the physical conditions and abundances of Ne VIII+O VI absorption line systems at z{sub abs} = 0.68381, 0.70152, 0.72478. In addition to Ne VIII and O VI, absorption lines from multiple ionization stages of oxygen (O II, O III, O IV) are detected and are well aligned with the more highly ionized species. We show that these absorbers are multiphase systems including hot gas (T Almost-Equal-To 10{sup 5.7} K) that produces Ne VIII and O VI, and the gas metallicity of the cool phase ranges from Z = 0.3 Z{sub Sun} to supersolar. The cool ( Almost-Equal-To 10{sup 4} K) phases have densities n{sub H} Almost-Equal-To 10{sup -4} cm{sup -3} and small sizes (<4 kpc); these cool clouds are likely to expand and dissipate, and the Ne VIII may be within a transition layer between the cool gas and a surrounding, much hotter medium. The Ne VIII redshift density, dN/dz{approx}7{sup +7}{sub -3}, requires a large number of these clouds for every L > 0.1 L* galaxy and a large effective absorption cross section ({approx}> 100 kpc), and indeed, we find a star-forming {approx}L {sup *} galaxy at the redshift of the z{sub abs} = 0.72478 system, at an impact parameter of 217 kpc. Multiphase absorbers like these Ne VIII systems are likely to be an important reservoir of baryons and metals in the circumgalactic media of galaxies.

  1. The head that wears the crown: Henry VIII and traumatic brain injury.

    PubMed

    Ikram, Muhammad Qaiser; Sajjad, Fazle Hakim; Salardini, Arash

    2016-06-01

    Henry VIII of England is one of the most controversial figures in European history. He was born on 28 June 1491 as the second son of Henry VII and Elizabeth of York and became the heir to the English throne after his elder brother died prematurely. A contradictory picture of Henry's character emerges from history: the young Henry was a vigorous, generous and intelligent king who saw early military and naval successes. In contrast, in his later years he became cruel, petty and tyrannical. His political paranoia and military misjudgements are in direct contrast to his earlier successes and promise. Several hypotheses have been put forward regarding his transformation from a renaissance king to a later medieval tyrant, including endocrinopathies, psychiatric illnesses and traumatic brain injury. In this paper we examine the historical evidence linking the change in Henry's personality and health problems to traumatic brain injury. To our knowledge this is the first systematic neurological study of traumatic brain injury in Henry VIII. PMID:26857293

  2. A novel family VIII carboxylesterase hydrolysing third- and fourth-generation cephalosporins.

    PubMed

    Jeon, Jeong Ho; Lee, Hyun Sook; Lee, Jung Hun; Koo, Bon-Sung; Lee, Chang-Muk; Lee, Sang Hee; Kang, Sung Gyun; Lee, Jung-Hyun

    2016-01-01

    A metagenomic library was constructed from a soil sample of spindle tree-rhizosphere. From this library, one clone with esterase activity was selected. The sequence analysis revealed an open reading frame (EstSTR1) encoded protein of 390 amino acids. EstSTR1 is a family VIII carboxylesterase and retains the S-X-X-K motif conserved in both family VIII carboxylesterases and class C β-lactamases. The estSTR1 gene was overexpressed in E. coli and the recombinant protein was purified by purified by metal chelating affinity chromatography and size-exclusion chromatography. EstSTR1 hydrolysed p-nitrophenyl esters, exhibited the highest activity toward p-nitrophenyl butyrate. Furthermore, EstSTR1 could hydrolyse third- and fourth-generation cephalosporins (cefotaxime and cefepime) as well as first-generation cephalosporin (cephalothin). Site-directed mutagenesis studies revealed that a catalytic residue, Ser71, of EstSTR1 plays an essential role in hydrolysing both antibiotics and p-nitrophenyl esters. We demonstrate that a metagenome-derived carboxylesterase displays β-lactam-hydrolysing activities toward third- and fourth-generation cephalosporins. PMID:27186489

  3. X-ray lines from MG VIII and SI X ions and their diagnostic use

    NASA Astrophysics Data System (ADS)

    Dwivedi, Bhola N.

    The solar X-ray emission lines from Mg VIII and Si X ions have been studied. The variation of the theoretical line-intensity ratios from Mg VIII and Si X as functions of electron density are found to be good density monitors of the emitting regions of solar plasma. The computed values of line intensity from these ions based on the Kopp and Orrall model have been used to derive the electron density of the quiet sun and coronal holes. Electron densities of 10 exp 9/cu cm and 4.6 x 10 exp 8/cu cm are estimated at the electron temperatures of 8 x 10 exp 5 K and 1.6 x 10 exp 6 K for the quiet sun whereas the respective values of 5.4 x 10 exp 8/cu cm and 1.7 x 10 exp 8/cu cm are obtained for the coronal holes. The line-intensity ratios studied here are independent of temperature variation and are therefore excellent candidates for electron-density diagnostics. However, observational data with improved spectral resolution is needed for using X-ray-line pairs studied for their diagnostic use.

  4. Significant changes in the Code rules in Section VIII, Division 1 concerning material and welding

    SciTech Connect

    Doty, W.D.

    1996-12-01

    Several very significant changes to the Code rules in Section VIII, Division 1 concerning materials and welding have been made since the 1950 Code. These changes include: (1) Improved steel making practice which has resulted in significant changes to the type of materials permitted for the construction of Section VIII vessels. (2) In 1950 no impact testing was required on any material for use at temperatures of {minus}20F and above. And when impact testing was required, the impact value was 15 ft-lb minimum average on Charpy type, keyhole, or U-notch specimens. (3) In the mid-1960`s, the impact test requirement was changed to Charpy type, V-notch specimens, a much more severe test. (4) In 1987 the Code published new toughness rules for carbon and low-alloy steels. (5) In regard to welding, a very significant improvement has been the use of low-hydrogen electrodes which started in the 1950`s. Few welding electrode specifications existed in the early editions of the Code. A recent improvement in electrode specification is moisture resistant electrodes.

  5. A DFT study to unravel the ligand exchange kinetics and thermodynamics of Os(VIII) oxo/hydroxido/aqua complexes in aqueous matrices.

    PubMed

    van Niekerk, Daniel M E; Gerber, Wilhelmus J; Koch, Klaus R

    2016-04-19

    The Os(VIII) oxo/hydroxido complexes that are abundant in mild to relatively concentrated basic aqueous solutions are Os(VIII)O4, [Os(VIII)O4(OH)](-) and two cis-[Os(VIII)O4(OH)2](2-) species. Os(VIII) complexes that contain water ligands are thermodynamically unfavoured w.r.t. the abovementioned species. Os(VIII)O4 reacts with hydroxide in two, consecutive, elementary coordination sphere expansion steps to form the [Os(VIII)O4(OH)](-) complex and then the cis-[Os(VIII)O4(OH)2](2-) species. The Gibbs energy of activation for both reactions, in the forward and reverse direction, are in the range of 6-12 kcal mol(-1) and are relatively close to diffusion-controlled. The thermodynamic driving force of the first reaction is the bonding energy of the Os(VIII)-OH metal-hydroxido ligand, while of the second reaction it is the relatively large hydration energy of the doubly-charged cis-[Os(VIII)O4(OH)2](2-) product compared to the singly-charged reactants. The DFT-calculated (PBE-D3 functional) in the simulated aqueous phase (COSMO) is -2.4 kcal mol(-1) for the first reaction and -0.6 kcal mol(-1) for the second reaction and agree to within 1 kcal mol(-1) with reported experimental values, at -2.7 and 0.3 kcal mol(-1) respectively. From QTAIM and EDA analyses it is deduced that the Os(VIII)[double bond, length as m-dash]O bonding interactions are ionic (closed-shell) and that Os(VIII)-OH bonding interactions are polar covalent (dative). In contrast to QTAIM, NCI analysis allowed for the identification of relatively weak intramolecular hydrogen bonding interactions between neighbouring oxo and hydroxido ligands in both [Os(VIII)O4(OH)](-) and cis-[Os(VIII)O4(OH)2](2-) complexes. PMID:26991070

  6. Lamina VIII interneurones interposed in crossed reflex pathways in the cat.

    PubMed Central

    Harrison, P J; Jankowska, E; Zytnicki, D

    1986-01-01

    The location of a group of interneurones projecting to contralateral motor nuclei has been established using retrograde transneuronal transport of horseradish peroxidase conjugated with wheat germ agglutinin (WGA-HRP). After labelling the motoneurones of semitendinosus, medial gastrocnemius or quadriceps muscles, interneurones which were secondarily labelled were found in lamina VIII and in the neighbouring narrow strip of lamina VII. They were found to be distributed from the 4th lumbar to the 1st sacral segments, with the highest concentration in the 6th and 7th lumbar segments and at the border between the 4th and 5th lumbar segments. The electrophysiological properties of lamina VIII interneurones of the 6th lumbar segment have been investigated using both extracellular and intracellular recording. Many of these interneurones could be antidromically activated following weak stimuli applied in contralateral motor nuclei. Post-synaptic potentials were evoked from a variety of primary afferents including group I muscle afferents. However, when present, the post-synaptic potentials (p.s.p.s) of group I origin were of considerably smaller amplitudes than p.s.p.s. evoked from higher threshold muscle or cutaneous afferents and smaller than p.s.p.s. which followed stimulation of the spinal cord at the thoracic level. P.s.p.s. from the latter two sources appear to constitute the main input to lamina VIII interneurones. Group I input has been found in forty lamina VIII interneurones. These were usually affected by either ipsilateral or contralateral group I afferents and only exceptionally by both. Excitatory post-synaptic potentials (e.p.s.p.s) from ipsilateral afferents were evoked in about twice as many neurones as e.p.s.p.s from the contralateral afferents. E.p.s.p.s were often accompanied by inhibitory post-synaptic potentials (i.p.s.p.s). Group Ia afferents appeared to contribute to both e.p.s.p.s and i.p.s.p.s, whether these were evoked from ipsilateral or from

  7. Towards personalised therapy for von Willebrand disease: a future role for recombinant products.

    PubMed

    Favaloro, Emmanuel J

    2016-05-01

    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

  8. Genetic polymorphism of LDLR (rs688) is associated with primary intracerebral hemorrhage.

    PubMed

    Lee, Jiann-Der; Hsiao, Kuang-Ming; Lee, Tsong-Hai; Kuo, Ya-Wen; Huang, Yen-Chu; Hsu, Huan-Lin; Lin, Ya-Hui; Wu, Chih-Ying; Huang, Ying-Chih; Lee, Meng; Yang, Hsin-Ta; Hsu, Chia-Yu; Pan, Yi-Ting

    2014-02-01

    Intracranial hemorrhage is the third most common cause of cerebrovascular disease. Some polymorphisms that affect clotting factors increase the risk of thrombosis. However, few reports have analyzed the effect of polymorphisms on the hemostatic state in bleeding disorders. The low-density lipoprotein receptor (LDLR) has been shown to contribute to factor VIII (FVIII) homeostasis, which represents a link between LDLR and hemostasis. FVIII plays a pivotal role in the coagulation cascade. Patients with high levels of FVIII are at an increased risk of arterial and venous thrombosis. On the other hand, patients with insufficient FVIII tend to bleed excessively, such as in hemophilia A. In a previous study, analysis of the genetic LDLR variant rs688 provided evidence suggesting that genetic polymorphisms of rs688 are associated with thrombotic cardiovascular diseases. The current study aimed to investigate the potential role of rs688 in primary intracerebral hemorrhage (PICH). This genetic association study was conducted within an isolated Taiwanese population (447 PICH patients and 430 controls). Genotypes C/C and C/T were used as the reference genotypes, and the genotype T/T was found to be associated with a 73% decreased risk of PICH. The preliminary evidence suggests that genetic polymorphisms of LDLR are associated with PICH. PMID:24295502

  9. 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. PMID:27405679

  10. 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. PMID:26728503

  11. 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

  12. 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. PMID:26838696

  13. Desmopressin in mild hemophilia A: indications, limitations, efficacy, and safety.

    PubMed

    Lethagen, Stefan

    2003-02-01

    Replacement therapy with blood products has long been the only available therapeutic option for patients with bleeding disorders. Plasma-derived cryoprecipitate and factor (F) VIII concentrates, which have been used for hemophilia A patients, involve the risk of transmitting blood-borne diseases. Both plasma-derived and recombinant FVIII concentrates are expensive, and there is a global shortage. The synthetic vasopressin analogue desmopressin acetate (1-deamino-[8-D-arginine]-vasopressin, DDAVP) increases plasma concentrations of coagulation FVIII and von Willebrand factor (vWF) two fold to six fold through endogenous release. The drug is an attractive therapeutic alternative because it carries no risk of transmission of infectious diseases. Desmopressin is today a widely used hemostatic agent not only in patients with mild hemophilia A or von Willebrand disease (vWD) but also in those with congenital or acquired platelet dysfunction. There is a long clinical experience with the drug because it has been used for prevention of bleedings in connection with invasive procedures and for treatment of bleedings since the mid-1970s. Not all hemophilia A patients can be treated. The clinical usefulness depends on the postdesmopressin plasma concentration of FVIII, which in turn depends on the patient's basal FVIII level. Therefore, a test dose is recommended in candidate patients. In general, only the mildest hemophilia A patients respond sufficiently. Optimal hemostatic effect is achieved with a dosage of 0.3 microg/kg given intravenously. An intranasal desmopressin spray is suitable for the home treatment. PMID:12640572

  14. 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.

  15. 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

  16. Paducah-Louisville Consortium. Project VIII: Focus on Dropouts... A New Design. Final Evaluation Report, 1971-72.

    ERIC Educational Resources Information Center

    Lyne, Evelyn

    Project VIII is an innovative behavioral science-oriented educational program for potential dropouts. It is designed to reduce the dropout rate in grades 9 through 12, increase attendance in grades 7 through 12, decrease discipline referrals and suspensions, increase reading and math achievement in grades 3 through 11, and improve the student's…

  17. 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....

  18. 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

    ..., Forest Supervisor, National Forest in Mississippi. BILLING CODE 3410-11-M ... 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...

  19. 78 FR 41783 - Notice of Proposed New Fee Sites; Federal Lands Recreation Enhancement Act, (Title VIII, Pub. L...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-07-11

    ... Forest Service Notice of Proposed New Fee Sites; Federal Lands Recreation Enhancement Act, (Title VIII, Pub. L. 108-447) AGENCY: Salmon-Challis National Forest, Forest Service, USDA. ACTION: Notice New Fee Sites. SUMMARY: The Salmon-Challis National Forest is proposing to begin charging fees at...

  20. 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

    .... Margrett L. Boley, Forest Supervisor, National Forests in Mississippi. BILLING CODE 3410-11-M ... 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....

  1. 77 FR 62215 - Notice of Proposed New Fee Site; Federal Lands Recreation Enhancement Act, (Title VIII, Pub. L...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-10-12

    ..., Forest Supervisor, Payette National Forest. BILLING CODE 3410-11-P ... Forest Service Notice of Proposed New Fee Site; Federal Lands Recreation Enhancement Act, (Title VIII, Pub. L. 108-447) AGENCY: Payette National Forest, Forest Service, USDA. ACTION: Notice of Proposed...

  2. 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...

  3. 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...

  4. 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...

  5. 76 FR 36518 - Notice of Meeting; Federal Lands Recreation Enhancement Act (Title VIII, Pub. L. 108-447)

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-06-22

    ... 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: Rocky Mountain Region, USDA Forest Service. ACTION: Notice of Meeting. SUMMARY: The...

  6. The effects of V/III gas ratios on the catalyst-assisted growths of InGaN nanowires

    NASA Astrophysics Data System (ADS)

    Tang, Wei-Che; Hong, Franklin Chau-Nan

    2015-11-01

    Single crystalline InGaN nanowires were grown on Si(1 0 0) using Au catalysts at 700 °C in a plasma-assisted chemical vapor deposition system. Under the low V/III (nitrogen radicals/(Ga + In vapor)) ratio in the gas phase, poor quality InGaN nanowires were grown along [0 0 0 1] orientation (c-axis) containing a high concentration of stacking faults and a low indium content of 12%. However, under the high V/III gas ratio, very high quality InGaN nanowires could be grown along [ 1 0 1 bar 0 ] orientation (m-axis) free of stacking faults containing a high indium content of 24%. The transformation of nanowire orientation was likely due to the decrease of indium and gallium compositions in gold catalysts from >85% to <60% with increasing the V/III gas ratios. Besides, the increase of V/III gas ratios enhanced the incorporation efficiency of indium into the nanowires and significantly improved the crystal quality of nanowires by stabilizing the formation of InN under a high concentration of nitrogen radicals for reversing the fast thermal decomposition reaction of InN at 700 °C.

  7. The immune tolerance induction (ITI) dose debate: does the International ITI Study provide a clearer picture?

    PubMed

    Ettingshausen, C Escuriola; Kreuz, W

    2013-01-01

    Among the proposed predictors for immune tolerance induction (ITI) outcome, the therapeutic regimen - specifically the dose and frequency of administered factor VIII (FVIII) as well as FVIII product type - is intensely debated. Are there any advantages for low-dose regimens (50 IU FVIII kg(-1) three times a week) over high-dose regimens (200 IU FVIII kg day(-1)) or vice versa? Are von Willebrand factor (VWF)-containing plasma-derived concentrates superior to recombinant FVIII concentrates for tolerance induction? A review of the available literature indicates that patients with good prognostic factors can achieve success with either low-dose or high-dose ITI regimens. Retrospective data suggest that patient characteristics such as maximum historical inhibitor titres and pre-ITI inhibitor titres are better predictors of treatment success than dose. Results of the prospective International ITI Study have recently become available. In inhibitor patients with good prognosis, success rates were similar between low-dose (50 IU FVIII kg(-1) three times a week) and high-dose (200 IU FVIII kg(-1) daily) regimens. However, patients receiving low-dose ITI took longer to achieve various ITI milestones and had a significantly higher bleed rate per month compared with the high-dose group (0.62 vs. 0.28; P = 0.00024), findings with important clinical implications. Inhibitor patients with poor prognostic features should be treated with a high-dose protocol. This conclusion is supported by a meta-analysis of the International Immune Tolerance Registry and North American Immune Tolerance Registry and by data from Germany showing good success rates with the high-dose, high-frequency Bonn protocol in poor prognosis patients. Type of concentrate also appears to have an influence on ITI success rates in this patient subgroup, with evidence suggesting an advantage for VWF-containing plasma-derived FVIII concentrates over recombinant or VWF-free concentrates. The ongoing prospective

  8. Immune tolerance induction for patients with severe hemophilia A: a critical literature review.

    PubMed

    Franchini, Massimo; Lippi, Giuseppe

    2011-11-01

    The development of inhibitors that neutralize the function of clotting factor VIII (FVIII) is currently the most challenging complication associated with the treatment of hemophilia A as it increases the disease-related morbidity and mortality. Immune tolerance induction (ITI) is the only documented strategy to eradicate persistent inhibitors in severe hemophilia A patients. Several studies have been conducted so far to identify patient- and treatment-related factors associated with greater ITI success. The currently available literature on ITI in hemophilia A will be critically reviewed in this article. In particular, we will focus on the role of the type of FVIII product on ITI outcome by analyzing the clinical and experimental data. PMID:21818664

  9. Clinical and laboratory diagnosis of von Willebrand disease: a synopsis of the 2008 NHLBI/NIH guidelines.

    PubMed

    Nichols, William L; Rick, Margaret E; Ortel, Thomas L; Montgomery, Robert R; Sadler, J Evan; Yawn, Barbara P; James, Andra H; Hultin, Mae B; Manco-Johnson, Marilyn J; Weinstein, Mark

    2009-06-01

    Von Willebrand factor (VWF) mediates blood platelet adhesion and accumulation at sites of blood vessel injury, and also carries coagulation factor VIII (FVIII) that is important for generating procoagulant activity. Von Willebrand disease (VWD), the most common inherited bleeding disorder, affects males and females, and reflects deficiency or defects of VWF that may also cause decreased FVIII. It may also occur less commonly as an acquired disorder (acquired von Willebrand syndrome). This article briefly summarizes selected features of the March 2008 evidence-based clinical and laboratory diagnostic recommendations from the National Heart, Lung, and Blood Institute (NHLBI) Expert Panel for assessment for VWD or other bleeding disorders or risks. Management of VWD is also addressed in the NHLBI guidelines, but is not summarized here. The VWD guidelines are available at the NHLBI Web site (http://www.nhlbi.nih.gov/guidelines/vwd). PMID:19415721

  10. Distribution of Lunar craters according to morphology from Ranger VIII and IX photographs

    USGS Publications Warehouse

    Trask, N.J.

    1967-01-01

    A classification of the craters photographed by the Ranger VIII and IX missions into four categories according to relative sharpness shows that at diameters of 100 meters, the predominant craters have broad rims and low depth-diameter ratios and are partly covered with smaller craters which generally have sharper rims and higher depth-diameter ratios but include all classes. If competing processes of crater formation and destruction are responsible for the mix of crater types observed, an abrupt increase in the proportion of sharp craters suggests an intense episode of crater destruction to produce a smoothed surface that was subsequently recratered. Data comparable to those presented can readily be obtained from Lunar Orbiter photographs to determine if an increase in the proportion of sharp craters is present over more extensive areas and to test various cratering histories that may account for it. ?? 1967.

  11. Adaptive evolution of cytochrome c oxidase subunit VIII in anthropoid primates

    PubMed Central

    Goldberg, Allon; Wildman, Derek E.; Schmidt, Timothy R.; Hüttemann, Maik; Goodman, Morris; Weiss, Mark L.; Grossman, Lawrence I.

    2003-01-01

    Cytochrome c oxidase (COX) is a 13-subunit protein complex that catalyzes the last step in mitochondrial electron transfer in mammals. Of the 10 subunits encoded by nuclear DNA (three are mtDNA products), some are expressed as tissue- and/or development-specific isoforms. For COX subunit VIII, previous work showed that expression of the contractile muscle-specific isoform gene, COX8H, is absent in humans and Old World monkeys, and the other isoform gene, COX8L, is expressed ubiquitously. Here, we show that COX8H is transcribed in most primate clades, but its expression is absent in catarrhines, that is, in Old World monkeys and hominids (apes, including humans), having become a pseudogene in the stem of the catarrhines. The ubiquitously expressed isoform, COX8L, underwent nonsynonymous rate acceleration and elevation in the ratio of nonsynonymous/synonymous changes in the stem of anthropoid primates (New World monkeys and catarrhines), possibly setting the stage for loss of the heart-type (H) isoform. The most rapidly evolving region of VIII-L is one that interacts with COX I, suggesting that the changes are functionally coadaptive. Because accelerated rates of nonsynonymous substitutions in anthropoids such as observed for COX8L are also shown by genes for at least 13 other electron transport chain components, these encoded amino acid replacements may be viewed as part of a series of coadaptive changes that optimized the anthropoid biochemical machinery for aerobic energy metabolism. We argue that these changes were linked to the evolution of an expanded neocortex in anthropoid primates. PMID:12716970

  12. Treatment of acquired von Willebrand syndrome in patients with monoclonal gammopathy of uncertain significance: comparison of three different therapeutic approaches.

    PubMed

    Federici, A B; Stabile, F; Castaman, G; Canciani, M T; Mannucci, P M

    1998-10-15

    Patients with monoclonal gammopathies of uncertain significance (MGUS) may develop an acquired bleeding disorder similar to congenital von Willebrand disease, called acquired von Willebrand syndrome (AvWS). In these patients, measures to improve hemostasis are required to prevent or treat bleeding episodes. We diagnosed 10 patients with MGUS and AvWS: 8 had IgGkappa (3) or lambda (5) MGUS and 2 IgM-kappa MGUS. Three therapeutic approaches were compared in them: (1) desmopressin (DDAVP), (2) factor VIII/von Willebrand factor (FVIII/vWF) concentrate, and (3) high-dose (1 g/kg/d for 2 days) intravenous Ig (IVIg). In patients with IgG-MGUS, DDAVP and FVIII/vWF concentrate increased factor VIII and von Willebrand factor in plasma, but only transiently. IVIg determined a more sustained improvement of the laboratory abnormalities and prevented bleeding during surgery (short-term therapy). In addition to the standard 2-day infusion protocol, a long-term IVIg therapy was performed in 2 patients with IgG-MGUS: repeated (every 21 days) single infusions of IVIg did improve laboratory abnormalities and stopped chronic gastrointestinal bleeding. On the other hand, IVIg failed to correct laboratories abnormalities in patients with IgM-MGUS. These comparative data obtained in a relative large and homogeneous group of patients with AvWS and MGUS confirm that DDAVP and FVIII/vWF concentrates improve the bleeding time (BT) and FVIII/vWF measurements only transiently, whereas IVIg provides a sustained treatment of AvWS associated with IgG-MGUS, but not with IgM-MGUS. PMID:9763553

  13. The burden of inhibitors in haemophilia patients.

    PubMed

    Walsh, Christopher E; Jiménez-Yuste, Víctor; Auerswald, Guenter; Grancha, Salvador

    2016-08-31

    The burden of disease in haemophilia patients has wide ranging implications for the family and to society. There is evidence that having a current inhibitor increases the risk of morbidity and mortality. Morbidity is increased by the inability to treat adequately and its consequent disabilities, which then equates to a poor quality of life compared with non-inhibitor patients. The societal cost of care, or `burden of inhibitors', increases with the ongoing presence of an inhibitor. Therefore, it is clear that successful eradication of inhibitors by immune tolerance induction (ITI) is the single most important milestone one can achieve in an inhibitor patient. The type of factor VIII (FVIII) product used in ITI regimens varies worldwide. Despite ongoing debate, there is in vitro and retrospective clinical evidence to support the use of plasma-derived VWF-containing FVIII concentrates in ITI regimens in order to achieve early and high inhibitor eradication success rates. PMID:27528280

  14. The influence of riboflavin photochemistry on plasma coagulation factors

    PubMed Central

    Larrea, Luis; Calabuig, María; Roldán, Vanesa; Rivera, José; Tsai, Han-Mou; Vicente, Vicente; Roig, Roberto

    2011-01-01

    Studies with riboflavin in the 1960s showed that it could be effective at inactivating pathogens when exposed to light. The principal mode of action is through electron transfer reactions, most importantly in nucleic acids. This suggested that it could act as a photosensitizer useful in the inactivation of pathogens found in blood products. Objective To study the influence of photo-inactivation with riboflavin on the coagulation factors of plasma. Methods The photo-inactivation procedure of riboflavin plus light was applied. Fifty isogroup pools of two plasmas were made from 100 U of plasma that were derived from whole blood products that had previously been held overnight. Pools were split into two bags. One of them was photo-inactivated, and post inactivation samples were obtained. The second bag was not photo-inactivated and samples were taken. Total protein, fibrinogen, FII, FV, FVII, FVIII, FIX, FX, FXI, FXIII, antithrombin III, PC, PS, α-2 antiplasmin and vWF:Ag, the multimeric structure of vWF and ADAMTS-13 were analyzed. Results In plasma, the proteins most sensitive to photo-inactivation were fibrinogen, FXI, FVIII, FV, and FIX (33%, 32%, 30%, 18% and 18% loss, respectively). Coagulation inhibitors, PS, antithrombin III and PC showed little decrease (all 2%). Retention of vWF and ADAMTS-13 were 99% and 88%, respectively. Conclusions As with other pathogen reduction procedures for plasma products, treatment with riboflavin and UV light resulted in reduction in the activity levels of several pro-coagulant factors. Coagulation inhibitors are well preserved. PMID:19782644

  15. Characterization of the Glycosyltransferase DesVII and Its Auxiliary Partner Protein DesVIII in the Methymycin/Pikromycin Biosynthetic Pathway†

    PubMed Central

    Borisova, Svetlana A.; Liu, Hung-wen

    2010-01-01

    The in vitro characterization of the catalytic activity of DesVII, the glycosyltransferase involved in the biosynthesis of the macrolide antibiotics methymycin, neomethymycin, narbomycin, and pikromycin in Streptomyces venezuelae, is described. DesVII is unique among glycosyltransferases in that it requires an additional protein component, DesVIII, for activity. Characterization of the metabolites produced by a S. venezuelae mutant lacking desVIII gene confirmed that desVIII is important for the biosynthesis of glycosylated macrolides, but can be replaced by at least one of the homologous genes from other pathways. The addition of recombinant DesVIII protein significantly improves the glycosylation efficiency of DesVII in the in vitro assay. When affinity-tagged DesVII and DesVIII proteins were co-produced in E. coli, they formed a tight (αβ)3 complex that is at least 103-fold more active than DesVII alone. The formation of the DesVII/DesVIII complex requires co-expression of both genes in vivo and cannot be fully achieved by mixing the individual protein components in vitro. The ability of DesVII/DesVIII system to catalyze the reverse reaction with the formation of TDP-desosamine was also demonstrated in a transglycosylation experiment. Taken together, our data suggest that DesVIII assists the folding of DesVII during protein production and remains tightly bound during catalysis. This requirement must be taken into consideration in the design of combinatorial biosynthetic experiments of new glycosylated macrolides. PMID:20695498

  16. 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

    2012-08-01

    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. PMID:22735894

  17. Photochemically induced intramolecular six-electron reductive elimination and oxidative addition of nitric oxide by the nitridoosmate(VIII) anion.

    PubMed

    Thornley, Wyatt A; Bitterwolf, Thomas E

    2015-02-01

    UV photolysis of the nitridoosmate(VIII) anion, OsO3 N(-) , in low-temperature frozen matrices results in nitrogen-oxygen bond formation to give the Os(II) nitrosyl complex OsO2 (NO)(-) . Photolysis of the Os(II) nitrosyl product with visible wavelengths results in reversion to the parent Os(VIII) complex. Formally a six-electron reductive elimination and oxidative addition, respectively, this represents the first reported example of such an intramolecular transformation. DFT modelling of this reaction proceeds through a step-wise mechanism taking place through a side-on nitroxyl Os(VI) intermediate, OsO2 (η(2) -NO)(-) . PMID:25537499

  18. 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.

  19. Lithium ionophore VIII as an extraordinarily strong receptor for the trivalent europium cation

    NASA Astrophysics Data System (ADS)

    Makrlík, Emanuel; Novák, Vít; Vaňura, Petr

    2015-01-01

    On the basis of extraction experiments and γ-activity measurements, the extraction constant corresponding to the equilibrium Eu3+(aq) + 3A-(aq) + 1(nb) ⇆ 1.Eu3+(nb) + 3A‑(nb) occurring in the two-phase water-nitrobenzene system (A- = CF3SO-3; 1 = lithium ionophore VIII; aq = aqueous phase, nb = nitrobenzene phase) was determined as log Kex (1.Eu3+, 3A‑) = 2.5 ± 0.1. Furthermore, the extremely high stability constant of the 1.Eu3+ complex in nitrobenzene saturated with water was calculated: log βnb (1.Eu3+) = 15.6 ± 0.1. Finally, by using DFT calculations, the most probable structure of the cationic complex species 1.Eu3+ was derived. In the resulting complex, the 'central' cation Eu3+ is bound by six very strong bond interactions to the corresponding six oxygen atoms of the parent ligand 1. It is evident that this exceptionally effective receptor 1 for the Eu3+ cation could be considered as a potential extraction agent for nuclear waste treatment.

  20. Lithium ionophore VIII as an extraordinarily effective receptor for the strontium cation: Experimental and theoretical study

    NASA Astrophysics Data System (ADS)

    Makrlík, Emanuel; Novák, Vít; Vaňura, Petr; Bouř, Petr

    2014-03-01

    From extraction experiments and γ-activity measurements, the extraction constant corresponding to the equilibrium Sr2+(aq) + 2A-(aq) + 1(nb) ⇔ 1ṡSr2+(nb) + 2A-(nb) taking place in the two-phase water-nitrobenzene system (A- = picrate, 1 = lithium ionophore VIII; aq = aqueous phase, nb = nitrobenzene phase) was evaluated as log Kex (1ṡSr2+, 2A-) = 7.8 ± 0.1. Further, the extremely high stability constant of the 1ṡSr2+ complex in nitrobenzene saturated with water was calculated for a temperature of 25 °C: log βnb (1ṡSr2+) = 16.9 ± 0.1. Finally, by using DFT calculations, the most probable structure of the cationic complex species 1ṡSr2+ was derived. In the resulting complex, the "central" cation Sr2+ is bound by six very strong bond interactions to the corresponding six oxygen atoms of the parent ligand 1; the interaction energy of this complex was found to be -959.9 kJ/mol.

  1. Identification of 31 novel mutations in the F8 gene in Spanish hemophilia A patients: structural analysis of 20 missense mutations suggests new intermolecular binding sites.

    PubMed

    Venceslá, Adoración; Corral-Rodríguez, María Angeles; Baena, Manel; Cornet, Mónica; Domènech, Montserrat; Baiget, Montserrat; Fuentes-Prior, Pablo; Tizzano, Eduardo F

    2008-04-01

    Hemophilia A (HA) is an X-linked bleeding disorder caused by a wide variety of mutations in the factor 8 (F8) gene, leading to absent or deficient factor VIII (FVIII). We analyzed the F8 gene of 267 unrelated Spanish patients with HA. After excluding patients with the common intron-1 and intron-22 inversions and large deletions, we detected 137 individuals with small mutations, 31 of which had not been reported previously. Eleven of these were nonsense, frameshift, and splicing mutations, whereas 20 were missense changes. We assessed the impact of the 20 substitutions based on currently available information about FV and FVIII structure and function relationship, including previously reported results of replacements at these and topologically equivalent positions. Although most changes are likely to cause gross structural perturbations and concomitant cofactor instability, p.Ala375Ser is predicted to affect cofactor activation. Finally, 3 further mutations (p.Pro64Arg, p.Gly494Val, and p.Asp2267Gly) appear to affect cofactor interactions with its carrier protein, von Willebrand factor, with the scavenger receptor low-density lipoprotein receptor-related protein (LRP), and/or with the substrate of the FVIIIapi*FIXa (Xase) complex, factor X. Characterization of these novel mutations is important for adequate genetic counseling in HA families, but also contributes to a better understanding of FVIII structure-function relationship. PMID:18184865

  2. Identification of 31 novel mutations in the F8 gene in Spanish hemophilia A patients: structural analysis of 20 missense mutations suggests new intermolecular binding sites

    PubMed Central

    Venceslá, Adoración; Corral-Rodríguez, María Ángeles; Baena, Manel; Cornet, Mónica; Domènech, Montserrat; Baiget, Montserrat; Fuentes-Prior, Pablo

    2008-01-01

    Hemophilia A (HA) is an X-linked bleeding disorder caused by a wide variety of mutations in the factor 8 (F8) gene, leading to absent or deficient factor VIII (FVIII). We analyzed the F8 gene of 267 unrelated Spanish patients with HA. After excluding patients with the common intron-1 and intron-22 inversions and large deletions, we detected 137 individuals with small mutations, 31 of which had not been reported previously. Eleven of these were nonsense, frameshift, and splicing mutations, whereas 20 were missense changes. We assessed the impact of the 20 substitutions based on currently available information about FV and FVIII structure and function relationship, including previously reported results of replacements at these and topologically equivalent positions. Although most changes are likely to cause gross structural perturbations and concomitant cofactor instability, p.Ala375Ser is predicted to affect cofactor activation. Finally, 3 further mutations (p.Pro64Arg, p.Gly494Val, and p.Asp2267Gly) appear to affect cofactor interactions with its carrier protein, von Willebrand factor, with the scavenger receptor low-density lipoprotein receptor–related protein (LRP), and/or with the substrate of the FVIIIapi•FIXa (Xase) complex, factor X. Characterization of these novel mutations is important for adequate genetic counseling in HA families, but also contributes to a better understanding of FVIII structure-function relationship. PMID:18184865

  3. Tailoring hemostatic therapies to lower inhibitor development in previously untreated patients with severe hemophilia A.

    PubMed

    Mannucci, P M; Mancuso, M E; Franchini, M

    2016-07-01

    After technological progress provided safer therapeutic products for patients with hemophilia A, the development of alloantibodies (inhibitors) neutralizing the coagulant activity of infused factor VIII (FVIII) remains the most serious complication of replacement therapy, predisposing patients to greater morbidity and causing higher treatment costs. The pathogenesis of inhibitors, which develop at a high rate in previously untreated children with severe hemophilia A, is multifactorial, resulting from complex interactions between genetic and environmental factors. Among non-genetic determinants, a key role is played by treatment-related factors, including the source of FVIII product (i.e., plasma derived or recombinant) and the mode of replacement therapy delivery (i.e., intensity, prophylaxis vs. on demand). We review the potential interventions on these modifiable factors that may help to lower the rate of inhibitor development. In addition, interest is currently directed toward the potential for lesser immunogenicity of novel hemostatic agents designed to decrease the dosing frequency or avoid/delay the need of FVIII replacement therapy. PMID:27155314

  4. Development of a reliable analytical method for the precise extractive spectrophotometric determination of osmium(VIII) with 2-nitrobenzaldehydethiocarbohydrazone: Analysis of alloys and real sample.

    PubMed

    Zanje, Sunil B; Kokare, Arjun N; Suryavanshi, Vishal J; Waghmode, Duryodhan P; Joshi, Sunil S; Anuse, Mansing A

    2016-12-01

    The proposed method demonstrates that the osmium(VIII) forms complex with 2-NBATCH from 0.8molL(-1) HCl at room temperature. The complex formed was extracted in 10mL of chloroform with a 5min equilibration time. The absorbance of the red colored complex was measured at 440nm against the reagent blank. The Beer's law was obeyed in the range of 5-25μgmL(-1), the optimum concentration range was 10-20μgmL(-1) of osmium(VIII) as evaluated by Ringbom's plot. Molar absorptivity and Sandell's sensitivity of osmium(VIII)-2NBATCH complex in chloroform is 8.94×10(3)Lmol(-1)cm(-1) and 0.021μgcm(-2), respectively. The composition of osmium(VIII)-2NBATCH complex was 1:2 investigated from Job's method of continuous variation, Mole ratio method and slope ratio method. The interference of diverse ions was studied and masking agents were used wherever necessary. The present method was successfully applied for determination of osmium(VIII) from binary, ternary and synthetic mixtures corresponding to alloys and real samples. The validity of the method was confirmed by finding the relative standard deviation for five determinations which was 0.29%. PMID:27380306

  5. Indication of increase of the lecithin/sphingomyelin (L/S) ratio in lung fluid of lambs maternally treated with metabolite VIII of Bisolvon.

    PubMed

    Egberts, J; Fontijne, P; Wamsteker, K

    1976-01-01

    Metabolite VIII of Bisolvon (Met. VIII) was administered to six pregnant ewes (i.m. daily 5 or 10 mg/kg for 7-9 days). The treatment was started at the 116th/117th or 117th/118th day of gestation and in one case the drug was given to the fetus (i.v. 2 mg/ml/h). Fetal lung fluid obtained from chronically implanted catheters was used for the estimation of the lecithin/sphingomyelin ratio. The maternal treatment with Met. VIII resulted in a significantly increased growth of the L/S ratio compared to the control group (n=4). In three lambs a sharp increase of the L/S ratio was noticed during the last days of Met. VIII treatment. The time gap between first treatment and noticeable effect was 6-8 days. It is postulated that the noticed increase of L/S ratio is the consequence of Met. VIII treatment and very unlikely the result of a release of glucocorticoticoids. PMID:974190

  6. Novel repair activities of AlkA (3-methyladenine DNA glycosylase II) and endonuclease VIII for xanthine and oxanine, guanine lesions induced by nitric oxide and nitrous acid

    PubMed Central

    Terato, Hiroaki; Masaoka, Aya; Asagoshi, Kenjiro; Honsho, Akiko; Ohyama, Yoshihiko; Suzuki, Toshinori; Yamada, Masaki; Makino, Keisuke; Yamamoto, Kazuo; Ide, Hiroshi

    2002-01-01

    Nitrosation of guanine in DNA by nitrogen oxides such as nitric oxide (NO) and nitrous acid leads to formation of xanthine (Xan) and oxanine (Oxa), potentially cytotoxic and mutagenic lesions. In the present study, we have examined the repair capacity of DNA N-glycosylases from Escherichia coli for Xan and Oxa. The nicking assay with the defined substrates containing Xan and Oxa revealed that AlkA [in combination with endonuclease (Endo) IV] and Endo VIII recognized Xan in the tested enzymes. The activity (Vmax/Km) of AlkA for Xan was 5-fold lower than that for 7-methylguanine, and that of Endo VIII was 50-fold lower than that for thymine glycol. The activity of AlkA and Endo VIII for Xan was further substantiated by the release of [3H]Xan from the substrate. The treatment of E.coli with N-methyl-N′-nitro-N-nitrosoguanidine increased the Xan-excising activity in the cell extract from alkA+ but not alkA– strains. The alkA and nei (the Endo VIII gene) double mutant, but not the single mutants, exhibited increased sensitivity to nitrous acid relative to the wild type strain. AlkA and Endo VIII also exhibited excision activity for Oxa, but the activity was much lower than that for Xan. PMID:12434002

  7. Effect of V/III ratio on the growth of (11 2 bar 2) AlGaN by metalorganic vapour phase epitaxy

    NASA Astrophysics Data System (ADS)

    Dinh, Duc V.; Alam, S. N.; Parbrook, P. J.

    2016-02-01

    The effect of V/III ratio on the growth and properties of AlGaN layers grown on (112bar2) AlN templates grown on (101bar0) sapphire by metalorganic vapour phase epitaxy was studied. The surface morphology of the (112bar2) AlGaN layers and the (112bar2) AlN templates showed an undulation along [ 1 1 bar 00 ] AlGaN , AlN. The Al-content and thickness of the layers increased with decreasing V/III ratio due to a reduction in the parasitic reactions of the precursors. The Al-content of the (112bar2) layers was found to be in the range of 29.5-47.9%, which is lower than the composition of the simultaneously grown (0001) reference layers (30.4-58.0%). This was attributed to a higher density of cation (nitrogen) dangling bonds on the (112bar2) surface. Low temperature photoluminescence measurements of the (112bar2) layers showed an emission wavelength that shifts gradually from 273 nm to 306 nm with increasing V/III ratio. A decreased PL intensity of the layers with decreasing V/III ratio was attributed to an increase in cation vacancies. The Stokes-shift of the (112bar2) layers was estimated to be about 60-194 meV, and this shift increases with increasing Al-content (decreasing V/III ratio) correlated to an increased exciton localization.

  8. ABO blood group and von Willebrand factor: biological implications.

    PubMed

    Franchini, Massimo; Crestani, Silvia; Frattini, Francesco; Sissa, Cinzia; Bonfanti, Carlo

    2014-09-01

    ABO blood group antigens are complex carbohydrate molecules expressed on the surface of red blood cells and a variety of human cells and tissues. It is well known that ABO blood type exerts a profound influence on hemostasis, being a major determinant of von Willebrand factor (VWF), and consequently factor VIII, plasma levels. In this review, we will focus on the molecular mechanisms underlying the interaction between ABO blood group and VWF in normal and pathological conditions. PMID:24945431

  9. Love and/in psychoanalysis: a commentary on Lacan's reading of Plato's Symposium in Seminar VIII: Transference.

    PubMed

    Fink, Bruce

    2015-02-01

    What is love and what part does it play in psychoanalysis? Where are the analyst and the analysand situated in relation to the roles defined as those of the "lover" and the "beloved"? Jacques Lacan explores these and other questions in his soon-to-be-published Seminar VIII: Transference by providing an extensive commentary on Plato's most famous dialogue on love, the Symposium. This paper outlines some of the major points about love that grow out of Lacan's reading of the dialogue and examines their relevance to the analytic setting. Can the analyst be characterized as a sort of modern-day Socrates? PMID:25688680

  10. Wilate use in 47 children with von Willebrand disease: the North London paediatric haemophilia network experience.

    PubMed

    Khair, K; Batty, P; Riat, R; Bowles, L; Burgess, C; Chen, Y-H; Hart, D; Platton, S; Pasi, J; Liesner, R

    2015-01-01

    Children with von Willebrand disease (VWD) in whom DDAVP is ineffective or contraindicated require treatment with a coagulation factor concentrate containing von Willebrand factor (VWF) and factor VIII (FVIII). The aim of this study was to monitor the safety, efficacy and tolerability of Wilate(®) (a VWF:FVIII concentrate with a 1:1 ratio) used across the North London Paediatric Haemophilia Network since May 2010. In total, 47 children (aged 0.0-17.0 years) with type 1 (n = 28), type 2 (n = 7), type 3 (n = 10) and acquired VWS (n = 2) have been treated for bleeds, surgery and/or prophylaxis using 260 000 IU Wilate(®). Analysis of dose and frequency of treatment show expected responses to treatment with mean doses of 55, 50 and 50 IU kg(-1) for bleeds, surgery and prophylaxis respectively. Most bleeds responded to a single treatment. Surgical procedures were covered with clinician approved dosing schedules with 95% (39/41) reported as having excellent or good efficacy. There was no accumulation of FVIII or VWF and no thromboembolic events. This case series confirms the efficacy, safety and tolerability of Wilate(®) in neonates, children and adolescents when used on-demand, prophylactically and in the surgical setting. PMID:25112927

  11. Acquired von Willebrand syndrome: an underdiagnosed and misdiagnosed bleeding complication in patients with lymphoproliferative and myeloproliferative disorders.

    PubMed

    Federici, Augusto B

    2006-01-01

    Acquired von Willebrand syndrome (AVWS) is a rare bleeding disorder with laboratory findings similar to those for congenital von Willebrand disease (VWD). Unlike the congenital disease, AVWS usually occurs in individuals with no personal or family history of bleeding. The prevalence of AVWS in the general population is unknown because data from large prospective studies of this syndrome are not available. Although AVWS is particularly frequent in lymphoproliferative or myeloproliferative disorders, it can also be associated with solid tumors, immunologic and cardiovascular disorders, and other miscellaneous conditions. Diagnosis of AVWS is based on assays measuring the activity of von Willebrand factor (VWF). This tends to be abnormally low, but factor VIII (FVIII) coagulant activity can sometimes be normal. FVIII/VWF inhibiting activity is found in only a minority of cases. Bleeding episodes in patients with AVWS are mostly of the mucocutaneous type and can be managed with desmopressin, plasma-derived FVIII/VWF concentrates, and intravenous immunoglobulin (IVIg). Recombinant activated factor VII can be useful in patients unresponsive to standard therapy. An updated version of the International Registry on AVWS, recently available online, will provide more information on this rare, but underdiagnosed and misdiagnosed, disorder. PMID:16427386

  12. Genetic tracing of arterial graft flow surface endothelialization in allogeneic marrow transplanted dogs.

    PubMed

    Shi, Q; Wu, M H; Fujita, Y; Ishida, A; Wijelath, E S; Hammond, W P; Wechezak, A R; Yu, C; Storb, R F; Sauvage, L R

    1999-01-01

    In order to trace genetically the source of fallout endothelialization on arterial grafts, six beagle dogs with successful autologous bone marrow transplantation received composite tandem aortic grafts with an isolated, totally impervious Dacron graft and a porous Dacron graft for 12 weeks. For impervious segments, five of 12 fresh tissue samples were Factor VIII/von Willebrand factor + (FVIII/vWF) and seven had faint or negative signals; three of the FVIII/vWF + samples had alpha-actin + smooth muscle cells. Polymerase chain reaction (PCR) study showed eight had a pure donor DNA genotype and four had donor/host mixed, with the donor predominant. Of 12 AgNO3-stained samples, 11 showed pure donor type and one had donor/host mixed, with the donor predominant. For porous segments, all 12 fresh samples had positive flow surface FVIII/vWF and alpha-actin cells. PCR showed all these samples and all 12 AgNO3-stained samples had donor/host mixed type, but the host pattern was predominant. Porous graft healing appears to involve both cellular fallout and tissue ingrowth, and bone-marrow-derived cells may be a source for fallout. PMID:10073768

  13. Successful Phenotype Improvement following Gene Therapy for Severe Hemophilia A in Privately Owned Dogs

    PubMed Central

    Callan, Mary Beth; Haskins, Mark E.; Wang, Ping; Zhou, Shangzhen; High, Katherine A.; Arruda, Valder R.

    2016-01-01

    Severe hemophilia A (HA) is an inherited bleeding disorder characterized by <1% of residual factor VIII (FVIII) clotting activity. The disease affects several mammals including dogs, and, like humans, is associated with high morbidity and mortality. In gene therapy using adeno-associated viral (AAV) vectors, the canine model has been one of the best predictors of the therapeutic dose tested in clinical trials for hemophilia B (factor IX deficiency) and other genetic diseases, such as congenital blindness. Here we report our experience with liver gene therapy with AAV-FVIII in two outbred, privately owned dogs with severe HA that resulted in sustained expression of 1–2% of normal FVIII levels and prevented 90% of expected bleeding episodes. A Thr62Met mutation in the F8 gene was identified in one dog. These data recapitulate the improvement of the disease phenotype in research animals, and in humans, with AAV liver gene therapy for hemophilia B. Our experience is a novel example of the benefits of a relevant preclinical canine model to facilitate both translational studies in humans and improved welfare of privately owned dogs. PMID:27011017

  14. Successful Phenotype Improvement following Gene Therapy for Severe Hemophilia A in Privately Owned Dogs.

    PubMed

    Callan, Mary Beth; Haskins, Mark E; Wang, Ping; Zhou, Shangzhen; High, Katherine A; Arruda, Valder R

    2016-01-01

    Severe hemophilia A (HA) is an inherited bleeding disorder characterized by <1% of residual factor VIII (FVIII) clotting activity. The disease affects several mammals including dogs, and, like humans, is associated with high morbidity and mortality. In gene therapy using adeno-associated viral (AAV) vectors, the canine model has been one of the best predictors of the therapeutic dose tested in clinical trials for hemophilia B (factor IX deficiency) and other genetic diseases, such as congenital blindness. Here we report our experience with liver gene therapy with AAV-FVIII in two outbred, privately owned dogs with severe HA that resulted in sustained expression of 1-2% of normal FVIII levels and prevented 90% of expected bleeding episodes. A Thr62Met mutation in the F8 gene was identified in one dog. These data recapitulate the improvement of the disease phenotype in research animals, and in humans, with AAV liver gene therapy for hemophilia B. Our experience is a novel example of the benefits of a relevant preclinical canine model to facilitate both translational studies in humans and improved welfare of privately owned dogs. PMID:27011017

  15. A Cross-sectional Study of Bleeding Phenotype in Haemophilia A Carriers

    PubMed Central

    Paroskie, Allison; Gailani, Dave; DeBaun, Michael R.; Sidonio, Robert F.

    2015-01-01

    Summary Haemophilia A carriers have historically been thought to demonstrate normal haemostasis. However, recent data demonstrates that despite normal factor VIII, haemophilia A carriers demonstrate an increased bleeding tendency. We tested the hypothesis that obligate haemophilia carriers demonstrate an increase in bleeding symptoms. A cross sectional study was performed comparing haemophilia A carriers to normal women. Questionnaire assessment included a general bleeding questionnaire, condensed MCMDM-1VWD bleeding assessment tool and Pictorial Bleeding Assessment Chart (PBAC). Laboratory assessment included complete blood count, prothrombin time, activated partial thromboplastin time, fibrinogen activity, FVIII activity (FVIII:C), von Willebrand factor antigen level, ristocetin cofactor, platelet function analyser-100™ and ABO blood type. 44 haemophilia A carriers and 43 controls were included. Demographic features were similar. Laboratory results demonstrated a statistically significant difference only in FVIII:C (82.5 versus 134%, p value < 0.001). Carriers reported a higher number of bleeding events, and both condensed MCMDM-1 VWD bleeding scores (5 versus 1, p value < 0.001) and PBAC scores (423 versus 182.5, p value = 0.018) were significantly higher in carriers. Haemophilia A carriers exhibit increased bleeding symptoms when compared to normal women. Further studies are necessary to fully understand the bleeding phenotype in this population and optimize clinical management. PMID:25832012

  16. Proceedings of the EMU Conference on Foreign Languages for Business and the Professions (Dearborn, Michigan, April 5-7, 1984). Part VIII: Spanish for Business and the Professions.

    ERIC Educational Resources Information Center

    Voght, Geoffrey M., Ed.

    Part VIII of the proceedings includes 16 presentations. They are: "Strengthening Internationalism through the Establishment of a Center for Spanish Language Training for Engineering Students" (David C. Kraft, Pamela J. Madl, Robert C. Spires, and Rusty McClanahan); "Training Court Interpreters: A Practitioner's Perspective" (Linda E. Haughton);…

  17. Revising and Extending Title VIII of the Public Health Service Act. Conference Report to Accompany S. 2574. House of Representatives, 98th Congress, Second Session.

    ERIC Educational Resources Information Center

    Congress of the U.S., Washington, DC. House.

    Revisions and extensions of Title VIII of the Public Health Service Act are presented in this conference report of the U.S. House of Representatives. It is proposed that the Act be cited as the "Public Health Service Act Amendments of 1984." The texts of proposed amendments, including revisions of the language of the legislation, are presented for…

  18. 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

    ... Code (incorporated by reference, see 46 CFR 54.01-1), as limited, modified, or replaced by specific... 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...

  19. 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

    ... Code (incorporated by reference, see 46 CFR 54.01-1), as limited, modified, or replaced by specific... 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...

  20. 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

    ... Code (incorporated by reference, see 46 CFR 54.01-1), as limited, modified, or replaced by specific... 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...

  1. 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

    ... Code (incorporated by reference, see 46 CFR 54.01-1), as limited, modified, or replaced by specific... 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...

  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

    ... Code (incorporated by reference, see 46 CFR 54.01-1), as limited, modified, or replaced by specific... 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...

  3. 75 FR 33760 - Notice of a New Fee Site; Federal Lands Recreation Enhancement Act (Title VIII, Pub. L. 108-447...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-15

    .... Anthony Erba, Chequamegon-Nicolet National, Acting Forest Supervisor. BILLING CODE 3410-11-P ... Forest Service Notice of a New Fee Site; Federal Lands Recreation Enhancement Act (Title VIII, Pub. L. 108-447); Chequamegon-Nicolet National Forest AGENCY: Forest Service, USDA. ACTION: Notice....

  4. 75 FR 33761 - Notice of a New Fee Site; Federal Lands Recreation Enhancement Act (Title VIII, Pub. L. 108-447)

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-15

    ... Forest Service Notice of a New Fee Site; Federal Lands Recreation Enhancement Act (Title VIII, Pub. L. 108-447) AGENCY: USDA Forest Service, Chequamegon-Nicolet National Forest USDA. ACTION: Notice. SUMMARY: The Chequamegon-Nicolet National Forest is proposing to begin charging a $5.00 daily user fee...

  5. Monte Carlo investigation of the influence of V/III flux ratio on GaAs/GaAs(001) submonolayer epitaxy

    NASA Astrophysics Data System (ADS)

    Ageev, O. A.; Solodovnik, M. S.; Balakirev, S. V.; Mikhaylin, I. A.

    2016-07-01

    The influence of the V/III flux ratio on the submonolayer growth of GaAs on the GaAs (001) surface is simulated by the Monte Carlo method. Growth is carried out using the method of molecular beam epitaxy at different values of process parameters. The surface density of islands against the V/III flux ratio is calculated. The saturation value of the surface density at 580°C is found to be 2 × 1012 cm-2, which is in agreement with the experimental data. The V/III flux ratio influences the island density most strongly at a reduced temperature (550°C) and an elevated growth rate (a monolayer per second), when the arsenic desorption is weak. The fraction of arsenic atoms in the growing film is estimated under different process conditions. It has been shown that, as the surface coverage rises, the influence of the V/III flux ratio on the fraction of arsenic atoms becomes weaker.

  6. Is Octavalent Pu(VIII) Possible? Mapping the Plutonium Oxyfluoride Series PuO(n)F(8-2n) (n = 0-4).

    PubMed

    Huang, Wei; Pyykkö, Pekka; Li, Jun

    2015-09-01

    While the oxidation state Pu(VIII) is shown to be less stable than Pu(V) in the PuO4 molecule, it is not clear if the more electronegative fluorine can help to stabilize Pu(VIII). Our calculations on PuO(n)F(8-2n) (n = 0-4) molecules notably confirm that PuO2F4 has both (1)D(4h) and (5)C(2v) minima with the oxidation states Pu(VIII) and Pu(V), respectively, with the latter having lower energy. The hybrid-DFT, CCSD(T), and CASSCF methods all give the same result. The results conform to a superoxide ligand when n ≥ 2. PuF8 in a (1)O(h) state can decompose to PuF6 and F2, and PuOF6 in a (1)C(2v) state also can break down to PuF6 and 1/2 O2. The Pu(VIII) anion PuO2F5(-) does have a D(5h) minimum, which also lies above a (5)C(2v) Pu(V) peroxide structure. However, the energy differences between the different minima are not large, indicating that metastable species with oxidation states higher than Pu(V) cannot be completely excluded. PMID:26309065

  7. Fatal Ruptured Blood Blister-like Aneurysm of Middle Cerebral Artery Associated with Ehlers-Danlos Syndrome Type VIII (Periodontitis Type)

    PubMed Central

    Cıkla, Ulaş; Sadighi, Alireza; Bauer, Andrew; Başkaya, Mustafa K.

    2014-01-01

    Objective To illustrate a unique instance of Ehlers-Danlos syndrome type VIII (EDS VIII) with blood blister–like aneurysm of the middle cerebral artery. Design This is a single patient case report. Setting University of Wisconsin-Madison Hospital. Participants A 42-year-old woman with diagnosis of EDS VIII presented with a sudden onset severe headache and altered mental status. She was diagnosed with Hunt and Hess grade IV subarachnoid hemorrhage. Angiography demonstrated a blood blister–like aneurysm of the left middle cerebral artery. After an unsuccessful coiling attempt in another facility, the patient was operated on with the intention to perform extracranial to intracranial bypass and trapping of the diseased segment of the artery. Results The patient's neurologic condition remained poor after surgery. On postoperative day 2, her neurologic examination unchanged, and care was withdrawn per the family's request. Conclusions Individuals with EDS VIII may be at risk for catastrophic vascular events. PMID:25485215

  8. Ground-satellite observation of Pc 4 pulsations by MAGDAS/CPMN and ETS-VIII geosynchronous orbit satellite

    NASA Astrophysics Data System (ADS)

    Ikeda, A.; Yumoto, K.; Koga, K.; Obara, T.; Baishev, D. G.; Shevtsov, B. M.; Uozumi, T.; Abe, S.; Shishime, A.

    2011-12-01

    Electromagnetic pulsations in ULF range have been studied extensively using ground and satellite observations. However, how Pc 4 pulsations (6.7-22.2 mHz) propagate from the magnetosphere to the ground is not fully understand. Especially the propagation to low latitudes is unclear. We examined data obtained by the ETS-VIII satellite at the geosynchronous orbit (G.M.Lat. -12 degree, G.G.Lon. 146.0 degree) (Koga et al., 2010). We also analyzed ground data of MAGDAS/CPMN (Yumoto and the MAGDAS Group, 2006). The ground data were obtained at high-latitude CHD station (G.M.Lat. 64.9 degree, G.M.Lon. 212.7 degree) and at low-latitude KUJ station (G.M.Lat. 26.1 degree, G.M.Lon. 203.0 degree). The magnetic longitudes of these ground stations are almost same as that of the ETS-VIII. Pc 4 events at ETS-VIII were selected by an automated routine using FFT method which was developed by Takahashi and Ukhorskiy (2007). These Pc 4 events were classified into 2 types. One type is a poloidal Pc 4, in which Hp (northward) component is dominant. Another type is a toroidal mode, in which Hn (eastward) component is dominant. About 10 % of the poloidal/toroidal Pc 4 pulsations, the peak frequency is identical with that of ground Pc 4 pulsations, and the coherence between pulsations observed aboard ETS-VII and on the ground stations is high at the peak frequencies. Thus, about 10 % of the poloidal/toroidal Pc 4 in the magnetosphere can be concluded to transmit to high-latitude ground stations as well as low-latitude stations. For such Pc 4 events, H (horizontal northward) and D (horizontal eastward) components at CHD showed higher amplitude (delta(H)/delta(Hn) = 6.8, delta(H)/delta(Hp) = 10.8, delta(D)/delta(Hn) = 6.8, delta(D)/delta(Hp) = 7.4) than that at the geosynchronous orbit. On the other hand, H and D components at KUJ was attenuated considerably (delta(H)/delta(Hn) = 0.66, delta(H)/delta(Hp) = 0.89, delta(D)/delta(Hn) = 0.35, delta(D)/delta(Hp) = 0.37).

  9. Biosafety Assessment of Site-directed Transgene Integration in Human Umbilical Cord–lining Cells

    PubMed Central

    Sivalingam, Jaichandran; Krishnan, Shruti; Ng, Wai Har; Lee, Sze Sing; Phan, Toan Thang; Kon, Oi Lian

    2010-01-01

    Biosafety and efficacy considerations that impede clinical application of gene therapy could be addressed by nonviral ex vivo cell therapy, utilizing transgenic cells that have been comprehensively pre-evaluated for genotoxic potential and transgene expression. We evaluated the genotoxic potential of phiC31 bacteriophage integrase-mediated transgene integration in cord-lining epithelial cells (CLECs) readily cultured from the outer membrane of human umbilical cords, by sequencing and mapping integration sites, spectral karyotyping, high-resolution genome copy number, transcriptome, and transgene copy number analyses and in vivo tumorigenicity. Of 44 independent integration events, <5% were exonic and 85% of modified cells had integrated ≤2 transgene(s). Expression of 95.6% of genes was unaltered in modified cells. Only three small regions showed genome copy number changes that did not correlate with altered gene expression or integration sites. Spectral karyotyping revealed rare nonrecurrent occurrence of three different translocations. Integrase-modified cells were not tumorigenic in immunocompromised mice for at least 4 months. Stable integration of a human factor VIII (FVIII) construct conferred durable FVIII secretion in vitro. Xenoimplantation of FVIII-secreting CLECs in immunocompetent hemophilic mice achieved significant phenotypic correction. Pre-evaluated clonal populations of phiC31 integrase–modified CLECs could be useful as bioimplants for monogenic diseases such as hemophilia. PMID:20424600

  10. Acquired Hemophilia A: A Frequently Overlooked Autoimmune Hemorrhagic Disorder

    PubMed Central

    Takeda, Tomohiro

    2014-01-01

    Acquired hemophilia A (AHA) is a rare hemorrhagic disease in which autoantibodies against coagulation factor VIII- (FVIII-) neutralizing antibodies (inhibitors) impair the intrinsic coagulation system. As the inhibitors developed in AHA are autoantibodies, the disease may have an autoimmune cause and is often associated with autoimmune disease. Although acute hemorrhage associated with AHA may be fatal and is costly to treat, AHA is often unrecognized or misdiagnosed. AHA should thus be considered in the differential diagnosis particularly in postpartum women and the elderly with bleeding tendency or prolonged activated partial thromboplastin time. Cross-mixing tests and measurement of FVIII-binding antibodies are useful to confirm AHA diagnosis. For treatment of acute hemorrhage, hemostatic therapy with bypassing agents should be provided. Unlike in congenital hemophilia A with inhibitors, in which immune tolerance induction therapy using repetitive infusions of high-dose FVIII concentrates is effective for inhibitor eradication, immune tolerance induction therapy has shown poor efficacy in treating AHA. Immunosuppressive treatment should thus be initiated to eradicate inhibitors as soon as the diagnosis of AHA is confirmed. PMID:24741588

  11. Effects of Mg/Ga and V/III source ratios on hole concentration of N-polar (000\\bar{1}) p-type GaN grown by metalorganic vapor phase epitaxy

    NASA Astrophysics Data System (ADS)

    Nonoda, Ryohei; Shojiki, Kanako; Tanikawa, Tomoyuki; Kuboya, Shigeyuki; Katayama, Ryuji; Matsuoka, Takashi

    2016-05-01

    The effects of growth conditions such as Mg/Ga and V/III ratios on the properties of N-polar (000\\bar{1}) p-type GaN grown by metalorganic vapor phase epitaxy were studied. Photoluminescence spectra from Mg-doped GaN depended on Mg/Ga and V/III ratios. For the lightly doped samples, the band-to-acceptor emission was observed at 3.3 eV and its relative intensity decreased with increasing V/III ratio. For the heavily doped samples, the donor-acceptor pair emission was observed at 2.8 eV and its peak intensity monotonically decreased with V/III ratio. The hole concentration was maximum for the Mg/Ga ratio. This is the same tendency as in group-III polar (0001) growth. The V/III ratio also reduced the hole concentration. The higher V/III ratio reduced the concentration of residual donors such as oxygen by substituting nitrogen atoms. The surface became rougher with increasing V/III ratio and the hillock density increased.

  12. Recombination line intensities for hydrogenic ions. II - Case B calculations for C VI, N VII and O VIII

    NASA Technical Reports Server (NTRS)

    Storey, P. J.; Hummer, D. G.

    1988-01-01

    The intensities of recombination lines formed in extended, optically thim, photoionized plasmas such as those found in PN, H II regions, and winds of certain hot stars are an important source of information on chemical abundances and can sometimes provide estimates of electron temperature. In this paper, the intensities of the recombination lines of C VI, N VII, and O VIII are calculated, accounting for both electron and heavy particle collisions and assuming case B of Baker and Menzel. The computational procedure is explained. The intensities of lines formed by transitions n(u) - n(l) are tabulated for n(u) of 50 or less, n(l) of 29 or less, at log N(e) = 4(1)13 and 10 values of electron temperature in the interval 10,000 K to 500,000 K.

  13. [The beneficial effect of mineral waters of Fiuggi on Pope Bonifacio VIII and Michelangelo: two prominent calculous patients].

    PubMed

    Androutsos, Georges

    2005-09-01

    Going back to the dawn of time, we deal with two historical figures, that of Pope Bonifacio VIII, and that of the hierophant of art, Michelangelo Buonarroti. The texts and the codes of the British Museum (Department of Manuscripts), of the Biblioteca Medica Laurenziana (Archivio Buonarroti) of Florence, and the Biblioteca Apostolica of Vatican, convincing detalled evidence that both figures were urological patients. It concerns two clinical cases with especially pertain to the history of lithiasis of urinary tract. Even though, both of them--the first with nephrolithiasis or gravel, and the second with a bladder stone--were historically bound with the well-known cures of Fiuggi in Italy. PMID:16459705

  14. Factor IX Amagasaki: A new mutation in the catalytic domain resulting in the loss of both coagulant and esterase activities

    SciTech Connect

    Miyata, Toshiyuki; Iwanaga, Sadaaki ); Sakai, Toshiyuki; Sugimoto, Mitsuhiko; Naka, Hiroyuki; Yamamoto, Kazukuni; Yoshioka, Akira; Fukui, Hiromu ); Mitsui, Kotoko; Kamiya, Kensyu; Umeyama, Hideaki )

    1991-11-26

    Factor IX Amagasaki (AMG) is a naturally occurring mutant of factor IX having essentially no coagulant activity, even though normal levels of antigen are detected in plasma. Factor IX AMG was purified from the patient's plasma by immunoaffinity chromatography with an anti-factor IX monoclonal antibody column. Factor IX AMG was cleaved normally by factor VIIa-tissue factor complex, yielding a two-chain factor IXa. Amino acid composition and sequence analysis of one of the tryptic peptides isolated from factor IX AMG revealed that Gly-311 had been replaced by Glu. The authors identified a one-base substitution of guanine to adenine in exon VIII by amplifying exon VIII using the polymerase chain reaction method and sequencing the product. This base mutation also supported the replacement of Gly-311 by Glu. In the purified system, factor IXa AMG did not activate for factor X in the presence of factor VIII, phospholipids, and Ca{sup 2+}, and no esterase activity toward Z-Arg-p-nitrobenzyl ester was observed. The model building of the serine protease domain of factor IXa suggests that the Gly-311 {yields} Glu exchange would disrupt the specific conformational state in the active site environment, resulting in the substrate binding site not forming properly. This is the first report to show the experimental evidence for importance of a highly conserved Gly-142 (chymotrypsinogen numbering) located in the catalytic site of mammalian serine proteases so far known.

  15. Alteration in lymphocyte responses, cytokine and chemokine profiles in chickens infected with genotype VII and VIII velogenic Newcastle disease virus.

    PubMed

    Rasoli, Mehdi; Yeap, Swee Keong; Tan, Sheau Wei; Moeini, Hassan; Ideris, Aini; Bejo, Mohd Hair; Alitheen, Noorjahan Banu Mohamed; Kaiser, Pete; Omar, Abdul Rahman

    2014-01-01

    Newcastle disease (ND) is a highly contagious avian disease and one of the major causes of economic losses in the poultry industry. The emergence of virulent NDV genotypes and repeated outbreaks of NDV in vaccinated chickens have raised the need for fundamental studies on the virus-host interactions. In this study, the profiles of B and T lymphocytes and macrophages and differential expression of 26 immune-related genes in the spleen of specific-pathogen-free (SPF) chickens, infected with either the velogenic genotype VII NDV strain IBS002 or the genotype VIII NDV strain AF2240, were evaluated. A significant reduction in T lymphocyte population and an increase in the infiltration of IgM+ B cells and KUL01+ macrophages were detected in the infected spleens at 1, 3 and 4 days post-infection (dpi) (P<0.05). The gene expression profiles showed an up-regulation of CCLi3, CXCLi1, CXCLi2 (IL-8), IFN-γ, IL-12α, IL-18, IL-1β, IL-6, iNOS, TLR7, MHCI, IL-17F and TNFSF13B (P<0.05). However, these two genotypes showed different cytokine expression patterns and viral load. IBS002 showed higher viral load than AF2240 in spleen at 3 and 4dpi and caused a more rapid up-regulation of CXCLi2, IFN-γ, IL-12α, IL-18, IL-1β, iNOS and IL-10 at 3dpi. Meanwhile, the expression levels of CCLI3, CXCLi1, IFN-γ, IL-12α, IL-1β and iNOS genes were significantly higher in AF2240 at 4dpi. In addition, the expression levels of IL-10 were significantly higher in the IBS002-infected chickens at 3 and 4dpi. Hence, infection with velogenic genotype VII and VIII NDV induced different viral load and production of cytokines and chemokines associated with inflammatory reactions. PMID:24225159

  16. MEASUREMENT OF THE Fe VIII-Fe XVI 3-3 EMISSION IN THE EXTREME ULTRAVIOLET AND COMPARISON WITH CHIANTI

    SciTech Connect

    Beiersdorfer, P.; Lepson, J. K.

    2012-08-01

    Laboratory measurements of the n = 3 to n = 3 emission from M-shell iron ions are presented and compared to synthetic spectra from the CHIANTI spectral model. The measurements cover the range 170-290 Angstrom-Sign and are made at an electron density of about 10{sup 11} cm{sup -3}. Emission from Fe VIII through Fe XVI has been identified. Excellent agreement with CHIANTI predictions is found for most lines. Twenty weaker features are noted in the laboratory data that are either absent in CHIANTI or have recently been added and correspond to lines that have not been verified by experimental measurements. A few of these lines may have already been observed (but not yet identified) in the Sun. The features are attributed to emission from various charge states of iron, notably Fe IX and Fe XIII, and two features have been identified as transitions in Fe VIII, i.e., the 3p{sup 6}3d {sup 2}D{sub 5/2}-3p {sup 5}3d{sup 2} {sup 2}P{sub 3/2} and the 3p {sup 6}3d {sup 2}D{sub 3/2}-3p {sup 5}3d{sup 2} {sup 2}P{sub 1/2} transitions at 225.25 {+-} 0.12 and 226.35 {+-} 0.10 Angstrom-Sign , respectively. Seven lines in Fe XI, Fe XII, and Fe XIII between 200 and 205 Angstrom-Sign are noted for which the wavelengths in the CHIANTI database disagree with those in the current database of the National Institute of Standards and Technology. Our measurements of five of these lines appear to agree with the assignments used in CHIANTI.

  17. The hemostatic role of factor XI.

    PubMed

    Puy, Cristina; Rigg, Rachel A; McCarty, Owen J T

    2016-05-01

    Coagulation factor (F)XI has been described as a component of the early phase of the contact pathway of blood coagulation, acting downstream of factor XII. However, patients deficient in upstream members of the contact pathway, including FXII and prekallikrein, do not exhibit bleeding complications, while FXI-deficient patients sometimes experience mild bleeding, suggesting FXI plays a role in hemostasis independent of the contact pathway. Further complicating the picture, bleeding risk in FXI-deficient patients is difficult to predict because bleeding symptoms have not been found to correlate with FXI antigen levels or activity. However, recent studies have emerged to expand our understanding of FXI, demonstrating that activated FXI is able to activate coagulation factors FX, FV, and FVIII, and inhibit the anti-coagulant tissue factor pathway inhibitor (TFPI). Understanding these activities of FXI may help to better diagnose which FXI-deficient patients are at risk for bleeding. In contrast to its mild hemostatic activities, FXI is known to play a significant role in thrombosis, as it is a demonstrated independent risk factor for deep vein thrombosis, ischemic stroke, and myocardial infarction. Recent translational approaches have begun testing FXI as an antithrombotic, with one promising clinical study showing that an anti-sense oligonucleotide against FXI prevented venous thrombosis in elective knee surgery. A better understanding of the varied and complex role of FXI in both thrombosis and hemostasis will help to allow better prediction of bleeding risk in FXI-deficient patients and also informing the development of targeted agents to inhibit the thrombotic activities of FXI while preserving hemostasis. PMID:27207433

  18. Comprehensive evaluation of haemostatic function in von Willebrand disease patients using a microchip-based flow chamber system.

    PubMed

    Ogiwara, K; Nogami, K; Hosokawa, K; Ohnishi, T; Matsumoto, T; Shima, M

    2015-01-01

    The diagnosis of von Willebrand disease (VWD) is difficult due to the wide spectrum of clinical phenotypes associated with this disorder. We have analysed and characterized haemostatic function in VWD patients using a microchip-based flow chamber system. Microchips coated with either collagen [platelet (PL)-chip] or collagen/thromboplastin [atherome (AR)-chip] were used to evaluate platelet thrombus formation at 1000 s(-1) and fibrin-rich platelet thrombus formation at 240 s(-1) respectively. Blood samples from an asymptomatic patient with VWD type 1 [von Willebrand factor (VWF): RCo 3.2%; bleeding score (BS 2] displayed normal thrombus formation in both PL- and AR-chips, whereas blood from a symptomatic type 1 patient (VWF: RCo 14%, BS 9) had significantly delayed capillary occlusion. Nearly complete suppression of the flow pressure increase was observed in symptomatic patients with VWD type 2A (BS 13) and 2N (BS 27), whereas no flow pressure was found for the type 3 patient (BS 6). Fibrin-rich platelet thrombus formation was only weakly increased by the in vitro addition of factor VIII (FVIII) to blood samples from the type 3 patient, but was normalized by the addition of VWF/FVIII. The in vivo effects of treatment with desmopressin or VWF/FVIII for the symptomatic patients were analysed using two types of microchips. The PL-chip was highly sensitive for patients' VWF-mediated platelet functions, whereas the AR-chip allowed assessment of overall haemostatic ability, including sensitivity to both VWF and FVIII. The combined analysis with PL- and AR-chips may be potentially useful for the diagnosis of VWD based on clinical phenotypes, and for monitoring drug effects. PMID:25545301

  19. Complete cDNA and derived amino acid sequence of human factor V

    SciTech Connect

    Jenny, R.J.; Pittman, D.D.; Toole, J.J.; Kriz, R.W.; Aldape, R.A.; Hewick, R.M.; Kaufman, R.J.; Mann, K.G.

    1987-07-01

    cDNA clones encoding human factor V have been isolated from an oligo(dT)-primed human fetal liver cDNA library prepared with vector Charon 21A. The cDNA sequence of factor V from three overlapping clones includes a 6672-base-pair (bp) coding region, a 90-bp 5' untranslated region, and a 163-bp 3' untranslated region within which is a poly(A)tail. The deduced amino acid sequence consists of 2224 amino acids inclusive of a 28-amino acid leader peptide. Direct comparison with human factor VIII reveals considerable homology between proteins in amino acid sequence and domain structure: a triplicated A domain and duplicated C domain show approx. 40% identity with the corresponding domains in factor VIII. As in factor VIII, the A domains of factor V share approx. 40% amino acid-sequence homology with the three highly conserved domains in ceruloplasmin. The B domain of factor V contains 35 tandem and approx. 9 additional semiconserved repeats of nine amino acids of the form Asp-Leu-Ser-Gln-Thr-Thr/Asn-Leu-Ser-Pro and 2 additional semiconserved repeats of 17 amino acids. Factor V contains 37 potential N-linked glycosylation sites, 25 of which are in the B domain, and a total of 19 cysteine residues.

  20. Benefits of prophylaxis versus on-demand treatment in adolescents and adults with severe haemophilia A: the POTTER study.

    PubMed

    Tagliaferri, Annarita; Feola, Giulio; Molinari, Angelo Claudio; Santoro, Cristina; Rivolta, Gianna Franca; Cultrera, Dorina Bianca; Gagliano, Fabio; Zanon, Ezio; Mancuso, Maria Elisa; Valdré, Lelia; Mameli, Luciana; Amoresano, Susanna; Mathew, Prasad; Coppola, Antonio

    2015-07-01

    Rigorous evidence is lacking on long-term outcomes of factor VIII (FVIII) prophylaxis initiated in adolescent or adult patients with severe haemophilia A. The prospective, open-label Prophylaxis versus On-demand Therapy Through Economic Report (POTTER) study (ClinicalTrials.gov NCT01159587) compared long-term late secondary prophylaxis (recombinant FVIII-FS 20-30 IU/kg thrice weekly) with on-demand treatment in patients aged 12 to 55 years with severe haemophilia A. The annual number of joint bleeding episodes (primary endpoint), total bleeding episodes, orthopaedic and radiologic (Pettersson) scores, health-related quality of life (HRQoL), pharmacoeconomic impact, and safety were evaluated over a > 5-year period (2004-2010). Fifty-eight patients were enrolled at 11 centres in Italy; 53 (27 prophylaxis, 26 on demand) were evaluated and stratified into 2 age subgroups (12-25 and 26-55 years). Patients receiving prophylaxis experienced a significantly lower number of joint bleeding episodes vs the on-demand group (annualised bleeding rate, 1.97 vs 16.80 and 2.46 vs 16.71 in younger and older patients, respectively; p=0.0043). Results were similar for total bleeding episodes. Prophylaxis was associated with significantly fewer target joints (p< 0.001), better orthopaedic (p=0.0019) and Pettersson (p=0.0177) scores, better HRQoL, and fewer days of everyday activities lost (p< 0.0001) but required significantly higher FVIII product consumption. The POTTER study is the first prospective, controlled trial documenting long-term benefits of late secondary prophylaxis in adolescents and adults with severe haemophilia A. The benefits of reduced bleeding frequency, improved joint status, and HRQoL may offset the higher FVIII consumption and costs. PMID:25855376

  1. Recombinant factor VIIa.

    PubMed

    Aitken, Michael G

    2004-01-01

    Human coagulation factor (F) VII is a single chain protease that circulates in the blood as a weakly active zymogen at concentrations of approximately 10 nmol/L. When converted to the active 2 chain form (FVIIa), it is a powerful initiator of haemostasis. Recombinant factor VIIa (rFVIIa, eptacog alfa, NovoSeven) is a genetically engineered product that was first introduced in 1988 for the treatment of patients with haemophilia A and B with high inhibitory antibody titres to factors VIII and IX. Recent reports in the form of case studies and series, and early trial data, have suggested a role for rFVIIa across a diverse range of indications including bleeding associated with trauma, surgery, thrombocytopaenia, liver disease and oral anticoagulant toxicity. This review describes the physiology of the coagulation pathway and in particular the role of recombinant factor VIIa. It will also focus on the emerging role of rFVIIa in both trauma and non-trauma bleeding and its potential use in the ED. PMID:15537408

  2. In pursuit of clean air: a data book of problems and strategies at the state level. Volume 5. Federal Regions VIII, IX, and X

    SciTech Connect

    Garvey, D.B.; Streets, D.G.

    1980-02-01

    The following material is provided for each state in Federal Regions VIII, IX, and X: state title page (lists nonattainment areas for each pollutant, the number of monitors with valid readings for a particular averaging time for a pollutant, and the number of monitors that recorded a violation of the standard); revised State Implementation Plan (SIP) outline (covers sources of the problems, the proposed strategies for achieving attainment, and new state review procedures); maps of nonattainment areas, as designated; SAROAD (Storage and Retrieval of Aerometric Data) data; SAROAD data maps; power plant data; power plant maps; and county maps. States in Federal Region VIII are Colorado, Montana, North Dakota, South Dakota, Utah, and Wyoming. Federal Region IX includes Arizona, California, and Nevada. Federal Region X includes Idaho, Oregon, and Washington. (JGB)

  3. Kinetics and Thermodynamics of Type VIII β-Turn Formation: A CD, NMR, and Microsecond Explicit Molecular Dynamics Study of the GDNP Tetrapeptide

    PubMed Central

    Fuchs, Patrick F. J.; Bonvin, Alexandre M. J. J.; Bochicchio, Brigida; Pepe, Antonietta; Alix, Alain J. P.; Tamburro, Antonio M.

    2006-01-01

    We report an experimental and theoretical study on type VIII β-turn using a designed peptide of sequence GDNP. CD and NMR studies reveal that this peptide exists in equilibrium between type VIII β-turn and extended conformations. Extensive MD simulations give a description of the free energy landscape of the peptide in which we retrieve the same two main conformations suggested by the experiments. The free energy difference between the two conformational states is very small and the transition between them occurs within a few kT at 300 K on a nanosecond timescale. The equilibrium is mainly driven by entropic contribution, which favors extended conformations over β-turns. This confirms other theoretical studies showing that β-turns are marginally stable in water solution because of the larger entropy of the extended state unless some stabilizing interactions exist. Our observations may be extended to any type of β-turn and have important consequences for protein folding. A comparison of our MD and CD results also suggests a possible type VIII β-turn CD signature indicated by one main band at 200 nm, close to that of random coil, and a fairly large shoulder at 220 nm. Last, our results clearly show that the XXXP motif can only fold into a type VIII β-turn, which is consistent with its fairly strong propensity for this type of turn. This important finding may help for peptide design and is in line with recent studies on bioactive elastin peptides. PMID:16443656

  4. Preparation and Thermoelectric Properties of Sn-Based Type VIII Single-Crystalline Clathrate Via a-Sn Flux Method

    NASA Astrophysics Data System (ADS)

    Cheng, Feng; Shen, Lanxian; Li, Decong; Liu, Hongxia; Wang, Jinsong; Deng, Shukang

    2016-06-01

    In this study, type VIII Ba8Ga16Sn30 single-crystal clathrates with different stoichiometric ratios were prepared using the a-Sn self-flux method. The structures of the samples were also investigated. Results indicate that n-/p-type single-crystal clathrates can be obtained by adjusting the initial content of Ga. Samples were determined to be n-type when the initial Ga content was X = 12, 13, and 14; and p-type when X = 16 and 18. The samples exhibited high melting points and large lattice parameters, as well as increasing Ga content. The electrical conductivities of the samples were lower than those prepared using the Sn self-flux method because of the low carrier concentration. The elemental composition of the sample was near the theoretical value when the Ga content was X = 14 and 16, which also yielded the highest Seebeck values of -523 μV/K and 331 V/K, respectively. Finally, the sample with X = 14 and 18 exhibited the highest ZT values of 0.82 and 0.46, respectively, at a temperature of 480 K in n-/p-type.

  5. Preparation and Thermoelectric Properties of Sn-Based Type VIII Single-Crystalline Clathrate Via a-Sn Flux Method

    NASA Astrophysics Data System (ADS)

    Cheng, Feng; Shen, Lanxian; Li, Decong; Liu, Hongxia; Wang, Jinsong; Deng, Shukang

    2016-04-01

    In this study, type VIII Ba8Ga16Sn30 single-crystal clathrates with different stoichiometric ratios were prepared using the a-Sn self-flux method. The structures of the samples were also investigated. Results indicate that n-/p-type single-crystal clathrates can be obtained by adjusting the initial content of Ga. Samples were determined to be n-type when the initial Ga content was X = 12, 13, and 14; and p-type when X = 16 and 18. The samples exhibited high melting points and large lattice parameters, as well as increasing Ga content. The electrical conductivities of the samples were lower than those prepared using the Sn self-flux method because of the low carrier concentration. The elemental composition of the sample was near the theoretical value when the Ga content was X = 14 and 16, which also yielded the highest Seebeck values of -523 μV/K and 331 V/K, respectively. Finally, the sample with X = 14 and 18 exhibited the highest ZT values of 0.82 and 0.46, respectively, at a temperature of 480 K in n-/p-type.

  6. Venom Concentrations and Clotting Factor Levels in a Prospective Cohort of Russell’s Viper Bites with Coagulopathy

    PubMed Central

    Isbister, Geoffrey K.; Maduwage, Kalana; Scorgie, Fiona E.; Shahmy, Seyed; Mohamed, Fahim; Abeysinghe, Chandana; Karunathilake, Harendra; O’Leary, Margaret A.; Gnanathasan, Christeine A.; Lincz, Lisa F.

    2015-01-01

    Background Russell’s viper envenoming is a major problem in South Asia and causes venom induced consumption coagulopathy. This study aimed to investigate the kinetics and dynamics of venom and clotting function in Russell’s viper envenoming. Methodology/Principal Findings In a prospective cohort of 146 patients with Russell’s viper envenoming, we measured venom concentrations, international normalised ratio [INR], prothrombin time (PT), activated partial thromboplastin time (aPTT), coagulation factors I, II, V, VII, VIII, IX and X, and von Willebrand factor antigen. The median age was 39y (16–82y) and 111 were male. The median peak INR was 6.8 (interquartile range[IQR]:3.7 to >13), associated with low fibrinogen [median,<0.01g/L;IQR:<0.01–0.9g/L), low factor V levels [median,<5%;IQR:<5–4%], low factor VIII levels [median,40%;IQR:12–79%] and low factor X levels [median,48%;IQR:29–67%]. There were smaller reductions in factors II, IX and VII over time. All factors recovered over 48h post-antivenom. The median INR remained >3 at 6h post-antivenom but had reduced to <2, by 24h. The aPTT had also returned to close to normal (<50sec) at 24h. Factor VII, VIII and IX levels were unusually high pre-antivenom, median peak concentrations of 393%, 307% and 468% respectively. Pre-antivenom venom concentrations and the INR (r = 0.20, p = 0.02) and aPTT (r = 0.19, p = 0.03) were correlated (non-parametric Spearman analysis). Conclusions Russell’s viper coagulopathy results in prolonged aPTT, INR, low fibrinogen, factors V, VIII and X which recover over 48h. Severity of clotting abnormalities was associated with venom concentrations. PMID:26296235

  7. Research and Evaluations of the Health Aspects of Disasters, Part VIII: Risk, Risk Reduction, Risk Management, and Capacity Building.

    PubMed

    Birnbaum, Marvin L; Loretti, Alessandro; Daily, Elaine K; O'Rourke, Ann P

    2016-06-01

    There is a cascade of risks associated with a hazard evolving into a disaster that consists of the risk that: (1) a hazard will produce an event; (2) an event will cause structural damage; (3) structural damage will create functional damages and needs; (4) needs will create an emergency (require use of the local response capacity); and (5) the needs will overwhelm the local response capacity and result in a disaster (ie, the need for outside assistance). Each step along the continuum/cascade can be characterized by its probability of occurrence and the probability of possible consequences of its occurrence, and each risk is dependent upon the preceding occurrence in the progression from a hazard to a disaster. Risk-reduction measures are interventions (actions) that can be implemented to: (1) decrease the risk that a hazard will manifest as an event; (2) decrease the amounts of structural and functional damages that will result from the event; and/or (3) increase the ability to cope with the damage and respond to the needs that result from an event. Capacity building increases the level of resilience by augmenting the absorbing and/or buffering and/or response capacities of a community-at-risk. Risks for some hazards vary by the context in which they exist and by the Societal System(s) involved. Birnbaum ML , Loretti A , Daily EK , O'Rourke AP . Research and evaluations of the health aspects of disasters, part VIII: risk, risk reduction, risk management, and capacity building. Prehosp Disaster Med. 2016;31(3):300-308. PMID:27025980

  8. A novel missense mutation in the FGB g. 3354 T>A (p. Y41N), Fibrinogen Caracas VIII

    PubMed Central

    Marchi, Rita; Rojas, Héctor; Meyer, Michael; Castillo, Oscar; De Sáez Ruiz, Arlette; Weisel, John W.

    2012-01-01

    Summary A novel dysfibrinogenemia with a replacement of Tyr by Asn at Bβ41 has been discovered (fibrinogen Caracas VIII). An asymptomatic 39 year-old male was diagnosed as having dysfibrinogenemia due to a mildly prolonged thrombin time (+ 5.8 sec); his fibrinogen concentration was in the low normal range, both by Clauss and gravimetric determination, 1.9 g/l and 2.1 g/l, respectively. The plasma polymerization process was slightly impaired, characterized by a mildly prolonged lag time and a slightly increased final turbidity. Permeation through the patients’ clots was dramatically increased, with the Darcy constant around 4 times greater than that of the control (22 ± 2 ×10−9 cm2 compared to 6 ± 0.5 ×10−9 cm2 in controls). The plasma fibrin structure of the patient, by scanning electron microscopy, featured a mesh composed of thick fibers (148 ± 50 nm vs. 120 ± 31 nm in controls, p<0.05) and larger pores than those of the control fibrin clot. The viscoelastic properties of the clot from the patient were also altered, as the storage modulus (G′, 310 ± 30) was much lower than in the control (831 ± 111) (p ≤ 0.005). The interaction of the fibrin clot with a monolayer of human microvascular endothelial cells, by confocal laser microscopy, revealed that the patients’ fibrin network had less interaction with the cells. These results demonstrate the significance of the amino terminal end of the β chain of fibrin in the polymerization process and its consequences on the clot organization on the surface of endothelial cells. PMID:21301788

  9. Effects of carbonic anhydrase-related protein VIII on human cells harbouring an A8344G mitochondrial DNA mutation.

    PubMed

    Wang, Tze-Kai; Cheng, Che-Kun; Chi, Tang-Hao; Ma, Yi-Shing; Wu, Shi-Bei; Wei, Yau-Huei; Hsieh, Mingli

    2014-04-01

    MERRF (myoclonus epilepsy associated with ragged-red fibres) is a maternally inherited mitochondrial encephalomyopathy with various syndromes involving both muscular and nervous systems. The most common mutation in MERRF syndrome, the A8344G mutation in mtDNA, has been associated with severe defects in the respiratory function of mitochondria. In the present study, we show that there is a significant decrease in CA8 (carbonic anhydrase-related protein VIII) in cybrids harbouring the MERRF A8344G mutation. CA8 deficiency and mutations were found to be associated with a distinctive lifelong gait disorder in wdl (Waddles) mice and novel syndromes characterized by cerebellar ataxia and mental retardation in humans. The results of the present study showed that overexpression of CA8 in MERRF cybrids significantly decreased cell death induced by STS (staurosporine) treatment, suggesting a protective function of CA8 in cells harbouring the A8344G mutation of mtDNA. Interestingly, an increase in the formation of LC3-II (microtubule-associated protein 1 light chain 3-II) was found in the cybrids with down-regulated CA8 expression, suggesting that reduced expression of CA8 leads to autophagy activation. Furthermore, cybrids exhibiting down-regulated CA8 showed increased cytosolic Ca2+ signals and reduced levels of phospho-Akt compared with those in the cybrids with overexpressed CA8, indicating that phospho-Akt is involved in the protection of cells by CA8. Our findings suggest that CA8 is involved in the autophagic pathway and may have a protective role in cultured cells from patients with MERRF. Targeting CA8 and the downstream autophagic pathway might help develop therapeutic agents for treatment of MERRF syndrome in the future. PMID:24476000

  10. Platelet and coagulation factors in proliferative diabetic retinopathy.

    PubMed Central

    Borsey, D Q; Prowse, C V; Gray, R S; Dawes, J; James, K; Elton, R A; Clarke, B F

    1984-01-01

    Plasma beta-thromboglobulin, platelet factor 4, fibrinogen, fibrinopeptide A, antithrombin III, factor VIII related antigen, alpha 2-macroglobulin, platelet count, and total glycosylated haemoglobin were measured in three well matched groups of subjects: non-diabetic controls, diabetics without retinopathy, and diabetics with proliferative retinopathy. beta-thromboglobulin and platelet factor 4 concentrations were significantly higher in the diabetics with retinopathy than in the controls and platelet factor 4 was also increased in the diabetics without retinopathy compared with controls. Fibrinogen concentration was raised in diabetics without retinopathy compared with controls, diabetics with retinopathy compared with controls, and diabetics with retinopathy compared with those without. Fibrinopeptide A concentration did not differ significantly between groups. Antithrombin III levels were increased in diabetics with retinopathy compared with controls, and in diabetics with retinopathy compared with those without. Factor VIII related antigen values were higher in both the diabetic groups when compared with the controls. Fibrinopeptide A concentration correlated with both beta-thromboglobulin and platelet factor 4 in each of the three groups. Haemostatic abnormalities in diabetes have been shown, although a hypercoagulable state has not been confirmed. These changes in platelet and coagulation function may be secondary to the development of microvascular disease and their role in the pathogenesis of retinopathy remains uncertain. PMID:6202721

  11. Plasmid pEC156, a Naturally Occurring Escherichia coli Genetic Element That Carries Genes of the EcoVIII Restriction-Modification System, Is Mobilizable among Enterobacteria

    PubMed Central

    Werbowy, Olesia; Kaczorowski, Tadeusz

    2016-01-01

    Type II restriction-modification systems are ubiquitous in prokaryotes. Some of them are present in naturally occurring plasmids, which may facilitate the spread of these systems in bacterial populations by horizontal gene transfer. However, little is known about the routes of their dissemination. As a model to study this, we have chosen an Escherichia coli natural plasmid pEC156 that carries the EcoVIII restriction modification system. The presence of this system as well as the cis-acting cer site involved in resolution of plasmid multimers determines the stable maintenance of pEC156 not only in Escherichia coli but also in other enterobacteria. We have shown that due to the presence of oriT-type F and oriT-type R64 loci it is possible to mobilize pEC156 by conjugative plasmids (F and R64, respectively). The highest mobilization frequency was observed when pEC156-derivatives were transferred between Escherichia coli strains, Enterobacter cloacae and Citrobacter freundii representing coliform bacteria. We found that a pEC156-derivative with a functional EcoVIII restriction-modification system was mobilized in enterobacteria at a frequency lower than a plasmid lacking this system. In addition, we found that bacteria that possess the EcoVIII restriction-modification system can efficiently release plasmid content to the environment. We have shown that E. coli cells can be naturally transformed with pEC156-derivatives, however, with low efficiency. The transformation protocol employed neither involved chemical agents (e.g. CaCl2) nor temperature shift which could induce plasmid DNA uptake. PMID:26848973

  12. Hydatid cyst surgery complicated by hemorrhage resistant to activated recombinant factor VII, in a hemophiliac A patient with an inhibitor.

    PubMed

    Nabil, Saad; Bentalha, Aziza; Jaiteh, Lamine; El Khorassani, Mohammed; El Koraichi, Alae; El Kettani, Salma E-C

    2016-09-01

    Factor VII is a new coagulation factor replacement therapy. It has permitted the practice of invasive procedures which were up until recently associated with a huge risk of bleeding in patients with hemophilia with inhibitors. Our case illustrates factor replacement therapy failure in a 13-year-old child operated on for hepatic cysts associated with a pelvic cyst. Major bleeding occurred postoperatively requiring several transfusions, an increase in dosage of factor VII, and administration of a heavy dose of factor VIII as a last resort. This case highlights the possibility of failure of factor replacement therapies constituting a life-threatening situation in which alternatives are few. PMID:26761579

  13. Band gap tunability of molecular beam epitaxy grown lateral composition modulated GaInP structures by controlling V/III flux ratio

    SciTech Connect

    Park, K. W.; Park, C. Y.; Lee, Y. T.

    2012-07-30

    Lateral composition modulated (LCM) GaInP structures were grown on (001) GaAs substrate by molecular beam epitaxy with different V/III flux ratios. Band gap of LCM structures could be tuned from 1.93 eV to 1.83 eV by decreasing flux ratio while maintaining the same photoluminescence intensity, enhanced light absorption, and widened absorption spectrum. It is shown that for band gap tuning of LCM structures, flux ratio adjustment is a more viable method compared to growth temperature adjustment.

  14. TECHNICAL BASIS AND APPLICATION OF NEW RULES ON FRACTURE CONTROL OF HIGH PRESSURE HYDROGEN VESSEL IN ASME SECTION VIII, DIVISION 3 CODE

    SciTech Connect

    Rawls, G

    2007-04-30

    As a part of an ongoing activity to develop ASME Code rules for the hydrogen infrastructure, the ASME Boiler and Pressure Vessel Code Committee approved new fracture control rules for Section VIII, Division 3 vessels in 2006. These rules have been incorporated into new Article KD-10 in Division 3. The new rules require determining fatigue crack growth rate and fracture resistance properties of materials in high pressure hydrogen gas. Test methods have been specified to measure these fracture properties, which are required to be used in establishing the vessel fatigue life. An example has been given to demonstrate the application of these new rules.

  15. Ginsenoside Rd Attenuates DNA Damage by Increasing Expression of DNA Glycosylase Endonuclease VIII-like Proteins after Focal Cerebral Ischemia

    PubMed Central

    Yang, Long-Xiu; Zhang, Xiao; Zhao, Gang

    2016-01-01

    Background: Ginsenoside Rd (GSRd), one of the main active ingredients in traditional Chinese herbal Panax ginseng, has been found to have therapeutic effects on ischemic stroke. However, the molecular mechanisms of GSRd's neuroprotective function remain unclear. Ischemic stroke-induced oxidative stress results in DNA damage, which triggers cell death and contributes to poor prognosis. Oxidative DNA damage is primarily processed by the base excision repair (BER) pathway. Three of the five major DNA glycosylases that initiate the BER pathway in the event of DNA damage from oxidation are the endonuclease VIII-like (NEIL) proteins. This study aimed to investigate the effect of GSRd on the expression of DNA glycosylases NEILs in a rat model of focal cerebral ischemia. Methods: NEIL expression patterns were evaluated by quantitative real-time polymerase chain reaction in both normal and middle cerebral artery occlusion (MCAO) rat models. Survival rate and Zea-Longa neurological scores were used to assess the effect of GSRd administration on MCAO rats. Mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) damages were evaluated by the way of real-time analysis of mutation frequency. NEIL expressions were measured in both messenger RNA (mRNA) and protein levels by quantitative polymerase chain reaction and Western blotting analysis. Apoptosis level was quantitated by the expression of cleaved caspase-3 and terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling assay. Results: We found that GSRd administration reduced mtDNA and nDNA damages, which contributed to an improvement in survival rate and neurological function; significantly up-regulated NEIL1 and NEIL3 expressions in both mRNA and protein levels of MCAO rats; and reduced cell apoptosis and the expression of cleaved caspase-3 in rats at 7 days after MCAO. Conclusions: Our results indicated that the neuroprotective function of GSRd for acute ischemic stroke might be partially explained by the up

  16. Micro-chemical and metallurgical study of Samnite bronze belts from ancient Abruzzo (central Italy, VIII-IV BC)

    NASA Astrophysics Data System (ADS)

    Riccucci, Cristina; Ingo, Gabriel Maria; Faustoferri, Amalia; Pierigè, Maria Isabella; Parisi, Erica Isabella; Di Carlo, Gabriella; De Caro, Tilde; Faraldi, Federica

    2013-12-01

    The Samnite bronze belts and the chest disk cuirasses (VIII-IV BC) are the distinctive defensive weapons of the Samnite warriors having likely also a symbolic relevance. These artefacts were mainly found during the archaeological excavations of warriors' graves from ancient Abruzzo (central Italy). Their chemical composition, metallurgical features and corrosion products formed during the long-term burial have been studied by means of the combined use of analytical techniques such as optical microscopy (OM), scanning electron microscopy coupled with energy-dispersive X-ray micro-analysis (SEM-EDS), X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD). The micro-chemical and structural results show that the bronze belts have often been produced by using unusual high-tin bronze alloys achieving a silver-like appearance and by performing tailored cycles of thermal treatments under reducing conditions and hot mechanical working aimed to shape the high-tin alloys in the form of a thin bronze sheet. Furthermore, the investigation has shown that the main alloying elements have been transformed during the burial into mineral species giving rise to the formation of stratified structures constituted by different mineral phases such as tin oxides, cuprous oxide (Cu2O) and copper carbonates (azurite (Cu3(CO3)2(OH)2 and malachite (CuCO3Cu(OH)2)) as well as dangerous chlorine-based compounds such as nantokite (CuCl) and atacamite (Cu2(OH)3Cl) polymorphs. This information evidences the strict interaction of the alloying elements with the soil components as well as the occurrence of the copper cyclic corrosion as a post-burial degradation phenomenon. The present study confirms that the combined micro-chemical and micro-structural investigation techniques such as SEM-EDS, XPS, XRD and OM can be successfully used to investigate the technological production processes of the ancient artefacts and to achieve the detailed micro-chemical and structural description of the

  17. New and Emerging Agents for the Treatment of Hemophilia: Focus on Extended Half-Life Recombinant Clotting Proteins.

    PubMed

    Ragni, Margaret V

    2015-09-01

    Hemophilia A and B are X-linked disorders caused by deficient or defective clotting factor VIII (FVIII) or IX factor (FIX) proteins, and characterized by spontaneous or traumatic bleeding into joints and muscles. Previous use of plasma and plasma-derived clotting factors that lacked appropriate viral inactivation steps in manufacturing led to significant morbidity associated with transfusion-transmitted HIV and hepatitis C virus (HCV). The development of recombinant proteins revolutionized their treatment, and, with no new HIV or HCV infection via clotting proteins for nearly 30 years, greatly improved their lifespan, which now approaches that of the general population, and with the same risks for aging complications. Novel long-acting factor proteins are being licensed to extend FVIII and FIX half-life, thereby reducing infusion frequency and potentially bleed frequency and associated morbidity. Further, novel therapeutics which take advantage of new technologies, including siRNA, monoclonal antibody, and small peptide inhibition technologies, have the potential to simplify treatment and improve outcomes for those with inhibitors. PMID:26310188

  18. Bloodcurdling movies and measures of coagulation: Fear Factor crossover trial

    PubMed Central

    Nemeth, Banne; Scheres, Luuk J J; Lijfering, Willem M

    2015-01-01

    Objective To assess whether, as has been hypothesised since medieval times, acute fear can curdle blood. Design Crossover trial. Setting Main meeting room of Leiden University’s Department of Clinical Epidemiology, the Netherlands, converted to a makeshift cinema. Participants 24 healthy volunteers aged ≤30 years recruited among students, alumni, and employees of the Leiden University Medical Center: 14 were assigned to watch a frightening (horror) movie followed by a non-threatening (educational) movie and 10 to watch the movies in reverse order. The movies were viewed more than a week apart at the same time of day and both lasted approximately 90 minutes. Main outcome measures The primary outcome measures were markers, or “fear factors” of coagulation activity: blood coagulant factor VIII, D-dimer, thrombin-antithrombin complexes, and prothrombin fragments 1+2. The secondary outcome was participant reported fear experienced during each movie using a visual analogue fear scale. Results All participants completed the study. The horror movie was perceived to be more frightening than the educational movie on a visual analogue fear scale (mean difference 5.4, 95% confidence interval 4.7 to 6.1). The difference in factor VIII levels before and after watching the movies was higher for the horror movie than for the educational movie (mean difference of differences 11.1 IU/dL (111 IU/L), 95% confidence interval 1.2 to 21.0 IU/dL). The effect of either movie on levels of thrombin-antithrombin complexes, D-dimer, and prothrombin fragments 1+2 did not differ. Conclusion Frightening (in this case, horror) movies are associated with an increase of blood coagulant factor VIII without actual thrombin formation in young and healthy adults. Trial registration ClinicalTrials.gov NCT02601053. PMID:26673787

  19. Dependence of polarity inversion on V/III ratio in ‑c-GaN growth by oxide vapor phase epitaxy

    NASA Astrophysics Data System (ADS)

    Taniyama, Yuki; Yamaguchi, Yohei; Takatsu, Hiroaki; Sumi, Tomoaki; Kitamoto, Akira; Imade, Mamoru; Yoshimura, Masashi; Isemura, Masashi; Mori, Yusuke

    2016-05-01

    One of the issues in bulk c-GaN growth is the decrease in the diameter of crystals with an increase in thickness owing to the appearance of inclined \\{ 10\\bar{1}1\\} and \\{ 10\\bar{1}2\\} facets. In this study, we performed ‑c-GaN growth by oxide vapor phase epitaxy (OVPE). As a result, truncated-inverted-pyramidal crystals were successfully grown on dot-patterned ‑c-GaN substrates. The diameter of the top surface of crystals was larger than that of windows. We further investigated the dependence of the ratio of inversion-domain area to growth area (R ID) on growth temperature, V/III ratio, and growth rate. The remained results revealed that R ID decreased with increasing growth temperature and V/III ratio, and kept constant for growth rate. Additionally, an epitaxial layer on ‑c-GaN substrates with a growth rate of 12.4 µm/h and an R ID as low as 3.8% was obtained under an NH3 partial pressure (P NH3) of 83 kPa at 1200 °C.

  20. Factor eight inhibitor bypass activity (FEIBA) in the management of bleeds in hemophilia patients with high-titer inhibitors

    PubMed Central

    Tjønnfjord, Geir E; Andre Holme, Pål

    2007-01-01

    The development of high-titer inhibitors to FVIII and less often to other coagulation factors are the most serious complication of hemophilia therapy and makes treatment of bleeds very challenging. At present, bypassing agents, such as factor eight inhibitor bypass activity (FEIBA) and activated recombinant factor VII (rFVIIa) are the only coagulation factor concentrates available for the treatment of bleeds in inhibitor patients. Both products are effective and safe, and their efficacy has been found to be comparable (approximately 80%) in a recent prospective study. A significant number of patients report a better effect of one or the other of the products, and in a minority of the patients none of the products are particularly effective. The hemostatic efficacy of bypassing agents is not considered equal to that of coagulation factor replacement in patients without inhibitors by most physicians. An improvement in hemostatic efficacy may be achieved by optimizing the dosing of by passing agents. However, the lack of standardized and validated laboratory assays reflecting the hemostatic efficacy of the bypassing agents is an obstacle to this achievement. PMID:17969383

  1. Changes in Dietary Fat Content Rapidly Alters the Mouse Plasma Coagulation Profile without Affecting Relative Transcript Levels of Coagulation Factors

    PubMed Central

    van Diepen, Janna A.; Verhoef, Daniël; Voshol, Peter J.; Reitsma, Pieter H.; van Vlijmen, Bart J. M.

    2015-01-01

    Background Obesity is associated with a hypercoagulable state and increased risk for thrombotic cardiovascular events. Objective Establish the onset and reversibility of the hypercoagulable state during the development and regression of nutritionally-induced obesity in mice, and its relation to transcriptional changes and clearance rates of coagulation factors as well as its relation to changes in metabolic and inflammatory parameters. Methods Male C57BL/6J mice were fed a low fat (10% kcal as fat; LFD) or high fat diet (45% kcal as fat; HFD) for 2, 4, 8 or 16 weeks. To study the effects of weight loss, mice were fed the HFD for 16 weeks and switched to the LFD for 1, 2 or 4 weeks. For each time point analyses of plasma and hepatic mRNA levels of coagulation factors were performed after overnight fasting, as well as measurements of circulating metabolic and inflammatory parameters. Furthermore, in vivo clearance rates of human factor (F) VII, FVIII and FIX proteins were determined after 2 weeks of HFD-feeding. Results HFD feeding gradually increased the body and liver weight, which was accompanied by a significant increase in plasma glucose levels from 8 weeks onwards, while insulin levels were affected after 16 weeks. Besides a transient rise in cytokine levels at 2 weeks after starting the HFD, no significant effect on inflammation markers was present. Increased plasma levels of fibrinogen, FII, FVII, FVIII, FIX, FXI and FXII were observed in mice on a HFD for 2 weeks, which in general persisted throughout the 16 weeks of HFD-feeding. Interestingly, with the exception of FXI the effects on plasma coagulation levels were not paralleled by changes in relative transcript levels in the liver, nor by decreased clearance rates. Switching from HFD to LFD reversed the HFD-induced procoagulant shift in plasma, again not coinciding with transcriptional modulation. Conclusions Changes in dietary fat content rapidly alter the mouse plasma coagulation profile, thereby

  2. Autoantibodies with Enzymatic Properties in Human Autoimmune Diseases

    PubMed Central

    Wootla, Bharath; Lacroix-Desmazes, Sébastien; Warrington, Arthur E.; Bieber, Allan J.; Kaveri, Srini V.; Rodriguez, Moses

    2011-01-01

    Immunoglobulins (Ig) or antibodies are heavy plasma proteins, with sugar chains added to amino acid residues by N-linked glycosylation and occasionally by O-linked glycosylation. The versatility of antibodies is demonstrated by the various functions that they mediate such as neutralization, agglutination, fixation with activation of complement and activation of effector cells. In addition to this plethora of functions, some antibodies express enzymatic activity. Antibodies endowed with enzymatic properties have been described in human autoimmune manifestations for more than a decade in a variety of disorders such as autoimmune thyroiditis, systemic erythematosus (SLE), scleroderma, rheumatoid arthritis (RA), multiple sclerosis (MS) and acquired hemophilia (AH). Antibodies isolated from these conditions were able to specifically hydrolyze thyroglobulin, DNA, RNA, myelin basic protein (MBP), and factor VIII (FVIII) or factor IX (FIX), respectively. The therapeutic relevance of these findings is discussed. PMID:21624820

  3. Type 2M and Type 2A von Willebrand Disease: Similar but Different.

    PubMed

    Favaloro, Emmanuel J; Pasalic, Leonardo; Curnow, Jennifer

    2016-07-01

    Analogous to the differentiation between hemophilia A and B, respectively, reflecting deficiency in factor VIII (FVIII) and FIX, and increasing being recognized as reflecting clinically different disorders, types 2A and 2M von Willebrand disease (VWD) can also be shown to express both similarities and differences in their prevalence, genetic defects, laboratory test results, clinical features, and treatment responses. In this narrative review, we explore these two "subtypes" of type 2 VWD, identifying parallels and dissimilarities in various aspects of their presentation to clinicians and to scientists/laboratories. This differential will become increasingly important as we strive to provide personalized approaches to future management of patients with VWD, particularly in the emerging landscape of recombinant von Willebrand factor. PMID:27148841

  4. Systemic causes of excessive uterine bleeding.

    PubMed

    Lusher, J M

    1999-07-01

    In assessing a patient with excessive uterine bleeding, the clinician should consider systemic causes in the differential diagnosis. Both hereditary and acquired conditions can result in mucous membrane bleeding, including menorrhagia, epistaxis, and gum bleeding, as well as excessive bruising. Among hereditary conditions, von Willebrand disease (vWD) is by far the most common, affecting an estimated 1% of the population worldwide. It is important to consider the possibility of vWD, and to establish the proper diagnosis (including subtype), as safe, effective, and easy-to-use treatment is available for most persons with this disorder. This review also covers a number of other systemic conditions that can be manifested by excessive uterine bleeding, including congenital deficiency of factor XI, idiopathic thrombocytopenic purpura and other acquired platelet disorders, acquired autoantibodies against factor VIII (FVIII), and vitamin K deficiency states. PMID:10513767

  5. Longer-acting clotting factor concentrates for hemophilia.

    PubMed

    Powell, J S

    2015-06-01

    Hemophilia, when severe, leads to spontaneous life-threatening bleeding episodes. Current therapy requires frequent intravenous infusions. Most patients must limit their physical activities to avoid bleeding when the factor activity levels are below normal. In 2014, new therapeutic factor VIII and IX products were approved in Canada and the U.S. Over the next couple of years, other new factor products will likely be approved. These new factors have been engineered to have improved pharmacokinetic properties, including extended half-life in circulation, thus providing major therapeutic advances for patients with hemophilia. In the completed clinical trials, over 700 patients have successfully used these longer acting products regularly for more than one year. These promising new therapies should allow patients with hemophilia to use fewer infusions to prevent spontaneous bleeding or to treat bleeding episodes, and to provide appropriate clotting factor levels for different physical activities. PMID:26149018

  6. Cost–utility analysis of prophylaxis versus treatment on demand in severe hemophilia A

    PubMed Central

    Colombo, Giorgio L; Di Matteo, Sergio; Mancuso, Maria Elisa; Santagostino, Elena

    2011-01-01

    Background: Individuals with severe hemophilia A have reduced blood levels of clotting factor VIII (FVIII) leading to recurrent bleeding into joints and muscles. Primary prophylaxis with clotting factor concentrates started early in childhood prevents joint bleeds, thus avoiding joint damage and improving people’s quality of life. There remain significant differences in the implementation of primary prophylaxis worldwide mainly due to the cost of prophylaxis compared with treatment on demand. Objective: To evaluate the cost-effectiveness of primary prophylaxis with FVIII concentrates versus secondary prophylaxis, versus treatment on demand, and versus a “hybrid” (primary prophylaxis followed by on-demand treatment in adults) in individuals with severe hemophilia A. Methods: A Markov model was developed and run using different sources of clinical, cost, and utility data. The model was populated with a hypothetical cohort of 100 individuals with severe hemophilia A. The perspective of the Italian National Health System was used. Results: The baseline results showed that primary and secondary prophylaxis is cost-effective compared both with treatment on demand and with a hybrid strategy. The incremental costs per quality-adjusted life-year gained for individuals with hemophilia A receiving primary and secondary prophylaxis were €40,229 to €40,236 versus an on-demand strategy. However, the sensitivity analyses performed showed that the results were sensitive to the unit cost of clotting FVIII, bleeding frequency, and the discount rate. Conclusion: Although primary prophylaxis is a costly treatment, our results show that it is cost-effective compared with treatment on demand. PMID:21935333

  7. Effect of levothyroxine administration on hemostatic analytes in Doberman Pinschers with von Willebrand disease.

    PubMed

    Heseltine, Johanna C; Panciera, David L; Troy, Gregory C; Monroe, William E; Brooks, Marjory B; Feldman, Bernard F

    2005-01-01

    Levothyroxine administration has been suggested to be an effective treatment for canine von Willebrand disease (vWd), but evidence supporting this treatment is lacking. Effects of levothyroxine administration were evaluated in 8 euthyroid Doberman Pinschers with plasma von Willebrand factor (vWf) concentrations < 15%, characteristic of type 1 vWd. Levothyroxine (0.04 mg/kg PO q12h) and placebo were administered for 30 days in a 2-period, 2-treatment, double-blinded, crossover design with a 30-day washout period between treatments. Buccal mucosal bleeding time (BMBT), plasma vWf concentration (vWf: Ag), vWf collagen binding activity (vWf:CBA), factor VIII coagulant activity (FVIII:C), and serum concentrations of total thyroxine (T4), free thyroxine (fT4), 3,5,3'-triiodothyronine (T3), and thyroid-stimulating hormone (TSH) were measured on days 0, 2, and 30 of each treatment period. The 8 dogs (1 male, 7 females) had markedly low plasma vWf:Ag (mean, 8.9%; reference range, 70-180%) and vWf:CBA (mean, 11.1%; reference range, >70%). Response to placebo versus levothyroxine treatment was not significantly different between groups at day 0, 2, or 30 for BMBT, vWf:Ag, vWf:CBA, and FVIII:C. Serum T4, fT4, and T3 concentrations were significantly higher and serum TSH significantly lower in the levothyroxine-treated group than in the placebo group at days 2 and 30. Administration of levothyroxine at 0.04 mg/kg caused laboratory evidence of hyperthyroidism but did not affect plasma FVIII:C and vWf:Ag concentrations or vWf-dependent collagen binding and BMBT. The results of this study failed to identify a direct action of levothyroxine supplementation on plasma vWf concentration or activity in euthyroid Doberman Pinschers with vWd. PMID:16095169

  8. The use of desmopressin in von Willebrand disease: the experience of the first 30 years (1977-2007).

    PubMed

    Federici, A B

    2008-01-01

    The aim of the treatment for von Willebrand disease (VWD) is to correct the dual defect of haemostasis, i.e. the abnormal platelet adhesion as a result of reduced and/or dysfunctional von Willebrand factor (VWF) and the abnormal coagulation expressed by low levels of factor VIII (FVIII). Correction of both deficiencies can be achieved by administering the synthetic peptide desmopressin (DDAVP) or, in cases unresponsive to this agent, the plasma concentrates containing VWF and FVIII (VWF/FVIII). DDAVP is the treatment of choice for type 1 VWD because it can induce release of normal VWF from cellular compartments, but the drug can be clinically useful also in other VWD types, including acquired von Willebrand syndrome (AVWS). A test dose of DDAVP at the time of diagnosis is recommended to establish the individual patterns of biological response and to predict clinical efficacy during bleeding and surgery. DDAVP is not effective in VWD type 3 and in severe forms of VWD 1 and 2. It can induce transient thrombocytopenia in patients with VWD type 2B. The results of several retrospective studies on the use of DDAVP in VWD management have been reported by many authors in different countries for the last 30 years. However, despite the widespread use of DDAVP in the treatment of VWD, there are only a few prospective clinical trials in a large number of cases on DDAVP efficacy and safety aimed at determining benefits and limits of this therapeutic approach. An investigator-driven observational prospective study on clinical efficacy of DDAVP in 200 patients with VWD types 1 and 2 has been recently organized: the effectiveness and safety of DDAVP will be evaluated prospectively for 24 months during bleeding episodes and minor or major surgeries in the VWD patients who were exposed to an infusion trial at enrollment. PMID:18173689

  9. Diagnosis of von Willebrand disease.

    PubMed

    Ingerslev, J; Gürsel, T

    1999-05-01

    The haemorrhagic diathesis in von Willebrand disease (vWD) is caused by a quantitative deficiency or a qualitative defect in the von Willebrand factor (vWF) in plasma and/or platelets causing insufficient primary haemostasis. Since vWF binds and protects factor VIII (FVIII) towards random proteolysis, coagulation may also be impaired in patients with a low plasma level of vWF, and in instances where vWF displays insufficient binding capacity to FVIII. The entity of vWD displays a vast heterogeneity. Apart from rarely occurring acquired cases, vWD is an inherited disorder of autosomal linkage. The major clinical hallmark in vWD is an increased tendency to mucocutaneous bleeding that rarely reach life-threatening proportions, unless vWF is severely reduced or completely absent. Increased bleeding may also occur in sites such as muscles and joints when the level of FVIII is particularly low. Significant progress has recently been achieved through extensive molecular genetic exploration of various forms of vWD. In order to guide treatment and to form a platform for genetic investigation, however, accuracy in diagnosis and phenotypic characterization is important. By means of various laboratory methods, major subclasses of vWD can be differentiated, as presented in another article of this series. Whereas most of the cases of vWD can quite easily be diagnosed and classified using today's diagnostic methods, the most frequently occurring bleeding disorder of all, vWd type 1 of mild degree, continues to challenge clinicians and diagnostic laboratories. The aim of this paper is to review the laboratory methods most commonly used in diagnostic investigation of the patient suspected of vWD. PMID:23401900

  10. Evaluation of Potential Clinical Surrogate Markers of a Trauma Induced Alteration of Clotting Factor Activities

    PubMed Central

    Payas, Arzu; Schoeneberg, Carsten; Wegner, Alexander; Kauther, Max Daniel; Lendemans, Sven

    2016-01-01

    Objective. The aim of this study was to identify routinely available clinical surrogate markers for potential clotting factor alterations following multiple trauma. Methods. In 68 patients admitted directly from the scene of the accident, all soluble clotting factors were analyzed and clinical data was collected prospectively. Ten healthy subjects served as control group. Results. Patients showed reduced activities of clotting factors II, V, VII, and X and calcium levels (all P < 0.0001 to 0.01). Levels of hemoglobin and base deficit correlated moderately to highly with the activities of a number of clotting factors. Nonsurvivors and patients who needed preclinical intubation or hemostatic therapy showed significantly reduced factor activities at admission. In contrast, factor VIII activity was markedly elevated after injury in general (P < 0.0001), but reduced in nonsurvivors (P < 0.05). Conclusions. Multiple trauma causes an early reduction of the activities of nearly all soluble clotting factors in general. Initial hemoglobin and, with certain qualifications, base deficit levels demonstrated a potential value in detecting those underlying clotting factor deficiencies. Nevertheless, their role as triggers of a hemostatic therapy as well as the observed response of factor VIII to multiple trauma and also its potential prognostic value needs further evaluation. PMID:27433474

  11. Prophylaxis in von Willebrand Disease: Coming of Age?

    PubMed

    Saccullo, Giorgia; Makris, Mike

    2016-07-01

    Although in most cases von Willebrand disease (VWD) is a mild disorder, a subgroup of patients experience frequent bleeding. In contrast to severe hemophilia in which prophylaxis is the accepted standard of care, this is less frequently used in VWD. Most type 1 VWD patients can be adequately managed with episodic desmopressin and tranexamic acid. In patients with more severe disease, especially those with type 3 VWD, joint bleeds, epistaxis, menorrhagia, and gastrointestinal bleeding are problematic and usually require treatment with von Willebrand factor/factor VIII (VWF/FVIII) concentrate. While in the past these patients were managed with on-demand VWF/FVIII concentrate, several recent reports have demonstrated the value of prophylactic treatment. Despite some uncertainties about the economic impact of treatment of severe VWD, prophylaxis with VWF concentrate should now be considered as the standard of care for the more severe end of the spectrum of affected individuals. The recent introduction of recombinant VWF concentrate is likely to improve the acceptability of prophylaxis in VWD. PMID:27253087

  12. Distribution of AAV8 particles in cell lysates and culture media changes with time and is dependent on the recombinant vector

    PubMed Central

    Piras, Bryan A; Drury, Jason E; Morton, Christopher L; Spence, Yunyu; Lockey, Timothy D; Nathwani, Amit C; Davidoff, Andrew M; Meagher, Michael M

    2016-01-01

    With clinical trials ongoing, efficient clinical production of adeno-associated virus (AAV) to treat large numbers of patients remains a challenge. We compared distribution of AAV8 packaged with Factor VIII (FVIII) in cell culture media and lysates on days 3, 5, 6, and 7 post-transfection and found increasing viral production through day 6, with the proportion of viral particles in the media increasing from 76% at day 3 to 94% by day 7. Compared to FVIII, AAV8 packaged with Factor IX and Protective Protein/Cathepsin A vectors demonstrated a greater shift from lysate towards media from day 3 to 6, implying that particle distribution is dependent on recombinant vector. Larger-scale productions showed that the ratio of full-to-empty AAV particles is similar in media and lysate, and that AAV harvested on day 6 post-transfection provides equivalent function in mice compared to AAV harvested on day 3. This demonstrates that AAV8 production can be optimized by prolonging the duration of culture post-transfection, and simplified by allowing harvest of media only, with disposal of cells that contain 10% or less of total vector yield. Additionally, the difference in particle distribution with different expression cassettes implies a recombinant vector-dependent processing mechanism which should be taken into account during process development. PMID:27069949

  13. Comparison of stress-hemoconcentration correction techniques for stress-induced coagulation.

    PubMed

    Austin, Anthony W; Patterson, Stephen M

    2013-01-01

    When examining stress effects on coagulation, arithmetic correction is typically used to adjust for concomitant hemoconcentration but may be inappropriate for coagulation activity assays. We examined a new physiologically relevant method of correcting for stress-hemoconcentration. Blood was drawn from healthy men (N = 40) during baseline, mental stress, and recovery, and factor VII activity (FVII:C), factor VIII activity (FVIII:C), activated partial thromboplastin time (APTT), prothrombin time (PT%), fibrinogen, D-dimer, and plasma volume were determined. Three hemoconcentration correction techniques were assessed: arithmetic correction and two reconstitution techniques using baseline plasma or physiological saline. Area-under-the-curve (AUC) was computed for each technique. For FVII:C, uncorrected AUC was significantly greater than AUC corrected arithmetically. For PT%, uncorrected AUC was significantly greater than AUC corrected with saline or arithmetically. For APTT, uncorrected AUC was significantly less than AUC corrected with saline and greater than AUC corrected arithmetically. For fibrinogen, uncorrected AUC was significantly greater than AUC corrected with saline or arithmetically. For D-dimer, uncorrected AUC was significantly greater than AUC corrected arithmetically. No differences in AUC were observed for FVIII:C. Saline reconstitution seems most appropriate when adjusting for hemoconcentration effects on clotting time and activity. Stress-hemoconcentration accounted for the majority of coagulation changes. PMID:24222908

  14. Distribution of AAV8 particles in cell lysates and culture media changes with time and is dependent on the recombinant vector.

    PubMed

    Piras, Bryan A; Drury, Jason E; Morton, Christopher L; Spence, Yunyu; Lockey, Timothy D; Nathwani, Amit C; Davidoff, Andrew M; Meagher, Michael M

    2016-01-01

    With clinical trials ongoing, efficient clinical production of adeno-associated virus (AAV) to treat large numbers of patients remains a challenge. We compared distribution of AAV8 packaged with Factor VIII (FVIII) in cell culture media and lysates on days 3, 5, 6, and 7 post-transfection and found increasing viral production through day 6, with the proportion of viral particles in the media increasing from 76% at day 3 to 94% by day 7. Compared to FVIII, AAV8 packaged with Factor IX and Protective Protein/Cathepsin A vectors demonstrated a greater shift from lysate towards media from day 3 to 6, implying that particle distribution is dependent on recombinant vector. Larger-scale productions showed that the ratio of full-to-empty AAV particles is similar in media and lysate, and that AAV harvested on day 6 post-transfection provides equivalent function in mice compared to AAV harvested on day 3. This demonstrates that AAV8 production can be optimized by prolonging the duration of culture post-transfection, and simplified by allowing harvest of media only, with disposal of cells that contain 10% or less of total vector yield. Additionally, the difference in particle distribution with different expression cassettes implies a recombinant vector-dependent processing mechanism which should be taken into account during process development. PMID:27069949

  15. Desmopressin in treatment of haematological disorders and in prevention of surgical bleeding.

    PubMed

    Svensson, Peter J; Bergqvist, Peter B F; Juul, Kristian Vinter; Berntorp, Erik

    2014-05-01

    Stimulation with the vasopressin analogue desmopressin (DDAVP) of extrarenal arginine vasopressin (AVP) V2-receptors in endothelial cells and possible in platelets increases the circulating levels of coagulation factor VIII (FVIII), von Willebrand factor (VWF) and tissue plasminogen activator (t-PA). The purpose of this paper is to provide an updated review of current information on the efficacy and safety of DDAVP in the treatment of haemophilia, von Willebrand disease (VWD), uremia, liver cirrhosis, and in congenital or drug-induced platelet dysfunction--under surgical or non-surgical conditions. In summary, desmopressin is an effective haemostatic drug that when administered i.v., s.c. or intranasally increases plasma levels of FVIII and VWF 2-6 times and improves platelet function. It has a proven haemostatic efficacy in mild haemophilia A and VWD as well as in uremia, liver cirrhosis and in congenital and acquired, drug induced platelet dysfunction. Desmopressin has few side effects but observation is advised in small children and elderly. PMID:24703870

  16. Acquired von Willebrand syndrome associated with hypothyroidism: a mild bleeding disorder to be further investigated.

    PubMed

    Federici, Augusto B

    2011-02-01

    Acquired von Willebrand syndrome (AVWS) is a rare bleeding disorder with laboratory findings similar to those for congenital von Willebrand disease (VWD). Unlike VWD, AVWS usually occurs in individuals with no personal or family history of bleeding. The prevalence of AVWS in the general population is unknown because data from large prospective studies of this syndrome are not available. Although AVWS is more frequently associated with lympho-myeloproliferative and cardiovascular disorders, it can also occur in solid tumors and in immunological and other miscellaneous conditions. Among these miscellaneous conditions, hypothyroidism has been associated with AVWS type 1 with a frequency of ~2 to 5%. In the 47 cases reported in the literature, the low VWF is apparently due to a reduction in its synthesis and/or secretion. Diagnosis of AVWS is based on assays measuring the level and activity of von Willebrand factor (VWF). These tend to be low, whereas factor VIII (FVIII) coagulant activity can be normal. In patients with AVWS associated with hypothyroidism, mucocutaneous bleeding episodes are the most frequent and can be managed with local therapy and/or systemic administrations of antifibrinolytic agents and desmopressin. VWF/FVIII concentrates have been used in only a few patients. The use of thyroid hormones can reverse this abnormality. PMID:21305800

  17. Acquired von Willebrand syndrome: von Willebrand factor propeptide to von Willebrand factor antigen ratio predicts remission status

    PubMed Central

    Lee, Adrienne; Sinclair, Gary; Valentine, Karen; James, Paula

    2014-01-01

    We investigated a case of acquired von Willebrand syndrome (AVWS) secondary to a nonneutralizing anti-von Willebrand factor (VWF) antibody associated with an autoimmune disorder. At diagnosis, VWF activity (VWF:Act), antigen (VWF:Ag), multimers, and factor VIII coagulant activity were virtually absent. VWF propeptide (VWFpp) was elevated with an infinitely high VWFpp to VWF:Ag ratio (VWFpp:Ag) consistent with rapid VWF clearance. Immunosuppressive treatment resulted in phenotypic remission 1 with normalization of VWF/factor VIII levels and multimer pattern. However, VWFpp:Ag remained elevated (∼2× normal), consistent with ongoing VWF clearance by the remaining anti-VWF antibody still present by enzyme-linked immunosorbent assay. This suggests that increased VWF secretion was compensating for the incomplete remission state. Relapse occurred when VWFpp:Ag was again infinitely high, with associated decreased VWFpp but unchanged anti-VWF titers; switching the balance to favor VWF clearance over secretion. Complete remission with undetectable anti-VWF occurred only when VWFpp:Ag was normal. This case of relapsing-remitting AVWS demonstrates the use of VWFpp:Ag for predicting remission status. PMID:24951428

  18. Intensity of lines from low-lying levels in C II, N III, O IV, NE VI, MG VIII, SI X, and SI II

    NASA Astrophysics Data System (ADS)

    Chandra, S.

    1982-01-01

    The populations of the excited state 2P 0 3/2 relative to the ground state 2P 0 1/2 are obtained, in a study of line intensities of the transition between those two states in C II, N III, O IV, Ne VI, Mg VIII, Si X, and Si II in the chromosphere-corona transition region, by considering all the radiative and collisional transition processes. The collisional transitions to the higher states which cascade to the upper level are included among these processes. It is found that the intensity, which may be expressed as a function of temperature alone, increases in the transition region with the charge on the ion for a sequence.

  19. Crystal structures of brain group-VIII phospholipase A2 in nonaged complexes with the organophosphorus nerve agents soman and sarin.

    PubMed

    Epstein, Todd M; Samanta, Uttamkumar; Kirby, Stephen D; Cerasoli, Douglas M; Bahnson, Brian J

    2009-04-21

    Insecticide and nerve agent organophosphorus (OP) compounds are potent inhibitors of the serine hydrolase superfamily of enzymes. Nerve agents, such as sarin, soman, tabun, and VX exert their toxicity by inhibiting human acetycholinesterase at nerve synapses. Following the initial phosphonylation of the active site serine, the enzyme may reactivate spontaneously or through reaction with an appropriate nucleophilic oxime. Alternatively, the enzyme-nerve agent complex can undergo a secondary process, called "aging", which dealkylates the nerve agent adduct and results in a product that is highly resistant to reactivation by any known means. Here we report the structures of paraoxon, soman, and sarin complexes of group-VIII phospholipase A2 from bovine brain. In each case, the crystal structures indicate a nonaged adduct; a stereoselective preference for binding of the P(S)C(S) isomer of soman and the P(S) isomer of sarin was also noted. The stability of the nonaged complexes was corroborated by trypsin digest and electrospray ionization mass spectrometry, which indicates nonaged complexes are formed with diisopropylfluorophosphate, soman, and sarin. The P(S) stereoselectivity for reaction with sarin was confirmed by reaction of racemic sarin, followed by gas chromatography/mass spectrometry using a chiral column to separate and quantitate each stereoisomer. The P(S) stereoisomers of soman and sarin are known to be the more toxic stereoisomers, as they react preferentially to inhibit human acetylcholinesterase. The results obtained for nonaged complexes of group-VIII phospholipase A2 are compared to those obtained for other serine hydrolases and discussed to partly explain determinants of OP aging. Furthermore, structural insights can now be exploited to engineer variant versions of this enzyme with enhanced nerve agent binding and hydrolysis functions. PMID:19271773

  20. Platelet-delivered therapeutics.

    PubMed

    Lyde, R; Sabatino, D; Sullivan, S K; Poncz, M

    2015-06-01

    We have proposed that modified platelets could potentially be used to correct intrinsic platelet defects as well as for targeted delivery of therapeutic molecules to sights of vascular injury. Ectopic expression of proteins within α-granules prior to platelet activation has been achieved for several proteins, including urokinase, factor (F) VIII, and partially for FIX. Potential uses of platelet-directed therapeutics will be discussed, focusing on targeted delivery of urokinase as a thromboprophylactic agent and FVIII for the treatment of hemophilia A patients with intractable inhibitors. This presentation will discuss new strategies that may be useful in the care of patients with vascular injury as well as remaining challenges and limitations of these approaches. PMID:26149015

  1. Preparation of functional human factor V111 in mammalian cells using methotrexate based selection

    SciTech Connect

    Capon, D.J.; Lawn, R.M.; Levinson, A.D.; Vehar, G.A.; Wood, W.I.

    1990-10-23

    This patent describes a process for producing factor VII. It comprises: cotransfecting a mammalian host cell with a DNA sequence encoding factor VIII, a second DNA sequence encoding an amplifiable marker, and a third DNA sequence encoding a selectable marker; growing the transfected cell in a non-selection medium and selecting for such selectable marker resistant cells; and amplifying the amplifiable marker DNA sequence by culturing the selected cells in media containing increasing amounts of selection agent, wherein the host cell is not deficient in the amplifiable marker.

  2. Inhibitors in childhood hemophilia A: genetic and treatment-related risk factors for development and eradication.

    PubMed

    DiMichele, Donna M

    2013-01-01

    The development of neutralizing antibodies remains a serious complication of hemophilia replacement therapy. Factor VIII inhibiting antibodies (inhibitors) occur commonly following replacement therapy in hemophilia A, creating a significant burden of clinical disease. This article will review our current understanding of risk factors and their known impact on inhibitor development in previously untreated or minimally treated children with severe and mild hemophilia A. It will also explore how the most recently elucidated immunology of inhibitor development might hold important clues to more effective inhibitor eradication and prevention in this heavily impacted patient population. PMID:23109404

  3. Quality laboratory issues in bleeding disorders.

    PubMed

    Adcock, D M; Mammen, J; Nair, S C; de Lima Montalvão, S A

    2016-07-01

    Selected quality issues pertinent to the determination of accurate results in the haemostasis laboratory are discussed. Specifically, the implementation of a successful external quality-assessment scheme is described, including its impact on result accuracy as well as the programme's unique challenges and opportunities. Errors in the preanalytical phase of laboratory testing represent the greatest source for reporting incorrect test results. Some of the most common preanalytical errors are described including those that necessitate sample rejection. Analytical means to identify potential sources of error and analytical means to overcome particular interferences are described. Representing the most important clinical complication in the treatment of patients with haemophilia, quality issues related to determination of the presence of inhibitory antibodies against factor VIII (FVIII) are reviewed. Heat treatment of patient plasma prior to testing, particularly in patients receiving replacement FVIII concentrate or during induction of immune tolerance to achieve more accurate results is recommended, while screening activated partial thromboplastin time-based mixing tests to rule out inhibitor presence is discouraged. The initiatives presented in this review can be implemented in robust and resource restricted settings to improve the quality of laboratory testing in patients with bleeding disorders. PMID:27405682

  4. Heat treatment of samples improve the performance of the Nijmegen-Bethesda assay in hemophilia A patients undergoing immune tolerance induction.

    PubMed

    de Lima Montalvão, Silmara Aparecida; Tucunduva, Alini Camargo; de Almeida Sambo, Andrea Luísa; De Paula, Erich Vinicius; de Souza Medina, Samuel; Ozelo, Margareth Castro

    2015-12-01

    Nijmegen-Bethesda assay is the gold standard to assess inhibitory antibodies against factor (F) VIII. This method has some limitations, including high coefficient of variation and possible interference of residual endogenous or exogenous factor VIII. Heat-treatment of samples at 56 °C for 30 min could be a strategy to improve the sensitivity of this test. The aim of this study was to compare inhibitor quantification in hemophilia patients with and without inhibitor performed in previously heated and non-heated samples. A total of 109 analyses from 46 patients with severe hemophilia A were performed. Patients were divided into three groups: 20 patients with no history of inhibitor, recently and not recently exposed to FVIII (group I), 21 patients with history of inhibitor not exposed to FVIII (group II), and 5 patients (68 samples) undergoing an immune tolerance induction (ITI) protocol (group III). For patients with no history of inhibitor, heat-treatment did not modify the results (p=0.24). However, differences in inhibitor levels between heated and non-heated samples were observed in patients with history of inhibitor (group II, p<0.05) and in patients in ITI (group III, p<0.001). In 11 samples, inhibitor quantification shifted from negative to positive. Additionally, a longitudinal evaluation of each ITI patient showed similar trend line for the results of heated and non-heated samples. In this study, we demonstrated that heating samples increase sensitivity of Nijmegen-Bethesda assay, with no shift from negative to positive results in patients with no history of inhibitor. Furthermore, this procedure has an important role to patients undergoing an ITI protocol. PMID:26344704

  5. AN XMM-NEWTON SURVEY OF THE SOFT X-RAY BACKGROUND. II. AN ALL-SKY CATALOG OF DIFFUSE O VII AND O VIII EMISSION INTENSITIES

    SciTech Connect

    Henley, David B.; Shelton, Robin L.

    2012-10-15

    We present an all-sky catalog of diffuse O VII and O VIII line intensities, extracted from archival XMM-Newton observations. This catalog supersedes our previous catalog, which covered the sky between l = 120 Degree-Sign and l = 240 Degree-Sign . We attempted to reduce the contamination from near-Earth solar wind charge exchange (SWCX) emission by excluding times of high solar wind proton flux from the data. Without this filtering, we were able to extract measurements from 1868 observations. With this filtering, nearly half of the observations became unusable, and only 1003 observations yielded measurements. The O VII and O VIII intensities are typically {approx}2-11 and {approx}<3 photons cm{sup -2} s{sup -1} sr{sup -1} (line unit, L.U.), respectively, although much brighter intensities were also recorded. Our data set includes 217 directions that have been observed multiple times by XMM-Newton. The time variation of the intensities from such directions may be used to constrain SWCX models. The O VII and O VIII intensities typically vary by {approx}<5 and {approx}<2 L.U. between repeat observations, although several intensity enhancements of >10 L.U. were observed. We compared our measurements with models of the heliospheric and geocoronal SWCX. The heliospheric SWCX intensity is expected to vary with ecliptic latitude and solar cycle. We found that the observed oxygen intensities generally decrease from solar maximum to solar minimum, both at high ecliptic latitudes (which is as expected) and at low ecliptic latitudes (which is not as expected). The geocoronal SWCX intensity is expected to depend on the solar wind proton flux incident on the Earth and on the sightline's path through the magnetosheath. The intensity variations seen in directions that have been observed multiple times are in poor agreement with the predictions of a geocoronal SWCX model. We found that the oxygen lines account for {approx}40%-50% of the 3/4 keV X-ray background that is not due to

  6. The influence of V/III ratio in the initial growth stage on the properties of GaN epilayer deposited on low temperature AlN buffer layer

    NASA Astrophysics Data System (ADS)

    Zhao, D. G.; Jiang, D. S.; Zhu, J. J.; Liu, Z. S.; Zhang, S. M.; Yang, Hui; Liang, J. W.

    2007-05-01

    The V/III ratio in the initial growth stage of metalorganic chemical vapor deposition has an important influence on the quality of a GaN epilayer grown on a low-temperature AlN buffer layer and c-plane sapphire substrate. A weaker yellow luminescence, a narrower half-width of the X-ray diffraction peak, and a higher electron mobility result when a lower V/III ratio is taken. The intensity of in situ optical reflectivity measurements indicates that the film surface is rougher at the beginning of GaN growth, and a longer time is needed for the islands to coalesce and for a quasi-two dimensional mode growth to start. A comparison of front- and back-illuminated photoluminescence spectra confirms that many threading dislocations are bent during the initial stage, leading to a better structural quality of the GaN layer.

  7. Impact of CBr 4, V/III ratio, temperature and AsH 3 concentration on MOVPE growth of GaAsSb:C

    NASA Astrophysics Data System (ADS)

    Ostinelli, O.; Bolognesi, C. R.

    2009-03-01

    We investigate the impact of the carbon tetrabromine (CBr 4) and arsine (AsH 3) concentrations, the V/III ratio at the gas inlet ( Rinlet) and the temperature ( Tg) on the growth rate, the solid composition, the incorporated carbon (C) and free hole concentration in GaAsSb:C grown by metal-organic vapor-phase epitaxy (MOVPE). The introduction of CBr 4 deeply perturbs the growth when compared to when no C-doping is used. The growth of GaAsSb:C using CBr 4 behaves as if an effective V/III ratio ( Reff) were defined as the Rinlet leading to the same growth rate in the absence of CBr 4 under the same group V inlet flux. This consideration allows (and/or accounts) for the growth of high-quality layers with Rinlet≪1, and explains alloy composition and growth rate variations as well as non-monotonic variations of the free hole concentration with increasing CBr 4 flow. We show how Reff˜1 can be experimentally determined to optimize the growth conditions in MOVPE reactors. An increase of the CBr 4 flux enhances the solid arsenic (As) concentration and reduces the growth rate. The As concentration dependence on the CBr 4 flux becomes stronger at higher Rinlet. Although the incorporated C density monotonically increases with increasing CBr 4 flow, the hole concentration increases and then drops. This drop is attributed to the amphoteric character of C and/or to the formation of C-C bonds. The peak hole density is enhanced from 2.5×10 19 to over 1.6×10 20 cm -3 by reducing Rinlet from 0.72 to 0.25. Furthermore, the hole density decreases from 1.6×10 20 to 5.5×10 19 cm -3 if the AsH 3 concentration increases from 0.31 to 0.51. Thus, alloys with As-rich composition obtained by increasing the AsH 3 composition in the gas phase cannot achieve hole concentrations as high as 1×10 20 cm -3 at Tg=550 °C. However, we demonstrate that GaAs 0.7Sb 0.3 with doping levels of 1×10 20 cm -3 could only be achieved by decreasing Tg to 510 °C.

  8. Acyclic diterpene glycosides, capsianosides VIII, IX, X, XIII, XV and XVI from the fruits of Paprika Capsicum annuum L. var. grossum BAILEY and Jalapeño Capsicum annuum L. var. annuum.

    PubMed

    Lee, Jong-Hyun; Kiyota, Naoko; Ikeda, Tsuyoshi; Nohara, Toshihiro

    2006-10-01

    Paprika and Jalapeño are used as vegetables and spices. We have obtained six new acyclic diterpene glycosides, called capsianosides XIII (2), XV (3), IX (4), XVI (5), X (6) and VIII (7) together with known capsianoside II (1) from the fruits of the Paprika and Jalapeño. The structures of these compounds have been elucidated by the (1)H- and (13)C-NMR spectra and two-dimensional NMR methods. PMID:17015971

  9. Virus elimination during the recycling of chromatographic columns used during the manufacture of coagulation factors.

    PubMed

    Roberts, Peter L

    2014-07-01

    Various chromatographic procedures are used during the purification and manufacture of plasma products such as coagulation factors. These steps contribute to the overall safety of such products by removing potential virus contamination. Virus removal by two affinity chromatography procedures, i.e. monoclonal antibody chromatography and metal chelate chromatography (immobilised metal ion affinity chromatography), used during the manufacture of the high purity factor VIII (Replenate®) and factor IX (Replenine®-VF), respectively, has been investigated. In addition, as these columns are recycled after use, the effectiveness of the sanitisation procedures for preventing possible cross-contamination, has also been investigated. Both chromatographic steps proved effective for eliminating a range of model enveloped and non-enveloped viruses by 4 to >6 and 5 to >8 log for the monoclonal and metal chelate columns, respectively. The effectiveness of the relatively mild column sanitisation conditions used, i.e. ethanol for factor IX and acetic acid for factor VIII, was confirmed using non-spiked column runs. The chemicals used contributed to virus elimination by inactivation and/or by physical removal of the virus. In summary, these studies demonstrate that potential virus contamination between chromatographic runs can be prevented when an effective column recycling and sanitisation procedure is included. PMID:24981392

  10. Characterization of the clotting activities of structurally different forms of activated factor IX. Enzymatic properties of normal human factor IXa alpha, factor IXa beta, and activated factor IX Chapel Hill.

    PubMed Central

    Griffith, M J; Breitkreutz, L; Trapp, H; Briet, E; Noyes, C M; Lundblad, R L; Roberts, H R

    1985-01-01

    Two structurally different forms of activated human Factor IX (Factor IXa alpha and IXa beta) have been previously reported to have essentially identical clotting activity in vitro. Although it has been shown that activated Factor IX Chapel Hill, an abnormal Factor IX isolated from the plasma of a patient with mild hemophilia B, and normal Factor IXa alpha are structurally very similar, the clotting activity of activated Factor IX Chapel Hill is much lower (approximately fivefold) than that of normal Factor IXa beta. In the present study we have prepared activated Factor IX by incubating human Factor IX with calcium and Russell's viper venom covalently bound to agarose. Fractionation of the activated Factor IX by high-performance liquid chromatography demonstrated the presence of both Factors IXa alpha and IXa beta. On the basis of active site concentration, determined by titration with antithrombin III, the clotting activities of activated Factor IX Chapel Hill and IXa alpha were similar, but both activities were less than 20% of the clotting activity of Factor IXa beta. Activated Factor IX activity was also measured in the absence of calcium, phospholipid, and Factor VIII, by determination of the rate of Factor X activation in the presence of polylysine. In the presence of polylysine, the rates of Factor X activation by activated Factor IX Chapel Hill, Factor IXa alpha, and Factor IXa beta were essentially identical. We conclude that the clotting activity of activated Factor IX Chapel Hill is reduced when compared with that of Factor IXa beta but essentially normal when compared with that of Factor IXa alpha. PMID:3871202

  11. The effect of color on the production of responses to Rorschach cards VIII, IX, and X in age groups of 11-12 and 15-16 years.

    PubMed

    Silva, Danilo R; Ferreira, Ana Sousa

    2014-01-01

    The effect of color on the production of responses to the Rorschach task has been considered by investigators from 2 different positions: (a) that color has little effect on the production of responses, and (b) that color increases the number of responses. Some previous results found by the current investigators have supported the first position for the last 3 fully colored Rorschach cards (VIII, IX, and X), in children from 5 to 12 years old. Other studies of ours, however, have confirmed the second position for these same cards with a group of young adults 17 to 23 years old. As there was no increase of responses up to age 12, for this study we hypothesized a developmental effect in adolescence such that there would be an increase in the production of responses to the colored Rorschach cards at the age of 15 to 16 years, and this is what the results indicate. From a pragmatic standpoint, these results imply a revision of interpretive meaning for the Color and Affective Ratio variables in children's protocols. Our results also indicate that color cannot be regarded as a means of expression of affect at age 11 to 12 like it will be from age 15 to 16 and on. PMID:24456089

  12. Mapping Henry: Synchrotron-sourced X-ray fluorescence mapping and ultra-high-definition scanning of an early Tudor portrait of Henry VIII

    NASA Astrophysics Data System (ADS)

    Dredge, Paula; Ives, Simon; Howard, Daryl L.; Spiers, Kathryn M.; Yip, Andrew; Kenderdine, Sarah

    2015-11-01

    A portrait of Henry VIII on oak panel c. 1535 has recently undergone technical examination to inform questions regarding authorship and the painting's relationship to a group of similar works in the collections of the National Portrait Gallery, London, and the Society of Antiquaries. Due to previous conservation treatments of the painting, the conventional transmission X-radiograph image was difficult to interpret. As a result, the painting underwent high-definition X-ray fluorescence (XRF) elemental mapping on the X-ray fluorescence microscopy beamline of the Australian Synchrotron. Scans were conducted at 12.6 and 18.5 keV, below and above the lead (Pb) L edges, respectively. Typical scan parameters were 120 μm pixel size at 7 ms dwell time, with the largest scan covering an area 545 × 287 mm2 collected in 23 h (10.8 MP). XRF mapping of the panel has guided the conservation treatment of the painting and the revelation of previously obscured features. It has also provided insight into the process of making of the painting. The informative and detailed elemental maps, alongside ultra-high-definition scans of the painting undertaken before and after varnish and over-paint removal, have assisted in comparison of the finely painted details with the London paintings. The resolution offered by the combination of imaging techniques identifies pigment distribution at an extremely fine scale, enabling a new understanding of the artist's paint application.

  13. ASME Section VIII Recertification of a 33,000 Gallon Vacuum-jacketed LH2 Storage Vessel for Densified Hydrogen Testing at NASA Kennedy Space Center

    NASA Technical Reports Server (NTRS)

    Swanger, Adam M.; Notardonato, William U.; Jumper, Kevin M.

    2015-01-01

    The Ground Operations Demonstration Unit for Liquid Hydrogen (GODU-LH2) has been developed at NASA Kennedy Space Center in Florida. GODU-LH2 has three main objectives: zero-loss storage and transfer, liquefaction, and densification of liquid hydrogen. A cryogenic refrigerator has been integrated into an existing, previously certified, 33,000 gallon vacuum-jacketed storage vessel built by Minnesota Valley Engineering in 1991 for the Titan program. The dewar has an inner diameter of 9.5 and a length of 71.5; original design temperature and pressure ranges are -423 F to 100 F and 0 to 95 psig respectively. During densification operations the liquid temperature will be decreased below the normal boiling point by the refrigerator, and consequently the pressure inside the inner vessel will be sub-atmospheric. These new operational conditions rendered the original certification invalid, so an effort was undertaken to recertify the tank to the new pressure and temperature requirements (-12.7 to 95 psig and -433 F to 100 F respectively) per ASME Boiler and Pressure Vessel Code, Section VIII, Division 1. This paper will discuss the unique design, analysis and implementation issues encountered during the vessel recertification process.

  14. A probable case of gigantism/acromegaly in skeletal remains from the Jewish necropolis of "Ronda Sur" (Lucena, Córdoba, Spain; VIII-XII centuries CE).

    PubMed

    Viciano, Joan; De Luca, Stefano; López-Lázaro, Sandra; Botella, Daniel; Diéguez-Ramírez, Juan Pablo

    2015-01-01

    Pituitary gigantism is a rare endocrine disorder caused by hypersecretion of growth hormone during growing period. Individuals with this disorder have an enormous growth in height and associated degenerative changes. The continued hypersecretion of growth hormone during adulthood leads to acromegaly, a condition related to the disproportionate bone growth of the skull, hands and feet. The skeletal remains studied belong to a young adult male from the Jewish necropolis of "Ronda Sur" in Lucena (Córdoba, Spain, VIII-XII centuries CE). The individual shows a very large and thick neurocranium, pronounced supraorbital ridges, an extremely prominent occipital protuberance, and an extremely large and massive mandible. Additional pathologies include enlargement of the vertebral bodies with degenerative changes, thickened ribs, and a slight increased length of the diaphysis with an increased cortical bone thickness of lower limbs. Comparative metric analysis of the mandible with other individuals from the same population and a contemporary Mediterranean population shows a trend toward acromegalic morphology. This case is an important contribution in paleopathological literature because it is a rare condition that has not been widely documented in ancient skeletal remains. PMID:25776010

  15. p- to n-type conductivity transition in 1.0 eV GaInNAs solar cells controlled by the V/III ratio

    SciTech Connect

    Langer, Fabian Perl, Svenja; Kamp, Martin; Höfling, Sven

    2015-02-09

    In this work, we report a p- to n-type conductivity transition of GaInNAs (1.0 eV bandgap) layers in p-i-n dilute nitride solar cells continuously controlled by the V/III ratio during growth. Near the transition region, we were able to produce GaInNAs layers with very low effective electrically active doping concentrations resulting in wide depleted areas. We obtained internal quantum efficiencies (IQEs) up to 85% at 0.2 eV above the bandgap. However, the high IQE comes along with an increased dark current density resulting in a decreased open circuit voltage of about 0.2 V. This indicates the formation of non-radiant defect centers related to the p-type to n-type transition. Rapid-thermal annealing of the solar cells on the one hand helps to anneal some of these defects but on the other hand increases the effective doping concentrations.

  16. p- to n-type conductivity transition in 1.0 eV GaInNAs solar cells controlled by the V/III ratio

    NASA Astrophysics Data System (ADS)

    Langer, Fabian; Perl, Svenja; Höfling, Sven; Kamp, Martin

    2015-02-01

    In this work, we report a p- to n-type conductivity transition of GaInNAs (1.0 eV bandgap) layers in p-i-n dilute nitride solar cells continuously controlled by the V/III ratio during growth. Near the transition region, we were able to produce GaInNAs layers with very low effective electrically active doping concentrations resulting in wide depleted areas. We obtained internal quantum efficiencies (IQEs) up to 85% at 0.2 eV above the bandgap. However, the high IQE comes along with an increased dark current density resulting in a decreased open circuit voltage of about 0.2 V. This indicates the formation of non-radiant defect centers related to the p-type to n-type transition. Rapid-thermal annealing of the solar cells on the one hand helps to anneal some of these defects but on the other hand increases the effective doping concentrations.

  17. Coagulation disorders in severely and critically injured patients.

    PubMed

    Avikainen, V

    1977-01-01

    Forty-five patients with multiple injuries treated at an intensive care unit were studied prospectively. The patients were divided into two groups: the severely injured (no mortality) and critically injured (56% mortality). Treatment was started within two hours from the accident in all cases. The following coagulation parameters were measured for eight days: euglobulin lysis time (ELT), thromboelastography (TEG), vecalcification time (RECA), partial thromboplastin time (PTT), factor V, factor VIII, Normotest, Thrombotest, thrombin time, fibrinogen and platelets. Severe coagulation disorders were observed in one-third of the patients 12-48 hours after trauma. The abnormalities were more pronounced in patients who had sustained very severe injuries and arrived in a state of shock. The ELT was shortened 0-6 hours after the accident and accelerated coagulation was indicated simultaneously by decreased PTT, RECA, and r-values as well as by elevated Thrombotest and factor VIII values. The factor V and fibrinogen levels were initially lowered. Low platelet values at 2-4 days, prolonged thrombin and r-times, secondary decrease of fibrinogen FV, FVIII, and low Thrombotest values suggested disseminated intravascular coagulation associated with complications, such as fat embolism and "shock lung" syndromes. General bleeding tendency with high mortality was observed in 16% of the patients. PMID:603216

  18. Formation of tissue factor activity following incubation of recombinant human tissue factor apoprotein with plasma lipoproteins

    SciTech Connect

    Sakai, T.; Kisiel, W. )

    1990-11-01

    Incubation of recombinant human tissue factor apoprotein (Apo-TF) with human plasma decreased the recalcified clotting time of this plasma in a time-and dose-dependent manner suggesting relipidation of the Apo-TF by plasma lipoproteins. Incubation of Apo-TF with purified preparations of human very low density, low density and high density lipoproteins resulted in tissue factor activity in a clotting assay. The order of effectiveness was VLDL greater than LDL much greater than HDL. Tissue factor activity generated by incubation of a fixed amount of Apo-TF with plasma lipoproteins was lipoprotein concentration-dependent and saturable. The association of Apo-TF with lipoprotein particles was supported by gel filtration studies in which {sup 125}I-Apo-TF coeluted with the plasma lipoprotein in the void volume of a Superose 6 column in the presence and absence of calcium ions. In addition, void-volume Apo-TF-lipoprotein fractions exhibited tissue factor activity. These results suggest that the factor VIII-bypassing activity of bovine Apo-TF observed in a canine hemophilic model may be due, in part, to its association with plasma lipoproteins and expression of functional tissue factor activity.

  19. Evaluation of Consequences of Dust Positioned in Southwest of Iran on Coagulant Factors

    PubMed Central

    Saeb, Keivan; Sarizade, Gholamreza; Khodadi, Mohammad; Biazar, Esmaeil

    2013-01-01

    Background: Various regions in Iran, especially the Khuzestan Province, have been covered by dust and dirt during the past two years due to environmental changes in the Middle East. We sought to evaluate the effect of these pollutants on the coagulant factors of people residing in Abadan and Khoramshahr, two major cities of Khuzestan Province. Methods: One hundred twenty-nine healthy individuals were enrolled into this study, and their prothrombin time as well as fibrinogen, platelet, and Factor VIII levels were measured before and after climate changes. Results: After climate changes, the mean prothrombin time decreased, while the fibrinogen, platelet, and Factor VIII levels rose. Conclusion: The results of this study suggest that the pollutants deployed in the Middle East can affect prothrombin time as well as fibrinogen, platelet, and Factor VII levels considerably and increase coagulant state. The pollutants can, consequently, increase the risk of cardiovascular diseases. It seems that cooperation at government levels between Iran and its neighboring countries is required to reverse desertification and avoid inaccurate usage of subterranean water resources so as to lessen air pollution. PMID:23825886

  20. The Panchromatic Hubble Andromeda Treasury. VIII. A Wide-area, High-resolution Map of Dust Extinction in M31

    NASA Astrophysics Data System (ADS)

    Dalcanton, Julianne J.; Fouesneau, Morgan; Hogg, David W.; Lang, Dustin; Leroy, Adam K.; Gordon, Karl D.; Sandstrom, Karin; Weisz, Daniel R.; Williams, Benjamin F.; Bell, Eric F.; Dong, Hui; Gilbert, Karoline M.; Gouliermis, Dimitrios A.; Guhathakurta, Puragra; Lauer, Tod R.; Schruba, Andreas; Seth, Anil C.; Skillman, Evan D.

    2015-11-01

    We map the distribution of dust in M31 at 25 pc resolution using stellar photometry from the Panchromatic Hubble Andromeda Treasury survey. The map is derived with a new technique that models the near-infrared color-magnitude diagram (CMD) of red giant branch (RGB) stars. The model CMDs combine an unreddened foreground of RGB stars with a reddened background population viewed through a log-normal column density distribution of dust. Fits to the model constrain the median extinction, the width of the extinction distribution, and the fraction of reddened stars in each 25 pc cell. The resulting extinction map has a factor of ≳4 times better resolution than maps of dust emission, while providing a more direct measurement of the dust column. There is superb morphological agreement between the new map and maps of the extinction inferred from dust emission by Draine et al. However, the widely used Draine & Li dust models overpredict the observed extinction by a factor of ˜2.5, suggesting that M31's true dust mass is lower and that dust grains are significantly more emissive than assumed in Draine et al. The observed factor of ˜2.5 discrepancy is consistent with similar findings in the Milky Way by the Plank Collaboration et al., but we find a more complex dependence on parameters from the Draine & Li dust models. We also show that the the discrepancy with the Draine et al. map is lowest where the current interstellar radiation field has a harder spectrum than average. We discuss possible improvements to the CMD dust mapping technique, and explore further applications in both M31 and other galaxies.

  1. Ionizing radiation and genetic risks. VIII. The concept of mutation component and its use in risk estimation for multifactorial diseases.

    PubMed

    Denniston, C; Chakraborty, R; Sankaranarayanan, K

    1998-08-31

    Multifactorial diseases, which include the common congenital abnormalities (incidence: 6%) and chronic diseases with onset predominantly in adults (population prevalence: 65%), contribute substantially to human morbidity and mortality. Their transmission patterns do not conform to Mendelian expectations. The model most frequently used to explain their inheritance and to estimate risks to relatives is a Multifactorial Threshold Model (MTM) of disease liability. The MTM assumes that: (i) the disease is due to the joint action of a large number of genetic and environmental factors, each of which contributing a small amount of liability, (ii) the distribution of liability in the population is Gaussian and (iii) individuals whose liability exceeds a certain threshold value are affected by the disease. For most of these diseases, the number of genes involved or the environmental factors are not fully known. In the context of radiation exposures of the population, the question of the extent to which induced mutations will cause an increase in the frequencies of these diseases has remained unanswered. In this paper, we address this problem by using a modified version of MTM which incorporates mutation and selection as two additional parameters. The model assumes a finite number of gene loci and threshold of liability (hence, the designation, Finite-Locus Threshold Model or FLTM). The FLTM permits one to examine the relationship between broad-sense heritability of disease liability and mutation component (MC), the responsiveness of the disease to a change in mutation rate. Through the use of a computer program (in which mutation rate, selection, threshold, recombination rate and environmental variance are input parameters and MC and heritability of liability are output estimates), we studied the MC-heritability relationship for (i) a permanent increase in mutation rate (e.g., when the population sustains radiation exposure in every generation) and (ii) a one-time increase

  2. Photometric properties for selected algol-type binaries. VIII. The triple systems DI Peg and AF Gem revisited

    SciTech Connect

    Yang, Yuan-Gui; Li, Shu-Zheng; Yang, Ying E-mail: yangyg@chnu.edu.cn

    2014-06-01

    New extensive photometry for two triple binary stars, DI Peg and AF Gem, was performed from 2012 October to 2013 January, with two small telescopes at Xinglong station (XLs) of NAOC. From new multi-color observations and previously published ones in literature, the photometric models were (re)deduced using the updated Wilson-Devinney code. The results indicated that the low third lights exist in two classic Algol-type binaries, whose fill-out factors for the more massive components are f{sub p} = 78.2(± 0.4)% for DI Peg, and f{sub p} = 69.0(± 0.3)% for AF Gem, respectively. Through analyzing the O–C curves, the orbital periods for two binaries change in the complicated mode. The period of DI Peg possibly appears to show two light-time orbits, whose modulated periods are P {sub 3} = 54.6(± 0.5) yr and P {sub 4} = 23.0(± 0.6) yr, respectively. The inferred minimum masses for the inner and outer sub-stellar companions are M {sub in} = 0.095 M {sub ☉} and M {sub out} = 0.170 M {sub ☉}, respectively. Therefore, DI Peg may be a quadruple star. The orbital period of AF Gem appears to show a continuous period decrease or a cyclic variation; the latter may be preferable. The cyclic oscillation, with a period of 120.3(± 2.5) yr, may be attributed to the light-time effect due to the third body. This kind of additional companion may extract angular momentum from the central system, which may play a key role in the evolution of the binary.

  3. Photometric Properties for Selected Algol-type Binaries. VIII. The Triple Systems DI Peg and AF Gem Revisited

    NASA Astrophysics Data System (ADS)

    Yang, Yuan-Gui; Yang, Ying; Li, Shu-Zheng

    2014-06-01

    New extensive photometry for two triple binary stars, DI Peg and AF Gem, was performed from 2012 October to 2013 January, with two small telescopes at Xinglong station (XLs) of NAOC. From new multi-color observations and previously published ones in literature, the photometric models were (re)deduced using the updated Wilson-Devinney code. The results indicated that the low third lights exist in two classic Algol-type binaries, whose fill-out factors for the more massive components are fp = 78.2(± 0.4)% for DI Peg, and fp = 69.0(± 0.3)% for AF Gem, respectively. Through analyzing the O-C curves, the orbital periods for two binaries change in the complicated mode. The period of DI Peg possibly appears to show two light-time orbits, whose modulated periods are P 3 = 54.6(± 0.5) yr and P 4 = 23.0(± 0.6) yr, respectively. The inferred minimum masses for the inner and outer sub-stellar companions are M in = 0.095 M ⊙ and M out = 0.170 M ⊙, respectively. Therefore, DI Peg may be a quadruple star. The orbital period of AF Gem appears to show a continuous period decrease or a cyclic variation; the latter may be preferable. The cyclic oscillation, with a period of 120.3(± 2.5) yr, may be attributed to the light-time effect due to the third body. This kind of additional companion may extract angular momentum from the central system, which may play a key role in the evolution of the binary.

  4. Anatomy of the AGN in NGC 5548. VIII. XMM-Newton's EPIC detailed view of an unexpected variable multilayer absorber

    NASA Astrophysics Data System (ADS)

    Cappi, M.; De Marco, B.; Ponti, G.; Ursini, F.; Petrucci, P.-O.; Bianchi, S.; Kaastra, J. S.; Kriss, G. A.; Mehdipour, M.; Whewell, M.; Arav, N.; Behar, E.; Boissay, R.; Branduardi-Raymont, G.; Costantini, E.; Ebrero, J.; Di Gesu, L.; Harrison, F. A.; Kaspi, S.; Matt, G.; Paltani, S.; Peterson, B. M.; Steenbrugge, K. C.; Walton, D. J.

    2016-07-01

    In 2013, we conducted a large multi-wavelength campaign on the archetypical Seyfert 1 galaxy NGC 5548. Unexpectedly, this usually unobscured source appeared strongly absorbed in the soft X-rays during the entire campaign, and signatures of new and strong outflows were present in the almost simultaneous UV HST/COS data. Here we carry out a comprehensive spectral analysis of all available XMM-Newton observations of NGC 5548 (precisely 14 observations from our campaign plus three from the archive, for a total of ~763 ks) in combination with three simultaneous NuSTAR observations. We obtain a best-fit underlying continuum model composed by i) a weakly varying flat (Γ ~ 1.5-1.7) power-law component; ii) a constant, cold reflection (FeK + continuum) component; iii) a soft excess, possibly owing to thermal Comptonization; and iv) a constant, ionized scattered emission-line dominated component. Our main findings are that, during the 2013 campaign, the first three of these components appear to be partially covered by a heavy and variable obscurer that is located along the line of sight (LOS), which is consistent with a multilayer of cold and mildly ionized gas. We characterize in detail the short timescale (mostly ~ks-to-days) spectral variability of this new obscurer, and find it is mostly due to a combination of column density and covering factor variations, on top of intrinsic power-law (flux and slope) variations. In addition, our best-fit spectrum is left with several (but marginal) absorption features at rest-frame energies ~6.7-6.9 keV and ~8 keV, aswell as a weak broad emission line feature redwards of the 6.4 keV emission line. These could indicate a more complex underlying model, e.g. a P-Cygni-type emission profile if we allow for a large velocity and wide-angle outflow. These findings are consistent with a picture where the obscurer represents the manifestation along the LOS of a multilayer of gas, which is also in multiphase, and which is likely outflowing at

  5. Glucose-regulated protein 78 may play a crucial role in promoting the pulmonary microvascular remodeling in a rat model of hepatopulmonary syndrome

    PubMed Central

    Zhang, Huiying; Lv, Minli; Zhao, Zhongfu; Jia, Jiantao; Zhang, Lili; Xiao, Peng; Wang, Limin; Li, Chen; Ji, Jingquan; Tian, Xiaoxia; Li, Xujiong; Fan, Yimin; Lai, Lina; Liu, Yan; Li, Baohong; Zhang, Cuiying; Liu, Mingshe; Guo, Jianhong; Han, Dewu; Ji, Cheng

    2015-01-01

    Objective This study is to investigate the role of glucose-regulated protein 78 (GRP78) in the pulmonary microvascular remodeling during hepatopulmonary syndrome (HPS) development. Methods The rat models with liver cirrhosis and HPS were induced by multiple pathogenic factors for 4 to 8 wk. The concentrations of alanine transferase (ALT) and endotoxin in plasma were detected in the models, followed by the detection of GRP78 expression. RT-PCR, quantitative real-time PCR and Western blotting were employed to assess the mRNA and protein expression levels of vascular endothelial growth factor (VEGF), respectively. Immunohistochemistry staining was used to examine the expression of a specific vascular marker, factor VIII-related antigen (FVIII-RAg), and several cell proliferation- and apoptosis-related proteins, including CHOP/GADD153, caspase-12, Bcl-2 and nuclear factor (NF)-κB. Results The levels of endotoxin and ALT in plasma were gradually increased as the disease progressed, so did GRP78, which were in a positive correlation. The expression levels of VEGF (both mRNA and protein) and FVIII-RAg were significantly elevated in the HPS models, indicating active angiogenesis, which was also positively correlated with GRP78 expression. Furthermore, the expression levels of the pro-apoptotic proteins of CHOP/GADD153 and caspase-12 were dramatically decreased, while the anti-apoptotic proteins of Bcl-2 and NF-κB were significantly elevated, in the HPS models. There were also close correlations between these proteins and GRP78. Conclusions Over-expression of GRP78 in lungs may be the critical pathogenic factor for HPS. Through promoting cell proliferation and survival and inhibiting apoptosis, GRP78 may promote the pulmonary microvascular remodeling in HPS pathogenesis. Our results provide a potential therapeutic target for clinical prevention and treatment for HPS and related complications. PMID:24768185

  6. Testosterone, anastrozole, factor V Leiden heterozygosity and osteonecrosis of the jaws.

    PubMed

    Pandit, Ramesh S; Glueck, Charles J

    2014-04-01

    Our specific aim is to describe the development of thrombotic osteonecrosis of the jaws after testosterone-anastrozole therapy in a 55-year-old white man subsequently found to have previously undiagnosed factor V Leiden heterozygosity. Before the diagnosis of V Leiden heterozygosity, he was given testosterone gel, 50 mg/day, and on testosterone, serum testosterone (963 ng/dl) and estradiol were high (50 pg/ml). Anastrozole was started, and testosterone was continued. Six months later, osteonecrosis of the jaws was diagnosed. Exogenous testosterone is aromatized to estradiol and estradiol-induced thrombophilia, when superimposed on underlying familial thrombophilia, as in this case, may lead to thrombosis and osteonecrosis. We recommend that before giving testosterone, at a minimum, screening for the factor V Leiden and G20210A mutations, and factor VIII and XI activity be carried out, to avoid unanticipated thrombosis. PMID:24674881

  7. [Strongyloidosis. Part VIII. Parasitological diagnosis].

    PubMed

    Soroczan, Wiesław

    2002-01-01

    The effectiveness and safety of the methods of detecting Strongyloides stercoralis, by passing larvae from the faeces to water, in duodenal fluid (duodenal intubation, Enterotest), in sputum and other body fluids, have been estimated. The author recommend Baermann technique for detecting S. stercoralis in individual examinations and Dancescu technique in mass field examinations. The detection of S. stercoralis larvae by the two methods ought to be checked by Fülleborn agar Petri dish technique in order to identify parasite to the species level. PMID:16888939

  8. Physiological levels of blood coagulation factors IX and X control coagulation kinetics in an in vitro model of circulating tissue factor

    NASA Astrophysics Data System (ADS)

    Tormoen, Garth W.; Khader, Ayesha; Gruber, András; McCarty, Owen J. T.

    2013-06-01

    Thrombosis significantly contributes to cancer morbidity and mortality. The mechanism behind thrombosis in cancer may be circulating tissue factor (TF), as levels of circulating TF are associated with thrombosis. However, circulating TF antigen level alone has failed to predict thrombosis in patients with cancer. We hypothesize that coagulation factor levels regulate the kinetics of circulating TF-induced thrombosis. Coagulation kinetics were measured as a function of individual coagulation factor levels and TF particle concentration. Clotting times increased when pooled plasma was mixed at or above a ratio of 4:6 with PBS. Clotting times increased when pooled plasma was mixed at or above a ratio of 8:2 with factor VII-depleted plasma, 7:3 with factor IX- or factor X-depleted plasmas, or 2:8 with factor II-, V- or VIII-depleted plasmas. Addition of coagulation factors VII, X, IX, V and II to depleted plasmas shortened clotting and enzyme initiation times, and increased enzyme generation rates in a concentration-dependent manner. Only additions of factors IX and X from low-normal to high-normal levels shortened clotting times and increased enzyme generation rates. Our results demonstrate that coagulation kinetics for TF particles are controlled by factor IX and X levels within the normal physiological range. We hypothesize that individual patient factor IX and X levels may be prognostic for susceptibility to circulating TF-induced thrombosis.

  9. Laboratory tests used to help diagnose von Willebrand disease: an update.

    PubMed

    Favaloro, Emmanuel J; Pasalic, Leonardo; Curnow, Jennifer

    2016-06-01

    von Willebrand disease (VWD) is due to quantitative deficiencies and/or qualitative defects in von Willebrand factor (VWF), and is reportedly the most common inherited bleeding disorder. However, diagnosis of VWD is problematic, and is subject to over-, under-, and misdiagnosis. This is due to many factors, including limitations in current test procedures and an over-reliance on these imperfect test systems for clinical diagnosis. VWF is a complex plasma protein with multiple functions, but essentially acts to assist in the formation of a platelet thrombus to stop blood loss from sites of injury. VWF achieves this by several activities, including binding to platelets [primarily through the glycoprotein Ib (GPIb) receptor], binding to subendothelial matrix components (primarily collagen), and binding to factor VIII (FVIII), thus protecting FVIII from degradation and enabling its delivery to sites of vascular injury. Laboratory assessment of VWD entails performance of a battery of tests, some of which aim to mimic in vivo VWF activity. VWD is classified into six separate types, based on quantitative deficiencies [types 1 (partial deficiency) and 3 (total deficiency)] of VWF, or qualitative defects (type 2 VWD), which comprise four 'subtypes'. The current report briefly overviews the diagnosis of VWD, describing the currently available armamentarium of laboratory tests, as well as emerging options for laboratory-assisted diagnostics. Although some methodologies suffer from significant limitations that challenge the accurate diagnosis of VWD, newer methodologies and specific approaches can improve detection of this common bleeding disorder, and the appropriate characterisation and typing of patients. PMID:27131932

  10. Reauthorization of the Higher Education Act, Title II (College Libraries); Title VI (International Education); Title VIII (Cooperative Education); Title X (F.I.P.S.E.); Title XI (Urban Grant Universities), Volume 8. Hearings before the Subcommittee on Postsecondary Education of the Committee on Education and Labor. House of Representatives, Ninety-Ninth Congress, First Session (September 6 and 10, 1985).

    ERIC Educational Resources Information Center

    Congress of the U.S., Washington, DC. House Committee on Education and Labor.

    Hearings on reauthorization of Titles II, VI, VIII, X, and XI of the Higher Education Act are presented. For Title II, the college library programs, Congress seeks acceptable criteria for providing library aid based on need and to determine needs for traineeships and fellowships for professional and paraprofessional librarians. Considerations for…

  11. Inhibitor development in non-severe haemophilia across Europe.

    PubMed

    Fischer, Kathelijn; Iorio, Alfonso; Lassila, Riitta; Peyvandi, Flora; Calizzani, Gabriele; Gatt, Alex; Lambert, Thierry; Windyga, Jerzy; Gilman, Estelle A; Hollingsworth, R; Makris, Michael

    2015-10-01

    Evidence about inhibitor formation in non-severe haemophilia and the potential role for clotting factor concentrate type is scant. It was the aim of this study to report inhibitor development in non-severe haemophilia patients enrolled in the European Haemophilia Safety Surveillance (EUHASS) study. Inhibitors are reported quarterly and total treated patients annually. Incidence rates and 95% confidence intervals (95% CI) were calculated according to diagnosis and concentrate used. Between 1-10-2008 and 31-12-2012, 68 centres reported on 7,969 patients with non-severe haemophilia A and 1,863 patients with non-severe haemophilia B. For haemophilia A, 37 inhibitors occurred in 8,622 treatment years, resulting in an inhibitor rate of 0.43/100 treatment years (95% CI 0.30-0.59). Inhibitors occurred at a median age of 35 years, after a median of 38 exposure days (EDs; P25-P75: 20-80); with 72% occurring within the first 50 EDs. In haemophilia B, one inhibitor was detected in 2,149 treatment years, resulting in an inhibitor rate of 0.05/100 years (95% CI 0.001-0.26). This inhibitor developed at the age of six years, after six EDs. The rate of inhibitors appeared similar across recombinant and plasma derived factor VIII (FVIII) concentrates. Rates for individual concentrates could not be calculated at this stage due to low number of events. In conclusion, inhibitors in non-severe haemophilia occur three times more frequently than in previously treated patients with severe haemophilia at a rate of 0.43/100 patient years (haemophilia A) and 0.05/100 years (haemophilia B). Although the majority of inhibitors developed in the first 50 EDs, inhibitor development continued with increasing exposure to FVIII. PMID:26293381

  12. Whole-exome sequencing to identify genetic risk variants underlying inhibitor development in severe hemophilia A patients.

    PubMed

    Gorski, Marcin M; Blighe, Kevin; Lotta, Luca A; Pappalardo, Emanuela; Garagiola, Isabella; Mancini, Ilaria; Mancuso, Maria Elisa; Fasulo, Maria Rosaria; Santagostino, Elena; Peyvandi, Flora

    2016-06-01

    The development of neutralizing antibodies (inhibitors) against coagulation factor VIII (FVIII) is the most problematic and costly complication of FVIII replacement therapy that affects up to 30% of previously untreated patients with severe hemophilia A. The development of inhibitors is a multifactorial complication involving environmental and genetic factors. Among the latter, F8 gene mutations, ethnicity, family history of inhibitors, and polymorphisms affecting genes involved in the immune response have been previously investigated. To identify novel genetic elements underling the risk of inhibitor development in patients with severe hemophilia A, we applied whole-exome sequencing (WES) and data analysis in a selected group of 26 Italian patients with (n = 17) and without (n = 9) inhibitors. WES revealed several rare, damaging variants in immunoregulatory genes as novel candidate mutations. A case-control association analysis using Cochran-Armitage and Fisher's exact statistical tests identified 1364 statistically significant variants. Hierarchical clustering of these genetic variants showed 2 distinct patterns of homozygous variants with a protective or harmful role in inhibitor development. When looking solely at coding variants, a total of 28 nonsynonymous variants were identified and replicated in 53 inhibitor-positive and 174 inhibitor-negative Italian severe hemophilia A patients using a TaqMan genotyping assay. The genotyping results revealed 10 variants showing estimated odds ratios in the same direction as in the discovery phase and confirmed the association of the rs3754689 missense variant (OR 0.58; 95% CI 0.36-0.94; P = .028) in a highly conserved haplotype region surrounding the LCT locus on chromosome 2q21 with inhibitor development. PMID:27060170

  13. Cancers in Patients with von Willebrand Disease: A Survey from the Italian Association of Haemophilia Centres.

    PubMed

    Franchini, Massimo; Di Perna, Caterina; Santoro, Cristina; Castaman, Giancarlo; Siboni, Simona Maria; Zanon, Ezio; Linari, Silvia; Gresele, Paolo; Pasca, Samantha; Coppola, Antonio; Santoro, Rita; Napolitano, Mariasanta; Ranalli, Paola; Tagliaferri, Annarita

    2016-02-01

    Besides its essential role in hemostasis, there is growing evidence that von Willebrand factor (VWF) has an additional antitumor effect. To elucidate the clinical significance of this biological activity we conducted a retrospective study on cancers among Italian patients with von Willebrand disease (VWD) on behalf of the Italian Association of Haemophilia Centres (AICE). A questionnaire to collect demographic, clinical, and treatment data of VWD patients with cancer was sent to all the 54 Italian Haemophilia Treatment Centres (HTCs) members of AICE. Overall, 18 HTCs (33%) provided information on 92 VWD patients (61 alive and 31 deceased) with 106 cancers collected during the period 1981 to 2014. Of them, 19 (18%) were hematological cancers and 87 (82%) were solid cancers. A total of 61% of patients had type 1, 36% type 2 (12% type 2A, 14% type 2B, 9% type 2M, and 1% type 2N), and 3% type 3 VWD: this distribution was significantly different from that observed in the whole VWD population (79% type 1, 16% type 2 [8% type 2A, 4% type 2B, 2% type 2M, 2% type 2N], and 5% type 3; type 2 vs. non-type 2: p < 0.001). Overall, VWD patients with cancer underwent 52 invasive and 72 surgical procedures, were treated with VWF/factor VIII (FVIII) concentrates in 77 cases, with desmopressin (DDAVP) alone in 24 cases and with DDAVP and VWF/FVIII concentrates in 7 cases. Hemorrhagic complications were observed only rarely (2% of invasive procedures and radiotherapy and 6% of surgical interventions). The data collected by this survey document that a substantial number of cancers are recorded among VWD patients and that these patients are safely managed by HTC physicians through a multidisciplinary approach. PMID:26595151

  14. Orthopaedic surgery in patients with von Willebrand disease.

    PubMed

    Siboni, S M; Biguzzi, E; Solimeno, L P; Pasta, G; Mistretta, C; Mannucci, P M; Peyvandi, F

    2014-01-01

    Patients with von Willebrand disease (VWD) may need orthopaedic surgery because of disabling chronic arthropathy due to recurrent joint bleeding. They may also require this surgery independently of their haemostasis disorder. Knowledge regarding the management of orthopaedic surgery in VWD is limited. Description of management of orthopaedic surgery in patients with VWD, based upon retrospective data collection and analysis of 32 orthopaedic procedures carried out over a period of 33 years in 23 patients was the aim of this study. Of 32 procedures, six were minor (three hand surgery, one foot surgery, two others) and 26 were major (seven joint replacements, nine arthroscopic procedures, two foot surgery, eight others). Twenty-two procedures were performed using replacement therapy with plasma-derived concentrates containing both factor VIII (FVIII) and von Willebrand factor (VWF). Two procedures in patients with acquired von Willebrand syndrome (AWVS) were performed using FVIII-VWF concentrates associated with intravenous immunoglobulins, or desmopressin plus tranexamic acid. Seven procedures were performed using desmopressin alone and one using intravenous immunoglobulins in AVWS. Bleeding complications occurred in seven procedures (22%). In one patient, an anti-VWF antibody was diagnosed after surgery. Anticoagulant prophylaxis of venous thromboembolism was implemented in four cases only and in two instances there was excessive bleeding. In conclusion, control of surgical haemostasis was achieved in most patients with VWD undergoing orthopaedic surgery. The control of haemostasis combined with an adequate surgical technique and early post-operative rehabilitation are warranted for the successful performance of orthopaedic surgery in VWD, which requires the involvement of specialized haemophilia centres. PMID:23992395

  15. Evaluation of the Q analyzer, a new cap-piercing fully automated coagulometer with clotting, chromogenic, and immunoturbidometric capability.

    PubMed

    Kitchen, Steve; Woolley, Anita

    2013-01-01

    The Q analyzer is a recently launched fully automated photo-optical analyzer equipped with primary tube cap-piercing and capable of clotting, chromogenic, and immunoturbidometric tests. The purpose of the present study was to evaluate the performance characteristics of the Q analyzer with reagents from the instrument manufacturer. We assessed precision and throughput when performing coagulation screening tests, prothrombin time (PT)/international normalized ratio (INR), activated partial thromboplastin time (APTT), and fibrinogen assay by Clauss assay. We compared results with established reagent instrument combinations in widespread use. Precision of PT/INR and APTT was acceptable as indicated by total precision of around 3%. The time to first result was 3  min for an INR and 5  min for PT/APTT. The system produced 115 completed samples per hour when processing only INRs and 60 samples (120 results) per hour for PT/APTT combined. The sensitivity of the DG-APTT Synth/Q method to mild deficiency of factor VIII (FVIII), IX, and XI was excellent (as indicated by APTTs being prolonged above the upper limit of the reference range). The Q analyzer was associated with high precision, acceptable throughput, and good reliability. When used in combination with DG-PT reagent and manufacturer's instrument-specific international sensitivity index, the INRs obtained were accurate. The Q analyzer with DG-APTT Synth reagent demonstrated good sensitivity to isolated mild deficiency of FVIII, IX, and XI and had the advantage of relative insensitivity to mild FXII deficiency. Taken together, our data indicate that the Q hemostasis analyzer was suitable for routine use in combination with the reagents evaluated. PMID:23249565